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Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-beta1.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-beta1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial-mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for...
Source: Biological Procedures Online - September 11, 2009 Category: Molecular Biology Authors: Staršíchová A, Kubala L, Lincová E, Pernicová Z, Kozubík A, Souček K Tags: Biol Proced Online Source Type: journals

The Possible Cellular Mechanism for Extending Lifespan of Mice with Rapamycin.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19728006 [PubMed - as supplied by publisher] (Source: Biological Procedures Online)
Source: Biological Procedures Online - August 31, 2009 Category: Molecular Biology Authors: Li S Tags: Biol Proced Online Source Type: journals

MicroRNA Expression Profiling by Bead Array Technology in Human Tumor Cell Lines Treated with Interferon-Alpha-2a.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
MicroRNAs are positive and negative regulators of eukaryotic gene expression that modulate transcript abundance by specific binding to sequence motifs located prevalently in the 3' untranslated regions of target messenger RNAs (mRNA). Interferon-alpha-2a (IFNalpha) induces a large set of protein coding genes mediating antiproliferative and antiviral responses. Here we use a global microarray-based microRNA detection platform to identify genes that are induced by IFNalpha in hepatoma- or melanoma-derived human tumor cell lines. Despite the enormous differences in expression levels between these models, we were able to i...
Source: Biological Procedures Online - July 22, 2009 Category: Molecular Biology Authors: Siegrist F, Singer T, Certa U Tags: Biol Proced Online Source Type: journals

Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiv...
Source: Biological Procedures Online - July 13, 2009 Category: Molecular Biology Authors: Dismuke AD, Kohn AD, Moon RT, Wong MH Tags: Biol Proced Online Source Type: journals

Segmentation of Extrapulmonary Tuberculosis Infection Using Modified Automatic Seeded Region Growing.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In the image segmentation process of positron emission tomography combined with computed tomography (PET/CT) imaging, previous works used information in CT only for segmenting the image without utilizing the information that can be provided by PET. This paper proposes to utilize the hot spot values in PET to guide the segmentation in CT, in automatic image segmentation using seeded region growing (SRG) technique. This automatic segmentation routine can be used as part of automatic diagnostic tools. In addition to the original initial seed selection using hot spot values in PET, this paper also introduces a new SRG grow...
Source: Biological Procedures Online - July 13, 2009 Category: Molecular Biology Authors: Avazpour I, Saripan MI, Nordin AJ, Abdullah RS Tags: Biol Proced Online Source Type: journals

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter,...
Source: Biological Procedures Online - June 23, 2009 Category: Molecular Biology Authors: Pappas JJ, Toulouse A, Bradley WE Tags: Biol Proced Online Source Type: journals

A Framework for White Blood Cell Segmentation in Microscopic Blood Images Using Digital Image Processing.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Evaluation of blood smear is a commonly clinical test these days. Most of the time, the hematologists are interested on white blood cells (WBCs) only. Digital image processing techniques can help them in their analysis and diagnosis. For example, disease like acute leukemia is detected based on the amount and condition of the WBC. The main objective of this paper is to segment the WBC to its two dominant elements: nucleus and cytoplasm. The segmentation is conducted using a proposed segmentation framework that consists of an integration of several digital image processing algorithms. Twenty microscopic blood images wer...
Source: Biological Procedures Online - June 10, 2009 Category: Molecular Biology Authors: Sadeghian F, Seman Z, Ramli AR, Abdul Kahar BH, Saripan MI Tags: Biol Proced Online Source Type: journals

A Method for Structure-Activity Analysis of Quorum-Sensing Signaling Peptides from Naturally Transformable Streptococci.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Many species of streptococci secrete and use a competence-stimulating peptide (CSP) to initiate quorum sensing for induction of genetic competence, bacteriocin production, and other activities. These signaling molecules are small, unmodified peptides that induce powerful strain-specific activity at nano-molar concentrations. This feature has provided an excellent opportunity to explore their structure-function relationships. However, CSP variants have also been identified in many species, and each specifically activates its cognate receptor. How such minor changes dramatically affect the specificity of these peptides r...
Source: Biological Procedures Online - June 10, 2009 Category: Molecular Biology Authors: Tian X, Syvitski RT, Liu T, Livingstone N, Jakeman DL, Li YH Tags: Biol Proced Online Source Type: journals

Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An important part of characterizing any protein molecule is to determine its size and shape. Sedimentation and gel filtration are hydrodynamic techniques that can be used for this medium resolution structural analysis. This review collects a number of simple calculations that are useful for thinking about protein structure at the nanometer level. Readers are reminded that the Perrin equation is generally not a valid approach to determine the shape of proteins. Instead, a simple guideline is presented, based on the measured sedimentation coefficient and a calculated maximum S, to estimate if a protein is globular or elo...
Source: Biological Procedures Online - May 14, 2009 Category: Molecular Biology Authors: Erickson HP Tags: Biol Proced Online Source Type: journals

SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP) for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 ston...
Source: Biological Procedures Online - May 14, 2009 Category: Molecular Biology Authors: Wai-Hoe L, Wing-Seng L, Ismail Z, Lay-Harn G Tags: Biol Proced Online Source Type: journals

In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analy...
Source: Biological Procedures Online - April 9, 2009 Category: Molecular Biology Authors: Hojman P, Eriksen J, Gehl J Tags: Biol Proced Online Source Type: journals

Detection and Processing Techniques of FECG Signal for Fetal Monitoring.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Fetal electrocardiogram (FECG) signal contains potentially precise information that could assist clinicians in making more appropriate and timely decisions during labor. The ultimate reason for the interest in FECG signal analysis is in clinical diagnosis and biomedical applications. The extraction and detection of the FECG signal from composite abdominal signals with powerful and advance methodologies are becoming very important requirements in fetal monitoring. The purpose of this review paper is to illustrate the various methodologies and developed algorithms on FECG signal detection and analysis to provide efficien...
Source: Biological Procedures Online - March 27, 2009 Category: Molecular Biology Authors: Hasan MA, Reaz MB, Ibrahimy MI, Hussain MS, Uddin J Tags: Biol Proced Online Source Type: journals

Computational Biology Methods and Their Application to the Comparative Genomics of Endocellular Symbiotic Bacteria of Insects.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Comparative genomics has become a real tantalizing challenge in the postgenomic era. This fact has been mostly magnified by the plethora of new genomes becoming available in a daily bases. The overwhelming list of new genomes to compare has pushed the field of bioinformatics and computational biology forward toward the design and development of methods capable of identifying patterns in a sea of swamping data noise. Despite many advances made in such endeavor, the ever-lasting annoying exceptions to the general patterns remain to pose difficulties in generalizing methods for comparative genomics. In this review, we dis...
Source: Biological Procedures Online - March 11, 2009 Category: Molecular Biology Authors: Commins J, Toft C, Fares MA Tags: Biol Proced Online Source Type: journals

Competitive RT-PCR Strategy for Quantitative Evaluation of the Expression of Tilapia (Oreochromis niloticus) Growth Hormone Receptor Type I.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Quantization of gene expression requires that an accurate measurement of a specific transcript is made. In this paper, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) by competition for tilapia growth hormone receptor type I is designed and validated. This experimental procedure was used to determine the abundance of growth hormone receptor type I transcript in different tilapia tissues. The results obtained with this developed competitive RT-PCR were similar to real-time PCR results reported recently. This protocol provides a reliable alternative, but less expensive than real-time PCR to quanti...
Source: Biological Procedures Online - March 10, 2009 Category: Molecular Biology Authors: Rodríguez-Mallon A, Cárdenas Y, Lugo JM, Oliva A, Morales A, Estrada MP Tags: Biol Proced Online Source Type: journals

Comparison of Enzymatic and Non-Enzymatic Means of Dissociating Adherent Monolayers of Mesenchymal Stem Cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The dissociation of adherent mesenchymal stem cell (MSC) monolayers with trypsin and enzyme-free dissociation buffer was compared. A significantly lower proportion of viable cells were obtained with enzyme-free dissociation buffers compared to trypsin. Subsequently, the dissociated cells were re-seeded on new cell culture dishes and were subjected to the MTT assay 24 h later. The proportion of viable cells that reattached was significantly lower for cells obtained by dissociation with enzyme-free dissociation buffer compared to trypsin. Frozen-thawed MSC displayed a similar trend, yielding consistently higher cell viab...
Source: Biological Procedures Online - March 10, 2009 Category: Molecular Biology Authors: Heng BC, Cowan CM, Basu S Tags: Biol Proced Online Source Type: journals

Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use. PMID: 19495915 [PubMed - as supplied by publisher] (Source: Biological Procedures Online)
Source: Biological Procedures Online - March 6, 2009 Category: Molecular Biology Authors: Rodríguez-Casuriaga R, Geisinger A, López-Carro B, Porro V, Wettstein R, Folle GA Tags: Biol Proced Online Source Type: journals

Streptozotocin, Type I Diabetes Severity and Bone.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
As many as 50% of adults with type I (T1) diabetes exhibit bone loss and are at increased risk for fractures. Therapeutic development to prevent bone loss and/or restore lost bone in T1 diabetic patients requires knowledge of the molecular mechanisms accounting for the bone pathology. Because cell culture models alone cannot fully address the systemic/metabolic complexity of T1 diabetes, animal models are critical. A variety of models exist including spontaneous and pharmacologically induced T1 diabetic rodents. In this paper, we discuss the streptozotocin (STZ)-induced T1 diabetic mouse model and examine dose-dependen...
Source: Biological Procedures Online - March 6, 2009 Category: Molecular Biology Authors: Motyl K, McCabe LR Tags: Biol Proced Online Source Type: journals

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In this report, we detail the different approaches used for the isolation, the identification of a RNA binding protein and the validation of its role. PMID: 19048127 [PubMed - as supplied by publisher] (Source: Biological Procedures Online)
Source: Biological Procedures Online - December 3, 2008 Category: Molecular Biology Authors: Unterberger A, Torrisani J, Szyf M Tags: Biol Proced Online Source Type: journals

In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogen-induced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a well-known amphibian pat...
Source: Biological Procedures Online - July 7, 2008 Category: Molecular Biology Authors: Morales H, Robert J Tags: Biol Proced Online Source Type: journals

Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA).Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Calcitriol (1alpha, 25-dihydroxyvitamin D(3)) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot ...
Source: Biological Procedures Online - July 7, 2008 Category: Molecular Biology Authors: Moeenrezakhanlou A, Nandan D, Reiner NE Tags: Biol Proced Online Source Type: journals

Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We theref...
Source: Biological Procedures Online - July 7, 2008 Category: Molecular Biology Authors: Keating DH, Shulla A, Klein AH, Wolfe AJ Tags: Biol Proced Online Source Type: journals

Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried, the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique, samples are frozen, fractured and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Imm...
Source: Biological Procedures Online - July 7, 2008 Category: Molecular Biology Authors: Robenek H, Severs NJ Tags: Biol Proced Online Source Type: journals

Quantitative evaluation of signaling events in Drosophila s2 cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report, we describe a simple method for the quantification of JNK activation by Western blot analysis or directly in tissue culture plates. PMID: 18385808 [PubMed - in process] (Source: Biological Procedures Online)
Source: Biological Procedures Online - July 7, 2008 Category: Molecular Biology Authors: Bond D, Primrose DA, Foley E Tags: Biol Proced Online Source Type: journals

A method for detecting functional activity related expression in gross brain regions, specific brain nuclei and individual neuronal cell bodies and their projections.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of beta-galactosidase (betagal), the product of the lacZ gene. Furthermore, the underly...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Murphy M, Greferath U, Wilson YM Tags: Biol Proced Online Source Type: journals

Generation of shRNAs from randomized oligonucleotides.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries from either individually made gene specific sequences or cDNA libraries,...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Wu H, Dinh A, Mo YY Tags: Biol Proced Online Source Type: journals

Rapid site-directed domain scanning mutagenesis of enteropathogenic Escherichia coli espD.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We developed a rapid mutagenesis method based on a modification of the QuikChange(R) system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 - 22 bp to achieve melting temperatures greater than 80 degrees C. 2: the final concentrations of both primers were increased to 5-10 ng/mul and the final concentration of template to 1-2 ng/mul. 3: the annealing temperature was adjusted when necessary from 52 degrees C to 58 degrees C. We gene...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Deng Q, Luo W, Donnenberg MS Tags: Biol Proced Online Source Type: journals

Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing re...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Koo SH, Ong TC, Chong KT, Lee CG, Chew FT, Lee EJ Tags: Biol Proced Online Source Type: journals

Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (Sparus aurata L.).Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocyt...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Salinas I, Meseguer J, Esteban MA Tags: Biol Proced Online Source Type: journals

