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Biomedical subjects

Kevin G Becker

Publications and source records attributed to Kevin G Becker.

At least 37 records · Page 2Linked to original sources
PubMed2004

Identification and characterization of metallothionein-1 and -2 gene expression in the context of (+/-)3,4-methylenedioxymethamphetamine-induced toxicity to brain dopaminergic neurons.

In mice, the recreational drug (+/-)3,4-methylenedioxymethamphetamine [MDMA ("ecstasy")] produces a selective toxic effect on brain dopamine (DA) neurons. Using cDNA microarray technology in combination with an approach designed to facilitate recognition of relevant changes in gene expression, the present studies sought to identify genes potentially involved in murine MDMA-induced toxicity to DA neurons. Of 15,000 mouse cDNA fragments studied, metallothionein (Mt)-1 and Mt2 emerged as candidate genes possibly involved in MDMA-induced toxicity to DA neurons. Northern blot analysis confirmed the microarray findings and revealed a dynamic upregulation of Mt1 and Mt2 mRNA in the ventral midbrain within 4-12 hr after MDMA treatment. Western blot analysis showed a similar increase in MT protein levels, with peak times occurring subsequent to increases in mRNA levels. Mt1-2 double knock-out mice were more vulnerable to MDMA-induced toxicity to DA neurons than corresponding wild-type mice. Stimulation of endogenous expression of MT protein with zinc acetate conferred complete protection against MDMA-induced toxicity to DA neurons, and administration of exogenous MT protein afforded partial protection. Collectively, these results indicate that MDMA-induced toxicity to DA neurons is associated with increased Mt1 and Mt2 gene transcription and translation, possibly as part of a neuroprotective mechanism. The present findings may have therapeutic implications for neuropathological conditions involving DA neurons.

3,4-Methylenedioxyamphetamine↗
PubMed2004

Identification and analysis of plasticity-induced late-response genes.

The excitatory neurotransmitter, glutamate, activates N-methyl-d-aspartate (NMDA) receptors to induce long-lasting synaptic changes through alterations in gene expression. It is believed that these long-lasting changes contribute to learning and memory, drug tolerance, and ischemic preconditioning. To identify NMDA-induced late-response genes, we used a powerful gene-identification method, differential analysis of primary cDNA library expression (DAzLE), and cDNA microarray from primary cortical neurons. We report here that a variety of genes, which we have named plasticity-induced genes (PLINGs), are up-regulated with differential expression patterns after NMDA receptor activation, indicating that there is a broad and dynamic range of long-lasting neuronal responses that occur through NMDA receptor activation. Our results provide a molecular dissection of the activity-dependent long-lasting neuronal responses induced by NMDA receptor activation.

Animals↗
PubMed2004

CYFIP2 is highly abundant in CD4+ cells from multiple sclerosis patients and is involved in T cell adhesion.

DNA microarray profiling of CD4(+) and CD8(+) cells from non-treated relapsing and remitting multiple sclerosis (MS) patients determined that the cytoplasmic binding partner of fragile X protein (CYFIP2, also called PIR121) was increased significantly compared to healthy controls. Western analysis confirmed that CYFIP2 protein was increased approximately fourfold in CD4(+) cells from MS compared to inflammatory bowel disorder (IBD) patients or healthy controls. Because CYFIP2 acts as part of a tetrameric complex that regulates WAVE1 activation we hypothesized that high levels of CYFIP2 facilitate T cell adhesion, which is elevated in MS patients. Several findings indicated that increased levels of CYFIP2 facilitated adhesion. First, adenoviral-mediated overexpression of CYFIP2 in Jurkat cells increased fibronectin-mediated adhesion. Secondly, CYFIP2 knock-down experiments using antisense oligodeoxynucleotides reduced fibronectin-mediated binding in Jurkat and CD4(+) cells. Thirdly, inhibition of Rac-1, a physical partner with CYFIP2 and regulator of WAVE1 activity, reduced fibronectin-mediated adhesion in Jurkat and CD4(+) cells. Finally, inhibition of Rac-1 or reduction of CYFIP2 protein decreased fibronectin-mediated adhesion in CD4(+) cells from MS patients to levels similar to controls. These studies suggest that overabundance of CYFIP2 protein facilitates increased adhesion properties of T cells from MS patients.

Adult↗
PubMed2004

The common variants/multiple disease hypothesis of common complex genetic disorders.

