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 'Springer protocols feed by Molecular Medicine' source. Sat, 10 Oct 2009 19:37:19 +0100 http://www.medworm.com/index.php?rid=2819092&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_9 The necessity of genotyping high number of variations in extended sample sets has become apparent in the era of large genomic studies of common complex disorders, in cancer and in pharmacogenomics. The single nucleotide polymorphisms’ (SNPs) apparent advantages over other genetic markers such as high frequency, relative stability, and statistically random distribution across the genome have made them a method of choice for most of these genome-wide oriented applications. The requirement for simultaneous genotyping of high number of SNPs, keeping at the same time reasonable price and reliable accuracy, triggered the rise of the genotyping throughput, and led to the development of the array-based technologies. The present chapter briefly reviews the methodological and historical aspect... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819092 Fri, 31 Jul 2009 23:00:00 +0100 2819092 http://www.medworm.com/index.php?rid=2819091&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_8 We describe here in detail a protocol for adenoviral gene transfer into isolated mouse islets of the pancreas. Effective gene transfer into pancreatic islets using recombinant adenoviruses can be achieved with a multiplicity of infection (MOI) of 10. However, if the islets are not dispersed, adenoviral gene transfer is limited only to the cells on the periphery of the islets, which represent the glucagon-producing alpha cells in rodents. Dispersion of pancreatic islets with EGTA increases the efficiency of gene transfer into the cells within the core of the islets, which consist of insulin-producing beta cells. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819091 Fri, 31 Jul 2009 23:00:00 +0100 2819091 http://www.medworm.com/index.php?rid=2819090&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_7 In situ hybridization represents a powerful technique to localize DNA or RNA of interest at the chromosomal or cellular level. In endocrine tissues composed of diverse and varied cell types, in situ hybridization has allowed the identification of specific cells responsible for the expression of genes controlling the function of the tissue. Our laboratory has routinely used this approach to understand the cellular expression of genes associated with the growth of the ovarian follicle, rupture of the follicle, and transformation of the ruptured follicle into the corpus luteum. The current study outlines the procedural details of in situ detection of mRNA in tissues and illustrates the utility of this approach in identifying the ovarian cells expressing the matrix metalloproteinases and their... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819090 Fri, 31 Jul 2009 23:00:00 +0100 2819090 http://www.medworm.com/index.php?rid=2819089&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_6 Microarray technology has been used widely in gynecology. Numerous studies have used this method to address biological questions related to human endometrium. The cyclic changes of endometrium confer special characteristics that should be considered before genomic analysis. The present study reviews these considerations and the principles of transcriptomic analysis through an example of a comparison of three different phases of the menstrual cycle. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819089 Fri, 31 Jul 2009 23:00:00 +0100 2819089 http://www.medworm.com/index.php?rid=2819088&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_5 DNA microarray is an important discovery technology that allows the analysis of the expression of thousands of genes at a time. Data from DNA microarrays elucidate fundamental biological processes through discovery of differential expression of genes not previously known or predicted to be involved in a particular process. In the ovary and other hormone-responsive tissues, the technology can be used to examine the effects of gene mutations, pharmaceutical agents, disease, hormones, developmental changes, or changes in gene expression related to aging. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819088 Fri, 31 Jul 2009 23:00:00 +0100 2819088 http://www.medworm.com/index.php?rid=2819087&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_4 Lipid second messengers play important roles in many cell signaling cascades. Lipid signaling molecules allow for high specificity, rapid transduction, and rapid reversibility of localized stimulation events. Fluorescent sensors capable of detecting individual signaling lipids enable their production and degradation to be followed, revealing the nature and dynamics of signaling pathways. The following sections outline a method for using lipid sensors to monitor the production of signaling lipids on the plasma membrane of C2C12 myotubes in response to insulin signaling. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819087 Fri, 31 Jul 2009 23:00:00 +0100 2819087 http://www.medworm.com/index.php?rid=2819086&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_3 Glucocorticoids regulate a broad spectrum of physiologic functions essential for life and play an important role in the maintenance of basal and stress-related homeostasis. At the cellular level, the actions of glucocorticoids are mediated by the human glucocorticoid receptor α (hGRα), a ligand-dependent transcription factor ubiquitously expressed in almost all tissues and cells. The molecular mechanisms of hGRα action involve (a) binding to glucocorticoids, (b) cytoplasmic to nuclear translocation, (c) binding/association to DNA/chromatin, and (d) transcriptional activation or repression by interacting with cofactors and other transcription factors. Mutations or polymorphisms in the hGR gene may impair these molecular mechanisms of hGRα action, thereby altering tis... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819086 Fri, 31 Jul 2009 23:00:00 +0100 2819086 http://www.medworm.com/index.php?rid=2819085&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_2 The ability to precisely measure circulating levels of hormones is a foundation of modern endocrinology. For assisted reproductive technologies such as in vitro fertilization (IVF), accurate determination of circulating levels of estradiol are crucial for patient management, retrieval of fertilizable oocytes, and successful pregnancy outcome. For many years, circulating levels of estradiol were determined by radioimmunoassay. More recently, nonradioactive techniques such as ELISAs or chemiluminescent approaches have replaced traditional radioimmunoassays. In the current chapter, we outline the procedures for analysis of circulating levels of estradiol by both radioimmunoassay and chemiluminescent techniques. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819085 Fri, 31 Jul 2009 23:00:00 +0100 2819085 http://www.medworm.com/index.php?rid=2819084&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_25 In recent years, genome-wide association approaches have proven a powerful and successful strategy to identify genetic contributors to complex traits, including a number of endocrine disorders. Their success has meant that genome wide association studies (GWAS) are fast becoming the default study design for discovery of new genetic variants that influence a clinical trait or phenotype. This chapter focuses on a number of key elements that require consideration for the successful conduct of a GWAS. Although many of the considerations are common to any genetic study, the greater cost, extreme multiple testing, and greater openness to data sharing require specific awareness and planning by investigators. In the section on designing a GWAS, we reflect on ethical considerations, study design, s... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819084 Fri, 31 Jul 2009 23:00:00 +0100 2819084 http://www.medworm.com/index.php?rid=2819083&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_24 Oxidative stress (OS) is an imbalance between the amount of reactive oxygen species (ROS) produced and the ability of the antioxidants to scavenge these. OS has been established as a major etiological cause of male infertility. High levels of ROS are harmful and cause damage to sperm nuclear DNA. Evaluation of OS-related damage to spermatozoa is therefore highly relevant in assisted reproductive techniques (ART) such as intracytoplasmic sperm injection (ICSI). ICSI is an effective therapy for severe male factor infertility that bypasses the majority of reproductive tract deficiencies. Despite the controversial findings in the existing literature, there is now enough evidence to show that sperm DNA damage is detrimental to reproductive outcomes. In addition to impairment of fertility, such ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819083 Fri, 31 Jul 2009 23:00:00 +0100 2819083 http://www.medworm.com/index.php?rid=2819082&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_23 In recent years, evidence has emerged supporting the hypothesis that cancer is a stem cell disease. The cancer stem cell field was led by the discovery of leukemia stem cells (Tan, B.T., Park, C.Y., Ailles, L.E., and Weissman, I.L. (2006) The cancer stem cell hypothesis: a work in progress. Laboratory Investigation. 86, 1203–1207), and within the past few years cancer stem cells have been isolated from a number of solid tumor including those of breast and brain cancer among others (Al-Hajj M., Wicha M.S., Benito-Hernandez A., Morrison, S.J., and Clarke, M.F. (2003) Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100, 3983–3988; Singh, S.K., Clarke, I.D., Terasaki, M., Bonn, V.E., Hawkins, C., Squire, J., and Dirks, P.B. (2003) Id... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819082 Fri, 31 Jul 2009 23:00:00 +0100 2819082 http://www.medworm.com/index.php?rid=2819081&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_22 Introduction of foreign DNA into the mouse germ line is considered a major technical advancement in the fields of developmental biology and genetics. This technology now referred to as transgenic mouse technology has revolutionized virtually all fields of biology and provided new genetic approaches to model many human diseases in a whole animal context. Several hundreds of transgenic lines with expression of foreign genes specifically targeted to desired organelles/cells/tissues have been characterized. Further, the ability to spatio-temporally inactivate or activate gene expression in vivo using the “Cre-lox” technology has recently emerged as a powerful approach to understand various developmental processes including those relevant to molecular endocrinology. In this chapter,... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819081 Fri, 31 Jul 2009 23:00:00 +0100 2819081 http://www.medworm.com/index.php?rid=2819080&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_21 ES cells are a potential source for insulin producing cells (IPCs). However, two major handicaps are establishing reliable differentiation protocols and the lack of imaging techniques that allow monitoring of these cells post-transplantation. Here, we describe a new approach for monitoring the in vitro differentiation and real-time, non-invasive imaging of ES cell-derived IPCs in vivo. ES cells were molecularly engineered so that the rat insulin promoter (RIP) driven luciferase (Luc) expression was specifically turned on and up regulated following their differentiation into IPCs. The rationale underlying this approach is that the transcriptional activation of RIP leads to Luc expression in IPCs providing an extremely sensitive reporter for monitoring the earliest differentiation events in ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819080 Fri, 31 Jul 2009 23:00:00 +0100 2819080 http://www.medworm.com/index.php?rid=2819079&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_20 During the past decade the remarkable progress in molecular genetics and the possibility to engineer cells to express genes reporting on the activity of specific promoters has produced major changes in biological research. The description and validation of reporter mice for non-invasive assessment of biological and biochemical processes in living subjects and the results obtained with the models reporting on the activity of estrogen and peroxisome proliferator receptors clearly showed that such technologies have the potential to enhance our understanding of disease and drug activity. Although reporter-gene technology is in its infancy, reporter animals already represent a valuable tool for biomedical investigation. The present chapter aims at critically illustrating the methodology to be a... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819079 Fri, 31 Jul 2009 23:00:00 +0100 2819079 http://www.medworm.com/index.php?rid=2819078&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_1 Characterization of the binding of a hormone to its cognate receptor is a cornerstone of many studies in molecular and cellular endocrinology since this event represents the beginning of a specific cellular response, generally from a highly regulated extracellular messenger. The premise of hormone−receptor interaction follows from the law of mass action describing a reversible second-order reaction, hormone plus receptor, to give a non-covalently associated hormone-receptor complex. From this basic principle, a host of useful experimental parameters are available to the interested investigator. This chapter is focused on development of the experimental and mathematical underpinning of hormone−receptor interaction, with emphasis on a gonadotropin, chorionic gonadotropin (or lute... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819078 Fri, 31 Jul 2009 23:00:00 +0100 2819078 http://www.medworm.com/index.php?rid=2819077&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_19 Endometriosis, a common gynecological disorder responsible for infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites. Gene expression studies performed on endometriotic tissue homogenates have yielded results reflecting mRNA abundance in a mixture of cell types (including epithelial cells, stromal cells, fibrotic tissue, and muscle tissue). Therefore, a method for quantifying gene expression separately in individual cell populations is essential for identifying genetic markers. Laser capture microdissection is a technique for obtaining pure populations of cells from heterogeneous tissues. This chapter provides methods to obtain high-quality RNA suitable for a variety of different down stream applications from frozen endometrial ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819077 Fri, 31 Jul 2009 23:00:00 +0100 2819077 http://www.medworm.com/index.php?rid=2819076&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_18 Many organisms, including humans, have many more proteins than are actually coded for by their genes. This discrepancy is partially explained by the existence of alternative transcripts produced by the same gene. Multiple isoforms of the same gene sometimes perform completely different functions, and as such, knowing the sequence of one of the transcripts is not enough. Rapid Amplification of cDNA Ends (RACE) provides an inexpensive and powerful tool to quickly identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5’ and 3’ cDNA ends using the “New Race” technique. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819076 Fri, 31 Jul 2009 23:00:00 +0100 2819076 http://www.medworm.com/index.php?rid=2819075&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_17 Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating multiple protein functional properties including protein−protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Consequently, there has been intense interest in identifying new proteins that are targets of this modification and determining what role it plays in regulating their functions. This... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819075 Fri, 31 Jul 2009 23:00:00 +0100 2819075 http://www.medworm.com/index.php?rid=2819074&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_16 Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein−protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP2, a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET2 energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET2 assay as it is used to examine the physical interaction between the nuclear receptor ERα and the transcriptional coactivator SRC-1. Description of methods include selection of donor and acceptor combinations, fusion construct generation and vali... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819074 Fri, 31 Jul 2009 23:00:00 +0100 2819074 http://www.medworm.com/index.php?rid=2819073&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_15 Chromatin immunoprecipitation (ChIP) is a well-established procedure for protein−DNA interaction research. ChIP-chip, combining chromatin immunoprecipitation (ChIP) and microarray technology (Chip), enables scientists to survey genome-wide DNA binding sites for a given protein. The ChIP-chip technique has been used to identify transcription factor binding sites, explore epigenomic information and investigate factors in DNA replicate/repairs. Here we describe a protocol for ChIP-chip to study Pituitary Tumor Transforming Gene (PTTG1) in mammalian cells. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819073 Fri, 31 Jul 2009 23:00:00 +0100 2819073 http://www.medworm.com/index.php?rid=2819072&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_14 In order to fully understand the functions of a DNA-binding protein it is necessary to identify all of its binding sites in chromosomes and assess the role of each site in the overall biological function of the factor. An approach ChIP-on-Chip which combines the chromatin immunoprecipitation technique with chromosomal DNA microarray analysis, has proven to be a powerful means for the chromosome-wide identification of protein binding sites. This approach can also be used to characterize chromosome-wide variations in patterns of post-translational protein modifications, for example histone modifications. This chapter presents methodologies for the ChIP-on-Chip analysis, using as an example the identification of chromosome-wide binding sites for the TATA-binding protein in mitotic cells. (Sou... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819072 Fri, 31 Jul 2009 23:00:00 +0100 2819072 http://www.medworm.com/index.php?rid=2819071&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_13 Regulating gene expression is a complex process requiring the interaction of multiple transcription factors with their cognate recognition sequences. While these DNA-bound transcription factors are the primary drivers of gene expression, the capacity of a transcription factor to alter gene expression is tempered by its association with a host of coregulatory proteins that are recruited to the DNA-bound transcription factor. We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound estrogen receptor α (ERα) using an agarose-based electrophoretic mobility shift assay (EMSA). This method should be readily adapted to a variety of cultured cell lines, DNA sequences, and transcription factors and has the potential to provide valuable info... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819071 Fri, 31 Jul 2009 23:00:00 +0100 2819071 http://www.medworm.com/index.php?rid=2819070&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_12 As a transcriptional regulator, the androgen receptor (AR) regulates the expression of many genes that are essential for male sexual differentiation, including the development of both normal prostate and prostate cancer. The AR acts by binding to regulatory DNA sequences found on the promoters of regulated genes. The study of AR activity on such responsive promoters is greatly facilitated by the use of the reporter gene assay, which provides a quantitative and reproducible method for studying the activity of such promoters. Among the several reporter genes that can be used, the genes encoding luciferase (Luc) and chloramphenicol acetyltransferase (CAT) have been used most widely and successfully by researchers interested in AR-regulated promoters. Such studies have led to the identificatio... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819070 Fri, 31 Jul 2009 23:00:00 +0100 2819070 http://www.medworm.com/index.php?rid=2819069&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_11 Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and β-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subunit- and estrogen receptor α (ERα)-promoter activities. We have demonstrated that estrogen suppresses activin subunit gene promoter activity while activin inc... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819069 Fri, 31 Jul 2009 23:00:00 +0100 2819069 http://www.medworm.com/index.php?rid=2819068&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_10 The methylated DNA immunoprecipitation microarray (MeDIP-chip) is a genome-wide, high-resolution approach to detect DNA methylation in whole genome or CpG (cytosine base followed by a guanine base) islands. The method utilizes anti-methylcytosine antibody to immunoprecipitate DNA that contains highly methylated CpG sites. Enriched methylated DNA can be interrogated using DNA microarrays or by massive parallel sequencing techniques. This combined approach allows researchers to rapidly identify methylated regions in a genome-wide manner, and compare DNA methylation patterns between two samples with diversely different DNA methylation status. MeDIP-chip has been applied successfully for analyses of methylated DNA in the different targets including animal and plant tissues (1, 2). Here we pres... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2819068 Fri, 31 Jul 2009 23:00:00 +0100 2819068 http://www.medworm.com/index.php?rid=1902758&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_9 The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902758 Fri, 24 Oct 2008 15:23:10 +0100 1902758 http://www.medworm.com/index.php?rid=1902757&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_8 The mammalian inner ear forms from a thickened patch of head ectoderm called the otic placode. The placodal ectoderm invaginates to form a cup whose edges cinch together to establish a fluid-filled sac called the otic vesicle or otocyst. The progenitor cells lining the otocyst lumen will give rise to sensory and non-sensory cells of the inner ear. These formative stages of inner ear development are initiated during the first week of postimplantation embryonic development in the mouse. The inaccessibility of the inner ear in utero has hampered efforts to gain insight into the molecular mechanisms regulating essential developmental processes. An experimental embryological method to misexpress genes in the developing mammalian inner ear is presented. Expression plasmid encoding a gene of inte... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902757 Fri, 24 Oct 2008 15:23:10 +0100 1902757 http://www.medworm.com/index.php?rid=1902756&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_7 Helios® Gene Gun–mediated transfection is a biolistic method for mechanical delivery of exogenous DNA into cells in vitro or in vivo. The technique is based on bombardment of a targeted cellular surface by micron- or submicron-sized DNA-coated gold particles that are accelerated by a pressure pulse of compressed helium gas. The main advantage of Helios® Gene Gun–mediated transfections is that it functions well on various cell types, including terminally differentiated cells that are difficult to transfect, such as neurons or inner ear sensory hair cells, and cells in internal cellular layers, such as neurons in organotypic brain slices. The successful delivery of mRNA, siRNA, or DNA of practically any size can be achieved using biolistic transfection. This chapter provi... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902756 Fri, 24 Oct 2008 15:23:09 +0100 1902756 http://www.medworm.com/index.php?rid=1902755&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_6 Gene manipulation, specifically in the hair cells of the inner ear during development and adulthood in mice, is crucial for understanding the physiology of hearing and the pathology of deafness in humans. Recent advances have demonstrated that gene expression can be manipulated in developing mouse hair cells in a spatially and temporally controlled manner. The Cre–loxP system has been widely used for such purposes. Many laboratories, including ours, have developed and characterized transgenic mouse lines that express or induce Cre activity specifically in inner ear hair cells. These Cre lines have been used with high efficiency to inactivate several genes such as Rb in hair cells. Here we discuss the use of these Cre lines in inner ear research with emphasis on practical issues for r... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902755 Fri, 24 Oct 2008 15:23:09 +0100 1902755 http://www.medworm.com/index.php?rid=1902754&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_5 Greatly impacting the field of neural development are new technologies for generating fate maps in mice and thus for illuminating relationships between embryonic and adult brain structures. Until now, efforts in mammalian models such as the mouse have presented challenges because their in utero development limits the access needed for traditional methods involving tracer injection or cell transplantation. But access is no longer an obstacle. It is now possible to deliver cell lineage tracers via noninvasive genetic, rather than physical, means. The hinge-pin of these new “genetic fate mapping” technologies is a class of molecule called a site-specific recombinase. The most commonly used being Cre and Flp. Through the capacity to produce precise DNA excisions, Cre or Flp can act... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902754 Fri, 24 Oct 2008 15:23:04 +0100 1902754 http://www.medworm.com/index.php?rid=1902753&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_4 To understand the mechanisms of development of the inner ear, it is important to know the lineal relationships among the different cell types and the migrational boundaries of individual clones within the inner ear. This chapter details the basic methods for performing lineage analysis of the inner ear using replication-defective retroviral vectors in chicken embryos. Protocols are provided for generating avian retroviruses pseudotyped with vesicular stomatitis virus (VSV) envelopes to improve infectivity in early embryos. Moreover, we include the pioneering methods of the Cepko laboratory, whereby a library of DNA tags was developed to allow clonal relationships to be confirmed by PCR amplification and sequencing of the tag in dispersed clonal progeny. By varying the site and time of vira... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902753 Fri, 24 Oct 2008 15:23:04 +0100 1902753 http://www.medworm.com/index.php?rid=1902752&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_3 In situ hybridization is well suited to obtaining specific topological information on gene transcripts and thereby to relating such observations to a particular function. In spite of the technical and practical difficulties, the application of molecular biological techniques such as in situ hybridization to the cochlea can provide important insights. However, the rarity of gene products (mRNA and proteins) in the cochlea and its fragile structure require the refinement and adaptation of in situ hybridization methods. The present chapter provides a detailed in situ hybridization protocol adapted to frozen tissue sections collected from adult and neonatal stages of the vertebrate cochlea. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902752 Fri, 24 Oct 2008 15:23:03 +0100 1902752 http://www.medworm.com/index.php?rid=1902751&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_24 The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. The origin of this cochlear amplification is the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. The gene of this protein has been identified and termed prestin. We, herein, present a method for observation by atomic force microscopy (AFM) of prestin expressed in the Chinese hamster ovary (CHO) cell plasma membrane. To obtain a stable sample for AFM imaging in liquid, we used as an example in the protocol provide here, CHO cells transfected with prestin or FLAG-tagged prestin, and untransfected CHO cells (1). The cells attached to a substrate... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902751 Fri, 24 Oct 2008 15:23:03 +0100 1902751 http://www.medworm.com/index.php?rid=1902750&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_23 Regulation of important cellular functions via signaling pathways is a fundamental property of the cell. Intracellular Ca2+ is probably a best known second messenger in cell biology. In mechanosensory cells of the inner ear, the hair cells, intracellular Ca2+ participates in a variety of functions including mechano-electrical transduction, synaptic transmission, and efferent regulation of the outer hair cells, one of two types of hair cells in the mammalian cochlea. The outer hair cells are responsible for the amplification of sound-induced vibrations within the cochlea, which determines the sensitivity of mammalian hearing. Besides Ca2+, another intracellular ion, Cl- may have very specific function in the same outer hair cells. Intracellular Cl- is required for the motor function of pres... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902750 Fri, 24 Oct 2008 15:23:02 +0100 1902750 http://www.medworm.com/index.php?rid=1902749&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_22 As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we describe two fluorescence microscopic methodologies applied to the outer hair cell-specific membrane protein prestin: the intensity and fluorescence lifetime (FLIM) variants of FRET (Fluorescence Resonance Energy Transfer), used in the study of protein-protein interactions, and the Scanning Cysteine Accessibility Method (SCAM), used for the determination of protein conformation. The methods are readily adaptable to other proteins. (Source:... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902749 Fri, 24 Oct 2008 15:23:02 +0100 1902749 http://www.medworm.com/index.php?rid=1902748&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_21 Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902748 Fri, 24 Oct 2008 15:23:01 +0100 1902748 http://www.medworm.com/index.php?rid=1902747&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_20 Surface plasmon resonance is an optical technique utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light, reflected after polarized light impinges upon the film, is altered, monitored as a change in detector position for the dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. We have utilized surface plasmon resonance to study the interaction of proteins of hair cells. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902747 Fri, 24 Oct 2008 15:23:00 +0100 1902747 http://www.medworm.com/index.php?rid=1902746&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_2 Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to ∼ 500 bp fragments, suitable for... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902746 Fri, 24 Oct 2008 15:22:57 +0100 1902746 http://www.medworm.com/index.php?rid=1902745&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_19 Expression of almost every gene is regulated at the transcription level. Therefore, transcriptional factor Transcription factors, consequently, have marked effects on the fate of a cell by establishing the gene expression patterns that determine biological processes. In the auditory and vestibular systems, transcription factors have been found to be responsible for development, cell growth, and apoptosis. It is vital to identify the transcription factor target genes and the mechanisms by which transcription factors control and guide gene expression and regulation pathways. Compared with earlier methods devised to study transcription factor–DNA interactions, the advantage of the chromatin immunoprecipitation (ChIP) assay is that the interaction of a transcription factor with its targe... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902745 Fri, 24 Oct 2008 15:22:56 +0100 1902745 http://www.medworm.com/index.php?rid=1902744&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_18 Genomics has provided us with vast amounts of data and thus, the challenge to identify and characterize gene products. Proteomics analysis, using methods such as yeast two-hybrid screenings, isoelectric focusing, and mass spectroscopy, generate potentially useful information. To determine functional relationships between and among proteins, however, the initial data for putative protein interactions must first be validated. One technique, which is considered the gold standard, is coimmunoprecipitation. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902744 Fri, 24 Oct 2008 15:22:56 +0100 1902744 http://www.medworm.com/index.php?rid=1902743&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_17 Structure-function studies of membrane proteins present a unique challenge to researchers due to the numerous technical difficulties associated with their expression, purification and structural characterization. In the absence of structural information, rational identification of putative functionally important residues/regions is difficult. Phylogenetic relationships could provide valuable information about the functional significance of a particular residue or region of a membrane protein. Evolutionary Trace (ET) analysis is a method developed to utilize this phylogenetic information to predict functional sites in proteins. In this method, residues are ranked according to conservation or divergence through evolution, based on the hypothesis that mutations at key positions should coincid... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902743 Fri, 24 Oct 2008 15:22:55 +0100 1902743 http://www.medworm.com/index.php?rid=1902742&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_16 Functional proteomics comprises a wide range of technologies for the identification of novel protein-protein interactions and biological markers. Studies of protein-protein interactions have gained from the development of techniques and technologies such as immunoprecipitation, preparative two-dimensional (2-D) gel electrophoresis for peptide mass fingerprinting (PMF), using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These applications enabled the discovery of putative protein partners without a priori knowledge of which one(s) might be relevant. Here, we report the methods by which membrane proteins are isolated from cochlear tissues and prepared for identification by mass spectro... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902742 Fri, 24 Oct 2008 15:22:55 +0100 1902742 http://www.medworm.com/index.php?rid=1902741&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_15 We describe a protocol to screen for protein-protein interactions using the Gal-4 based yeast two-hybrid system. In this protocol, we describe serial transformation of bait into an already constructed cDNA library in yeast AH109 cells. We find this method to gives the most number of true interactions. Where a pre-made library in yeast cells is not available, the method outlined can be quickly adapted. AH109 cells can be first transformed with bait containing a vector followed by selection of yeast containing the bait. A second transformation of yeast cells is then accomplished with the cDNA library. The method is quick and can lead to the discovery of significant interactions. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902741 Fri, 24 Oct 2008 15:22:54 +0100 1902741 http://www.medworm.com/index.php?rid=1902740&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_14 We describe here the “twist-off” method of bundle isolation, where dissected inner-ear organs are embedded in agarose, then subjected to a mechanical disruption that shears off bundles and leaves them in agarose blocks. With care in the dissection and in clean-up of the isolated bundles, contamination from cell bodies can be kept to a minimum. Isolated bundles can be analyzed by a variety of techniques, including immunocytochemistry, SDS-PAGE, immunoblotting, and mass spectrometry. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902740 Fri, 24 Oct 2008 15:22:54 +0100 1902740 http://www.medworm.com/index.php?rid=1902739&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_13 The identification of the majority of the known causative genes involved in nonsyndromic sensorineural hearing loss (NSHL) started with linkage analysis as part of a positional cloning procedure. The human and mouse genome projects in combination with technical developments on genotyping, transcriptomics, proteomics, and the creation of animal models have greatly enhanced the speed of disease gene identification. In the present chapter, we first discuss the possibilities for exclusion of known NSHL loci and genes. Subsequently, methods are described to determine the genomic regions that contain the genetic defects. These include linkage analysis with genotyping and statistical evaluation and the determination of copy number variations. In the case of a large genomic region, candidate genes... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902739 Fri, 24 Oct 2008 15:22:53 +0100 1902739 http://www.medworm.com/index.php?rid=1902738&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_12 Most chronic bacterial infections are associated with biofilm formation wherein the bacteria attach to mucosal surfaces, wound tissue, or medical device surfaces in the human body via the formation of an extracellular matrix. Biofilms assume complex three-dimensional structures dependent on the species, the strain, and the prevailing environmental conditions and are composed of both the bacteria and the extracellular slime-like matrices, which surround the bacteria. Bacteria deep in the biofilm live under anaerobic conditions and must use alternatives to O2 as a terminal electron acceptor. Thus, the metabolic rates of these deep bacteria are greatly reduced, which renders them extremely resistant to antibiotic treatment, and for reasons not clearly understood, it is often very difficult to... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902738 Fri, 24 Oct 2008 15:22:52 +0100 1902738 http://www.medworm.com/index.php?rid=1902737&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_11 Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the three leading bacteria species associated with otitis media. Defining the molecular epidemiology of bacteria known to cause otitis media is of great importance, in both clinical and research settings. PFGE and MLST provide data for the characterization of isolates’ genetic relatedness, yet they differ in the types of studies for which they are most useful. Consequently, knowledge of both techniques is important for laboratories intending to study the molecular epidemiology of otitis media–associated bacterial pathogens. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902737 Fri, 24 Oct 2008 15:22:50 +0100 1902737 http://www.medworm.com/index.php?rid=1902736&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_10 Recent advances in molecular biology have led to a better understanding of the etiology of vestibular schwannomas. The underlying purpose of vestibular schwannoma research is the development of new treatment options; however, such options have not yet been established. A fundamental understanding of the underlying molecular events leading to tumor formation began when mutations in the neurofibromatosis type 2 (NF2) tumor suppressor gene were identified in vestibular schwannomas. The clinical characteristics of vestibular schwannomas and neurofibromatosis type 2 (NF2) syndromes have both been related to alterations in the NF2 gene. Genetic screening for NF2 is now available. When utilized with clinical screening, such as magnetic resonance imaging (MRI), conventional audiometry, and auditor... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902736 Fri, 24 Oct 2008 15:22:49 +0100 1902736 http://www.medworm.com/index.php?rid=1902735&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_1 The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for tra... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1902735 Fri, 24 Oct 2008 15:22:48 +0100 1902735 http://www.medworm.com/index.php?rid=2504171&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-448-3_9 Transdifferentiation is defined as an irreversible switch in postnatal life of one differentiated cell to another. Transdifferentiation from different cellular origins into pancreatic-like β-cells is of clinical significance since this approach may offer a potential cure for diabetes. In order to achieve this goal, the liver is considered as a suitable candidate due to its close developmental relationship to the pancreas, its large size and a well-documented regenerative capacity that could provide enough original tissues to initiate the transdifferentiation procedure. In this chapter, we describe a protocol to overexpress Pdx1, a master regulator essential for pancreas development in the cultured human liver cell line, HepG2. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=2504171 Thu, 31 Jul 2008 23:00:00 +0100 2504171 http://www.medworm.com/index.php?rid=1548055&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_9 Role of specific reactive lipids as well as amino acids in control of insulin signalling in adipose tissue is well recognized. Since it is practically impossible to measure the levels of all metabolites in the biological sample simultaneously with a single analytical platform, we utilize multiple platforms to study the lipids and metabolites of relevance to adipose tissue metabolism and insulin signalling. Two screening platforms cover a broad range of lipid molecular species (UPLC/MS based lipidomics platform) as well as organic acids and sterols (GCxGC-TOF platform). A targeted platform for amino acids (UPLC) is also applied. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548055 Sun, 01 Jun 2008 04:00:00 +0100 1548055 http://www.medworm.com/index.php?rid=1548054&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_8 Brown and white adipose tissues in mammals have a number of similar properties, such as lipid storage and adipokine production, but also distinctive properties. The energy-storing white adipose tissue has few mitochondria and low oxidative capacity. The heat-producing brown adipose tissue has a high density of mitochondria and high oxidative capacity. Mitochondrial function can be investigated in cells and organelles isolated from both brown and white adipose tissues. This chapter describes methods for successful isolation of suitable preparations of adipose tissues and their subsequent use. Questions concerning thermogenic capacity of the tissues, their potential influence on whole body metabolism, and specific properties of the mitochondria and their mode of function may be addressed usi... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548054 Sun, 01 Jun 2008 04:00:00 +0100 1548054 http://www.medworm.com/index.php?rid=1548053&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_7 Regulation of blood flow in tissues such as skeletal muscle, liver, and adipose tissue is needed to meet the changing local metabolic and physiological demands under varying conditions. In healthy individuals, adipose tissue blood flow (ATBF) is remarkably responsive to meal ingestion, but changes in ATBF in response to other physiological stimuli, such as stress and physical exercise, have also been noted. The ATBF response to nutrient intake may be of particular importance in the regulation of metabolism by facilitating transport of nutrients as well as signaling between adipose tissue and other metabolically active tissues. A reduction in both fasting and postprandial ATBF has been observed in obesity; this impairment is associated with insulin resistance. A better understanding of the ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548053 Sun, 01 Jun 2008 04:00:00 +0100 1548053 http://www.medworm.com/index.php?rid=1548052&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_6 Brown and white adipose tissue have recently gained prominence as key players in obesity and related health problems, such as type-2 diabetes and cardiovascular disease. Brown adipose tissue-dependent nonshivering thermogenesis significantly affects the body's energy balance. Originally considered as a passive store of lipids, white adipose tissue has recently been found to secrete a number of hormones and cytokines and to be thus involved in the control of body metabolism and energy balance at multiple sites. These findings have renewed the interest in adipose organ biology, including its innervation by the autonomic nervous system and sensory nerves. Here, we describe our protocols for detecting different types of adipose tissue nerves by light microscopy using peroxidase immunostaining ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548052 Sun, 01 Jun 2008 04:00:00 +0100 1548052 http://www.medworm.com/index.php?rid=1548051&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_5 Angiogenesis is required for the growth and expansion of both healthy and pathological tissues. The plasticity of the adipose tissue is reflected by its remarkable ability to expand or to reduce in size throughout the adult lifespan. We, and others, have recently shown that expansion of fat mass is dependent on angiogenesis, and suppression of angiogenesis might provide a novel therapeutic approach for prevention and treatment of obesity. Here, we outline two technical procedures for assessment of angiogenesis in adipose tissues. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548051 Sun, 01 Jun 2008 04:00:00 +0100 1548051 http://www.medworm.com/index.php?rid=1548050&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_4 Adipose tissue plays a critical role in energy homeostasis, not only in storing triglycerides, but also in responding to nutrient, neural, and hormonal signals, and secreting adipokines that control feeding, thermogenesis, immunity, and neuroendocrine function. It is conceivable that adipose tissue-specific gene expression would influence the aforementioned functions. A feasible approach to prepare adipose tissue-specific transgenic mouse models is necessary for such studies. Here, we report the preparation of adipose tissue-specific cholesteryl ester transfer protein transgenic mice. The general principle might apply to the establishment of other adipose tissue-specific transgenic mice models. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548050 Sun, 01 Jun 2008 04:00:00 +0100 1548050 http://www.medworm.com/index.php?rid=1548049&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_3 In vivo adipose tissue quantification is an important tool to characterize phenotypes of obesity, especially in the human. The amount and distribution of adipose tissue is associated with many of the adverse consequences of obesity. Recent studies suggest that adipose tissue is not a single homogeneous compartment. Regional adipose tissue depots vary in biological functions and individual adipose tissue compartments have stronger associations with metabolic conditions than does total adipose tissue mass. Currently there is intense and increasing interest in regional adipose tissue compartments. Computed tomography and magnetic resonance imaging often are used to quantify adipose tissue volumes or cross-sectional adipose tissue areas. Other modalities, including dual-energy absorpti-ometry ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548049 Sun, 01 Jun 2008 04:00:00 +0100 1548049 http://www.medworm.com/index.php?rid=1548048&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_24 Adipogenesis is an important component of adipose tissue development and growth. Alterations in adipogenesis may promote adipose tissue insulin resistance and inflammation. The ability of preadipocytes to differentiate into mature adipocytes depends on the activation of phosphoinositide 3-kinase (PI3K). This chapter describes the methodology used to measure the cellular accumulation of phosphoinositide products of PI3K. This approach involves labeling the cells with myo-[2—3H]-inositol, extraction and deacylation of the phosphoinositides, and HPLC separation of the deacylated derivatives. The assay of PI3K activity itself is also described in detail. The ability to analyze PI3K and its phosphoinositide products is a useful tool for ongoing endeavours to understand adipogenesis and ad... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548048 Sun, 01 Jun 2008 04:00:00 +0100 1548048 http://www.medworm.com/index.php?rid=1548047&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_23 Glucose is the main metabolic fuel in mammalian cells. Glucose entry into cells is facilitated by a family of ubiquitously expressed glucose transporter proteins. Typically, glucose transporters are localized on the plasma membrane. One notable exception is the glucose transporter isoform 4 (Glut4), which is specifically expressed in insulin sensitive tissues, i.e., skeletal muscle, heart muscle, and fat, and is responsible for the insulin effect on blood glucose clearance (1). Under basal conditions, Glut4 is compartmentalized in intracellular membrane vesicles and thus has no access to the extracellular space. Upon insulin administration, Glut4-containing vesicles fuse with the plasma membrane and deliver the transporter to its site of action. As a result, Glut4 content on the plasma mem... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548047 Sun, 01 Jun 2008 04:00:00 +0100 1548047 http://www.medworm.com/index.php?rid=1548046&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_22 Lipolysis involves the sequential breakdown of triglycerides into free fatty acids and glycerol. The extent of lipolysis is therefore a key determinant of the energy status of an individual and also dictates insulin resistance. Here, we describe a protocol for estimating lipolysis in murine adipocytes. Glycerol released during the lipolytic reaction is estimated radiometrically to determine the extent of lipolysis within the cell and the data are normalized to cell number. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548046 Sun, 01 Jun 2008 04:00:00 +0100 1548046 http://www.medworm.com/index.php?rid=1548045&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_21 When isolated from tissue, white adipose cells are round, and their interior is filled with a large (80–120 μm) droplet of stored triglyceride, leaving a thin (1–2-μm) layer of cytoplasm between the lipid droplet and the plasma membrane. Their three-dimensional architecture, together with the fact that these cells ordinarily float in medium, have created major challenges when one attempts to perform microscopy techniques with these cells. Adipocytes serve as the principal energy reservoir in the body, and it is essential to overcome these difficulties to be able to study hormone-mediated responses in real adipose cells, which convey physiological significance that cannot be readily duplicated by the use of cultured model adipocytes. This chapter focuses on the use of conf... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548045 Sun, 01 Jun 2008 04:00:00 +0100 1548045 http://www.medworm.com/index.php?rid=1548044&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_20 Adipose tissue synthesizes factors involved in the body's homeostasis. Thus, measurements of messenger ribonucleic acid (mRNA) concentrations are important to study the involvement of adipose tissue in various physiological and pathophysiological conditions, in particular in obesity. Because adipose tissue is highly heterogeneous, containing both a stromal and an adipocyte compartment, each one having different cellular composition and functional capacities, in situ hybridization is a powerful tool to analyze the discrete expression of the mRNAs coding for the various factors synthesized within this tissue. Presented here is a detailed protocol for in situ hybridization of mRNAs in adipose tissue using 35S-labeled single-stranded probes with sufficient details for the readers unfamiliar wi... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548044 Sun, 01 Jun 2008 04:00:00 +0100 1548044 http://www.medworm.com/index.php?rid=1548043&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_2 The importance and the role of adipose tissues are now largely expanded not only because the very high occurrence of obesity but also because the emerging view that adipose tissue could be a reservoir of therapeutic cells. A critical examination of the adipose tissue features according to their location shows that sampling is not as easy as previously thought and needs special attention to heterogeneity and differences. We discussed here these different points and give precise protocols to sample the different adipose tissues and manipulate them. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548043 Sun, 01 Jun 2008 04:00:00 +0100 1548043 http://www.medworm.com/index.php?rid=1548042&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_19 Adipose tissue has emerged as a major endocrine organ producing a wide spectrum of hormones and factors that play crucial roles in regulating cell turnover and function, not only locally within the adipose tissue but also in the brain and other key metabolic organ systems. It is known that gene activity is controlled at both transcriptional and post-transcriptional levels. Consequently, one of the most important means by which the activity of a gene is assessed is through the determination of levels of the corresponding messenger ribonucleic acid (mRNA). This process involves the isolation of total cellular RNA and subsequent analysis of the mRNA of interest. Given the unique nature of adipose tissue and adipocytes (i.e., containing high amounts of lipid), special RNA isolation techniques ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548042 Sun, 01 Jun 2008 04:00:00 +0100 1548042 http://www.medworm.com/index.php?rid=1548041&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_18 RNA interference (RNAi) is a powerful, quick, and easy technique to reduce the expression of a particular gene. However, investigators need to consider several steps for the experiments, including the design of the siRNA, an efficient delivery method, and a means for monitoring the biological effects of the siRNA introduced. Adipocytes have long been recognized as one of the most difficult cell types in which to perform gene delivery. In this chapter, three distinct transfec-tion methods to obtain high efficiency of gene delivery and gene knockdown are described. The transfection efficiency of siRNA and shRNA is also compared. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548041 Sun, 01 Jun 2008 04:00:00 +0100 1548041 http://www.medworm.com/index.php?rid=1548040&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_17 Chromatin, long thought to be no more than a scaffold supporting DNA compaction inside the cell nucleus, has emerged in the last few years as a major regulatory element involved in the control of gene expression both acutely during interphase and programmatically throughout complex processes of development and differentiation. Adipogenesis is the result of an intertwined network of transcription factors and coregulators with chromatin-modifying activities and offers an excellent model for the study of transcriptional regulation. In this regard, electrophoretic mobility shift assay and immunoprecipitation of chromatin are complementary methods that can be used to study the binding of nuclear proteins to DNA and to characterize how these proteins interact with and modify chromatin to regulat... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548040 Sun, 01 Jun 2008 04:00:00 +0100 1548040 http://www.medworm.com/index.php?rid=1548039&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_16 Adipose tissue contains cell types other than adipocytes that may contribute to complications linked to obesity. For example, macrophages have been shown to infiltrate adipose tissue in response to a high-fat diet. Isolation of the stromal-vascular fraction of adipose tissue allows one to use flow cytometry to analyze cell surface markers on leukocytes. Here, we present a technical approach to identify subsets of leukocytes that differentially express cell surface markers. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548039 Sun, 01 Jun 2008 04:00:00 +0100 1548039 http://www.medworm.com/index.php?rid=1548038&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_15 Much of the research devoted to understanding adipose tissue development is currently performed in vitro. Several cell culture models, including preadipocyte cell lines and primary culture of adipose-derived stromal vascular precursor cells, are commonly used to study molecular and cellular events and regulatory influences on preadipocyte proliferation and differentiation. Primary preadipocyte culture systems have several distinct advantages over preadipose cell lines. Because they have not been passaged continuously in culture, primary cultures of adipose derived stromal-vascular (SV) cells more closely reflect the in vivo characteristics of the tissue from which they are derived. In addition, primary cells can be obtained from various adipose tissue depots and from animals at different s... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548038 Sun, 01 Jun 2008 04:00:00 +0100 1548038 http://www.medworm.com/index.php?rid=1548037&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_14 Obesity is characterized by increased adiposity of visceral and subcutaneous depots as well as other organs, including the vasculature. These fat depots secrete various hormone-like proteins implicated in metabolic homeostasis (e.g., adiponectin, resistin), the central control of appetite (e.g., leptin) and the increased production of cytokines. These molecules act either in a paracrine or endocrine manner, contributing to the metabolic and cardiovascular complications of obesity. Explant cultures of white adipose tissue are an important step in analyzing the secretory mechanisms of adipose tissue by preserving the physiological in vivo cross-talk between the various types of cells. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548037 Sun, 01 Jun 2008 04:00:00 +0100 1548037 http://www.medworm.com/index.php?rid=1548036&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_13 The mouse embryo fibroblast cell lines 3T3-L1 and C3H10T1/2 differentiate to adipocytes that exhibit similar insulin regulation of lipogenesis. These cell lines, however, differ appreciably in the processes that produce the major regulator PPARγ. Each line is stimulated by a mixture of insulin, dexamethasone, and methylisobutylxanthine (IDM). In the first 24 h, IDM activates each cell type to produce similar regulatory changes and cell contraction. However, the increase in PPARγ is delayed by 24 h in typical 3T3-L1 cells compared with C3H10T1/2 cells. This delay is caused by the need for one or two rounds of cell division (clonal expansion) for PPARγ synthesis in 3T3-L1 cells. This expansion also occurs in C3H10T1/2 cells, but is not needed for PPARγ synthesis and d... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548036 Sun, 01 Jun 2008 04:00:00 +0100 1548036 http://www.medworm.com/index.php?rid=1548035&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_12 The broad definition of a stem cell is a cell that has the ability to self-renew and differentiate into one or more specialized terminally differentiated cell types. It has become evident that stem cells persist in, and can be isolated from, many adult tissues. Adipose tissue has been shown to contain a population of cells that retain a high proliferation capacity in vitro and the ability to undergo extensive differentiation into multiple cell lineages. These cells are referred to as adipose stem cells and are biologically similar, although not identical, to mesenchymal stem cells derived from the bone marrow. Differentiation causes stem cells to adopt the pheno-typic, biochemical, and functional properties of more terminally differentiated cells. This chapter will provide investigators wi... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548035 Sun, 01 Jun 2008 04:00:00 +0100 1548035 http://www.medworm.com/index.php?rid=1548034&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_11 Adipose tissue is increasingly recognized as a metabolically active endocrine organ with multiple functions beyond its lipid storage capability. Various constituents of the tissue, such as mature adipocytes and stromal vascular cells, have distinct functions. For example, they express and secrete different kinds of bioactive molecules collectively called adipokines. Altered adipokine secretion patterns characterize obesity and insulin resistance, which are major risk factors for type 2 diabetes mellitus. The contribution of dysregulated adipokine expression to these diseases may be assembled from transcriptomic profiles of the tissue and/or its cellular constituents. The gene expression profiles may also complement genetic approaches to identify disease susceptibility genes. Here, we descr... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548034 Sun, 01 Jun 2008 04:00:00 +0100 1548034 http://www.medworm.com/index.php?rid=1548033&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_10 Determination of the complex secretory proteome of adipocytes and its metabolic changes induced by drug treatment such as insulin or rosiglitazone is possible with the advanced proteomics technologies described herein. To study the secreted proteins of adipocytes, a 2D- liquid chromatography/mass spectrometry/mass spectrometry protocol has been established. With the use of reversed-phase high-performance liquid chromatography, intact proteins were separated in the first dimension into eight fractions, then digested with Lys-C and trypsin. Comparative differences after drug treatment were assessed using 18O proteolytic labeling strategies. With the advent of more and more sophisticated instrumentation and data analysis tools, protocols like this one will likely become standard tools for sci... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548033 Sun, 01 Jun 2008 04:00:00 +0100 1548033 http://www.medworm.com/index.php?rid=1548032&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_1 As the result of its apparent structural and histological simplicity, adipose tissue (AT) functions initially were limited to energy storage, insulation, and thermoregulation. Only decades later was the extraordinarily dynamic role of AT recognized, revealing its participation in a broad range of physiological processes, including reproduction, apoptosis, inflammation, angiogenesis, blood pressure, atherogenesis, coagulation, fibrinolysis, immunity and vascular homeostasis with either direct or indirect implications in the regulation of proliferation. The functional pleiotropism of AT relies on its ability to synthesize and, in some cases, secrete a large number of enzymes, hormones, growth factors, cytokines, complement factors, and matrix and membrane proteins, collectively termed adipok... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1548032 Sun, 01 Jun 2008 04:00:00 +0100 1548032 http://www.medworm.com/index.php?rid=1538953&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_25 Proteomics has brought with it the hope of identifying novel biomarkers for the fetal aneuploidies. This hope is built on the ability of proteomic technologies, such as two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients combined with protein identification by mass spectrometry (MS). The large dynamic range of plasma necessitates the effective removal of abundant plasma proteins to allow analysis of the lower concentration analytes. There are many factors that make this research very challenging, beginning with standardization of sample collection, consistent sample preparation, and continuing through the entire analytical process. Therefore, reproducible sample complexity reduction methods such as depletions or fractionations are an essential first step in biomarker d... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538953 Fri, 09 May 2008 04:00:00 +0100 1538953 http://www.medworm.com/index.php?rid=1538952&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_22 Circulating fetal RNA in maternal plasma has offered a new approach for noninvasive prenatal diagnosis and monitoring. Circulating fetal RNA markers could potentially be used for all pregnant women without being limited by fetal–maternal genetic polymorphisms and fetal gender. Over the past few years, encouraging findings have been reported on the detection and possible clinical applications of circulating fetal RNA. Placental-derived RNA has been shown to be easily detectable in maternal plasma during pregnancy and rapidly cleared after delivery. Such observations suggest that the placenta is an important organ for releasing fetal RNA into maternal plasma. Noninvasive prenatal gene expression profiling of the placenta also has been demonstrated to be feasible by analyzing the circul... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538952 Fri, 09 May 2008 04:00:00 +0100 1538952 http://www.medworm.com/index.php?rid=1538951&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_19 Recent studies have shown that cell-free fetal DNA in maternal plasma can be enriched by means of size fractionation. This technique makes use of the smaller size of fetal DNA fragments compared with maternal DNA fragments isolated simultaneously. On this basis, a highly improved detection of fetal single gene point mutations is permitted. Here, we introduce the use of agarose gel electrophoresis for the size fractionation of cell-free DNA from maternal plasma and the detection of fetal β-thalassemia mutations in the size-fractionated cell-free DNA by using peptide nucleic acid-clamping polymerase chain reaction (PCR) combined with an allele-specific real-time PCR assay. Matrix-assisted laser desorption ionization/time of flight mass spectrometry has been reliably used for detection o... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538951 Fri, 09 May 2008 04:00:00 +0100 1538951 http://www.medworm.com/index.php?rid=1538950&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_15 Cell-free fetal DNA present in the maternal circulation holds great potential for noninvasive prenatal diagnosis and analysis of fetal genetic traits. However, only approximately 3–6% of total DNA in the maternal plasma is of fetal origin. Because of its scarcity in the maternal circulation, various methods have been developed and tested to optimize the extraction of this rare material from plasma. Here, we first describe the commonly used protocol for separating plasma from whole blood samples. We also describe two commercially available methods for the extraction of cell-free DNA from maternal plasma, which we have found particularly straightforward and easy to use: a manual method using the High Pure PCR Template Preparation kit (Roche Diagnostics) and an automated system using th... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538950 Fri, 09 May 2008 04:00:00 +0100 1538950 http://www.medworm.com/index.php?rid=1538949&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_8 Multiplex ligation-dependent probe amplification (MLPA) is a new method to determine the copy number of up to 45 genomic DNA sequences in a single multiplex polymerase chain reaction (PCR)-based reaction. In contrast to standard multiplex PCR, only one pair of PCR primers is used. MLPA reactions with currently commercial available kits result in very reproducible gel patterns with fragments of 130 to 480 bp that can be analyzed by sequence type electrophoresis. Comparison of this gel pattern to that obtained from a control sample indicates which sequences show an aberrant copy number. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538949 Fri, 09 May 2008 04:00:00 +0100 1538949 http://www.medworm.com/index.php?rid=1538948&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_16 For fetal DNA extraction, in principle each DNA extraction method can be used; however, because most methods have been optimized for genomic DNA from leucocytes, we describe here the methods that have been optimized for the extraction of fetal DNA from maternal plasma and validated for this purpose in our laboratories. The use of the QIAamp DSP Virus kit (QIAGEN), the QIAamp DNA Blood Mini kit (QIAGEN), and the Magna Pure LC (Roche) is based on the kit components provided by the respective companies. However, we noticed that the yield of fetal DNA from maternal plasma can be increased when higher volumes are processed or some slight modifications of the protocols provided by the manufacturer are followed. Here, we also describe an in-house method that allows the specific capture of target ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538948 Fri, 09 May 2008 04:00:00 +0100 1538948 http://www.medworm.com/index.php?rid=1538947&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_3 The most common aneuploidies in prenatal diagnostics of the second trimenon are trisomies of chromosomes 13, 18, and 21 and gonosomal abnormalities. To detect these trisomies as quickly as possible after amniocentesis, besides using polymerase chain reaction, fluorescence in situ hybridization (FISH), applying corresponding centromeric or locus-specific probes, is the method of choice. Results of a rapid prenatal aneuploidy screening in uncultured amniocytes by using FISH are available within 24 hr or less. However, care has to be taken against possible pitfalls in connection with the commercially available probe sets and thus interpretation of results in general. Here, we explain how rapid prenatal aneuploidy screening is performed using the Food and Drug Administration-approved Aneu Vysi... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538947 Fri, 09 May 2008 04:00:00 +0100 1538947 http://www.medworm.com/index.php?rid=1538946&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_6 Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype. They are normally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14 days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48 h of sample receipt. Interphase fluorescence in si... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538946 Fri, 09 May 2008 04:00:00 +0100 1538946 http://www.medworm.com/index.php?rid=1538945&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_5 We describe the use of array CGH for prenatal diagnosis of a range of chromosomal syndromes associated with congenital malformations visible by ultrasound. The procedure is reproducible in a clinical setting and provides reliable results in a short period (~5 days). Thus, depending on the array used, array CGH may develop into an excellent tool for prenatal diagnosis. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538945 Fri, 09 May 2008 04:00:00 +0100 1538945 http://www.medworm.com/index.php?rid=1538944&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_2 Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be identified or characterized unambiguously by conventional banding cytogenetics alone, and they are generally equal in size or smaller than a chromosome 20 of the same metaphase spread. sSMC are reported in 0.043% of newborn infants and 0.075% of prenatal cases. Molecular cytogenetics is necessary to characterize the origin of an sSMC, and many highly sophisticated approaches are available throughout the literature for their comprehensive description. However, because in a prenatal diagnostic laboratory such techniques are not available, I suggest here a straightforward scheme to characterize at least the sSMC’s chromosomal origin as quickly as possible. Based on this scheme, it is possi... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538944 Fri, 09 May 2008 04:00:00 +0100 1538944 http://www.medworm.com/index.php?rid=1538943&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_11 Tay-Sachs disease is an autosomal recessive storage disease caused by the impaired activity of the lysosomal enzyme hexosaminidase A. In this fatal disease, the sphingolipid GM2 ganglioside accumulates in the neurons. Due to high carrier rates and the severity of the disease, population screening and prenatal diagnosis of Tay-Sachs disease are routinely carried out in Israel. Laboratory diagnosis of Tay-Sachs is carried out with biochemical and DNA-based methods in peripheral and umbilical cord blood, amniotic fluid, and chorionic villi samples. The assay of hexosaminidase A (Hex A) activity is carried out with synthetic substrates, 4-methylumbelliferyl-6-sulfo-N-acetyl-β-glucosaminide (4-MUGS) and 4-methylumbelliferil-N-acetyl-β-glucosamine (4-MUG), and the DNA-based analysis in... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538943 Fri, 09 May 2008 04:00:00 +0100 1538943 http://www.medworm.com/index.php?rid=1538942&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_24 Circulating cell-free fetal deoxyribonucleic acids (cffDNAs) are promising biomarkers with various potential clinical applications. Second and third trimester amniotic fluid (AF) is a rich source of cffDNAs. Further improvements to the original protocol for the extraction of cffDNAs from AF supernatant resulted in statistically significant higher yields of high-quality cffDNAs, allowing for a substantial majority of samples to be analyzed with subsequent molecular methods (e.g., comparative genomic hybridization microarrays) to further assess for genetic abnormalities. Several advantages have been realized with the optimized protocol. In addition to an improved yield from a greater proportion of samples compared with the original protocol, the current method, using large silico-membranes, ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538942 Fri, 09 May 2008 04:00:00 +0100 1538942 http://www.medworm.com/index.php?rid=1538941&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_21 The discovery of cell-free RNA in plasma and serum samples provides possibilities for noninvasive prenatal diagnosis. Quantitative alterations of cell-free placental-derived mRNA in the maternal circulation are associated with many pregnancy-related disorders, such as preeclampsia and preterm labor. Obtaining circulating cell-free RNA is the first and often the most critical step for analyzing the placental-derived mRNA in maternal blood. We have compared different protocols for the extraction of cell-free RNA from plasma samples, and we have found the protocol using TRIzol LS reagent (Invitrogen) as lysis buffer combined with RNeasy Mini kit (QIAGEN) to be the optimal method for extracting high-quality cell-free RNA in the highest quantities possible. This method is also amenable to the s... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538941 Fri, 09 May 2008 04:00:00 +0100 1538941 http://www.medworm.com/index.php?rid=1538940&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_14 Rhesus D (RhD) blood group incompatibility between mother and fetus can occasionally result in maternal alloimmunization where the resultant anti-D can cross the placenta and attack the fetal red cells, which in worse case scenarios can cause fetal anemia and ultimately death. Fetal RHD genotyping was introduced in the mid-1990s after the molecular characterization of the RH genes as an aid to the clinical management of these cases. Initially, these tests used fetal DNA extracted invasively from chorionic villus and amniocyte samples. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538940 Fri, 09 May 2008 04:00:00 +0100 1538940 http://www.medworm.com/index.php?rid=1538939&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_13 The electronic microchip is a recently developed technology for the fast and reliable detection of known single-nucleotide polymorphisms (SNPs) in the genome. The DNA fragment to be analyzed is directed electrophoretically into the chip, and then it is hybridized with fluorescent-tagged DNA probes specific for the mutant and wild-type sequences. The presence or absence of the mutation is detected by the fluorescence signal. Electronic stringency provides quality control for the hybridization process and ensures that any bound pairs of DNA are truly complementary; the microchip can be easily customized by the end user, allowing for assembly of specific probes onto the microchip to perform individualized analyses. Assays for 10 frequent mutations in the β-globin gene causing β-thal... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538939 Fri, 09 May 2008 04:00:00 +0100 1538939 http://www.medworm.com/index.php?rid=1538938&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_4 Since its discovery by Koch in 1989, primed in situ labeling (PRINS) reaction provides an alternative approach for direct detection of human chromosomes. The multiple color (multi)-PRINS technique can simultaneously and specifically display different chromosomes with different colors in the same metaphase or interphase nucleus by using sequential labeling of different chromosome targets. We developed a triple-PRINS reaction on uncultured amniotic cells by omitting the blocking step and taking advantage of mixing two fluorochromes (fluorescein and rhodamine) to create a third color for simultaneous detection in the same amniocytes of three different chromosome targets, e.g., chromosomes 18, X, and Y. Fluorescent signals corresponding to chromosomes 18, X, and Y were shown as yellow, red, an... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538938 Fri, 09 May 2008 04:00:00 +0100 1538938 http://www.medworm.com/index.php?rid=1538937&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_7 In prenatal analysis, one of the major concerns is the detection of fetal aneuploidies. Several molecular methods have been described recently for the rapid analysis of amniotic fluid and chorionic villi. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) of short tandem repeats are already implemented in prenatal laboratories and permit the evaluation of the major chromosomal aberrations within 24 h. However, both methods have their disadvantages. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538937 Fri, 09 May 2008 04:00:00 +0100 1538937 http://www.medworm.com/index.php?rid=1538936&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_10 The inherited disorders of hemoglobin synthesis constitute themost commonmonogenic diseases worldwide. The clinical severity of β-thalassemia major and the sickle cell syndromes targets themas priority genetic diseases for prevention programs,which incorpo- rates population screening to identify heterozygotes,with the option of prenatal diagnosis for carrier couples. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis. The application of real-time polymerase chain reaction (PCR) provides a means for rapid and potentially high-throughput assays, without compromising accuracy. It has several advantages over endpoint PCR analysis, including the elimination of post-PCR processing steps and a wide dynamic range of detecti... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538936 Fri, 09 May 2008 04:00:00 +0100 1538936 http://www.medworm.com/index.php?rid=1538935&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_17 The analysis of cell-free fetal DNA in the circulation of the pregnant woman plays the pivotal role in noninvasive prenatal research. Here, we describe an improved method for the quantification of male DNA, which is a valuable research tool for the quantification of fetal DNA. The quantification of fetal DNA serves two main purposes. First, the levels may indicate certain pregnancy-related disorders such as preeclampsia even before onset of the disease; thus, the quantification may serve as a marker for early detection. Second, extraction and enrichment strategies of the fetal DNA compartment are important factors in the development and implementation of clinical tests, such as detection of fetal sex, Rhesus D status, point mutations, and aneuploidies. In this context, the quantification o... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538935 Fri, 09 May 2008 04:00:00 +0100 1538935 http://www.medworm.com/index.php?rid=1538934&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_12 Arrayed primer extension reaction (APEX) is a straightforward and robust enzymatic genotyping method in which hundreds to thousands of variations in the genome are simultaneously analyzed in a single multiplexed reaction. It differs from allele-specific hybridization in that the genotype information in APEX is obtained by single base extension, performed by a specific DNA polymerase, together with four different dye terminators. This approach ensures highly specific discrimination without allele-specific hybridization, because the primer to be extended anneals just adjacent to the DNA base that needs to be identified. Selection of primers for specific sites or their consecutive placement in tiled format, shifting them by one base, permits single-nucleotide polymorphism analysis, mutation d... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538934 Fri, 09 May 2008 04:00:00 +0100 1538934 http://www.medworm.com/index.php?rid=1538933&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_1 The method of spectral karyotyping (SKY) is based on a combination of the technologies of charge-coupled device imaging and spectrometry. The engineering feasibility has been realized in the SpectraCube system from Applied Spectral Imaging Inc., and it allows the simultaneous identification of all 24 human chromosomes. This is performed by characterizing the spectral signature of every image pixel in relation to a fluorochrome combinatorial library translating the image and spectral information into chromosome classification. Applications for SKY include pre- and postnatal characterization of certain numerical and structural rearrangements and complex karyotypes and highly informative analysis of sample materials with only single or few cells available for investigation. (Source: Springer ... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538933 Fri, 09 May 2008 04:00:00 +0100 1538933 http://www.medworm.com/index.php?rid=1538932&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_18 Since the demonstration of cell-free fetal DNA in maternal circulation, its aberrational quantification has been explored and demonstrated in numerous clinical situations. Moreover, several centers have begun to use detection and quantification of fetal DNA to diagnose fetal genetic status with high reliability, such as fetal RhD. One of the methods for analyzing cell-free fetal DNA is quantification by using LightCycler technology. With the advent of various fluorescent reporters and greater stability of kit’s reagent, real-time quantitative polymerase chain reaction by using LightCycler technology is relatively simple and fast. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538932 Fri, 09 May 2008 04:00:00 +0100 1538932 http://www.medworm.com/index.php?rid=1538931&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_20 The discovery of cell-free fetal DNA in the plasma and serum of pregnant women has opened a new window for noninvasive prenatal diagnosis. Robust detection and quantification have been achieved when the fetal DNA sequence of interest does not have a maternal counterpart (e.g., Y chromosomal DNA, RhD gene when the mother is RhD negative) by techniques such as real-time polymerase chain reaction (PCR). However, detection of subtle fetal mutations is difficult due to the overwhelming maternal DNA background. A method combining PCR, base extension reaction, and matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) allowing DNA detection with single base specificity and single DNA molecule sensitivity is described. DNA sequence is amplified by PCR first. Th... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538931 Fri, 09 May 2008 04:00:00 +0100 1538931 http://www.medworm.com/index.php?rid=1538930&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_23 A novel in vitro method is described wherein gene expression profiling is reflective and informative for the way how syncytiotrophoblast cells shed RNA products in vivo in maternal plasma. After controlled denudation, RNA is obtained selectively from the syncytiotrophoblast cells of trisomy 21 placentae. cDNA copies are subsequently analyzed by microarray profiling and cDNA cloning with sequencing. Given the preponderance of 5′ mRNA fragments lacking a poly-A tail, the placental RNA products are amplified after polymerase A-mediated tailing by using a method originally designed for small-sized microRNAs. This approach, when combined with cDNA library or microarray expression screening, is a novel in vitro method to screen for syncytiotrophoblast-derived RNA products representative of... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538930 Fri, 09 May 2008 04:00:00 +0100 1538930 http://www.medworm.com/index.php?rid=1538929&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-066-9_9 Matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a tool currently under investigation for use in prenatal detection of abnormalities in chromosome number, such as trisomy 21. Because of its ability to detect extremely small differences in mass, even to the level of a single nucleotide difference, this method can be applied to the detection of single-nucleotide polymorphisms (SNPs), which when present in a heterozygous state, can yield quantitative information regarding chromosome status from diagnostic specimens such as amniotic fluid or chorionic villus samples. MALDI-TOF mass spectrometry has several potential advantages over traditional karyotyping methods, including its amenability to high-throughput analyses and its nonreliance on prior cel... Springer protocols feed by Molecular Medicine info http://www.medworm.com/rss/comments.php?id=1538929 Fri, 09 May 2008 04:00:00 +0100 1538929