MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Journal of Biochemistry' source. Wed, 17 Mar 2010 16:27:12 +0100 http://www.medworm.com/index.php?rid=3287147&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F445%3Frss%3D1 Human SALL1 is a homologue of the Drosophila region-specific homeotic gene sal, and is also known as a causative gene for Townes–Brocks syndrome, which is characterized by multi-organ malformations. We previously demonstrated that mouse Sall1 plays a crucial role in ureteric bud invasion during kidney development, and possibly in nephron progenitor cells in the metanephric mesenchyme. To gain insights into the Sall1 functions in the kidney and other tissues, we generated R26Sall1 mice, in which Rosa26 locus stop sequences flanked by two loxP sites were located upstream of the Sall1 cDNA. This allele allowed exogenous Sall1 expression in a Cre recombinase-dependent manner. R26Sall1 mice were first crossed with CAGCre mice, which expressed Cre recombinase ubiquitously during embryogene... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287147 Fri, 19 Feb 2010 07:29:26 +0100 3287147 http://www.medworm.com/index.php?rid=3287146&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F433%3Frss%3D1 In this study, a disturbing nucleotide sequence similar to a Shine–Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells. (Source: Journ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287146 Fri, 19 Feb 2010 07:29:26 +0100 3287146 http://www.medworm.com/index.php?rid=3287145&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F427%3Frss%3D1 We examined many stabilizers and found that 60% of a concentrated (2.5 mg/ml) unstable protein can be refolded using 40% glycerol as the best stabilizer. This procedure can be widely applicable for the refolding of unstable proteins. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287145 Fri, 19 Feb 2010 07:29:26 +0100 3287145 http://www.medworm.com/index.php?rid=3287144&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F415%3Frss%3D1 The accumulation of classical cadherins is essential for their function, but the mechanism is poorly understood. Hence, we investigated the accumulation of E- and N-cadherin and the formation of cell junctions in epithelial cells. Immunostaining revealed a scattered dot-like accumulation of E- and N-cadherin throughout the lateral membrane in MDCK II and other epithelial cells. Mutant E-cadherin lacking the β-catenin binding site accumulated granularly at cell–cell contact sites and showed weak cell aggregation activity in cadherin-deficient epithelial cells, MIA PaCa2 cells. Mutant E-cadherin lacking the p120-catenin binding site exhibited scattered punctate accumulation and strong cell adhesion activity in MIA PaCa2 cells. Electron microscopy demonstrated that MIA PaCa2 transf... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287144 Fri, 19 Feb 2010 07:29:26 +0100 3287144 http://www.medworm.com/index.php?rid=3287143&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F405%3Frss%3D1 The inhibition of tau fibrillation is a potential therapeutic target for Alzheimer’s and other neurodegenerative diseases. As a series of studies on inhibiting the transition of soluble monomeric tau into mature fibril, the effect of Tyr310 residue in the third repeat (R3) of the microtubule-binding domain (MBD) on the assembly of MBD was investigated using Tyr-substituted MBD mutants by fluorescence, circular dichroism spectroscopy and electron microscopy. Consequently, the importance of the Tyr residue located at position 310, not at other positions, was clearly shown. The conformational comparison of the Tyr310Ala-substituted R3 repeat peptide with the unsubstituted one showed that the Tyr residue contributes to the rigid extended structure of the N-terminal V306QIVYK311 sequence,... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287143 Fri, 19 Feb 2010 07:29:26 +0100 3287143 http://www.medworm.com/index.php?rid=3287142&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F393%3Frss%3D1 To identify functionally essential sequences and residues of CTLA-2, in vitro mutagenesis was carried out. The coefficient of inhibition (Ki) was determined towards rabbit cathepsin L using Z-Phe-Arg-MCA as the substrate. Recombinant CTLA-2 inhibited the enzyme potently (Ki = 15 nM). A truncated mutant, lacking the N- and C-terminal Ala1–Asp9 and Leu80–Glu109 regions, was also a potent inhibitor (Ki = 10 nM). Subsequent short deletions in the central region (Asn10–Ser79) showed three functionally essential distinct regions: Asn10–Phe19, His30–Ala44 and Ser55–Ser79. These regions cover sequences corresponding to three helices (1, 2 and 3) and sequences that interact with the cognate enzyme. Alanine scanning showed that replacement of one of three conserve... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287142 Fri, 19 Feb 2010 07:29:26 +0100 3287142 http://www.medworm.com/index.php?rid=3287141&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F381%3Frss%3D1 The Er(III) complexes are prepared from Er(NO3)3·6H2O and Schiff-base ligands derived from 8-hydroxyquinoline-2-carboxaldehyde with four aroylhydrazines, including benzoylhydrazine, 2-hydroxybenzoylhydrazine, 4-hydroxybenzoylhydrazine and isonicotinylhydrazine, respectively. X-ray crystal and other structural analyses indicate that Er(III) and every ligand can form a binuclear Er(III) complex with nine-coordination and 1: 1 metal-to-ligand stoichiometry at the Er(III) centre. All the Er(III) complexes can bind to calf thymus DNA through intercalation with the binding constants at the order of magnitude 106 M–1, and they may be used as potential anticancer drugs. All the Er(III) complexes have strong scavenging effects for hydroxyl radicals and superoxide radicals; however, com... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287141 Fri, 19 Feb 2010 07:29:26 +0100 3287141 http://www.medworm.com/index.php?rid=3287140&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F371%3Frss%3D1 Internal symmetry is a common feature of the tertiary structures of proteins and protein domains. Probably, because the genes of homo-oligomeric proteins duplicated and fused, their evolutionary descendants are proteins with internal symmetry. To identify any advantages that cause monomeric proteins with internal symmetry to be selected evolutionarily, we characterized some of the physical properties of a recombinant protein with a sequence consisting of two tandemly fused copies of the Escherichia coli Lac repressor C-terminal -helix. This polypeptide exists in solution mainly as dimer that likely maintains a four-helix bundle motif. Thermal unfolding experiments demonstrate that the protein is considerably more stable at elevated temperatures than is a homotetramer consisting of four non... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287140 Fri, 19 Feb 2010 07:29:26 +0100 3287140 http://www.medworm.com/index.php?rid=3287139&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F361%3Frss%3D1 The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a putative transcriptional regulator (Tk-Phr) that is orthologous to the Pyrococcus furiosus Phr (Pf-Phr). Pf-Phr, a transcriptional regulator, represses genes encoding the small heat shock protein (sHSP), AAA+ ATPase and Pf-Phr itself under normal growth temperatures. Here we constructed a gene disruption strain of Tk-Phr (strain KHR1). KHR1 cells showed similar specific growth rates with those of the wild-type strain under various temperatures. A whole genome microarray analysis was performed between KHR1 and wild-type cells grown at 80°C. Transcript levels of more than 20 genes were significantly higher in KHR1 cells. Most genes contained a sequence motif virtually identical to that of Pf-Phr in their 5'-flanking regi... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287139 Fri, 19 Feb 2010 07:29:26 +0100 3287139 http://www.medworm.com/index.php?rid=3287138&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F353%3Frss%3D1 Down-regulation of carcinogen detoxifying enzymes might be a critical factor in tumour formation by increasing the carcinogen concentration in the target organ. Previous reports revealed that the expression of UGT1A mRNA is either lost or decreased in certain human cancer tissues, including urinary bladder cancer. To elucidate this down-regulation mechanism, we used an N-nitrosobutyl (4-hydroxybutyl) amine (BBN)-induced mouse urinary bladder carcinogenesis model. Similar to human cancer, the expressions of Ugt1a6, Ugt1a9 and total Ugt1a mRNA in the BBN-induced bladder cancer were markedly decreased compared with those of normal mice. BBN down-regulated the basal Ugt1a mRNA expression in a time-dependent manner and this was reversible in the first 2 weeks of BBN treatment. However, after 4 ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287138 Fri, 19 Feb 2010 07:29:26 +0100 3287138 http://www.medworm.com/index.php?rid=3287137&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F345%3Frss%3D1 Catalases are essential components of the cellular equipment to cope with oxidative stress. Here we have purified a highly abundant catalase P of Paracoccidioides brasiliensis (PbCatP) that is preferentially expressed in the parasitic yeast phase. This oxidative stress-induced protein was isolated from yeast cells grown in the presence of 15 mM of hydrogen peroxide (H2O2). We have used consecutive steps of protein precipitation and gel filtration chromatography to achieve the purified protein. Protein purification was validated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and bioinformatics analysis. The purified enzyme showed strong similarity to small-subunit catalases. Like most monofunctional catalases, PbCatP is a homotetramer, resistant to inacti... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287137 Fri, 19 Feb 2010 07:29:26 +0100 3287137 http://www.medworm.com/index.php?rid=3287136&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F337%3Frss%3D1 The complex of protein O-mannosyltransferase 1 (POMT1) and POMT2 catalyzes the initial step of O-mannosyl glycan biosynthesis. The mutations in either POMT1 or POMT2 can lead to Walker–Warburg syndrome, a congenital muscular dystrophy with abnormal neuronal migration. Here, we used three algorithms for predicting transmembrane helices to construct the secondary structural models of human POMT1 and POMT2. In these models, POMT1 and POMT2 have seven- and nine-transmembrane helices and contain four and five potential N-glycosylation sites, respectively. To determine whether these sites are actually glycosylated, we prepared mutant proteins that were defective in each site by site-directed mutagenesis. Three of the POMT1 sites and all of the POMT2 sites were found to be N-glycosylated, s... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287136 Fri, 19 Feb 2010 07:29:26 +0100 3287136 http://www.medworm.com/index.php?rid=3287135&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F327%3Frss%3D1 This study demonstrated that the third FN type III repeat (FnIII3) and several fragments containing the repeat promote cell spreading and migration of human dermal fibroblasts (HDFs), whereas the fourth repeat (FnIII4) did not. A variety of cell types also spread on FnIII3 in a cell-type-specific manner, but not on FnIII4. Immunofluorescence assays revealed that FnIII3 induced the organization of focal contacts and stress fibres in HDFs. Cyclic [RGDFV] peptides with a D-Phe residue, which are selective inhibitors of cell adhesion to vitronectin, inhibited HDF spreading on FnIII3 equally with GRGDS, indicating little involvement of V-integrins in FnIII3 spreading. An anti-β1 integrin mAb inhibited cell spreading on FnIII3 and FN. To our knowledge, this is the first demonstration that a... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287135 Fri, 19 Feb 2010 07:29:25 +0100 3287135 http://www.medworm.com/index.php?rid=3287134&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F317%3Frss%3D1 Free haem is known to be toxic to organs, tissues and cells. It enhances permeability by binding to a cell membrane, which leads to cell death, and damages lipids, proteins and DNA through the generation of reactive oxygen species. Lysine- and arginine-specific gingipains (Kgp and RgpA/B) are major proteinases that play an important role in the pathogenicity of a black-pigmented periodontopathogen named Porphyromonas gingivalis. One of the adhesin domains of gingipain, HbR could bind haem as an iron nutrient source for P. gingivalis. Using erythrocyte and its membrane as a model, results from the present study demonstrate that recombinant HbR expressed in Escherichia coli could inhibit haem-induced haemolysis, probably through removing haem from the haem–membrane complex and lowering... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287134 Fri, 19 Feb 2010 07:29:25 +0100 3287134 http://www.medworm.com/index.php?rid=3287133&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F307%3Frss%3D1 The bacterium Deinococcus radiodurans is extremely resistant to the intense ionizing irradiation which causes extensive DNA double-strand breaks (DSBs). The deinococcal SbcCD complex (drSbcCD) is required for DSB repair. The drSbcC and drSbcD genes were cloned and overexpressed in Escherichia coli cells, respectively. The nearly homogeneous drSbcC and drSbcD proteins were purified and reconstituted to form a stable complex in vitro. The drSbcCD complex has an ATP-independent 3'->5' exonuclease activity to cleave both dsDNA and ssDNA substrates in the presence of either Mn2+ or Mg2+ ion. The drSbcCD complex also has an ATP-independent endonuclease activity. It can cleave the circular ssDNA, nick the supercoiled circular dsDNA, cleave the 3' flap DNA substrate at the site of the single-st... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287133 Fri, 19 Feb 2010 07:29:25 +0100 3287133 http://www.medworm.com/index.php?rid=3287132&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F3%2F297%3Frss%3D1 Cytochrome P450 enzyme system consists of P450 and its NAD(P)H-linked reductase or reducing system, and catalyses monooxygenation reactions. The most prevalent type in eukaryotic organisms is ‘microsomes type’, which consists of membrane-bound P450 and NADPH-P450 reductase. The second type is ‘mitochondria type’, in which P450 is bound to the inner membrane while the reducing system consisting of an NADPH-linked flavoprotein and a ferredoxin-type iron–sulphur protein is soluble in the matrix space. The third type is ‘bacteria type’, in which both P450 and the reducing system are soluble in the cytoplasm. In addition to these three types, several forms of P450-reductase fusion proteins have been found in prokaryotic organisms. On the other hand, som... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3287132 Fri, 19 Feb 2010 07:29:25 +0100 3287132 http://www.medworm.com/index.php?rid=3227129&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F291%3Frss%3D1 The topology of 4-pyridoxic acid dehydrogenase in the Escherichia coli cell membrane was examined with transformed E. coli cells overexpressing the enzyme from Mesorhizobium loti. The recombinant enzymes with a His6-tag either in the N-terminal region or at the C-terminus were localized on the E. coli cell membrane like the wild-type enzyme without a His6-tag. The His6-tags were labelled with Ni–NTA AP conjugate only when the E. coli protoplast cells were broken. The membrane-bound enzyme in the intact protoplast cells was not digested by trypsin, although the one in the gently broken protoplast cells was almost totally digested. Thus, 4-pyridoxic acid dehydrogenase was an integral monotopic protein, protruding into a cytoplasm side from the bacterial membrane. The deletion or mutati... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227129 Mon, 01 Feb 2010 13:26:23 +0100 3227129 http://www.medworm.com/index.php?rid=3227128&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F279%3Frss%3D1 The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with Kd in the range 1.93–6.44 µM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227128 Mon, 01 Feb 2010 13:26:23 +0100 3227128 http://www.medworm.com/index.php?rid=3227127&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F273%3Frss%3D1 In this study, we performed structural characterization of Thr35-phosphoroylated inhibitor-1 by using multi-dimensional heternuclear NMR spectroscopy. The result of structural comparison between Thr35-phosphoroylated and non-phosphorylated inhibitor-1 indicated that PKA-phosphorylation has no significant effect on the global conformation of free-state inhibitor-1. This finding may support the inference that regulation of the interactions between inhibitor-1 and PP1 through PKA-phosphorylation mainly depends on the phosphate group instead of phosphorylation-induced conformational change. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227127 Mon, 01 Feb 2010 13:26:23 +0100 3227127 http://www.medworm.com/index.php?rid=3227126&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F265%3Frss%3D1 Developmental pluripotency-associated 2 (Dppa2) gene is one of the genes recently identified to be expressed specifically in pluripotent cells. To investigate the role of Dppa2 in mouse embryonic stem (ES) cells, we examined its expression during differentiation and performed knockdown of Dppa2 in mouse ES cells. Our results showed that the expression of Dppa2 decreased markedly in differentiated cells. Dppa2 knockdown induced the differentiation of mouse ES cells, as indicated by reduced alkaline phosphatase activity, slightly downregulated expression of the putative pluripotency marker genes Oct4 and Nanog and increased expression of early differentiation marker genes, such as Fst and Psx1. Moreover, reduced expression of Dppa2 also repressed cell proliferation activity as shown by the 5... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227126 Mon, 01 Feb 2010 13:26:23 +0100 3227126 http://www.medworm.com/index.php?rid=3227125&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F257%3Frss%3D1 Histamine dehydrogenase from Nocardioides simplex (nHmDH) is a homodimer containing one 6-S-cysteinyl FMN (CFMN) and one [4Fe–4S] cluster per monomer. nHmDH catalyses the oxidative deamination of histamine to ammonia and imidazole acetaldehyde, but histamine inhibits its catalytic activity at high concentrations. We mutated gene-encoded residues (Tyr180, Gly268 and Asp269) near CFMN to understand the biophysical meaning of the substrate inhibition. Three mutants Y180F, G268D/D269C and Y180F/G268D/D269C were expressed by considering the DNA sequence alignment of histamine dehydrogenase from Rhizobium sp. 4-9 (rHmDH), which does not suffer from the substrate inhibition. The Y180F/G268D/D269C mutation to mimic rHmDH successfully suppressed the inhibition, although the catalytic activity... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227125 Mon, 01 Feb 2010 13:26:23 +0100 3227125 http://www.medworm.com/index.php?rid=3227124&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F245%3Frss%3D1 This study is the first report on the separation and identification of positional isomers of endogenous phosphatidylcholine using reversed-phase LC-ESIMS/MS. The separation of positional isomers in PC was achieved by using ultra performance LC, which uses a high-resolution HPLC system. To identify positional isomers in individual PC species, their lyso-PC-related fragments and fatty acids, which were obtained by MS/MS analysis in the negative ion mode, were used. From the application results of biological samples, the lipid extracts of mouse brain were found to be abundant in PC containing 22:6 at the sn-1 position of the glycerol backbone. However, the lipid extracts from mouse heart and liver were not abundant in positional isomers. This achievement demonstrates that the relative amounts... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227124 Mon, 01 Feb 2010 13:26:23 +0100 3227124 http://www.medworm.com/index.php?rid=3227123&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F237%3Frss%3D1 Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0–3.0 Å from the NW i... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227123 Mon, 01 Feb 2010 13:26:23 +0100 3227123 http://www.medworm.com/index.php?rid=3227122&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F225%3Frss%3D1 We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na+/Ca2+ exchanger (NCX) in the ER. Treatment of the ER with Ca2+ (5 mM) dissociates m-calpain–calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral transmembrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pre-treatment of the ER with calpain inhibitors, calpeptin (10 µM) or MDL28170 (10 µM), or Ca2+ chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227122 Mon, 01 Feb 2010 13:26:23 +0100 3227122 http://www.medworm.com/index.php?rid=3227121&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F213%3Frss%3D1 In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560–CaM). K560–CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca2+-dependent reversible binding of K560–CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560–CaM in the absence of Ca2+, while both dimeric and tetrameric configurations in the presence of Ca2+. Further, Ca2+-dependent change in the configuration o... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227121 Mon, 01 Feb 2010 13:26:23 +0100 3227121 http://www.medworm.com/index.php?rid=3227120&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F201%3Frss%3D1 In this study, the roles of Glu607, Glu609 and Glu610 in Tb OPB were investigated by site-directed mutagenesis. A striking effect on kcat/Km was obtained following mutation of Glu607 to glutamine. In contrast, the heat stability of Tb OPB decreased markedly following the single mutation of Glu610 to glutamine, although this mutation had significantly less effect on catalytic properties compared with the Glu607 mutation. Although no differences were found in the tertiary and secondary structures between wild-type (WT) OPB and the E610Q mutant prior to heat treatment, the E610Q mutant is inactivated more rapidly than WT OPB following heat treatment in a manner correlating with its attendant structural changes. Trypsin digestion showed that the boundary regions between the β-propeller an... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227120 Mon, 01 Feb 2010 13:26:23 +0100 3227120 http://www.medworm.com/index.php?rid=3227119&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F191%3Frss%3D1 Human serum albumin (HSA) plays important roles in transport of fatty acids and binding a variety of drugs and organic compounds in the circulatory system. This protein experiences several conformational transitions by the change of pH, and the resulting conformations were essential for completing the physiological roles in vivo. Steady-state and time-resolved fluorescence spectroscopy was applied to single tryptophan residue solely arranged in HSA to study subtle conformational change around single tryptophan residue in HSA at various pH. The results showed the characteristic feature of local conformation around tryptophan residue in domain II responding to the change in entire structure. The study of time-resolved area-normalized fluorescence emission spectra (TRANES) also showed the pec... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227119 Mon, 01 Feb 2010 13:26:23 +0100 3227119 http://www.medworm.com/index.php?rid=3227118&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F185%3Frss%3D1 Calcineurin (CN), the Ca2+/calmodulin (CaM)-dependant protein phosphatase, is an integral enzyme involved in activation of T cells. It is also the target of various inhibitors such as cyclosporine A (CsA) and FK506 both of which have been widely used as immunosuppressants. We show that the novel CN inhibitor, quercetin (QC), associates with CN both in vitro and in Jurkat cells, and that it causes non-competitive inhibition of phosphatase activity. Unlike CsA and FK506, QC does not require a matchmaker protein for CN inhibition. It acts directly on the catalytic domain and its inhibitory effect was increased by the presence of CNB. Using semi-quantitative and real-time RT–PCR, we show that QC inhibits IL-2 gene expression in activated Jurkat cells. The physiological inhibitory activit... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227118 Mon, 01 Feb 2010 13:26:23 +0100 3227118 http://www.medworm.com/index.php?rid=3227117&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F175%3Frss%3D1 The molecular mechanisms of the endogenous circadian clocks that allow most animals to adapt to environmental cycles have recently been uncovered. The draft genome of the ascidian, Ciona intestinalis, a model animal that is close to vertebrates, has been described. However, the C. intestinalis genome lacks the canonical clock genes such as Per, Bmal and Clock that are shared by vertebrates and insects. Here, we found the circadian rhythms at the physiological and molecular levels. The oxygen consumption rate was lower during the light phase and higher during the dark phase during a day, and the rhythm highly damped and continued under constant darkness. From the microarray analysis, the 396 spots (1.8% of the total; corresponding to 388 clones) were extracted as candidates for circadian ex... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227117 Mon, 01 Feb 2010 13:26:23 +0100 3227117 http://www.medworm.com/index.php?rid=3227116&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F167%3Frss%3D1 Porcine pepsin A and bovine chymosin are typical models of aspartic proteinases. The hydrolytic specificities of these proteinases, along with those of human pepsin A and monkey chymosin, were investigated with 29 peptide substrates that included various P1' variants of seven parent peptides. From these peptides, AFPLEFFREL was preferred by pepsin A and chymosin, while its P1' variant, AFPLEFEREL was preferred by bovine chymosin. Porcine and human pepsin A showed similar hydrolytic specificities, strongly preferring a hydrophobic/aromatic residue at P1’ of any type of peptide. This specificity is well explained by the very hydrophobic nature of the S1' subsite that consists of Tyr189, Ile213, Ile300, Met289, Val/Leu291 and Leu298. The first three residues are well conserved in pepsin... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227116 Mon, 01 Feb 2010 13:26:22 +0100 3227116 http://www.medworm.com/index.php?rid=3227115&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F157%3Frss%3D1 In this study, as a first step to elucidate the physiological role of free N-glycans and the de-N-glycosylation machinery working in developing plant cells, we have succeeded in expressing a cDNA from tomato fruits in Pichia pastoris and identified an acidic peptide:N-glycanase in the culture supernatant. The PNGase-gene-encoded protein is a single polypeptide chain of 588 amino acids with a predicted molecular mass of 65.8 kDa. The deduced amino acid sequence showed 57.9% similarity with almond PNGase A. The recombinant tomato PNGase showed optimum activity at pH 4.5 and 40°C. It did not require any metal ions for full enzymatic activity and could release the complex-type N-glycan from glycopeptides. Our phylogenetic analysis reveals that the plant acidic PNGase is completely differen... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227115 Mon, 01 Feb 2010 13:26:22 +0100 3227115 http://www.medworm.com/index.php?rid=3227114&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F2%2F153%3Frss%3D1 Here, we communicate that the design of the anti-oxidant consisting of amphiphilic manganese porphyrin with one dimethylimidazolium group and three phenyl groups for mitochondrion targeting. The resulting Mn-porphyrin MnMImP3P exhibited high partition coefficient (logPow = +4.78) as well as significant superoxide dismutase and peroxynitrite decomposition activities. Accordingly, the MnMImP3P exhibited a little increase in fluorescence intensity attributed to 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide [diS-C3-(5)], a tracer dye to assess the mitochondrial membrane potential, which suggested the interaction of the MnMImP3P, leading to the release of the fluorescence dye from the mitochondrial membrane, with the mitochondria. As a result, the MnMImP3P rescued the cell death under oxidative ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3227114 Mon, 01 Feb 2010 13:26:22 +0100 3227114 http://www.medworm.com/index.php?rid=3141818&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F143%3Frss%3D1 Heme oxygenase (HO) catalyzes oxidative breakdown of heme, and constitutes two isozymes, HO-1 and HO-2. Here, we explored the tissue-specific regulation of expression of HO-1 and HO-2 under hypoxemia. There was no significant change in the overall expression levels of HO-1 and HO-2 mRNAs and proteins in the lung during adaptation of C57BL/6 mice to normobaric hypoxia (10% O2). However, immunohistochemical analysis revealed the increased expression of HO-1 and HO-2 proteins after 28 days of normobaric hypoxia in the pulmonary venous myocardium that is the extension of the left atrial myocardium into pulmonary venous walls. Moreover, the expression of HO-2 protein was increased in the sub-endocardial myocardium of ventricles under hypoxia, while HO-1 protein level was increased in the full-t... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141818 Tue, 05 Jan 2010 12:35:26 +0100 3141818 http://www.medworm.com/index.php?rid=3141817&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F135%3Frss%3D1 We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly4-Ser)3. The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m5dCyd and weakly with 5-methylcytidine (m5Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Altho... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141817 Tue, 05 Jan 2010 12:35:26 +0100 3141817 http://www.medworm.com/index.php?rid=3141816&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F127%3Frss%3D1 Epitopes are located at the surface of allergens with which antibodies specifically bind. On the assumption that fragments unique to allergens have common, characteristic amino acid sequences, we compared the amino acid sequences of allergens with those of non-allergens. Segments around fragments unique to allergens showed wavelet-like distributions for several amino acids. Charged residues, alanine and glycine had positive peaks at the centre of the unique segments with small valleys on both sides, while aromatic residues, proline and cysteine showed the inverse distribution. Furthermore, the wavelet-like distribution of amino acids could be represented by a universal distribution function together with an index characterizing the intensity of the wavelet. Using the universal distribution... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141816 Tue, 05 Jan 2010 12:35:26 +0100 3141816 http://www.medworm.com/index.php?rid=3141815&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F117%3Frss%3D1 We cloned full-length cDNAs of 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium and successfully expressed 70 isoforms in Saccharomyces cerevisiae. To elucidate substrate specificity of P. chrysosporium P450s, we examined various substrates including steroid hormones, several drugs, flavonoids and polycyclic aromatic hydrocarbons using the recombinant S. cerevisiae cells. Of these P450s, two CYPs designated as PcCYP50c and PcCYP142c with 14% identity in their amino acid sequences catalyse 3'-hydroxylation of flavone and O-deethylation of 7-ethoxycoumarin. Kinetic data of both enzymes on both reactions fitted not to the Michaelis–Menten equation but to Hill’s equation with a coefficient of 2, suggesting that two substrates bind to the active site. Molecular ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141815 Tue, 05 Jan 2010 12:35:26 +0100 3141815 http://www.medworm.com/index.php?rid=3141814&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F109%3Frss%3D1 This study provides insight into the early stage of the Prx reaction. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141814 Tue, 05 Jan 2010 12:35:26 +0100 3141814 http://www.medworm.com/index.php?rid=3141813&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F95%3Frss%3D1 Nicotinamide (NM) phosphoribosyltransferase (NMPRTase) catalyzes the reaction of NM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to form NM mononucleotide (NMN) and pyrophosphate (PPi) in the pathway of NAD-biosynthesis. Monitoring the 1H and 31P NMR spectra of the reaction mixture, we found that this reaction is reversible as dictated by the equilibrium constant K = [NMN][PPi]/([NM][PRPP]) = 0.14, which agreed well with the ratio of second-order rate constants for forward and backward reactions, K = 0.16. The crystal structures of this enzyme in the free form and bound to NM and PRPP at the resolution of 2.0–2.2 Å were essentially identical to that of the complex with NMN, except for some variations that could facilitate the substitution reaction by fixing the nucleophile and... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141813 Tue, 05 Jan 2010 12:35:26 +0100 3141813 http://www.medworm.com/index.php?rid=3141812&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F83%3Frss%3D1 The methods of homologous high-level expression and simple large-scale purification for coenzyme B12-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer 6β6. Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B12, the binding of 1 mol of the inhibitor per mol of the β unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141812 Tue, 05 Jan 2010 12:35:26 +0100 3141812 http://www.medworm.com/index.php?rid=3141811&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F73%3Frss%3D1 Neurotrophins such as NGF promote neuronal survival and differentiation via the cell surface TrkA neurotrophin receptor. Compounds with neurotrophic actions that are low in molecular weight and can permeate the blood–brain barrier are promising therapeutic agents against neurodegenerative diseases such as Alzheimer’s disease. Carnosic acid (CA), an electrophilic compound in rosemary, activates antioxidant responsive element (ARE)-mediated transcription via activation of Nrf2. In the present study, we discovered that CA strongly promotes neurite outgrowth of PC12h cells. NGF as well as CA activated Nrf2, whereas CA and NGF-mediated neuronal differentiation was suppressed by Nrf2 knockdown. On the other hand, CA activated TrkA-downstream kinase Erk1/2 independently of Nrf2. CA-in... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141811 Tue, 05 Jan 2010 12:35:26 +0100 3141811 http://www.medworm.com/index.php?rid=3141810&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F63%3Frss%3D1 MicroRNAs (miRNAs) are non-coding small RNAs that have been found in various kinds of eukaryotes and viruses. Recently, adenovirus non-coding RNAs, VA RNAs, have been reported to generate miRNAs. Here, we developed a lentiviral vector for monitoring adenovirus-derived miRNAs in living cells. By using red and green fluorescent proteins under the control of bi-directional two distinct promoters, adenoviral infection and consequent miRNA expression was successfully visualized and quantified by the reduction in green fluorescence when 3'-untranslated regions were connected to the target sequences of the adenovirus-derived miRNAs. Our functional analysis using a lentiviral vector is a useful method to examine the activity of miRNA in living mammalian cells. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141810 Tue, 05 Jan 2010 12:35:26 +0100 3141810 http://www.medworm.com/index.php?rid=3141809&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F53%3Frss%3D1 To characterize the structure of jaw muscle fibres expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibres of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibres were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibres, the peak of the first myosin layer line of masseter fibres was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibres are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the pe... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141809 Tue, 05 Jan 2010 12:35:26 +0100 3141809 http://www.medworm.com/index.php?rid=3141808&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F35%3Frss%3D1 Bone morphogenetic proteins (BMPs) exhibit broad spectra of biological activities in various tissues, including bone, cartilage, blood vessels, heart, kidney, neurons, liver and lung. BMPs are members of the transforming growth factor-β (TGF-β) family that bind to type II and type I serine-threonine kinase receptors, and transduce signals through Smad and non-Smad signalling pathways. Recent findings have revealed that BMP signalling is finely tuned by various mechanisms in both positive and negative fashions. Perturbations of BMP signalling pathways are linked to a wide variety of clinical disorders, including vascular diseases, skeletal diseases and cancer. Administration of recombinant BMP ligands and increasing endogenous expression of BMPs provide therapeutic effects on some... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141808 Tue, 05 Jan 2010 12:35:26 +0100 3141808 http://www.medworm.com/index.php?rid=3141807&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F27%3Frss%3D1 The unfolded protein response is an adaptive stress response that responds to the imbalance between the entry of newly synthesized unfolded proteins and the inherent folding capacity in the endoplasmic reticulum (ER). Various environmental stresses and changes in physiological conditions can result in the accumulation of unfolded proteins in the ER, which is sensed through ER transmembrane protein sensors named inositol requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6), and the sensed signals are transduced to the cytosol and the nucleus. IRE1 is a prototype ER stress sensor that is evolutionarily conserved from yeast to humans. Higher eukaryotes have evolved two other sensors, PERK and ATF6. This review focuses on the current progress in our... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141807 Tue, 05 Jan 2010 12:35:26 +0100 3141807 http://www.medworm.com/index.php?rid=3141806&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F19%3Frss%3D1 The folding of secretory and membrane proteins takes place in the endoplasmic reticulum (ER). The quality of the proteins folded in the ER is carefully monitored by an ER quality control mechanism that allows only correctly folded proteins to be transported to their final destination, and misfolded or unassembled proteins to be retained in the ER and subsequently degraded in a process termed ‘ER-associated degradation’ (ERAD). The ERAD pathway is conserved from yeast to mammals, and plays an essential role in the maintenance of ER homeostasis, as well as in the prevention of various diseases that arise from the accumulation of aberrant proteins in the ER. In the ERAD pathway, molecular chaperones and lectin-like proteins are involved in the identification of misfolded proteins,... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141806 Tue, 05 Jan 2010 12:35:26 +0100 3141806 http://www.medworm.com/index.php?rid=3141805&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F9%3Frss%3D1 Akira Kobata is a pioneer of the glycobiology of N-linked sugar chains. He established the basis of glycobiology by developing a series of reliable methods to analyse the structures of N-linked sugar chains. The sensitive methods established by him greatly contributed to our understanding of the structural characteristics of the sugar chains and the biosynthetic mechanisms responsible for the production of such characteristics. He also provided new aspects that the sugar chains of glycoproteins play an important role in cell-to-cell recognition, and that the structures of sugar chains are altered under physiological and pathological conditions, including many tumours and diseases on a structural basis. In this article, the author would like to sketch out Kobata’s main contributions t... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141805 Tue, 05 Jan 2010 12:35:26 +0100 3141805 http://www.medworm.com/index.php?rid=3141804&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F3%3Frss%3D1 Sen-itiroh Hakomori has been involved for the past ~40 years in studies on structure and function of glycosphingolipids (GSLs). The following is a brief summary of highlights of his research, performed in association with his colleagues. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141804 Tue, 05 Jan 2010 12:35:26 +0100 3141804 http://www.medworm.com/index.php?rid=3141803&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F147%2F1%2F1%3Frss%3D1 (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3141803 Tue, 05 Jan 2010 12:35:26 +0100 3141803 http://www.medworm.com/index.php?rid=3043873&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F917%3Frss%3D1 The steroids synthesized in the central nervous system (CNS) are the neurosteroids. Since little information is currently available concerning the roles of the retinoic acids (RAs) during steroidogenesis in the CNS, we investigated the effects of RAs upon their synthesis in our current study. Specifically, we analyzed the effects of all-trans-retinoic acid (ATRA) upon the expression of neurosteroid biosynthesis genes in the human glial cell line GI-1, in which the major steroidogenic genes are expressed. Treatment with ATRA (10 µM) induced a 4.9-fold increase in the expression of the cytochrome P450scc (CYP11A1) gene, the product of which cleaves the cholesterol side chain, a rate-limiting step during steroidogenesis. ATRA also strongly induced the expression of steroidogenic acute r... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043873 Tue, 01 Dec 2009 15:10:08 +0100 3043873 http://www.medworm.com/index.php?rid=3043872&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F905%3Frss%3D1 In this study, we examined the interaction of Syntaxin 5L (Syx5L), a Syx5 isoform that has an N-terminal extension containing a di-arginine ER-retrieval motif, with presenilin (PS) and its effects on the processing of β-amyloid precursor protein (βAPP). Similar to Syx5, Syx5L bound to PS1 holoprotein but not to its N- or C-terminal fragments. Unlike Syx5, Syx5L overexpression did not cause marked accumulation of intracellular βAPP holoprotein, and did not inhibit amyloid β peptide (Aβ) secretion. Analyses using deletion mutants of Syx5L revealed that, in addition to the difference in the intracellular localization between the isoforms, the presence of the N-terminal extension in Syx5L was critical for suppressing its inhibition of βAPP processing. Treatment of... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043872 Tue, 01 Dec 2009 15:10:08 +0100 3043872 http://www.medworm.com/index.php?rid=3043871&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F895%3Frss%3D1 Using 100-fold molar excess of succinic anhydride, about 99% of lysine residues of hen egg white lysozyme (HEWL) were modified. Succinylated (S99) HEWL showed both charge and size homogeneity as judged by PAGE and gel filtration, respectively. Hydrodynamic parameters such as Stokes radius and frictional ratio (f/fo) showed more expanded conformation of S99 HEWL compared to native HEWL as evident from the increase in Stokes radius (from 1.36 to 1.86 nm) and f/fo (from 0.86 to 1.15) values. Guanidine hydrochloride (GdnHCl) denaturation studies using fluorescence spectroscopy connoted a marked decrease in conformational stability of HEWL upon succinylation. Complete denaturation of S99 HEWL was achieved at lower GdnHCl concentration (~3.8 M) compared to native HEWL (~5 M). Furthermore, free e... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043871 Tue, 01 Dec 2009 15:10:07 +0100 3043871 http://www.medworm.com/index.php?rid=3043870&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F887%3Frss%3D1 This work shows that the DNA cationic probe, ethidium bromide (EtBr), induces the transition from selective to non-selective mitochondrial permeability. This statement is based on the findings, indicating: (i) EtBr induced the release of accumulated Ca2+ through a mechanism sensitive to cyclosporin A and octylguanidine; (ii) EtBr induced the release of cytochrome c and (iii) EtBr induced mitochondrial swelling. Interestingly, mersalyl inhibited, in a non-competitive fashion, EtBr uptake, which would indicate that the uptake may be carried out through a protein membrane system. This work also shows that the effect of the dye on permeability transition was stimulated by carboxyatractyloside. Taking into account the facts that EtBr inhibited the ADP exchange reaction and increased the binding... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043870 Tue, 01 Dec 2009 15:10:07 +0100 3043870 http://www.medworm.com/index.php?rid=3043869&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F881%3Frss%3D1 Fibroblast growth factor-1 (FGF-1) is released from astrocytes in stress and stimulates MEK/ERK and PI3K/Akt pathways in autocrine fashion to increase synthesis of cholesterol and 25-OH-cholesterol, and to induce transport and secretion of apoE, respectively. FGF-1-induced phosphorylation of Src, and phosphorylation of MEK, ERK and Ark was inhibited by Src inhibitors in rat astrocytes. Src inhibitors also suppressed FGF-1-induced increase of biosynthesis and release of cholesterol and increase of apolipoprotein E (apoE) secretion. The results were reproduced in rat astrocytoma cells transfected by rat apoE and in 3T3-L1 cells. Down-regulation of Src expression reduced FGF-1-induced phosphorylation of the signalling protein and subsequent reactions. Increase by FGF-1 of messages of apoE and... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043869 Tue, 01 Dec 2009 15:10:07 +0100 3043869 http://www.medworm.com/index.php?rid=3043868&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F875%3Frss%3D1 Although detergents have been widely used in G-protein studies to increase solubility and stability of the protein, we noticed that detergents modulate the nucleotide-binding properties of G-proteins. Hence, we analysed the effects of detergents on guanine nucleotide exchange reactions of Gi1. Lubrol PX, a non-ionic detergent, which has been widely used in nucleotide dissociation/binding assays, was found to accelerate both GDP dissociation and GTPS binding from/to G in parallel at above its critical micelle concentration (cmc). Sodium cholate, an anionic detergent, which have been used to extract G-proteins from animal tissues, decelerated and accelerated GDP dissociation below and above its cmc, respectively. Surprisingly, micellar cholate decelerated GTPS binding, and the binding rate c... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043868 Tue, 01 Dec 2009 15:10:07 +0100 3043868 http://www.medworm.com/index.php?rid=3043867&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F867%3Frss%3D1 In this study, we attempted to alter the specificity of BNC by insertion of biotin-acceptor peptide (BAP), which is efficiently biotinylated using biotin ligase BirA from Escherichia coli. Using streptavidin as a linker, biotinylated BNC could be display various biotinylated ligands that are otherwise difficult to fuse with BNC, such as antibodies, synthetic peptides and functional molecules. BAP-fused BNC was efficiently biotinylated and effectively displayed streptavidin. Furthermore, we demonstrated that biotinylated BNC was internalized into targeted cells via biotinylated Nanobody displayed on the BNC surface. Biotinylated BNC permit display of diverse ligands, and thus have potential as a versatile carrier for drug delivery to a variety of target cells. (Source: Journal of Biochemist... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043867 Tue, 01 Dec 2009 15:10:07 +0100 3043867 http://www.medworm.com/index.php?rid=3043866&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F857%3Frss%3D1 Cytochromes b561 constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane -helices and two haem groups. Of particular interest is the presence of a large number of plant homologues having putative ascorbate- and monodehydroascorbate radical-binding sites. We conducted a diethylpyrocarbonate-modification study employing Zea mays cytochrome b561 heterologously expressed in Pichia pastoris cells. Pre-treatment of cytochrome b561 with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His86, His159 and Lys83, leading to a drastic inhibition of the electron transfer from ascorbate. The activity was protected by the inclusion of ascorbate during the treatment. However, midpoint potentials of two haem cen... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043866 Tue, 01 Dec 2009 15:10:07 +0100 3043866 http://www.medworm.com/index.php?rid=3043865&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F845%3Frss%3D1 In this study, we constructed two mutant strains, lacking of either subunit I of the cytochrome c oxidase (ctaD) or subunits I and II of the quinol oxidase (cydAB), and also plasmids to complement the deficient genes to investigate their effects on energy conservation and cell growth. We measured H+/O ratios of C. glutamicum wild-type and mutant cells grown aerobically. The H+/O ratio of the wild-type cells grown in the semi-synthetic medium was 3.94 ± 0.30, while the value was 2.76 ± 0.25 for the ctaD mutant. In contrast, the value was 5.23 ± 0.36 for the cydAB mutant. The cells grown in the LB medium showed higher value compared to that of cells grown in the semi-synthetic medium. The ctaD mutant grew less than the wild-type in LB medium, while they grew about equall... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043865 Tue, 01 Dec 2009 15:10:07 +0100 3043865 http://www.medworm.com/index.php?rid=3043864&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F839%3Frss%3D1 Hypoxia-inducible factor 1 (HIF-1) is a master regulator for hypoxic activation of genes for angiogenesis, hormone synthesis, glycolysis and cell survival. In addition to hypoxic stimulus, various effectors and reagents were reported to affect HIF-1 activity. Here, we show that cyclic AMP (cAMP) down-regulates the HIF-1 activity in pheochromocytoma PC12 cells but not in Hep3B and HeLa cells. Hypoxia response element-dependent reporter activity was decreased by the addition of dibutyryl cAMP. Expression of protein kinase A (PKA) catalytic -subunits repressed the HIF-1 activity. HIF-1 and HLF (HIF-2 or EPAS1) protein levels were decreased by the treatment with dibutyryl cAMP. Although CREB was served as a negative factor for the HIF-1 activity, it may not be a major PKA target in the cAMP-de... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043864 Tue, 01 Dec 2009 15:10:07 +0100 3043864 http://www.medworm.com/index.php?rid=3043863&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F833%3Frss%3D1 We examined the effect of knockdown of PER1 on apoptosis in pancreatic cancer (MIA PaCa-2 and PANC-1) and hepatocellular carcinoma (HepG2) cells. Transfection of siRNA against PER1 into these cells increased the cleaved forms of caspases and poly-ADP-ribose-polymerase and induced apoptosis in all three cell lines. In the two pancreatic cancer cell lines, PER1 knockdown resulted in upregulation of Bax and downregulation of Bcl-2. Expression of p53 was not altered in the two pancreatic cancer cell lines containing mutated p53, but was upregulated in the HepG2 cells containing wild-type p53. Cell proliferation of MIA PaCa-2 and HepG2 was inhibited by PER1 knockdown. We also examined, by immunohistochemical staining, the expression of PER1 in pancreatic cancer tissue and found that PER1 was st... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043863 Tue, 01 Dec 2009 15:10:07 +0100 3043863 http://www.medworm.com/index.php?rid=3043862&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F821%3Frss%3D1 Upon activation, NF-B translocates into the nucleus and initiates biological events. This NF-B signalling is mainly regulated by the protein kinase IKKβ. Early in this signalling pathway, IKKβ is phosphorylated for activation by several factors, such as pro-inflammatory cytokines and the Tax oncoprotein of HTLV-1. In cells infected by HTLV-1, IKKβ is persistently phosphorylated and conjugated with monoubiquitin due to Tax expression. Although this Tax-induced monoubiquitination appears to be an important regulation system for IKKβ, how the monoubiquitination occurs is unknown and its role in NF-B signalling is still unclear. Here, we show that an E3-ubiquitin ligase Ro52 interacts weakly with wild-type IKKβ but strongly with a phosphomimetic mutant IKKβ to con... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043862 Tue, 01 Dec 2009 15:10:07 +0100 3043862 http://www.medworm.com/index.php?rid=3043861&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F815%3Frss%3D1 In this study, we showed that tyrosine sulphation of S1P1 is necessary for S1P-induced Src phosphorylation and migration in human umbilical vein endothelial cells (HUVECs). Both substitution of phenylalanine (F) for tyrosine (Y) in S1P1 and inhibition of tyrosine sulphation blocked c-Src phosphorylation and migration in HUVECs. In addition, overexpression of mutant (F19, 22F) S1P1, lacking tyrosine sulphation sites, suppressed native S1P1 effects on migration, actin rearrangement and lamellipodia formation. Therefore, tyrosine sulphation of S1P1 is required for its optimal transduction of signals from S1P in HUVECs. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043861 Tue, 01 Dec 2009 15:10:07 +0100 3043861 http://www.medworm.com/index.php?rid=3043860&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F805%3Frss%3D1 Amyloid formation is the hallmark of many diseases. The propensity of a protein to aggregate depends on a number of biological factors like the presence of sulphated polysaccharides termed as glycosaminoglycans (GAGs). Here we assessed whether the polymeric nature of GAGs is responsible for their protein aggregation-promoting effect. We studied the effect of different monosaccharide derivatives, featuring the main characteristics of heparin and heparan sulphate (HS) building blocks, on the aggregation kinetics of human muscle acylphosphatase (mAcP), a useful model protein for these studies. We observed that while heparin and HS changed the mAcP aggregation kinetic profile, the monosaccharide derivatives had no effect, whatever their concentration could be and both when they are studied sep... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043860 Tue, 01 Dec 2009 15:10:07 +0100 3043860 http://www.medworm.com/index.php?rid=3043859&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F797%3Frss%3D1 The nucleotide sequence of IStaqTZ2 are available in the DDBJ/EMBL/GenBank databases under the accession number AB063392. A novel insertion sequence (IStaqTZ2) was transposed from the genome of Thermus thermophilus TZ2 to that of the thermophilic bacteriophage IN93. The complete nucleotide sequence of IStaqTZ2 was determined and was found to be 1,258 bp in length and to contain an open reading frame (ORF1179), which is predicted to encode a transposase. IStaqTZ2 was also found to contain two terminal inverted repeats with 48 and 52 bp, respectively. Based on homology analysis, IStaqTZ2 was classified as a member of the IS256 family. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043859 Tue, 01 Dec 2009 15:10:07 +0100 3043859 http://www.medworm.com/index.php?rid=3043858&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F787%3Frss%3D1 Mitochondrial fusion and fission processes play a role in a variety of cell functions, including energy metabolism, cell differentiation and programmed cell death. Still, it is not clear how these processes contribute to the cell functions. Here, we investigated the role of mitochondrial remodelling on lipid metabolism in adipocytes. In 3T3-L1 pre-adipocytes, the morphology of mitochondria is organized as a continuous reticulum. Upon differentiation of adipocytes manifested by cellular triacylglycerol (TG) accumulation, mitochondrial morphology altered from filamentous to fragmented and/or punctate structures. When the mitochondrial fusion was induced in adipocytes by silencing of mitochondrial fission proteins including Fis1 and Drp1, the cellular TG content was decreased. In contrast, th... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043858 Tue, 01 Dec 2009 15:10:07 +0100 3043858 http://www.medworm.com/index.php?rid=3043857&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F775%3Frss%3D1 We have determined the complete nucleotide sequence of the phage IN93 is 19,604-bp long and contains 39 putative open reading frames. The functions for 20% of IN93 gene products are similar to those expressed by other known phages and bacteria, and include peptidase, lytic enzymes, integrase, repressor protein and replication protein. The structural proteins of the IN93 virion were identified through sodium dodecyl sulphate–polyacrylamide gel electrophoresis and found to have no similarity to those of other phages. We also determined the transcription initiation sites and classified four transcription units using the primer extension method. Three transcription units were transcribed in the same direction as part of the lytic cycle, while the remaining unit was transcribed in the opp... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043857 Tue, 01 Dec 2009 15:10:07 +0100 3043857 http://www.medworm.com/index.php?rid=3043856&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F771%3Frss%3D1 The Commd1 gene is imprinted in the adult mouse brain and is predominantly expressed from the maternal allele. A paternally expressing imprinted gene, U2af1-rs1, resides in the first intron of Commd1 in an antisense orientation. We found that RNA polymerase II phosphorylated at serine 2 of the carboxyl-terminal domain repeats, a marker of transcription elongation, is enriched on the paternal allele than on the maternal allele in the Commd1 promoter. The Commd1 promoter harbours no allelic differences in DNA methylation and histone modifications. These results strongly suggested that imprinting of Commd1 is generated by interference with paternal Commd1 transcription by the oppositely directed U2af1-rs1 transcription. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043856 Tue, 01 Dec 2009 15:10:06 +0100 3043856 http://www.medworm.com/index.php?rid=3043855&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F767%3Frss%3D1 Sulphatase family enzymes hydrolyse the sulphate ester, found on the pathogens cell surface and playing an important role for host–pathogen interaction. The AtsG, homologue of arylsulphatase, predicted in the Mycobacterium tuberculosis genomic data, was successfully expressed in Escherichia coli. The recombinant AtsG protein exhibited hydrolysis of para-nitrophenyl sulphate and para-nitrocatechol sulphate, and binding affinity to the heparin–sepharose resin. This is the first report of molecular evidence for an arylsulphatase activity of the AtsG protein. The maximum activity was detected at pH 8.0 and 37°C. As EDTA completely inhibited this activity, a divalent cation was required for the activity. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043855 Tue, 01 Dec 2009 15:10:06 +0100 3043855 http://www.medworm.com/index.php?rid=3043854&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F757%3Frss%3D1 Cells induce the hypoxia responses to adapt to the environment when organisms are exposed to a low oxygen environment. The hypoxia response leads to the activation of multiple cellular signalling pathways involved in regulation of respiration, metabolism, cell survival and so forth. Hypoxia-Inducible-Factor (HIF) pathway plays a central role during the hypoxia response as its expression and activity are regulated in an oxygen-dependent manner and it also regulates the expression of multiple hypoxia responsive genes. The expression of HIF is regulated by proline hydroxylation, which is mediated by HIF prolyl-hydroxylase named PHD. The hydroxylated HIF- subunit is degraded via the ubiquitin–proteasome pathway. The PHD activity needs to be strictly regulated to ensure the stabilization ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043854 Tue, 01 Dec 2009 15:10:06 +0100 3043854 http://www.medworm.com/index.php?rid=3043853&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F751%3Frss%3D1 Diverse human diseases, including various neurodegenerative disorders and amyloidoses, are thought to result from the misfolding and aggregation of disease-causative proteins, and thus are collectively called protein misfolding diseases. Natively folded disease-causative proteins generally undergo a β-sheet conformational transition through an energetically unfavourable process, and further polymerize into amyloid fibrils. In the case of β2-microglobulin-related amyloidosis, an extracellular protein misfolding disease, many kinds of biological molecules including glycosaminoglycans, proteoglycans and lipids partially unfold β2-microglobulin and catalyse its subsequent nucleus formation. After amyloid fibrils are formed, these biological molecules stabilize the β2-microg... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043853 Tue, 01 Dec 2009 15:10:06 +0100 3043853 http://www.medworm.com/index.php?rid=3043852&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F6%2F743%3Frss%3D1 The accumulation of unfolded proteins in the endoplasmic reticulum (ER) under ER stress conditions activates a series of homoeostatic responses collectively termed the unfolded protein response (UPR). The UPR is unique in which the molecular mechanisms it uses to transmit signals from the ER lumen to the nucleus are completely different to those used for signalling from the plasma membrane. An ER stress signal is sensed and transmitted across the membrane by a transmembrane protein(s) in the ER. Interestingly, the number of such functional sensors/transducers, ubiquitously expressed, has increased with evolution, for example, one in Saccharomyces cerevisiae, two in Caenorhabditis elegans and Drosophila melanogaster, and three in mammals. Accordingly, mammalian cells are able to cope with E... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=3043852 Tue, 01 Dec 2009 15:10:06 +0100 3043852 http://www.medworm.com/index.php?rid=2958292&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F733%3Frss%3D1 The assembly of FtsZ is considered to be a fundamental process during the bacterial cytokinesis. We used several complimentary techniques to probe the assembly of recombinant Escherichia coli FtsZ (EcFtsZ) and Mycobacterium tuberculosis FtsZ (MtbFtsZ) proteins in vitro. As documented earlier, EcFtsZ was found to polymerize at much faster rate than MtbFtsZ. Interestingly, we found that MtbFtsZ produced higher sedimentable polymerized mass than that of the EcFtsZ and that MtbFtsZ formed thicker protofilaments than that of the EcFtsZ. The results indicated that the EcFtsZ polymers are more labile than the MtbFtsZ polymers. Further, divalent calcium exerted strikingly different effects on the assembly of EcFtsZ and MtbFtsZ. Divalent calcium strongly enhanced the assembly of EcFtsZ and promoted... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958292 Wed, 04 Nov 2009 14:24:50 +0100 2958292 http://www.medworm.com/index.php?rid=2958291&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F725%3Frss%3D1 We have identified a novel mitochondrial protein, termed M19, by proteomic analysis of mitochondrial membrane proteins from HeLa cells. M19 is highly conserved among vertebrates, and possesses no homologous domains with other known proteins. By northern and western blotting, mouse M19 was shown to be expressed in various tissues, and to be especially abundant in the brain. Human M19 (hM19) is present in mitochondria, and protease-protection experiment showed it to be sublocalized in the matrix space. Carboxy-terminally tagged hM19 appeared as spotted signals within mitochondria and co-localized with signals arising from mitochondrial DNA (mtDNA), suggesting the inclusion of M19 in the mtDNA–protein complex (mitochondrial nucleoids). Fractionation of mitochondrial nucleoids from HeLa ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958291 Wed, 04 Nov 2009 14:24:50 +0100 2958291 http://www.medworm.com/index.php?rid=2958290&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F713%3Frss%3D1 Periostin is a matricellular protein participating in the tissue remodelling of damaged cardiac tissue after acute myocardial infarction and of the periodontal ligament in mice. However, further studies on the periostin protein have been limited by the intrinsic difficulty of purifying this protein produced in Escherichia coli due to its insolubility. Here, we demonstrate the expression of recombinant periostin protein with high solubility and monodispersity in E. coli. Periostin is composed of an amino-terminal EMI domain, a tandem repeat of 4 fas1 domains (RD1-4), and a carboxyl-terminal region (CTR). We expressed the RD4-CTR region tagged with GST at amino-terminal and 6x Histidine at carboxyl-terminal end in E. coli. The recombinant protein was purified by using GSH-Sepharose and nicke... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958290 Wed, 04 Nov 2009 14:24:50 +0100 2958290 http://www.medworm.com/index.php?rid=2958289&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F705%3Frss%3D1 This study further investigated the components involved in this actin reorganization at pH 3.0. Gene deletions on the Sln1p branch of the HOG pathway completely blocked actin depolarization, suggesting that Hog1p activation depends mainly on the osmosensor Sln1p. The protein-synthesis inhibitor cycloheximide did not influence the time course of actin depolarization, suggesting that the depolarization is a direct effect of the HOG pathway. Deletion of the scaffolding protein, Spa2p, or the Spa2p-interacting protein Pea2p, markedly inhibited the depolarization, and further deletion of the formin protein, Bni1p, notably delayed actin repolarization. Our results suggest the involvement of polarisome proteins, such as Spa2p, Pea2p and Bni1p, but not Bud6p, in Hog1p-dependent reorganization of t... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958289 Wed, 04 Nov 2009 14:24:50 +0100 2958289 http://www.medworm.com/index.php?rid=2958288&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F693%3Frss%3D1 The objective of this study was to investigate different transport systems for FA and MTX, and to delineate their uptake mechanism in MOLT4, K562, Hut78 leukemia cells and normal human T cells. In MOLT4, uptake of MTX was higher than FA, similar to that of K562, Hut78 and normal T cells. In MOLT4 cells, MTX uptake was maximum at pH 7.4 whereas FA uptake was maximum at pH 4.5. Uptake of FA and MTX was significantly inhibited by anions, suggesting anion-dependent transport system. FA uptake was found to be energy dependent whereas MTX uptake was energy independent. RT-PCR and immunofluorescence results demonstrated the presence of reduced folate carrier as well as proton coupled folate transporter and absence of FR in MOLT4 and normal T cells. These data suggest the existence of two separate... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958288 Wed, 04 Nov 2009 14:24:50 +0100 2958288 http://www.medworm.com/index.php?rid=2958287&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F683%3Frss%3D1 Krüppel-like factor 5 (KLF5) and c-Jun are involved in angiotensin II (Ang II)-induced cell proliferation and play an important role in p21 expression. But the direct and functional implications of KLF5 and c-Jun in regulating p21 expression in vascular smooth muscle cells (VSMCs) are unclear. Here, we show that Ang II upregulated KLF5 and c-Jun expression and inhibited p21 expression in VSMCs, and silencing of KLF5 expression by KLF5-specific small interfering RNA (siRNA) neutralized the inhibitory effects of Ang II on p21 expression. Exposure of VSMCs to Ang II rapidly and strongly stimulated KLF5 phosphorylation, which results in an increase of the interaction of KLF5 with c-Jun. Treating VSMCs with PD98059, the ERK inhibitor, inhibited ERK activation and KLF5 phosphorylation as we... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958287 Wed, 04 Nov 2009 14:24:50 +0100 2958287 http://www.medworm.com/index.php?rid=2958286&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F675%3Frss%3D1 Ghrelin was originally isolated from rat stomach as an endogenous ligand for the GH secretagogue receptor. The major active form of ghrelin is a 28-amino acid peptide modified by an n-octanoic acid on the serine 3 residue, and this lipid modification is essential for the biological activity of ghrelin. However, it is not clear whether prohormone convertase (PC) and ghrelin O-acyltransferase (GOAT) are the minimal requirements for synthesis of acyl-modified ghrelin in cultured cells. By using three cultured cell lines, TT, AtT20 and COS-7, in which the expression levels of processing proteases and GOAT vary, we examined the processing patterns of ghrelin precursor. We found that not only PC1/3 but also both PC2 and furin could process proghrelin to the 28-amino acid ghrelin. Moreover, the p... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958286 Wed, 04 Nov 2009 14:24:50 +0100 2958286 http://www.medworm.com/index.php?rid=2958285&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F667%3Frss%3D1 The HP0827 protein is an 82-residue protein identified as a putative ss-DNA-binding protein 12RNP2 Precursor from Helicobacter pylori. Here, we have determined 3D structure of HP0827 using Nuclear Magnetic Resonance. It has a ferredoxin-like fold, β1–1–β2–β3–2–β4 (; -helix and β; β-sheet) and ribonucleoprotein (RNP) motifs which are thought to be important in RNA binding. By using structural homologues search and analyzing electrostatic potential of surface, we could compared HP0827 with other RNA-binding proteins (sex-lethal, T-cell restricted intracellular antigen-1, U1A) to predict RNA-binding sites of HP0827. We could predict that β sheets of HP0827, especially β1 and β3, are primary region for RNA binding. Cons... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958285 Wed, 04 Nov 2009 14:24:50 +0100 2958285 http://www.medworm.com/index.php?rid=2958284&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F659%3Frss%3D1 Trehalose dimycolate (TDM) is a major surface-exposed mycolyl glycolipid that contributes to the hydrophobic cell wall architecture of mycobacteria. Nevertheless, because of its potent adjuvant functions, pathogenic mycobacteria appear to have evolved an evasive maneuver to down-regulate TDM expression within the host. We have shown previously that Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av), replace TDM with glucose monomycolate (GMM) by borrowing host-derived glucose as an alternative substrate for the FbpA mycolyltransferase. Mycobacterium leprae (M.le), the causative microorganism of human leprosy, is also known to down-regulate TDM expression in infected tissues, but the function of its mycolyltransferases has been poorly analysed. We found that, unlike M.tb and M... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958284 Wed, 04 Nov 2009 14:24:50 +0100 2958284 http://www.medworm.com/index.php?rid=2958283&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F651%3Frss%3D1 In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel β-sheet, designated as a ‘platform’, on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)5. A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp m... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958283 Wed, 04 Nov 2009 14:24:50 +0100 2958283 http://www.medworm.com/index.php?rid=2958282&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F643%3Frss%3D1 Precise regulation of the number and placement of flagella is critical for the mono-flagellated bacterium Vibrio alginolyticus to swim efficiently. We previously proposed a model in which the putative GTPase FlhF determines the polar location and generation of the flagellum, the putative ATPase FlhG interacts with FlhF to prevent FlhF from localizing to the pole, and thus FlhG negatively regulates the flagellar number in V. alginolyticus cells. To investigate the role of the GTP-binding motif of FlhF, we generated a series of alanine-replacement mutations at the positions that are highly conserved among homologous proteins. The results indicate that there is a correlation between the polar localization and the ability to produce flagella in the mutants. We investigated whether the mutation... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958282 Wed, 04 Nov 2009 14:24:50 +0100 2958282 http://www.medworm.com/index.php?rid=2958281&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F633%3Frss%3D1 β-1,3-Xylanase from Vibrio sp. strain AX-4 (XYL4) is a modular enzyme composed of an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules (CBMs) belonging to family 31 in the C-terminal region. To investigate the functions of these three modules, five deletion mutants lacking individual modules were constructed. The binding assay of these mutants showed that a repeating unit of the CBM was a non-catalytic β-1,3-xylan-binding module, while the catalytic module per se was not likely to contribute to the binding activity when insoluble β-1,3-xylan was used for the assay. The repeating CBMs were found to specifically bind to insoluble β-1,3-xylan, but not to β-1,4-xylan, Avicel, β-1,4-mannan, curdlan,... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958281 Wed, 04 Nov 2009 14:24:50 +0100 2958281 http://www.medworm.com/index.php?rid=2958280&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F627%3Frss%3D1 Organic compounds are used as templates to regulate the morphology of inorganic nanostructures. In the present study, we used intermediate filaments (IFs), the major cytoskeleton component of most eukaryotic cells, as a template for hollow silica nanotube preparation. Sol–gel polymerization of tetraethoxysilane proceeded preferentially on the surface of IFs assembled from vimentin protein in vitro, resulting in silica-coated fibres. After removing IFs by calcination, electron microscopy revealed hollow silica nanotubes several micrometers long, with outer diameters of 35–55 nm and an average inner diameter of 10 nm (comparable to that of IFs). Furthermore, the silica nanotubes exhibited a gnarled surface structure with an 18–26 nm repeating pattern (comparable to the 21-n... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958280 Wed, 04 Nov 2009 14:24:50 +0100 2958280 http://www.medworm.com/index.php?rid=2958279&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F623%3Frss%3D1 Hydrogen sulfide (H2S) has been recognized as a smooth muscle relaxant. Cystathionine -lyase, which is localized to smooth muscle, is thought to be the major H2S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H2S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H2S from cysteine and -ketoglutarate. The present study provides a new insight into the production and release of H2S as a smooth muscle relaxant from vascular endothelium. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958279 Wed, 04 Nov 2009 14:24:50 +0100 2958279 http://www.medworm.com/index.php?rid=2958278&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F617%3Frss%3D1 H+-translocating pyrophosphatase converts energy from hydrolysis of pyrophosphate to active H+ transport across biomembranes. Mutational analysis of Streptomyces coelicolor A3(2) enzyme revealed that amino acid substitution of Phe-388 and Ala-514 altered the enzyme activity. Both residues are located at the interface between the transmembrane domains and cytosolic loops, in which the catalytic domain exists. Systematic amino acid substitution was carried out using the Escherichia coli heterologous expression system. Two of the 38 mutant enzymes, F388Y and A514S, showed a high ratio of H+-pump to substrate hydrolysis without decrease in the substrate hydrolysis activity, indicating high energy-coupling efficiency. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958278 Wed, 04 Nov 2009 14:24:50 +0100 2958278 http://www.medworm.com/index.php?rid=2958277&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F609%3Frss%3D1 Misfolded proteins are toxic to cells and the accumulation of toxic species can lead to protein misfolding diseases, such as neurodegenerative disorders. The toxicity of misfolded proteins is thought to result from the presence of exposed hydrophobic surfaces, which mediate unnecessary binding to normal proteins, interrupting essential interactions between cellular proteins. To prevent toxicity, quality control systems monitor protein folding and remove misfolded species in the cytosol. Molecular chaperones recognize and mask hydrophobic surfaces of misfolded monomers, and transfer them to the ubiquitin–proteasome system and chaperone-mediated autophagy. To eliminate soluble aggregates of misfolded proteins, the macroautophagy–lysosome system is thought to degrade proteasome-re... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958277 Wed, 04 Nov 2009 14:24:50 +0100 2958277 http://www.medworm.com/index.php?rid=2958276&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F599%3Frss%3D1 Proteins of the intermembrane space (IMS) of mitochondria fulfil crucial functions in cellular processes, such as transport of proteins and metal ions, ATP production and apoptotic cell death. All IMS proteins are synthesized in the cytosol and then transported across the mitochondrial outer membrane. A subset of these proteins contains disulphide bonds. For their import into the IMS, they employ a disulphide relay system, made up of two essential proteins, Mia40/Tim40 and the flavin-dependent sulfhydryl-electron transferase Erv1. The disulphide relay system introduces disulphide bonds in substrate proteins triggering their folding. The oxidative folding traps substrates in the IMS and thereby drives their net import into the IMS. Thus, protein import is coupled to oxidative protein foldin... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958276 Wed, 04 Nov 2009 14:24:50 +0100 2958276 http://www.medworm.com/index.php?rid=2958275&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F5%2F591%3Frss%3D1 The biological kingdoms have evolved elaborate systems that ensure the catalysis of protein disulfide bond (dsb) formation in the cell. Coexisting in the periplasm of Escherichia coli are the DsbA–DsbB disulfide-introducing and DsbC–DsbD disulfide-isomerizing pathways, which promote the oxidative folding of secreted proteins. Recent structural studies of DsbB have illuminated conformational dynamics involved in the effective oxidation of the extremely reduction-prone oxidase, DsbA, as well as the structure of the reaction centre involved in protein Dsb formation de novo in conjunction with ubiquinone. Extensive genetic and biochemical analysis has recently provided insight into how DsbD transports electrons from cytosolic thioredoxin to periplasmic DsbC. To a great extent, the ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2958275 Wed, 04 Nov 2009 14:24:50 +0100 2958275 http://www.medworm.com/index.php?rid=2865097&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F581%3Frss%3D1 Calmodulin (CaM), a physiologically important Ca2+-binding protein, participates in numerous cellular regulatory processes. It is dumbbell shaped and contains two globular domains connected by a short -helix. Each of the globular domains has two Ca2+-binding sites, the EF hands. CaM undergoes a conformational change upon binding to Ca2+, which enables it to bind to specific proteins for specific responses. Here, we successfully photocontrolled CaM binding to its target peptide using the photochromic compound N-(4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis–trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. In order to specifically incorporate PAM, CaM mutants having reactive cysteine residues in the functional region were prepared; PAM... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865097 Tue, 06 Oct 2009 05:50:51 +0100 2865097 http://www.medworm.com/index.php?rid=2865096&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F571%3Frss%3D1 In order to explore the mechanism of myoglobinuric renal toxicity, detection and identification of free radicals was performed for the reaction mixtures of bovine kidney microsomes. EPR measurements showed prominent signals for the control reaction mixture containing 2.0 mg protein/ml bovine kidney microsomes, 5 mM NADPH, 0.1 M 4-POBN and 29 mM phosphate buffer (pH 7.4). Addition of myoglobin (Mb) to the control reaction mixture resulted in increase of EPR peak height. The result indicates that Mb enhances the radical formation. An HPLC–EPR measurement showed three peaks with retention times of 29.4 min (P1), 32.4 min (P2) and 46.6 min (P3). HPLC–EPR–MS analyses of P1 and P2 gave ions at m/z 282. The results show that 4-POBN/hydroxypentyl radical adducts form in the react... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865096 Tue, 06 Oct 2009 05:50:51 +0100 2865096 http://www.medworm.com/index.php?rid=2865095&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F563%3Frss%3D1 Ubiquitin is a globular protein with a highly conserved sequence. Sequence conservation and compact structure make it an ideal protein for structure–function studies. One of the atypical secondary structural features found in ubiquitin is a parallel G1 β-bulge. Glutamate at 64 is the first residue of this β-bulge and the third residue in a type II turn. However, glycine is seen in these positions in several proteins. To understand the effects of substitution of glutamate64 by glycine on the structure, stability and function of ubiquitin, mutant UbE64G has been constructed and characterized in Saccharomyces cerevisiae. The secondary and tertiary structures of UbE64G mutant protein are only marginally different from wild-type protein (UbWt) and fluorescent form of ubiquitin (... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865095 Tue, 06 Oct 2009 05:50:51 +0100 2865095 http://www.medworm.com/index.php?rid=2865094&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F549%3Frss%3D1 This study provides the structural evidence that directly supports our proposed mechanism of the substrate synergism in the SPT reaction. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865094 Tue, 06 Oct 2009 05:50:51 +0100 2865094 http://www.medworm.com/index.php?rid=2865093&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F541%3Frss%3D1 In this study, we further investigated the physiological importance of membrane localization of PTP1B in vivo. We found that transient liver-specific overexpression of wild-type PTP1B (PTP1B-WT) using adenovirus-mediated gene transfer was associated with hypertriglyceridaemia and enhanced hepatic SREBP-1 gene expression in mice. However, overexpression of the C-terminal truncated PTP1B (PTP1BCT) failed to increase hepatic SREBP-1 expression or serum triglyceride levels, despite causing insulin resistance. Our results indicate that activation of PTP1B in the liver could induce hypertriglyceridaemia and that anchoring of PTP1B to the membrane is crucial for its action. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865093 Tue, 06 Oct 2009 05:50:51 +0100 2865093 http://www.medworm.com/index.php?rid=2865092&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F535%3Frss%3D1 The HP0062 gene encodes a small acidic protein of 86 amino acids with a theoretical pI of 4.6. The crystal structure of hypothetical protein HP0062 from Helicobacter pylori has been determined at 1.65 Å by molecular-replacement method. The crystallographic asymmetric unit contains dimer, in which HP0062 monomer folds into a helix–hairpin–helix structure. The two protomers are primarily held together by extensive hydrophobic interactions in an antiparallel arrangement, forming a four helix bundle. Aromatic residues located at a or g position in the heptad leucine zipper are not major contributor required for HP0062 dimerization but important for the thermostability of this protein. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865092 Tue, 06 Oct 2009 05:50:51 +0100 2865092 http://www.medworm.com/index.php?rid=2865091&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F527%3Frss%3D1 This study demonstrates that although members of the plant UGlcAE isoforms are highly conserved, the in vitro enzymatic activity of specific Poaceae isoform(s) may be regulated differently by specific nucleotide or nucleotide sugar. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865091 Tue, 06 Oct 2009 05:50:51 +0100 2865091 http://www.medworm.com/index.php?rid=2865090&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F523%3Frss%3D1 In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are converted to CG base pairs by the very short patch (VSP) repair pathway. DNA Polymerase I removes and resynthesizes the mismatched T starting from a 5'-nick created by the Vsr endonuclease. We used limited trypsinolysis to probe conformational changes in the N-terminal domain of Vsr in response to DNA binding, DNA cleavage and interaction with the polymerase. Our data show that the domain becomes trypsin resistant only under conditions that allow DNA cleavage, while interaction with the polymerase restores trypsin sensitivity. We suggest that the domain changes its conformation as a result of DNA nicking, and that DNA Pol I releases Vsr from the nick by reversing that conformational change. (Sou... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865090 Tue, 06 Oct 2009 05:50:51 +0100 2865090 http://www.medworm.com/index.php?rid=2865089&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F509%3Frss%3D1 The Glu54 residue of the norovirus 3C-like protease was implicated in proteolysis as a third-member carboxylate of the catalytic triad. The E54L mutant protease cleaved the sequence 133LSFE/AP between the 3B and 3C regions of norovirus polyprotein, but did not cleave the sequence 198ATSE/GK between the 3A and 3B. The 3BC junction mutation (3B-L133A or 3B-F135S) hampered the cleavage by the E54L protease, whereas the 3AB junction mutation (3A-A198L, S200F) allowed the E54L protease to digest. These results indicate that the E54L mutant protease is a substrate-specificity mutant and requires large hydrophobic amino acid residues at both P4 and P2 positions of the substrate. It was notable that the 3A-S200F P2 position mutation caused tight interaction between the wild-type protease and the C... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865089 Tue, 06 Oct 2009 05:50:51 +0100 2865089 http://www.medworm.com/index.php?rid=2865088&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F501%3Frss%3D1 To unravel the growth inhibition mechanism of Polo-like kinase 3 (Plk3), the effect of overexpression of Plk3 was examined in 293T cells. Cell rounding, changes in actin organization and cellular detachment were induced by Plk3 transfection in a kinase activity-dependent manner. Although apoptosis was not observed, Plk3 overexpression suppressed cellular growth in a long-term colony-forming assay. Because both Plk3 and Ras affect F-actin organization, the effect of co-transfection of Plk3 and Ras was evaluated. Adhesion was synergistically lost by co-transfection of these two genes, compared with transfection of Plk3 alone. Furthermore, overexpression of Plk3 caused long-term growth suppression in Ras-transformed NIH3T3. Collectively, Plk3 activation might cause cytoskeleton re-organizatio... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865088 Tue, 06 Oct 2009 05:50:51 +0100 2865088 http://www.medworm.com/index.php?rid=2865087&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F491%3Frss%3D1 Tuberculosis is the leading cause of death due to a single infectious agent in the world and the emergence of multidrug-resistant strains prompted us to develop new drugs with novel targets and mechanism. Here, we screened a natural antibiotics library with Mycobacterium smegmatis membrane-bound dehydrogenases and identified polymyxin B (cationic decapeptide) and nanaomycin A (naphtoquinone derivative) as inhibitors of alternative NADH dehydrogenase [50% inhibitory concentration (IC50) values of 1.6 and 31 µg/ml, respectively] and malate: quinone oxidoreductase (IC50 values of 4.2 and 49 µg/ml, respectively). Kinetic analysis on inhibition by polymyxin B showed that the primary site of action was the quinone-binding site. Because of the similarity in Km value for ubiquinone-1 a... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865087 Tue, 06 Oct 2009 05:50:51 +0100 2865087 http://www.medworm.com/index.php?rid=2865086&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F481%3Frss%3D1 Short hairpin RNAs (shRNA) targeting viral or cellular genes can effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication. This inhibition, however, may induce mutations in the targeted gene, leading to rapid escape from the shRNA-induced inhibition. We generated a lymphoid cell line that stably expressed a 19-bp shRNA targeting a well-conserved dimerization initiation site (DIS) of HIV-1, which strongly inhibited viral replication, thereby delaying virus escape. Furthermore, treatment of HIV-1 infection with DIS- and vif-shRNA combination therapy resulted in superior anti-viral responses compared to vif-shRNA monotherapy. Continuous challenge with HIV-1, however, generated virus mutants that could overcome the RNA interference restriction. Such anti-genes may be promisi... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865086 Tue, 06 Oct 2009 05:50:51 +0100 2865086 http://www.medworm.com/index.php?rid=2865085&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F471%3Frss%3D1 Peptidylarginine deiminases (PADs) consist of five enzymes which are widely distributed in human and rodent tissues. The two types of enzymes are found in human peripheral blood cells; PAD4 mainly in granulocytes and monocytes and PAD2 in lymphocytes and macrophages. Little is known about the regulation of PAD expression in macrophages. Here, we report that PAD2 is expressed in human monocytic leukaemia THP-1 cells during differentiation into macrophages by 12-O-tetradecanoylphorbol-13-acetate. During this differentiation, the levels of PAD2 mRNA and protein increased concomitantly, indicating the transcriptional regulation of PAD2 gene expression in the cells. The treatment of THP-1-derived macrophages with calcium ionophore A23187 generated vimentin deimination and resulted in the disrup... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865085 Tue, 06 Oct 2009 05:50:51 +0100 2865085 http://www.medworm.com/index.php?rid=2865084&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F463%3Frss%3D1 The chloroplast originated from endosymbiosis of photosynthetic bacteria. Thus, mechanisms essential for chloroplast biogenesis/homeostasis (protein synthesis, import from cytosol, assembly, and degradation) are predominantly governed by prokaryotic systems. Among these, the quality control system is crucial, because light energy constantly damages photosynthetic proteins and excessive light often limits plant growth by irreversibly inactivating the photosynthetic apparatuses. Here, we overview prokaryotic proteases (FtsH and Deg) which are two enzymes that play critical roles in this system. We particularly focus on Photosystem II (PSII) in thylakoid membranes, which is composed of more than 20 subunits. Among the subunits is one of the intrinsic reaction centre proteins (D1) which is con... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865084 Tue, 06 Oct 2009 05:50:51 +0100 2865084 http://www.medworm.com/index.php?rid=2865083&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F455%3Frss%3D1 Mitochondria are crucial for both life and death of eukaryotic cells. Compromised mitochondrial integrity has severe cellular consequences and is linked to senescence and neurodegenerative disorders in humans. To maintain the functionality of proteins in mitochondria, quality-control mechanisms including signal transduction pathways counteracting mitochondrial stress have evolved. A network of molecular chaperones and proteases monitors protein integrity and prevents accumulation of damaged proteins. In this review, the current knowledge of elaborate defence strategies within mitochondria is summarized. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865083 Tue, 06 Oct 2009 05:50:51 +0100 2865083 http://www.medworm.com/index.php?rid=2865082&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F4%2F449%3Frss%3D1 In Escherichia coli, like in any organism, the cytoplasmic (inner or plasma) membrane proteins play essential roles in transport of small and macro-molecules as well as in transmission of environmental signals across the membrane. Their quality control is critically important for growth and survival of the cell. However, our knowledge about the players and mechanisms of the system is still limited. This review focuses on proteolytic quality control of membrane proteins, in which two membrane-integrated proteases, FtsH and HtpX, with different modes of action, play central roles. The prohibitin family membrane protein complexes (HflKC and QmcA) contribute to the quality control system as a regulatory factor of FtsH and also as a possible membrane-chaperone. Failure of the quality control sy... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2865082 Tue, 06 Oct 2009 05:50:51 +0100 2865082 http://www.medworm.com/index.php?rid=2737192&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F439%3Frss%3D1 Previously, we histochemically examined the kidney of the MCC strain of mastomys (Praomys coucha) and found the storage of gangliosides. In the present studies, the lipid-bound sialic acid content of gangliosides in the MCC kidney was about 9- to 14-fold higher than that of the control (MWC strain). In the MCC kidney, sialic acids of male gangliosides were composed of N-acetylneuraminic acid at 91.5%; sialic acids of female gangliosides, however, were composed almost entirely of N-glycolylneuraminic acid. TLC of gangliosides showed that the MCC kidney contained four abundant gangliosides (two gangliosides each in males and females). These gangliosides isolated by HPLC were identified to be GM2(NeuAc) and fucosyl GM1(NeuAc) in the male MCC kidney and GM2(NeuGc) and fucosyl GM1(NeuGc) in the... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737192 Tue, 25 Aug 2009 23:00:00 +0100 2737192 http://www.medworm.com/index.php?rid=2737191&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F429%3Frss%3D1 ELOVL6, a member of the elongation of very long-chain fatty acids (ELOVL) family, has recently been identified as the rate-limiting enzyme for the elongation of palmitoyl-CoA. ELOVL6 deficient mice are protected from high-fat diet induced insulin resistance, suggesting that ELOVL6 might be a promising target for the treatment of metabolic disorders. Despite the increasing interest in Elovl6 as a therapeutic target, the lack of chemical tools for this enzyme has limited further elucidation of the biochemical and pharmacological properties of ELOVL6. We have identified Compound-A, a potent inhibitor for ELOVL6, by screening our company library and subsequently optimizing hit compounds. Compound-A potently inhibited human and mouse ELOVL6 and displayed >100-fold greater selectivity for ELO... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737191 Tue, 25 Aug 2009 23:00:00 +0100 2737191 http://www.medworm.com/index.php?rid=2737190&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F417%3Frss%3D1 CDK11p58, a CDK11 family Ser/Thr kinase, is a G2/M specific protein and contributed to regulation of cell cycle, transcription and apoptotic signal transduction. Recently, CDK11p58 has been reported to exert important functions in mitotic process, such as the regulation of bipolar spindle formation and sister chromatid cohesion. Here, we identified p21 activated kinase 1 (PAK1) as a new CDK11p58 substrate and we mapped a new phosphorylation site of Ser174 on PAK1. By mutagenesis, we created PAK1174A and PAK1174E, which mimic the dephosphorylated and phosphorylated form of PAK1; further analysis showed PAK1174E could be recruited to myosin V motor complex through binding to dynein light chain 2 (DLC2). PAK1174E could accelerate the mitosis progression in a nocodazole blocked cell model, whi... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737190 Tue, 25 Aug 2009 23:00:00 +0100 2737190 http://www.medworm.com/index.php?rid=2737189&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F407%3Frss%3D1 Most growth factors stimulate myoblast proliferation and prevent differentiation, whereas insulin-like growth factors (IGFs) promote myoblast differentiation through the phosphatidylinositol 3-kinase (PI3K) pathway. Subtilisin-like proprotein convertases (SPCs) are involved in cell growth and differentiation via activation of pro-growth factors. However, the role of SPCs in myogenesis remains poorly understood. Here we show that PACE4, a member of the SPC family, plays a critical role in myogenic differentiation of C2C12 cells. PACE4 mRNA levels increased markedly during myogenesis, whereas the expression of other member of SPC family, furin and PC6, remained unchanged. The expression pattern of pro-IGF-II, which is processed extracellularly by SPCs, was similar to that of PACE4. The expre... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737189 Tue, 25 Aug 2009 23:00:00 +0100 2737189 http://www.medworm.com/index.php?rid=2737188&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F399%3Frss%3D1 By searching the mouse EST database, we identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (RIKEN cDNA 2410078J06). Sequence analysis revealed that this new SULT belongs to the cytosolic SULT6 gene family. The recombinant form of this newly identified SULT, designated SULT6B1, was expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Purified mouse SULT6B1 exhibited sulfonating activity toward thyroxine and bithionol among a variety of endogenous and xenobiotic compounds tested as substrates. pH optimum of purified mouse SULT6B1 was determined to be 8.0. Tissue-specific expression of mouse and human SULT6B1 was examined by RT–PCR. While human SULT6B1 was specifically expressed in kidney and testis... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737188 Tue, 25 Aug 2009 23:00:00 +0100 2737188 http://www.medworm.com/index.php?rid=2737187&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F389%3Frss%3D1 Endo--N-acetylgalactosaminidase (endo--GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyses the O-glycosidic bonds in mucin-type O-glycan between -GalNAc and Ser/Thr. Endo--GalNAc-ase from Bifidobacterium longum JCM1217 (EngBF) is highly specific for the core 1-type O-glycan to release the disaccharide Galβ1-3GalNAc (GNB), whereas endo--GalNAc-ase from Clostridium perfringens (EngCP) exhibits broader substrate specificity. We determined the crystal structure of EngBF at 2.0 Å resolution and performed automated docking analysis to investigate possible binding modes of GNB. Mutational analysis revealed important residues for substrate binding, and two Trp residues (Trp748 and Trp750) appeared to form stacking interactions with the β-faces of sugar ring... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737187 Tue, 25 Aug 2009 23:00:00 +0100 2737187 http://www.medworm.com/index.php?rid=2737186&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F383%3Frss%3D1 To identify antibiotics targeting to respiratory enzymes, we carried out matrix screening of a structurally varied natural compound library with Pseudomonas aeruginosa membrane-bound respiratory enzymes. We identified a succinate dehydrogenase inhibitor, siccanin (IC50, 0.9 µM), which is a potent antibiotic against some pathogenic fungi like Trichophyton mentagrophytes and inhibits their mitochondrial succinate dehydrogenase. We found that siccanin was effective against enzymes from P. aeruginosa, P. putida, rat and mouse mitochondria but ineffective or less effective against Escherichia coli, Corynebacterium glutamicum, and porcine mitochondria enzyme. Action mode was mixed-type for quinone-dependent activity and noncompetitive for succinate-dependent activity, indicating the proxim... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737186 Tue, 25 Aug 2009 23:00:00 +0100 2737186 http://www.medworm.com/index.php?rid=2737185&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F375%3Frss%3D1 Tumour necrosis factor receptor-associated factor (TRAF)-interacting protein with a forkhead-associated domain (TIFA) activates TRAF6 to induce NF-B activation. TIFA-related protein, TIFAB, is highly expressed in the spleen and inhibits TIFA-mediated TRAF6 activation. However, little is known about cell types that express TIFAB and its function in those cells. Here, we show that TIFAB is mainly expressed in B cells rather than T cells in the spleen and that the expression level was much higher in dendritic cells (DCs) and macrophages than that in splenic lymphocytes. TIFAB expression was downregulated when B cells, DCs or macrophages were stimulated by TRAF6-mediated proliferative or maturation signals including those emanating from CD40, sIgM and TLRs. Furthermore, microinjection experime... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737185 Tue, 25 Aug 2009 23:00:00 +0100 2737185 http://www.medworm.com/index.php?rid=2737184&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F369%3Frss%3D1 Human C21orf63 is a type-1 transmembrane protein of hitherto unknown function, with two repeats of putative ‘galactose-binding lectin domains'. By using glycan microarray analysis and other assays, we found that human C21orf63 interacts with heparin and to a lesser extent with heparan sulphate. The C-terminal galactose-binding lectin domain of C21orf63 is necessary for heparin binding. The inability of other human proteins with galactose-binding lectin domains to interact with heparin suggests that heparin binding is a unique property of C21orf63. Results of real-time polymerase chain reaction and tissue immunostaining imply that C21orf63 is expressed on epithelia of various human tissues. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737184 Tue, 25 Aug 2009 23:00:00 +0100 2737184 http://www.medworm.com/index.php?rid=2737183&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F359%3Frss%3D1 The interactions of recombinant human eIF4A (4A) and its N- and C-terminal side domains (AN and AC, respectively) with the middle- and C-terminal-domain-linked fragment (GMC) of eIF4G and its middle and C-terminal domains (GM and GC, respectively) were investigated by surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC). It is remarkable that the kinetic parameter-dependent SPR profile observed for the 4A–GMC pair was quite different from the steady affinity profiles of the 4A–GM/GC pairs, suggesting the simultaneous contribution of the middle and C-terminal domains of eIF4G for the binding with eIF4A. On the other hand, ITC yielded the enthalpy energies of –1.5 x 104 to –2.5 x 104 J/mol for the domain–domain interactions of 4A w... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737183 Tue, 25 Aug 2009 23:00:00 +0100 2737183 http://www.medworm.com/index.php?rid=2737182&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F351%3Frss%3D1 The interactions of acyl-CoA with medium-chain acyl-CoA dehydrogenases (MCADs) reconstituted with artificial FADs—i.e. 8-CN-, 7,8-Cl2-, 8-Cl-, 8-OCH3- and 8-NH2-FAD—were investigated by UV-visible absorption and FT-IR measurements. Although 8-NH2-FAD-MCAD did not oxidize acyl-CoA the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs binding. Thus, 8-NH2-FAD-MCAD is one of the attractive materials for investigation of enzyme–substrate (ES) interaction in ES complex (the complex of oxidized MCAD with acyl-CoA). FT-IR difference spectra between non-labelled and [1-13C]-labelled acyl-CoA free in solution and bound to oxidized 8-NH2-FAD-MCAD were obtained. The broad 1668-cm–1 band of free octanoyl-CoA assigned to the C(1) = O stretching vibratio... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737182 Tue, 25 Aug 2009 23:00:00 +0100 2737182 http://www.medworm.com/index.php?rid=2737181&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F343%3Frss%3D1 A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analysed by gas chromatography, mass spectroscopy and nuclear magnetic resonance spectroscopy. Five major fatty acids were identified as hexadecanoic acid (palmitic acid, PA), cis-9-hexadecenoic acid (palmitoleic acid), 9,10-methylenehexadecanoic acid, cis-11-octadecenoic acid (vaccenic acid) and 11,12-methyleneoctadecanoic acid (lactobacillic acid). The composition of the bound fatty acids was similar to the fatty acid composition of E. coli extract, suggesting that the binding affinities are similar for these fatty acids. The urea-induced and thermal-unfolding transitions of the holoprotein (nondelipidated), apoprotein (delipidated) and PA-bound protein were observed by circular dichroism.... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737181 Tue, 25 Aug 2009 23:00:00 +0100 2737181 http://www.medworm.com/index.php?rid=2737180&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F337%3Frss%3D1 Apart from inhibiting RecA activity through protein–protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other anti-oxidant proteins. To identify the repertoire of proteins regulated by RecX, comparative proteomic studies were undertaken on a wild-type strain (R1) and recX null mutant (RecX–). Two-dimensional electrophoresis followed by MALDI-TOF identification revealed 35 differentially expressed proteins, including 12 up-regulated and 23 down-regulated proteins in the mutant. The 12 up-regulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation-induced proteins. The 23 down-regulated proteins are mainly involved in cellular me... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737180 Tue, 25 Aug 2009 23:00:00 +0100 2737180 http://www.medworm.com/index.php?rid=2737179&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F327%3Frss%3D1 Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'–5' direction. These results suggest that the h... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737179 Tue, 25 Aug 2009 23:00:00 +0100 2737179 http://www.medworm.com/index.php?rid=2737178&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F317%3Frss%3D1 Bem1 and Cdc24 of the budding yeast Saccharomyces cerevisiae interact with each other through PB1–PB1 heterodimer formation to regulate the establishment of cell polarity. Here we present the tertiary structure of the heterodimer of Bem1 and Cdc24 PB1 domains determined by NMR spectroscopy. To avoid ambiguity in the NMR spectral analysis, we first prepared a mutant of the Cdc24 PB1 domain that had truncated loops. The mutant provided well dispersed spectra without spectral overlapping, thus allowing unambiguous spectral assignments for structure determination. We confirmed that the loop deletion-mutant was quite similar to the wild-type in both 3D structure and binding affinity. The NMR structure of the heterodimer of the deletion-mutant of Cdc24 PB1 and Bem1 PB1 was determined using... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737178 Tue, 25 Aug 2009 23:00:00 +0100 2737178 http://www.medworm.com/index.php?rid=2737177&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F3%2F307%3Frss%3D1 Nestin is an intermediate filament protein mainly expressed in muscle and neural progenitors. Recently, we reported that nestin is expressed in rat vascular smooth muscle cells (VSMCs), disappears after serum-deprivation and then is re-expressed again following EGF stimulation. As the function of nestin in VSMCs remains unknown, its anti-apoptotic function was investigated in this study. We first showed that cell viability of nestin-depleted cells following H2O2 treatments decreased by nestin RNAi. Further DNA laddering analysis and flow cytometry results demonstrated that this loss of cell viability was mediated through apoptosis. In addition, caspase-9, caspase-3 and PARP were activated in nestin-depleted VSMCs following H2O2 treatments, indicating that nestin has an upstream inhibitory ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2737177 Tue, 25 Aug 2009 23:00:00 +0100 2737177 http://www.medworm.com/index.php?rid=2663735&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F295%3Frss%3D1 A lactose-specific lectin with a molecular mass of about 25 kDa was purified from the skin mucus of a cartilaginous fish—the common skate (Raja kenojei). The complementary DNA sequence of the lectin was 1540 bp long and contained a reading frame encoding 226 amino acids, which showed ~38% identity to pentraxins of mammals and teleosts. Gene expression was observed in the skin, gill, stomach and intestine in the healthy skate. We also identified an isotype gene from the liver whose deduced amino-acid sequence shared 69.0% identity with the skin type gene. The antiserum detected protein in the skin, where the lectin is localized in the epidermal cells, and in the blood plasma. The lectin genes are multicopied in the common skate genome. Although pentraxins are acute phase proteins, mRN... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663735 Sun, 02 Aug 2009 23:00:00 +0100 2663735 http://www.medworm.com/index.php?rid=2663734&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F283%3Frss%3D1 Lysophosphatidic acid (LPA) is a simple phospholipid but has numerous biological effects through a series of G-protein-coupled receptors specific to LPA. In general, LPA is short-lived when applied in vivo, which hinders most pharmacological experiments. In our continuing study to identify stable LPA analogues capable of in vivo applications, we identified here lysophosphatidylmethanol (LPM) as a stable and pan-LPA receptor agonist. A synthetic LPM activated all five LPA receptors (LPA1–5), and stimulates both cell proliferation and LPA-receptor-dependent cell motility. In addition, LPM showed a hypertensive effect in rodent when applied in vivo. We found that, when fetal calf serum was incubated in the presence of methanol, formation of LPM occurred rapidly, whereas it was completel... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663734 Sun, 02 Aug 2009 23:00:00 +0100 2663734 http://www.medworm.com/index.php?rid=2663733&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F273%3Frss%3D1 We reported previously that the activated enzyme was produced in the conditioned medium when full-length rat matriptase was expressed in monkey kidney COS-1 cells. The present study aimed to address when and where the matriptase activation occurs. COS-1 cells expressing matriptase were labelled with a membrane-impermeable biotin derivative and then solubilized with Triton. Both activated and non-activated matriptase molecules were detected in the avidin precipitants of Triton extracts, whereas only the non-activated molecules were detected in the flow-through fraction of avidin-precipitation procedure. Single-chain matriptase has been thought to have an inherent activity. Indeed, a secreted single-chain variant of recombinant matriptase bearing mutation at the activation-cleavage site was ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663733 Sun, 02 Aug 2009 23:00:00 +0100 2663733 http://www.medworm.com/index.php?rid=2663732&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F263%3Frss%3D1 In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663732 Sun, 02 Aug 2009 23:00:00 +0100 2663732 http://www.medworm.com/index.php?rid=2663731&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F251%3Frss%3D1 Recently, riboswitches and other structures discovered on mRNAs have been reported as examples of functional RNA structures, motifs. Such motifs were shown to be present as single-form valid structures but they are obscured among other less-valid structures. Here, I present a novel, practical virtual spectrometry (the GenoPoemicsTM Spectrometry) visualizing motifs on mRNA strands as spectra at-a-glance. Every motif along with validity of their existences could be observed on the spectra in human-friendly manners, and whole structures of mRNAs could be overviewed. Therefore, the spectra helped distinguish valid and less valid motifs. The spectrometry was applied to variety of mRNAs such as ADM, rPolβ and CD83 to identify structures of high validity on them, previously reported function... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663731 Sun, 02 Aug 2009 23:00:00 +0100 2663731 http://www.medworm.com/index.php?rid=2663730&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F241%3Frss%3D1 The KMSKS motif is the ATP binding motif for aminoacylation process of class I aminoacyl-tRNA synthetases. Although researches based on natural proteins inform us about the contribution of natural amino acid sequences for the catalysis, they have difficulties in discussing the other alternative sequences and prohibited sequences for the motif to maintain the catalytic ability. In order to reveal such the conditions for the alternative and prohibited sequences, it is important to investigate a library of various mutants for the motif. For that purpose, we build a library of more than 200 mutants substituting the KMSSS loop, Lys204-Met205-Ser206-Ser207-Ser208, in tyrosyl-tRNA synthetase of Methanococcus jannaschii, and their catalytic abilities were examined by the Amber suppression method. ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663730 Sun, 02 Aug 2009 23:00:00 +0100 2663730 http://www.medworm.com/index.php?rid=2663729&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F231%3Frss%3D1 The role of lipopolysaccharides (LPSs) in the biogenesis of outer membrane proteins have been investigated in several studies. Some of these analyses showed that LPS is required for correct and efficient folding of outer membrane proteins; other studies support the idea of independence of outer membrane proteins biogenesis from LPS structure. In this article, we investigated the involvement of LPS structure in the anomalous aggregation of outer membrane proteins in a E. coli mutant strain (S17-1(pir)). To achieve this aim, the LPS structure of the mutant strain was carefully determined and compared with the E. coli K-12 one. It turned out that LPS of these two strains differs in the inner core for the absence of a heptose residue (HepIII). We demonstrated that this difference is due to a m... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663729 Sun, 02 Aug 2009 23:00:00 +0100 2663729 http://www.medworm.com/index.php?rid=2663728&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F219%3Frss%3D1 The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought to be anchored to the peptidoglycan by the C-terminal conserved peptidoglycan-binding (PGB) motif of MotB. To clarify the role of the C-terminal region, we performed systematic cysteine mutagenesis and constructed a chimeric MotB protein which was replaced with the peptidoglycan-associated lipoprotein Pal. Although this chimera could not restore motility to a motB strain, we were able to isolate two motile revertants. One was F172V in the Pal region and the other was P159L in the MotB region. Furthermore, we attempted to map the MotB Cys mutations in the crystal structure of Escherichia coli Pal. We found that the MotB mutations that affected motility nearly overlapped with the predicted PG-binding resid... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663728 Sun, 02 Aug 2009 23:00:00 +0100 2663728 http://www.medworm.com/index.php?rid=2663727&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F209%3Frss%3D1 We found that CEL-I, a GalNAc-specific C-type lectin isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), induces inducible nitric oxide synthase (iNOS) expression and NO production in RAW264.7 cells. The NO production was inhibited by an iNOS inhibitor, L-NAME, but was not by a lipopolysaccharide (LPS) inhibitor, polymyxin B. In the presence of 0.1-M GalNAc, increased NO production by CEL-I-treated RAW264.7 cells was observed rather than the inhibition. Bovine serum albumin (BSA) significantly inhibited the CEL-I-induced NO production as well as the binding of FITC-labelled CEL-I on RAW264.7 cells. Three MAP kinase inhibitors (specific to extra-cellular regulated kinase, c-jun NH2-terminal kinase and p38 MAP kinase) inhibited CEL-I-induced NO production with different... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663727 Sun, 02 Aug 2009 23:00:00 +0100 2663727 http://www.medworm.com/index.php?rid=2663726&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F201%3Frss%3D1 In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base. At 37°C incubation for 1 h, BglII and EcoRI showed higher DNA cleavage reactivity on Hyp-containing DNA substrates than on normal ones, whereas BamHI showed lower values on Hyp-containing substrates. Such high cleavage performance of BglII and EcoRI on Hyp-containing DNA substrates is in contrast to the results obtained 20 years ago, in which short DNA substrates (8- or 10-mer) and low reaction temperatures (15–20°C) were employed. The... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663726 Sun, 02 Aug 2009 23:00:00 +0100 2663726 http://www.medworm.com/index.php?rid=2663725&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F193%3Frss%3D1 In this study, a systematic analysis of the conformation and stability changes induced by these two metal ions was reported. The spectra of protein intrinsic fluorescence were used to measure the equilibrium unfolding of the p53DBD protein. The stability against chemical denaturation increased in the order apo < Mg2+ < Zn2+. The thermal stability monitored by DSC scans showed that the binding of metal ions to p53DBD increased the thermal stability of the protein. To explore additional information of structural changes after the binding of metal ions, we used the fluorescent probes to evaluate the hydrophobic surface exposure. The results established that metal ions binding increased hydrophobic exposure on the surface of p53DBD. Analysis of acrylamide quenching experiments revealed t... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663725 Sun, 02 Aug 2009 23:00:00 +0100 2663725 http://www.medworm.com/index.php?rid=2663724&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F185%3Frss%3D1 In the digestive tract of mice (HR-1 strain), glycolipids belonging to the ganglio-series were revealed to be expressed in region-specific manners, i.e. FGA1 and FGM1 in the stomach, GA1 in the small intestine, and FGA1 and sulphatides in the cecum. The amount of GA1 as a receptor glycolipid for Lactobacilli was especially higher in the small intestine than in the other regions, it comprising 1.6–2.8 µg/mg dry weight. On immunization of rabbits with Lactobacillus johnsonii and Lactobacillus intestinalis, both of which are murine intestinal bacteria, antibodies toward bacterial glycolipids, i.e. Gal1–2Glc1–3DG, and tri- and tetrahexaosyl DGs, were effectively generated and, in addition, they were found to cross-react with GA1 and GalCer, but not with structurally rel... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663724 Sun, 02 Aug 2009 23:00:00 +0100 2663724 http://www.medworm.com/index.php?rid=2663723&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F173%3Frss%3D1 Due to instability of pyrimidine motif triplex nucleic acid under physiological pH and low magnesium ion concentration, stabilization of the triplex under the physiological condition is crucial in improving its therapeutic potential to artificially control gene expression in vivo. To this end, we investigated the thermodynamic and kinetic effects of morpholino (MOR) modification of triplex-forming oligonucleotide (TFO) on the triplex formation under the physiological condition. The thermodynamic analyses indicated that the MOR modification of TFO not only significantly increased the thermal stability of the triplex but also increased the binding constant for the triplex formation by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters suggested that the ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663723 Sun, 02 Aug 2009 23:00:00 +0100 2663723 http://www.medworm.com/index.php?rid=2663722&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F167%3Frss%3D1 Tumour necrosis factor (TNF) is an important cytokine that induces an inflammatory response predominantly through the TNF receptor-1 (TNFR1). A crucial strategy for the treatment of many autoimmune diseases, therefore, is to block the binding of TNF to TNFR1. We previously identified a TNFR1-selective antagonistic mutant TNF (R1antTNF) from a phage library containing six randomized amino acid residues at the receptor-binding site (amino acids 84–89). Two R1antTNFs, R1antTNF-T2 (A84S, V85T, S86T, Y87H, Q88N and T89Q) and R1antTNF-T8 (A84T, V85P, S86A, Y87I, Q88N and T89R), were successfully isolated from this library. Here, we analysed R1antTNF-T8 using surface plasmon resonance spectroscopy and X-ray crystallography to determine the mechanism underlying the antagonistic activity of R... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663722 Sun, 02 Aug 2009 23:00:00 +0100 2663722 http://www.medworm.com/index.php?rid=2663721&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F157%3Frss%3D1 Iron is essential to the unicellular green alga Chlamydomonas, but the molecular mechanism for response to iron deficiency remains largely unknown. In previous studies, we have identified FOX1 and ATX1 FEREs (Fe deficiency-responsive elements) as important regulation components of iron response in this organism. Here we present another iron regulated gene FEA1, which promoter was analysed by using a 5'-and 3'-end deletion and a scanning mutagenesis assay. The results reveal that the co-existence of –273/–188 and –118/–49 regions from transcriptional start site of FEA1 were sufficient and necessary for Fe deficiency-induced expression. Further deletion analysis indicates both –273/–253 and –103/–85 regions are essential for inducible expressio... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663721 Sun, 02 Aug 2009 23:00:00 +0100 2663721 http://www.medworm.com/index.php?rid=2663720&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F2%2F149%3Frss%3D1 Tamio Yamakawa isolated a glycosphingolipid from horse erythrocyte membrane, named it hematoside, and reported the results in Journal of Biochemistry. This was the first paper to report that glycosphingolipids are located in the cell membrane. He also isolated a glycosphingolipid, globoside, from human erythrocytes and demonstrated for the first time that ABO blood group antigens are glycosphingolipids in the erythrocytes. He reported the correct chemical structure of sulfatide, and found seminolipid, which is unexpectedly a glyceroglycolipid, as the major glycolipid of mammalian testis and spermatozoa. He started and developed the research of glycolipid biochemistry and established the basis for the further development of glycobiology. He published most of his original work in Journal of ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2663720 Sun, 02 Aug 2009 23:00:00 +0100 2663720 http://www.medworm.com/index.php?rid=2575855&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F141%3Frss%3D1 Presenilin 1 (PS1) has been identified as a causative gene for the early-onset of familial Alzheimer's disease, and it is mainly localized in the endoplasmic reticulum and the Golgi membrane as a multiple membrane-spanning protein. In the cell, PS1 is proteolytically processed to a 30-kDa N-terminal fragment and a 20-kDa C-terminal fragment (CTF), both of which exist as a stable high-molecular-weight protein complex, together with other components of -secretase. However, as there has been no report about the precise amount of PS1 expressed in mammalian tissues, the aim of this study was to quantitatively determine PS1–CTF amounts in various tissues such as liver, kidney, brain and heart of rat by western blotting using a [35S]-methionine-labelled PS1-CTF as a standard synthesized in ... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575855 Sun, 05 Jul 2009 23:00:00 +0100 2575855 http://www.medworm.com/index.php?rid=2575854&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F133%3Frss%3D1 PTK6 (also known as Brk) is an intracellular tyrosine kinase whose expression is up-regulated in several tumour types. Because localization of protein tyrosine kinases plays an important role in the development of cancers, we investigated the relationship between subcellular localization of PTK6 and its oncogenic properties. PTK6 was targeted to the plasma membrane or the nucleus of HEK 293 cells using the Src myristoylation signal (Myr) or SV40 T-antigen nuclear localization signal (NLS), respectively. The profile of cellular proteins phosphorylated by Myr-PTK6 was quite different from those phosphorylated by NLS-PTK6. Localization of PTK6 to the plasma membrane enhanced the ability of PTK6 to promote proliferation, cell survival and migration and to permit anchorage-independent colony fo... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575854 Sun, 05 Jul 2009 23:00:00 +0100 2575854 http://www.medworm.com/index.php?rid=2575853&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F123%3Frss%3D1 This study demonstrated the importance of CHOP-10 in coordinating individual gene expression in response to the mitochondrial stress. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575853 Sun, 05 Jul 2009 23:00:00 +0100 2575853 http://www.medworm.com/index.php?rid=2575852&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F113%3Frss%3D1 Estrogen-related receptor (ERR), one of the 48 human nuclear receptors, has a fully active conformation with no ligand. We recently demonstrated that ERR binds strongly bisphenol A (BPA), one of the nastiest endocrine disruptors, and thus retaining ERR's high basal constitutive activity. A report that BPA accumulates in the human maternal–fetal placental unit has led us to hypothesize that a large amount of ERR might exist in the human placenta. Here we report evidence that placenta indeed expresses ERR exceptionally strongly. We first ascertained the presence of nine different ERR mRNA variants and the resulting three ERR protein isoforms. By real-time PCR, we estimated the relative amount of ERR mRNA using total RNA extracts from human reproductive tissues. Placenta was found to ex... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575852 Sun, 05 Jul 2009 23:00:00 +0100 2575852 http://www.medworm.com/index.php?rid=2575851&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F103%3Frss%3D1 In this study, the DNA-binding property of the STPR domain in FMBP-1 was examined. Use of reagents selecting the DNA groove and an oligonucleotide in which the dA:dT pairs of the probe were replaced with dI:dC pairs in mobility shift assay demonstrated that FMBP-1 approaches DNA from the major groove. Permutation electrophoresis using probes of the same length but containing the FMBP-1-binding site at different positions showed that FMBP-1 bends DNA through its binding. To induce the sharp bend of DNA, the STPR domain alone was insufficient and the long N-terminal extending region was necessary. Moreover, the basic region extending from the N-terminus of the STPR domain stabilized the DNA binding of the STPR domain. These results suggested that DNA-binding properties of the STPR domain are... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575851 Sun, 05 Jul 2009 23:00:00 +0100 2575851 http://www.medworm.com/index.php?rid=2575850&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F95%3Frss%3D1 l-Threonine dehydrogenase, a key enzyme in the l-threonine metabolism, catalyses the NAD+-dependent conversion of l-threonine to 2-amino-3-ketobutyrate, that non-enzymically decarboxylates to aminoacetone. A search of the genome sequence of hyperthermophilic archaeon, Thermococcus kodakaraensis revealed the presence of a closely related orthologue (TK0916) of archaeal and bacterial l-threonine dehydrogenase genes. Expression in Escherichia coli, purification and characterization of the TK0916 gene product revealed that this gene actually coded for a protein with high levels of l-threonine dehydrogenase activity (7.26 U mg–1). The enzyme exhibited highest activity at pH 12 and 90°C. The Km values for l-threonine and NAD+ at 50°C were 1.6 mM and 0.028 mM, respectively. The enzy... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575850 Sun, 05 Jul 2009 23:00:00 +0100 2575850 http://www.medworm.com/index.php?rid=2575849&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F87%3Frss%3D1 In the present study, we clarified that transforming growth factor β (TGF-β) induces cellular senescence in human normal diploid cells, TIG-1, and identified protein kinase Cs (PKCs) as downstream mediators of TGF-β-induced cellular senescence. Among PKCs, we showed that PKC- induced cellular senescence in TIG-1 cells and was activated in replicatively and prematurely senescent TIG-1 cells. The causative role of PKC- in cellular senescence programs was demonstrated using a kinase negative PKC- and small interfering RNA against PKC-. Furthermore, PKC- was shown to function in human telomerase reverse transcriptase (hTERT) gene repression. These results indicate that PKC- plays a key role in cellular senescence programs, and suggest that the induction of senescence and hTERT r... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575849 Sun, 05 Jul 2009 23:00:00 +0100 2575849 http://www.medworm.com/index.php?rid=2575848&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F77%3Frss%3D1 Site-specific recognition and DNA-binding activity of p53 are crucial for its tumour suppressor function. Previous reports have shown that metal ions can affect the specific recognition and DNA-binding activity of p53DBD. Here we firstly report that magnesium ion can bind to the protein and influence its DNA-binding activity. To elucidate the nature and the effect of metal ions in the reaction chemistry, we utilized endogenous tryptophan fluorescence to quantitate the interaction between p53DBD and metal ions. The Ka value for the binding of Mg2+ to the protein is 1.88 x 103 M–1. Analysis of the CD data clearly suggested that the binding of magnesium ion induced a subtle conformational change rather than a radical modification of the overall protein architecture. Based on the results... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575848 Sun, 05 Jul 2009 23:00:00 +0100 2575848 http://www.medworm.com/index.php?rid=2575847&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F71%3Frss%3D1 In this study, we developed a highly sensitive and convenient assay for GP activity by analysing its chain-lengthening action on a fluorogenic maltooligosaccharide derivative in a glucose-1-phosphate-rich medium. Characterization of the substrate specificity of GP using pyridylaminated (PA-) maltooligosaccharides of various sizes revealed that a maltotetraosyl (Glc4) residue comprising the non-reducing-end of a PA-maltooligosaccharide is indispensable for the chain-lengthening action of GP, and PA-maltohexaose is the most suitable substrate for the purpose of this study. By using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, PA-maltoheptaose produced by the chain elongation of PA-maltohexaose could be isolated and quantified at 10 fmol. This method... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575847 Sun, 05 Jul 2009 23:00:00 +0100 2575847 http://www.medworm.com/index.php?rid=2575846&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F61%3Frss%3D1 Retaining glycosyl hydrolases, which catalyse both glycosylation and deglycosylation in a concerted manner, are the most abundant hydrolases. To date, their visualization has tended to be focused on glycosylation because glycosylation reactions can be visualized by inactivating deglycosylation step and/or using substrate analogues to isolate covalent intermediates. Furthermore, during structural analyses of glycosyl hydrolases with hydrolytic reaction products by the conventional soaking method, mutarotation of an anomeric carbon in the reaction products promptly and certainly occurs. This undesirable structural alteration hinders visualization of the second step in the reaction. Here, we investigated X-ray crystallographic visualization as a possible method for visualizing the conformatio... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575846 Sun, 05 Jul 2009 23:00:00 +0100 2575846 http://www.medworm.com/index.php?rid=2575845&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F51%3Frss%3D1 In this study, we performed the expression analysis of both proteins in humans and wild-type mice. The results of expression analysis indicated that salvage pathway II worked in human liver; however, it did not act in human brain or in mouse liver and brain. For this reason, a patient with SPR deficiency may show progressive neurological deterioration without HPA, and SPR knockout mice may exhibit HPA and abnormal locomotion activity. (Source: Journal of Biochemistry) Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575845 Sun, 05 Jul 2009 23:00:00 +0100 2575845 http://www.medworm.com/index.php?rid=2575844&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F43%3Frss%3D1 Many anticoagulant proteins have been found from snake venoms. Recently, L-amino acid oxidase (LAO) from the venom of Gloydius blomhoffi, M-LAO, was reported to inhibit coagulation factor IX; however, the mechanism of its anticoagulant activity is still unclear. Here, we re-evaluated the anticoagulant activity of M-LAO. We first purified M-LAO from the venom of G. blomhoffi, and examined the effect of LAO inhibitors and the hydrogen peroxide scavenger, catalase, on the anticoagulant activity of M-LAO. We found that the isolated M-LAO fraction prolongs the APTT, PT and fibrinogen clotting time and cleaves the A-chain of fibrinogen. LAO inhibitors or catalase did not inhibit these effects. Detailed analysis revealed that the M-LAO fraction contained a small amount of 39-kDa metalloproteinase... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575844 Sun, 05 Jul 2009 23:00:00 +0100 2575844 http://www.medworm.com/index.php?rid=2575843&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F33%3Frss%3D1 In this report, we developed a novel linker molecule, named ‘f-mono’, which has both of these properties: simple immobilization chemistry and a fluorescent label. Since the molecule contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved using the well-established reductive amination reaction. This conjugate of sugar chain and fluorescent linker (fluorescent ligand-conjugate, FLC) has fluorescent properties (ex. 335 nm, em. 380 nm), and as little as 1 µg of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. As a +2 Da larger MS peak ([M + H + 2]+ ion) was always associated with the theoretical MS peak ([M + H]+) (due to the reduction of the thioctic acid... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575843 Sun, 05 Jul 2009 23:00:00 +0100 2575843 http://www.medworm.com/index.php?rid=2575842&cid=s_32012_60_f&fid=32012&url=http%3A%2F%2Fjb.oxfordjournals.org%2Fcgi%2Fcontent%2Fshort%2F146%2F1%2F23%3Frss%3D1 The amino-acid sequence of a putative tryptophan monooxygenase (PTMO) from Ralstonia solanacearum is homologous with that of proenzyme (proPAO) of l-Phe oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp. P-501 in their overall sequences. PTMO was expressed in E. coli and purified, but had no catalytic activity to oxidize l-Phe. By treating PTMO with various proteases, the Pronase-treated PTMO (PTMOp) showed a relatively high activity to oxidize l-Phe, l-Trp, l-Tyr and l-Met. Studies on the stoichiometry of the reaction showed that l-Phe and l-Tyr were mostly oxygenated, that l-Met was mostly oxidized, and both oxygenation and oxidation of l-Trp was observed. Initial velocity patterns were a ping-pong type with l-Phe and l-Tyr, and a sequential type with l-Trp and l-Met as... Journal of Biochemistry journals http://www.medworm.com/rss/comments.php?id=2575842 Sun, 05 Jul 2009 23:00:00 +0100 2575842