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At least 253 records · Page 14Linked to original sources
PubMedDate not supplied

Immune mechanisms in HIV infection.

Recent advances in immunobiology have led to a greater understanding of the healthy immune system and the complex pathogenesis of human immunodeficiency virus (HIV) infection. Knowledge of the mechanisms underlying the decrease in CD4+ T cells is rapidly evolving as a result of new assays, genetic advances, and recombinant DNA technologies. Studying the immune responses of long-term non-progressors is also providing insight into the immunopathology of HIV. Trials using highly active antiretroviral therapy and immune modulators have shown that it may be possible to reverse damage to the immune system and increase CD4+ T-cell numbers. Current and future findings might provide the knowledge necessary to identify effective HIV drugs and vaccines with acceptable toxicity profiles and to determine whether it will be possible to fully restore immune system function in patients with HIV disease.

AIDS Vaccines↗
PubMed2000

Use of microarrays to identify targets in cardiovascular disease.

The year 2000 will long be remembered in biology for the completion of the basic sequence of the human genome. This remarkable achievement comes at a time when the technology for exploiting differential expression of genes in cells and tissues is rapidly progressing. Although many methods have been available over the past two decades for exploring differential expression of genes, DNA microarray technology has already proven revolutionary. The capability of monitoring expression of thousands of genes simultaneously has already yielded unique insight into many physiologic and pathophysiologic processes. For cardiovascular disease, microarray technology promises to provide new understanding of mechanisms of cardiac hypertrophy, heart failure and vascular disease. As the availability of this technology expands and the costs decrease, microarray technology will be an essential method for identifying novel therapeutic and diagnostic targets in cardiovascular disease. In addition to identifying new targets, microarrays have the potential to decrease the long research and development time required to bring a concept to the bedside. Although microarray technology will help fuel the imminent explosion of novel approaches to cardiovascular disease, the technology also requires cautious interpretation and well-considered validation strategies.

Journal Article↗
PubMed2005

[Molecular unraveling of disease by means of DNA-microarrays].

Determination of the human genome sequence and the development of microarray technologies allowing the rapid measurement of all genes in the genome have generated new perspectives for our current biomedical research. Gene expression analysis will make a major contribution to our insight into the underlying biology of disease and will lead to improved methods for diagnostics, prognosis and treatment. Microarray studies create the possiblity to subclassify patients with diseases such as rheumatoid arthritis, diffuse large B-cell lymphoma and breast cancer, with both prognostic and therapeutic consequences. The simultaneous quantification of the activity of all genes in tissues or cells from patients by microarray technology, linked to the clinical parameters, creates a large number of data points, which cannot be analysed without the aid of the advanced application of bioinformatics. As a result, genomic research has become, in part, a bioinformatics discipline that will be integrated with clinical medicine. The microarray technology makes it possible to develop personalized medicine, with a more accurate diagnosis and prognosis for every patient and subsequently a tailored treatment strategy.

Arthritis, Rheumatoid↗
PubMed2006

Detection of resistance to acetolactate synthase inhibitors in weeds with emphasis on DNA-based techniques: a review.

Resistance to herbicides inhibiting acetolactate synthase (ALS) has been increasing at a faster rate than in any other herbicide group. The great majority of these cases are due to various single-nucleotide polymorphisms in the ALS gene endowing target site resistance. Many diagnostic techniques have been devised in order to confirm resistance and help producers to adopt the best management strategies. Recent advances in DNA technologies coupled with the knowledge of sequence information have allowed the development of accurate and rapid diagnostic tests. While whole plant-based diagnostic techniques such as seedling bioassays or enzyme-based in vitro bioassays provide accurate results, they tend to be labour- and/or space-intensive and will only respond to the particular herbicides tested, making resolution of cross-resistance patterns more difficult. Successful DNA-based diagnosis of ALS inhibitor resistance has been achieved with three main techniques, (1) restriction fragment length polymorphism, (2) polymerase chain reaction amplification of specific alleles and (3) denaturing high-performance liquid chromatography. All DNA-based techniques are relatively rapid and provide clear identification of the mutations causing resistance. Resistance based on non-target mechanisms is not identified by these DNA-based methods; however, given the prevalence of target site-based ALS inhibitor resistance, this is a minor inconvenience.

