Enhanced DNA fragmentation in the thymus of spontaneously hypertensive rats.
(25/2595)
The mechanisms contributing to organ injury in hypertension have been incompletely defined. The thymus gland of the spontaneously hypertensive rat (SHR) shows significant atrophy at the age of 15 wk compared with its normotensive control, the Wistar-Kyoto rat (WKY). The aim of the present study was to examine the thymus of SHR for evidence of DNA nicking as one of the mechanisms for thymic atrophy. SHR and WKY were subjected to adrenalectomy or sham surgery at 12 wk and studied at 15 wk. Adrenalectomy served to normalize the blood pressure in the SHR. DNA nicking was detected by in situ nick-end labeling (ISEL) of fixed tissue sections. Tissue sections were treated with proteolysis, and terminal deoxyribonucleotidyl transferase was used to incorporate biotinylated deoxynucleotides into DNA nick end in situ. Separately, DNA fragmentation was evaluated by measuring the level of released mono- and oligonucleosomes to the cytoplasm. A higher number of thymic ISEL-positive cells and a higher level of cytoplasmic mono- and oligonucleosomes were observed in SHR than in WKY. After adrenalectomy the enhanced level of ISEL and cytoplasmic mono- and oligonucleosomes in SHR was reduced to the level in WKY. Dexamethasone treatment (0.05 mg. kg-1. day-1) in WKY serves to decrease the thymus weight and significantly elevate the level of mono- and oligonucleosomes. Thus increased DNA fragmentation represents one of the mechanisms associated with thymic atrophy, a feature that reflects immune suppression in SHR. (+info)
Molecular pathology in the preclinical development of biopharmaceuticals.
(26/2595)
Advances in cell and molecular biology have engendered a wide range of techniques that can be used to study the molecular events that underlie the cause of disease, thus producing a new field of study called "molecular pathology." These techniques can be either slide-based or non-slide-based (solution-based). The slide-based techniques include immunohistochemistry, in situ hybridization, and in situ polymerase chain reaction; pathologists play a unique role in the administration of these techniques because of their ability to interpret the end product (i.e., the slide). In this manuscript, we briefly discussed the use and impact of these slide-based techniques within all phases of drug development in the pharmaceutical industry. (+info)
Revolution through genomics in investigative and discovery toxicology.
(27/2595)
The remarkable technologic and methodologic advances spurred on by the Human Genome Project are being applied throughout the life sciences. In the field of toxicology, high-resolution assays now make it possible to discover virtually all the differences in gene expression brought on by exposure to a particular xenobiotic. There are 2 principal approaches used to build a catalog of changes in gene expression: hybridization microarrays and gel-based methods, such as differential display and AFLP-based mRNA finger-printing. The power of such approaches is exemplified by the identification of more than 300 genes that differ in expression level by at least 2-fold in response to the nongenotoxic rodent liver carcinogen phenobarbital. (+info)
Mechanisms of disease and injury: utilization of mutants, monoclonals, and molecular methods.
(28/2595)
Rapid advances in our ability to localize and quantify macromolecular changes in health and disease are being brought about by the availability of genetically altered animals (mutants), purified reagents such as monoclonal antibodies, and new molecular methods. Targeted gene deletion (knockouts) and gene insertions (transgenics) in animals can allow identification of the importance and function of macromolecules. Monoclonal antibodies and fluorescent labels coupled with advances in microscopy provide exacting and multi-dimensional information about localization and cellular changes in proteins, carbohydrates, and lipids using immunohistochemistry, fluorescent activated cell sorting, and immunoprecipitation. Similarly, new applications of molecular methods can be used to identify and localize nucleic acids in tissues via in situ hybridization, polymerase chain reaction (PCR), reverse transcription (RT) PCR, differential display RT-PCR, RNase protection assays, and microchip arrays. The ligand for CD40 (CD40L), an important immunoregulatory molecule, is an example of the successful application of mutants, monoclonal antibodies, and molecular methods to cloning and biological characterization of new molecules. CD40L knockout mice, monoclonal antibodies, and several molecular methods were used to identify mutations in CD40L as the genetic basis for hyper-IgM syndrome in humans, to provide new insights into the pathobiology of Pneumocystis carinii infection, and to evaluate CD40L for immunotherapy of tumors and opportunistic infections. (+info)
Generalized neighbor-joining: more reliable phylogenetic tree reconstruction.
