Methods in molecular medicine最新文献

Proteomics technology allows a comprehensive and efficient analysis of the proteome and has become an indispensable tool in biomedical research. Since the late 80s, advances on mass spectrometry (MS) instrumentation and techniques have revolutionized the way proteins can be analyzed. Such analysis would only be possible with a proper sample preparation and separation ahead of the MS step. Different gel and nongel-based methods are available for protein separation. This chapter will focus on the use of two-dimensional gel electrophoresis (2-DE) in proteomics and its application to platelet research. 2-DE separates proteins according to their isoelectric point (pI) and size (molecular weight) and allows the detection of thousands of proteins at a time. Platelets are enucleated cells that play a critical function in the control of bleeding and wound healing. As platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. This chapter presents a protocol for an efficient sample preparation, protein separation by 2-DE, and protein digestion ahead of the MS analysis. The experimental approach, already successfully applied to the study of the platelet proteome, includes recommendations for an efficient platelet preparation for proteomics studies.

Stem cell therapy is a progressive approach to a pervasive clinical problem; cardiovascular disease is the number 1 killer in the USA and other developed countries, and aspects of it are amenable to stem cell therapy. Many types of stem cells have been used in treating the heart during myocardial infarction, and here, we describe our approach of direct myocardial injection of bone marrow-derived mesenchymal stem cells into the infarct of rats. We will also briefly introduce the methods we have used to inject neonatal cardiomyocytes into the aorta as a first step in attempting to produce an external cardiac pump. Proper surgical technique and postoperative care are as important as adequate injection of the cells and will greatly improve the survival of the animal after surgery. By carefully following the methods presented in this chapter, the reader will be able to perform direct myocardial and vascular injection of stem cells into rats.

Isolation, quantification, and genetic analysis of circulating plasma DNA have clinical applications in prenatal diagnosis, oncology, organ transplantation, posttrauma monitoring, and infectious disease. Recent technology has allowed the rapid isolation and purification of DNA from whole blood, plasma, serum, buffy coat, tissues, stool, and urine. With the advent of real-time polymerase chain reaction (PCR) amplification, extracted DNA not only can be easily identified to aid in clinical diagnoses, but also can be readily quantified to analyze ongoing clinical dynamics and aid in the medical prognoses of patients. Historically, identification of unique cell-free fetal DNA sequences has relied on the detection of paternally specific Y chromosome sequences owing to their relative ease in identification. However, any DNA sequence that is unique to the fetus has the potential to be amplified and quantified using real-time PCR. Our laboratory specializes in extraction of fetal DNA from maternal plasma with subsequent quantification with real-time PCR of paternally inherited sequences, such as the Y chromosome gene, SRY. The successful isolation and quantification of this DNA from plasma is dependent on three distinct protocols: plasma harvesting from whole blood, DNA extraction from cell-free plasma, and real-time PCR amplification and quantification of the SRY sequence.

Fluorescence in situ hybridization (FISH) is the technique of choice for preimplantation genetic diagnosis (PGD) selection of female embryos in families with X-linked disease, for which there is no mutation-specific test. FISH with target-specific DNA probes is also the primary technique used for PGD detection of chromosome imbalance associated with Robertsonian translocations, reciprocal translocations, inversions, and other chromosome rearrangements, because the DNA probes, labeled with different fluorochromes or haptens, detect the copy number of their target loci. The methods described outline strategies for PGD for sex determination and chromosome rearrangements. These methods are assessment of reproductive risks, the selection of suitable probes for interphase FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring using directly labeled and indirectly labeled probes.

Heart disease is a leading cause of death in western society. Despite the success of heart transplantation, a chronic shortage of donor organs, along with the associated immunological complications of this approach, demands that alternative treatments be found. One such option is to repair, rather than replace, the heart with engineered cardiac tissue. Multiple studies have shown that to attain functional tissue, assembly signaling cues must be recapitulated in vitro. In their native environment, cardiomyocytes are directed to beat in synchrony by propagation of pacing current through the tissue. Recently, we have shown that electrical stimulation directs neonatal cardiomyocytes to assemble into native-like tissue in vitro. This chapter provides detailed methods we have employed in taking this "biomimetic" approach. After an initial discussion on how electric field stimulation can influence cell behavior, we examine the practical aspects of cardiac tissue engineering with electrical stimulation, such as electrode selection and cell seeding protocols, and conclude with what we feel are the remaining challenges to be overcome.