Acetolactate Synthase↗
PubMedDate not supplied

DNA microarray technology and application.

DNA microarrays (collections of DNA probes arranged on a shared base) have recently enlarged the spectrum of commercially available laboratory-ready kits in molecular biology. They are powerful new tools for the investigation of global changes in gene expression profiles in cells and tissues. Their assembly process is automatized and the DNA microarrays are further miniaturized. The DNA microarrays are used in search for various specific genes (e.g. connected with an infectious agent) or in gene polymorphism and expression analysis. They will be widely used to investigate expression of various genes connected with various diseases in order to find causes of these diseases and to enable their accurate treatment. Since the DNA microarray assembly technology has been based on methods widely used in the semiconductor industry, we can expect a rapid onset of the routine use of this revolutionary device.

Base Sequence↗
PubMed2001

[Identification of proteome molecules by proteomics using two-dimensional gel electrophoresis and MALDI-TOF MS].

Genomic technologies have enabled rapid accumulation of information from complex biological systems over the last two decades. The complete DNA sequence is now known for many organisms and the informational database obtained from genome sequencing projects has provided the base for the specification of proteome - the protein complement of genome. Genomic functions can be inferred from the analysis of gene structure and gene expression profiles because proteins are the functional molecules of an organism. Integrated technologies including protein separation, identification, characterization and information manage system are essential to analyze the proteins in complex cellular matrix. This study is focusing on the strategies of proteome analysis using sample preparation, 2-dimensional gel electrophoresis, processing of protein spots and identification of proteins, protein-protein interaction and posttranslational modification using MALDI-TOF-MS. 2-D gel electrophoresis is currently the most powerful protein separation technique and MALDI-TOF MS is powerful identification technique for protein and peptides as a sensitive, rapid, and high resolution analytical method. The developed integrated proteome technologies are very useful to understand the biological phenomena at molecular level by identifying the new molecules and their modifications in various cellular processes, and can be applied for biotechnology including medical science.

Databases, Protein↗
PubMed2003

Protein micro- and macroarrays: digitizing the proteome.

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.

Protein Array Analysis↗
PubMed2000

[Molecular biological approaches to lung cancer].

Advances in lung cancer research have come from modern technology, allowing the establishment of lung cancer cell lines and experimental model systems, recombinant DNA technology, robust biological and biochemical functional assays in addition to classical methods such as immunohistochemistry. Such work indicates that the aberrant activation of protooncogenes and inactivation of tumor suppressor genes are fundamental processes for lung carcinogenesis. Further improvements such as DNA array technology will continue to unravel the genetic and epigenetic defects in a far more rapid and comprehensive way that was previously possible. This knowledge will lead to a genetic characterization of lung cancer cells and its specific subtypes, which will hopefully translate to new diagnostic and therapeutic gene based strategies. This review focuses on molecular biological techniques that have been used on lung cancer research and discusses future technological approaches.

Animals↗
PubMedDate not supplied

Microarray analysis of normal and dystrophic skeletal muscle.

The development and increasingly common use of DNA microarrays for comprehensive RNA expression analysis has had a substantial impact on the study of molecular pathology. DNA microarrays are orderly, high-density arrangements of nucleic acid spots that can be used as substrates for global gene expression analysis. Prior to their development, technical limitations necessitated that the molecular mechanisms underlying biological processes be broken down into their component parts and each gene or protein studied individually. This approach, focused as it is on a single aspect of a scientific phenomenon, does not allow appreciation or understanding of the fact that biological pathways do not exist in isolation, but are influenced by numerous factors. Enormous technological advances have been made over the past decade and now high-density DNA microarrays can provide rapid measurement of thousands of distinct transcripts simultaneously. These experiments raise the exciting opportunity to examine biological pathways in all their complexity and to compare the hypotheses deduced from the study of histological pathology with the findings of molecular pathology. This review focuses on how microarray technology has been used to interrogate muscular gene expression and, in particular, on how data generated from differential expression analysis of dystrophic and normal skeletal muscle has contributed to understanding the molecular pathophysiological pathways of muscular dystrophy.

Animals↗
PubMed2000

DNA vaccines: capacity to induce auto-immunity and tolerance.