An in vitro method to study the effects of hematopoietic regulators during immune and blood cell development.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1I and the neuropeptide, substance P (SP). SDF-1I production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1I levels stimulate HSC functions through direct and indirect mechanisms. The indirect meth...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Patel N, Castillo M, Rameshwar P Tags: Biol Proced Online Source Type: journals

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions. PMID: 18385803 [PubMed - in process] (Source: Biological Procedures Online)
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Poon HF, Abdullah L, Reed J, Doore SM, Laird C, Mathura V, Mullan M, Crawford F Tags: Biol Proced Online Source Type: journals

Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x10(6) purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilizati...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Antony P, Hoek K, Sarmah B, Khan WN Tags: Biol Proced Online Source Type: journals

Development of human gene reporter cell lines using rAAV mediated homologous recombination.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Understanding mechanisms of gene regulation has broad therapeutic implications for human disease. Here we describe a novel method for generating human cell lines that serve as reporters of transcriptional activity. This method exploits the ability of recombinant adeno-associated virus to mediate the insertion of exogenous DNA sequences into specific genomic loci through homologous recombination. To overcome the severe size limitation of the rAAV for carrying exogenous DNA, an enhanced green fluorescent protein (EGFP)-Luciferase fusion gene was used as both a selectable marker and gene expression reporter. EGFP was used...
Source: Biological Procedures Online - January 1, 2007 Category: Molecular Biology Authors: Fernandez SL, Russell DW, Hurlin PJ Tags: Biol Proced Online Source Type: journals

Rapid and simple comparison of messenger RNA levels using real-time PCR.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species. PMID: 16446781 [PubMed] (Source: Biological Procedures Online)
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Dussault AA, Pouliot M Tags: Biol Proced Online Source Type: journals

Techniques of EMG signal analysis: detection, processing, classification and applications.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Electromyography (EMG) signals can be used for clinical/biomedical applications, Evolvable Hardware Chip (EHW) development, and modern human computer interaction. EMG signals acquired from muscles require advanced methods for detection, decomposition, processing, and classification. The purpose of this paper is to illustrate the various methodologies and algorithms for EMG signal analysis to provide efficient and effective ways of understanding the signal and its nature. We further point up some of the hardware implementations using EMG focusing on applications related to prosthetic hand control, grasp recognition, and...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Raez MB, Hussain MS, Mohd-Yasin F Tags: Biol Proced Online Source Type: journals

Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell ...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Yamada HY, Gorbsky GJ Tags: Biol Proced Online Source Type: journals

Use of chromatin immunoprecipitation (ChIP) to detect transcription factor binding to highly homologous promoters in chromatin isolated from unstimulated and activated primary human B cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report, we demonstrate the selective in vivo association of NF-kappaB, p300 and CREB with the human Igamma1 promoter located in the intronic region upstream of the Cgamma1 exons in the immunoglobulin heavy chain locus. These methods have the ability to extend ChIP analysis to promoters with a high degree of homology. PMID: 16799696 [PubMed] (Source: Biological Procedures Online)
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Dryer RL, Covey LR Tags: Biol Proced Online Source Type: journals

Direct application of plasmid DNA containing type I interferon transgenes to vaginal mucosa inhibits HSV-2 mediated mortality.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The application of naked DNA containing type I interferon (IFN) transgenes is a promising potential therapeutic approach for controlling chronic viral infections. Herein, we detail the application of this approach that has been extensively used to restrain ocular HSV-1 infection, for antagonizing vaginal HSV-2 infection. We show that application of IFN-alpha1, -alpha5, and -beta transgenes to vaginal mouse lumen 24 hours prior to HSV-2 infection reduces HSV-2 mediated mortality by 2.5 to 3-fold. However, other type I IFN transgenes (IFN- alpha4, -alpha5, -alpha6, and -alpha9) are non effectual against HSV-2. We further...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Austin BA, James CM, Härle P, Carr DJ Tags: Biol Proced Online Source Type: journals

Practical three color live cell imaging by widefield microscopy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be ima...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Xia J, Kim SH, Macmillan S, Truant R Tags: Biol Proced Online Source Type: journals

Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this work we describe protocols for the generation of gene deletions and gene replacements using a temperature sensitive plasmid in Escherichia coli O157:H7. This technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. A number of examples are used to illustrate the flexibility of the system which has been used to create novel gene replacements including fusions for protein localisation work an...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Emmerson JR, Gally DL, Roe AJ Tags: Biol Proced Online Source Type: journals

Chromatin immunoprecipitation assay detects ERalpha recruitment to gene specific promoters in uterus.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chromatin immunoprecipitation (ChIP) technique allows detection of proteins that bind to chromatin. While this technique has been applied extensively in cell-based studies, its tissue-based application remains poorly explored. We are specifically interested in examining estrogen-dependent transcriptional mechanism in respect of recruitment of estrogen receptor-alpha (ERalpha), a ligand-activated transcription factor, to uterine gene promoters in mice. Recent gene-array studies, utilizing ERalpha knock-out vs. wild-type mice, have revealed that estrogen regulates numerous uterine genes temporally and most importantly vi...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Ray S, Das SK Tags: Biol Proced Online Source Type: journals

Combining genetic and biochemical approaches to identify functional molecular contact points.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins an...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Badtke MP, Cao F, Tavis JE Tags: Biol Proced Online Source Type: journals

Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time - calculations that comm...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Gallup JM, Ackermann MR Tags: Biol Proced Online Source Type: journals

A statistical framework for genetic association studies of power curves in bird flight.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
How the power required for bird flight varies as a function of forward speed can be used to predict the flight style and behavioral strategy of a bird for feeding and migration. A U-shaped curve was observed between the power and flight velocity in many birds, which is consistent to the theoretical prediction by aerodynamic models. In this article, we present a general genetic model for fine mapping of quantitative trait loci (QTL) responsible for power curves in a sample of birds drawn from a natural population. This model is developed within the maximum likelihood context, implemented with the EM algorithm for estima...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Lin M, Zhao W, Wu R Tags: Biol Proced Online Source Type: journals

Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (rho=0.708, p<0.0001, n=277) using these platforms. The contribution of factors including up- vs. down-regulation, spot intensity, rho...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Morey JS, Ryan JC, Van Dolah FM Tags: Biol Proced Online Source Type: journals

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described fo...
Source: Biological Procedures Online - January 1, 2006 Category: Molecular Biology Authors: Harrison JJ, Ceri H, Yerly J, Stremick CA, Hu Y, Martinuzzi R, Turner RJ Tags: Biol Proced Online Source Type: journals

A rapid and sensitive assay for quantification of siRNA efficiency and specificity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed ag...
Source: Biological Procedures Online - January 1, 2005 Category: Molecular Biology Authors: Smart N, Scambler PJ, Riley PR Tags: Biol Proced Online Source Type: journals

An in vitro method to select malignant cells from surgical biopsies of breast cancer patients.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This study describes a reliable and reproducible method for isolating and expanding malignant cells from surgical breast tissues. The method uses co-cultures with BM stroma to select for the cancer cells while the healthy cells undergo rapid cell death. Studies are described to show the cloning efficiencies and sensitivity of the method using surgical samples of varying sizes, different stages of BC, and samples from needle biopsies. PMID: 15678170 [PubMed - as supplied by publisher] (Source: Biological Procedures Online)
Source: Biological Procedures Online - January 1, 2005 Category: Molecular Biology Authors: Corcoran KE, Patel PS, Rameshwar P Tags: Biol Proced Online Source Type: journals

Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected...
Source: Biological Procedures Online - January 1, 2005 Category: Molecular Biology Authors: Lefrancois S, Canuel M, Zeng J, Morales CR Tags: Biol Proced Online Source Type: journals

Rapid cryopreservation of five mammalian and one mosquito cell line at -80 degrees C while attached to flasks in a serum free cryopreservative.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at -80 degrees C while attached to the substratum of 25 cm2 tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines. PMID: 16136221 [PubMed] (Source: Biological Procedures Online)
Source: Biological Procedures Online - January 1, 2005 Category: Molecular Biology Authors: Corsini J, Mann E Tags: Biol Proced Online Source Type: journals

An improved method for constructing and selectively silanizing double-barreled, neutral liquid-carrier, ion-selective microelectrodes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and emp...
Source: Biological Procedures Online - January 1, 2005 Category: Molecular Biology Authors: Deveau JS, Lindinger MI, Grodzinski B Tags: Biol Proced Online Source Type: journals