Unlike simple rare Mendelian disorders, the genetic basis for common disorders is unclear. A general model of the genetics of common complex disorders is proposed which emphasizes the shared nature of common alleles in related common disorders, such as schizophrenia and bipolar disorder, Type II diabetes and obesity, and among autoimmune diseases. This model, the common variants/multiple disease hypothesis, emphasizes that many disease genes may not be disease specific. Common deleterious alleles, found at a relatively high frequency in the population may play a role in related clinical phenotypes in the context of different genetic backgrounds and under different environmental conditions.

Chromosome Mapping↗
PubMed2004

Transcriptional profiling in an MPNST-derived cell line and normal human Schwann cells.

cDNA microarrays were utilized to identify abnormally expressed genes in a malignant peripheral nerve sheath tumor (MPNST)-derived cell line, T265, by comparing the mRNA abundance profiles with that of normal human Schwann cells (nhSCs). The findings characterize the molecular phenotype of this important cell-line model of MPNSTs, and elucidate the contribution of Schwann cells in MPNSTs. In total, 4608 cDNA sequences were screened and hybridizations replicated on custom cDNA microarrays. In order to verify the microarray data, a large selection of differentially expressed mRNA transcripts were subjected to semi-quantitative reverse transcription PCR (LightCycler). Western blotting was performed to investigate a selection of genes and signal transduction pathways, as a further validation of the microarray data. The data generated from multiple microarray screens, semi-quantitative RT-PCR and Western blotting are in broad agreement. This study represents a comprehensive gene-expression analysis of an MPNST-derived cell line and the first comprehensive global mRNA profile of nhSCs in culture. This study has identified ~900 genes that are expressed abnormally in the T265 cell line and detected many genes not previously reported to be expressed in nhSCs. The results provide crucial information on the T265 cells that is essential for investigation using this cell line in experimental studies in neurofibromatosis type I (NF1), and important information on normal human Schwann cells that is applicable to a wide range of studies on Schwann cells in cell culture.

Journal Article↗
PubMed2004

DSCR1(Adapt78) modulates expression of SOD1.

DSCR1(Adapt78) is a stress responsive gene that can be induced by multiple stresses. We have previously demonstrated that acute DSCR1(Adapt78) overexpression can transiently protect cells against oxidative stress and calcium-mediated stresses, while its chronic overexpression is associated with neurofibrillary tangles, Alzheimer disease, and Down's syndrome. It seems that a delicate balance of DSCR1(Adapt78) expression is maintained in cells, and this gene can have either protective or damaging effects, depending on both its level and duration of expression. The mechanisms by which DSCR1(Adapt78) can protect or harm cells are poorly understood. Here, we tried to identify pathways and targets affected by the DSCR1(Adapt78) gene using regulated expression of DSCR1(Adapt78) in PC-12 cells, followed by microarray analysis of mRNAs from these cells. We found that DSCR1(Adapt78) expression stimulates SOD1 (intracellular Cu,Zn superoxide dismutase) gene expression and increased sod 1 enzyme activity. Previous studies have indicated that sod 1 can either protect or damage cells, depending on its levels. Our findings suggest that sod 1 may also be involved in both the acute protective and the chronic damaging effects of DSCR1(Adapt78) expression. These data also have importance for our understanding of Down's syndrome, Alzheimer's disease, and other human pathologies.

Animals↗
PubMed2004

Antigen-specific gene expression profiles of peripheral blood mononuclear cells do not reflect those of T-lymphocyte subsets.

Advances in microarray technology have allowed for the monitoring of thousands of genes simultaneously. This technology is of particular interest to immunologists studying infectious diseases, because it provides tremendous potential for investigating host-pathogen interactions at the level of immune gene expression. To date, many studies have focused either on cell lines, where the physiological relevance is questionable, or on mixed cell populations, where the contributions of individual subpopulations are unknown. In the present study, we perform an intrasubject comparison of antigen-stimulated immune gene expression profiles between a mixed population of peripheral blood mononuclear cells (PBMC) and the two predominant cell types found in PBMC, CD4+ and CD8+ T lymphocytes. We show that the microarray profiles of CD4+ and CD8+ T lymphocytes differ from each other as well as from that of the mixed cell population. The independence of the gene expression profiles of different cell types is demonstrated with a ubiquitous antigen (Candida albicans) as well as with a disease-specific antigen (human immunodeficiency virus p24). This study has important implications for microarray studies of host immunity and underscores the importance of profiling the expression of specific cell types.