DNA technology has facilitated the development of plasmid-based vaccines designed to prevent viral, bacterial and parasitic infections. The rapid transition of these novel vaccines from the laboratory to the clinic raises important safety concerns. Our review examines whether DNA vaccines (i) are likely to induce systemic or organ-specific auto-immune disease and (ii) have the potential to induce tolerance rather than immunity.

Animals↗
PubMed2005

High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs.

Mutation of the human genome ranges from single base-pair changes to whole-chromosome aneuploidy. Karyotyping, fluorescence in situ hybridization, and comparative genome hybridization are currently used to detect chromosome abnormalities of clinical significance. These methods, although powerful, suffer from limitations in speed, ease of use, and resolution, and they do not detect copy-neutral chromosomal aberrations--for example, uniparental disomy (UPD). We have developed a high-throughput approach for assessment of DNA copy-number changes, through use of high-density synthetic oligonucleotide arrays containing 116,204 single-nucleotide polymorphisms, spaced at an average distance of 23.6 kb across the genome. Using this approach, we analyzed samples that failed conventional karyotypic analysis, and we detected amplifications and deletions across a wide range of sizes (1.3-145.9 Mb), identified chromosomes containing anonymous chromatin, and used genotype data to determine the molecular origin of two cases of UPD. Furthermore, our data provided independent confirmation for a case that had been misinterpreted by karyotype analysis. The high resolution of our approach provides more-precise breakpoint mapping, which allows subtle phenotypic heterogeneity to be distinguished at a molecular level. The accurate genotype information provided on these arrays enables the identification of copy-neutral loss-of-heterozygosity events, and the minimal requirement of DNA (250 ng per array) allows rapid analysis of samples without the need for cell culture. This technology overcomes many limitations currently encountered in routine clinical diagnostic laboratories tasked with accurate and rapid diagnosis of chromosomal abnormalities.

Chromosome Aberrations↗
PubMed2001

Total chemical synthesis of human activin beta(A)[12-116] and related large-loop polypeptides.

We report here the synthesis, purification, and characterization of several large polypeptides related to the human activin beta(A) subunit and their cyclic counterparts. In particular, we describe for the first time the total chemical synthesis of a 105-mer polypeptide, des[1-11] activin beta(A), and related large-loop polypeptide, by an optimized solid phase synthetic protocol based on 9-flouroenylmethyoxycarbonyl (Fmoc) chemistry. These studies show that automated chemical synthesis utilizing Fmoc-based solid phase synthetic strategies provides a practical alternative to recombinant DNA technology for the production of activin-related subunits, with the opportunity to rapidly provide different analogues and structural variants for subsequent structure-function and associated biophysical investigations.

Activins↗
PubMed2000

Development of a blocking enzyme-linked immunosorbent assay for the detection of avian polyomavirus-specific antibodies.

Avian polyomavirus, described originally as budgerigar fledgling disease virus, has been associated with devastating contagious disease outbreaks in budgerigar aviaries. At present, this virus affects a wide range of psittacine and non-psittacine birds worldwide, and the serum neutralisation test is used for the serodiagnosis of avian polyomavirus infections. A blocking enzyme-linked immunosorbent assay was developed for the screening of large numbers of sera collected from various avian species. The assay employs a monoclonal antibody directed against the major structural protein VP1 as a blocking antibody in a sandwich blocking procedure. Either purified avian polyomavirus particles or avian polyomavirus VP1 expressed in recombinant baculovirus-infected Sf9 cells were used as antigen. The specificity of the blocking enzyme-linked immunosorbent assay was evaluated by testing sera directed against mammalian polyomaviruses. Using sera obtained from chicken infected experimentally with avian polyomavirus and a collection of psittacine field-origin sera, a good correlation was observed between the results of the blocking enzyme-linked immunosorbent assay and the serum neutralisation test. However, the blocking enzyme-linked immunosorbent assay is more rapid and more economic. Both, avian polyomavirus particles and VP1 produced by recombinant DNA technology proved to be suitable antigens.

Animals↗
PubMed2002

Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery.

Streptococcus pneumoniae is a useful tool for the discovery of broad-spectrum antibiotics because of its genetic malleability and importance as a pathogen. Recent publications of complete chromosomal DNA sequences for S. pneumoniae facilitate rapid and effective use of genomics-based technology to identify essential genes encoding new targets for antibacterial drugs. These methods include computational comparative genomics, gene disruption studies to determine essentiality or identify essential genes, and gene expression analysis using microarrays and gel-based proteomics. We review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it the best species for the identification and validation of new antibiotic targets.