Tubulin heterogeneity within eukaryotic cells is generated by differential gene expression and posttranslational modification of alpha- and beta-tubulin gene products, either as heterodimers or when polymerized into microtubules. The characterization of posttranslationally modified tubulins from the crustacean Artemia franciscana is presented, although tubulins from other sources can be studied with these procedures. Tubulin is prepared from cell free extracts by taxol-induced assembly and centrifugation of microtubules through sucrose cushions, which also yields microtubule-associated proteins, or it is purified to apparent homogeneity by relatively simple chromatographic procedures and assembly/disassembly steps. To detect posttranslationally modified tubulins protein samples are electrophoresed in sodium dodecyl sulfate (SDS) polyacrylamide gels, blotted to nitrocellulose membranes and probed with isoform-specific antibodies. Isotubulins, for which gene-encoded amino acid differences and posttranslational modifications generate charge variations, are resolved in two-dimensional gels using isoelectric focusing followed by SDS polyacrylamide gel electrophoresis, a procedure useful for resolution of microtubule-associated proteins. Isoforms patterns are visualized by Coomassie blue and/or silver staining and individual isoforms are identified by antibody reactivity on Western blots. Tubulin isoforms are localized in Artemia by immunofluorescent staining of larvae. The focus of this chapter is the purification of tubulin from a nonneural source and characterization of tyrosinated, detyrosinated, and nontyrosinatable alpha-tubulins using polyclonal antibodies made to carboxy-terminal peptides of each isoform.

In the last decade, the analysis of gene expression in tissues and cells has evolved from the analysis of a selected set of genes to an efficient high throughput whole-genome screening approach of potentially all genes expressed. Development of sophisticated methodologies such as microarray technology allows an open-ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and that define the samples' unique biology. By a global analysis of the genes that are expressed in cells and tissues of an individual under different conditions and during disease, we can build up "gene expression profiles (signatures)" which characterize the dynamic functioning of the genome under pathophysiological conditions. This strategy also provides the means to subdivide patients that suffer from a complex heterogeneous disease into more homogeneous subgroups. Such discovery-based research identifies biological processes that may include new genes with unknown function or genes not previously known to be involved in this process. The latter category may hold surprises that sometimes urge us to redirect our thinking. We have used microarrays to disclose the heterogeneity of rheumatoid arthritis (RA) patients at the level of gene expression of the affected synovial tissues. Analysis of the expression profiles of synovial tissues from different patients with RA revealed considerable variability, resulting in the identification of at least two molecularly distinct forms of RA tissues. One is characterized by genes that indicate an active inflammatory infiltrate with high immunoglobulin production, whereas the other type shows little immune activation and instead shows a higher stromal cell activity. These results confirm the heterogeneous nature of RA and suggest the existence of distinct pathogenic mechanisms that contribute to RA. The differences in expression profiles provide opportunities to stratify patients for intervention therapies based on molecular criteria.

B-cells and antibody-secreting plasma cells are key players in protective immunity, but also in autoimmune disease. To understand their various functions in the initiation and maintenance of autoimmune pathology, a detailed dissection of their functional diversity is mandatory. This requires a detailed phenotypic classification of the diversity of B-cells. Here, technologies of immunocytometry and ELISpot are described in detail, and their value for phenotypic characterization of cells of the B lineage, as well as for preparative cell sorting, to further characterize them functionally and on the molecular level are described.

A critical step in working with adenovirus (Ad) and its vectors is the accurate, reproducible, sensitive, and rapid measurement of the amount of virus present in a stock. Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. Determining the infectious titer of a virus stock by plaque assay has important limitations, including cell line-, researcher-, and laboratory-dependent variation in titer, and the length of time required to perform the assay (2-4 wk). A major drawback of particle titration methods is the lack of consistent correlation between the resultant titer and the infectious titer. To overcome these problems, a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay was developed that detects encapsidated full-length genomes. Importantly, there is a linear correlation between the titer determined by the realtime PCR assay and the infectious titer determined by a plaque assay. This chapter provides step-by-step guidance for preparing viral DNA, conducting the real-time PCR assay, and using the resultant data to calculate a viral titer.

Advances in amplification techniques have revolutionized the ability to detect viruses both quantitatively and qualitatively and to study viral load. Real-time polymerase chain reaction (PCR) amplification depends on the ability to detect and quantify a fluorescent reporter molecule whose signal increases in proportion to the amount of amplification product generated. Recent advances have been made by using probes, such as TaqMan probes, to detect amplified products. Use of these probes offers confirmation of specificity of the PCR product. Here we describe a sensitive real-time PCR assay to quantify subgroup C adenoviral DNA in human lymphocytes derived from mucosal tissues removed in routine tonsillectomy or adenoidectomy. This chapter will describe in detail the methods used for these analyses.