(29/2595)
We have developed a phylogenetic tree reconstruction method that detects and reports multiple topologically distant low-cost solutions. Our method is a generalization of the neighbor-joining method of Saitou and Nei and affords a more thorough sampling of the solution space by keeping track of multiple partial solutions during its execution. The scope of the solution space sampling is controlled by a pair of user-specified parameters--the total number of alternate solutions and the number of alternate solutions that are randomly selected--effecting a smooth trade-off between run time and solution quality and diversity. This method can discover topologically distinct low-cost solutions. In tests on biological and synthetic data sets using either the least-squares distance or minimum-evolution criterion, the method consistently performed as well as, or better than, both the neighbor-joining heuristic and the PHYLIP implementation of the Fitch-Margoliash distance measure. In addition, the method identified alternative tree topologies with costs within 1% or 2% of the best, but with topological distances of 9 or more partitions from the best solution (16 taxa); with 32 taxa, topologies were obtained 17 (least-squares) and 22 (minimum-evolution) partitions from the best topology when 200 partial solutions were retained. Thus, the method can find lower-cost tree topologies and near-best tree topologies that are significantly different from the best topology. (+info)
An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications.
(30/2595)
Transposons are mobile genetic elements and have been utilized as essential tools in genetics over the years. Though highly useful, many of the current transposon-based applications suffer from various limitations, the most notable of which are: (i) transposition is performed in vivo, typically species specifically, and as a multistep process; (ii) accuracy and/or efficiency of the in vivo or in vitro transposition reaction is not optimal; (iii) a limited set of target sites is used. We describe here a genetic analysis methodology that is based on bacteriophage Mu DNA transposition and circumvents such limitations. The Mu transposon tool is composed of only a few components and utilizes a highly efficient and accurate in vitro DNA transposition reaction with a low stringency of target preference. The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies. The Mu methodology is readily applicable in a variety of current and emerging transposon-based techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins. (+info)
Precise developmental regulation of Ets family transcription factors during specification and commitment to the T cell lineage.
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Ets family transcription factors control the expression of a large number of genes in hematopoietic cells. Here we show strikingly precise differential expression of a subset of these genes marking critical, early stages of mouse lymphocyte cell-type specification. Initially, the Ets family member factor Erg was identified during an arrayed cDNA library screen for genes encoding transcription factors expressed specifically during T cell lineage commitment. Multiparameter fluorescence-activated cell sorting for over a dozen cell surface markers was used to isolate 18 distinct primary-cell populations representing discrete T cell and B cell developmental stages, pluripotent lymphoid precursors, immature NK-like cells and myeloid hematopoietic cells. These populations were monitored for mRNA expression of the Erg, Ets-1, Ets-2, Fli-1, Tel, Elf-1, GABPalpha, PU.1 and Spi-B genes. The earliest stages in T cell differentiation show particularly dynamic Ets family gene regulation, with sharp transitions in expression correlating with specification and commitment events. Ets, Spi-B and PU.1 are expressed in these stages but not by later T-lineage cells. Erg is induced during T-lineage specification and then silenced permanently, after commitment, at the beta-selection checkpoint. Spi-B is transiently upregulated during commitment and then silenced at the same stage as Erg. T-lineage commitment itself is marked by repression of PU.1, a factor that regulates B-cell and myeloid genes. These results show that the set of Ets factors mobilized during T-lineage specification and commitment is different from the set that maintains T cell gene expression during thymocyte repertoire selection and in all classes of mature T cells. (+info)
Genetics: a touch of elegance with RNAi.
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RNA-mediated gene interference (RNAi), a rapid, convenient tool for inhibiting gene function in Caenorhabditis elegans, has recently been shown to work in other organisms. (+info)