Anti-Bacterial Agents↗
PubMed2001

Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.

We evaluated real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA arrays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavirus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than twofold difference, and the other 144 genes as expressed uniquely or with more than twofold difference between the two subclones. Real-time RT-PCR used LightCycler-based SYBR Green I dye detection and melting curve analysis to validate the relative change in gene expression. Real-time RT-PCR confirmed the change in expression of 17 of 24 (71%) genes identified by high-density filter arrays. Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR. This data suggests that (i) both hybridization intensity and the level of differential expression determine the likelihood of validating high-density filter array results and (ii) genes identified by DNA arrays with a two- to fourfold difference in expression cannot be eliminated as false nor be accepted as true without validation. Real-time RT-PCR based on LightCycler technology is well-suited to validate DNA array results because it is quantitative, rapid, and requires 1000-fold less RNA than conventional assays.

Computer Systems↗
PubMed2000

Detection of the causal mutation for canine leukocyte adhesion deficiency (CLAD) using pyrosequencing.

A missense mutation in the ITGB2 gene causes canine leukocyte adhesion deficiency (CLAD) in Irish setters. We constructed a diagnostic test to identify heterozygous CLAD carriers based on a newly developed technology termed pyrosequencing. Although primarily designed for high-speed generation of DNA sequence in a gel-free system, the technology can be applied to rapid single-nucleotide polymorphism analysis in a clinical setting. The testing of 339 dogs originating from a total of 10 countries was conducted and CLAD carriers were identified within every country where more than one sample was analysed. This indicates that the CLAD mutation is widespread and that there is a strong need for a robust diagnostic test.

Animals↗
PubMed2005

Influence of royal jelly on mouse hepatic gene expression and safety assessment with a DNA microarray.

We used a DNA microarray to compare the gene expression profiles in liver among three groups of mice fed a diet containing 5% royal jelly (RJ), a diet containing 5% RJ stored at 40 degrees C for 7 d (40-7d RJ) or a control diet which provides the same total energy as RJ. Expression of 267 genes was increased or decreased by 1.8-fold or more in animals given the RJ diet for 14 d as compared with control diet, though serum total cholesterol, triglyceride, phospholipid, glucose, insulin and leptin levels were unaffected. Many genes involved in cell growth, signal transduction, energy metabolism and transcription regulation were responsive to the RJ diet. Among the 267 genes whose expression was altered by RJ, 60% showed no change or a reduced change in response to 40-7d RJ diet. The 40-7d RJ diet contained little 57-kDa protein, identified as a possible freshness marker of RJ. Furthermore, the RJ diet did not influence the gene expression of cytochrome P450 enzymes and detoxifying enzymes, whereas the 40-7d RJ diet increased the gene expression of glutathione S-transferase and glutathione peroxidase. Indeed, the RJ diet decreased the gene expression of cytochrome P450 4A14 (CYP4A14), which catalyzes peroxidation of endogenous lipids that is associated with nonalcoholic steatohepatitis and alcoholic liver disease, while the 40-7d RJ diet was not effective to decrease the gene expression of CYP4A14. The results indicate that the efficacy of RJ decreased and the toxicity of RJ increased during storage at high temperature. We suggest that application of DNA microarray technology to the biochemical evaluation of food safety may be effective for rapid and precise quality control.

Animals↗
PubMed1995

Hepatocyte transplantation and gene therapy.

Trials of liver-directed gene therapy are a clinical reality. Technology is rapidly developing for the isolation of large numbers of human hepatocytes. Recombinant DNA techniques are available to engineer recombinant retroviruses which express genes of clinical relevance. Familial hypercholesterolemia serves as the clinical paradigm for liver-directed gene therapy, and the clinical protocol is underway. Early results are promising. Techniques for hepatocyte delivery, optimization of gene transfer, and safety considerations are important considerations in designing a human trial. Other diseases which are likely to be among the first treated with this new approach are ornithine transcarbamylase deficiency, hemophilia B, and Crigler-Najjar syndrome.

Amino Acid Metabolism, Inborn Errors↗