Antigens↗
PubMed2004

Survival of retinal pigment epithelium after exposure to prolonged oxidative injury: a detailed gene expression and cellular analysis.

PURPOSE: To detail, by DNA microarrays and cellular structure labeling, the in vitro responses of retinal pigment epithelial (RPE) cells to a nonlethal dose of the oxidant agent hydroquinone (HQ). METHODS: The viability of growth-quiescent ARPE-19 cells after treatment with HQ was measured by XTT conversion, (3)H-leucine incorporation, trypan blue exclusion, and the presence of DNA laddering. The effect of a nonlethal dose of HQ on the localization of apoptosis-induced factor (AIF) and phosphorylation of stress-activated kinase-2/p38 (SAPK2/p38) was detected by immunocytochemistry. Actin structures were visualized by phalloidin staining. Cell membrane blebbing was detected using GFP-membrane-labeled RPE cells (ARPE-GFP-c'-rRas). Changes in gene expression patterns of RPE cells within 48 hours of prolonged treatment with a nonlethal dose of HQ were evaluated by microarray analysis and confirmed by Northern blotting. RESULTS: The viability of RPE after a prolonged sublethal injury dose of HQ was determined by multiple assays and confirmed by the absence of AIF translocation or DNA laddering. Prolonged exposure (16 hours) of RPE cells to a nonlethal dose of HQ resulted in actin rearrangement into globular aggregates and cell membrane blebbing. Kinetic microarray analysis at several time points over a 48-hour recovery period revealed significant upregulation of genes involved in ameliorating the oxidative stress, chaperone proteins, anti-apoptotic factors, and DNA repair factors, and downregulation of pro-apoptotic genes. Genes involved in extracellular matrix functions were also dysregulated. Recovery of RPE cells after the injury was confirmed by the normalization of gene expression dysregulation back to baseline levels within 48 hours. CONCLUSIONS: RPE cells avoided cell death and recovered from prolonged oxidative injury by activating a host of defense mechanisms while simultaneously triggering genes and cellular responses that may be involved in RPE disease development.

Actins↗
PubMed2004

Global analysis of HuR-regulated gene expression in colon cancer systems of reducing complexity.

HuR, a protein that binds to target mRNAs and can enhance their stability and translation, is increasingly recognized as a pivotal regulator of gene expression during cell division and tumorigenesis. We sought to identify collections of HuR-regulated mRNAs in colon cancer cells by systematic, cDNA array-based assessment of gene expression in three systems of varying complexity. First, comparison of gene expression profiles among tumors with different HuR abundance revealed highly divergent gene expression patterns, and virtually no changes in previously reported HuR target mRNAs. Assessment of gene expression patterns in a second system of reduced complexity, cultured colon cancer cells expressing different HuR levels, rendered more conserved sets of HuR-regulated mRNAs. However, the definitive identification of direct HuR target mRNAs required a third system of still lower complexity, wherein HuR-RNA complexes immunoprecipitated from colon cancer cells were subject to cDNA array hybridization to elucidate the endogenous HuR-bound mRNAs. Comparison of the transcript sets identified in each system revealed a strikingly limited overlap in HuR-regulated mRNAs. The data derived from this systematic analysis of HuR-regulated genes highlight the value of low-complexity, biochemical characterization of protein-RNA interactions. More importantly, however, the data underscore the broad usefulness of integrated approaches comprising systems of low complexity (protein-nucleic acid) and high complexity (cells, tumors) to comprehensively elucidate the gene regulatory events that underlie biological processes.

Animals↗
PubMed2003

Methamphetamine-induced gene expression profiles in the striatum of male rat pups exposed to the drug in utero.

Methamphetamine is a neurotoxic pychostimulant which affects monoaminergic and non-monoaminergic systems in the brain. Clinical studies in humans have found that exposure to methamphetamine in the developing embryo can cause significant behavioral and cognitive anomalies later in life. Exposure of animals to methamphetamine (METH) in utero can cause neurobehavioral effects that do not become apparent until young adulthood. In the present study, we sought to determine the effects of in utero METH exposure on the striata of perinatal rat pups using a recently developed 17 k cDNA microarray. We found that METH administration caused alterations in 913 genes according to strict criteria. These alterations include changes in genes that participate in signal transduction, heat shock responses and neuronal development. The majority of the changes in gene expression were more prominent at the 7-day time point. These observations suggest that in utero METH exposure might initiate molecular programs that significantly impact gene expression during the developmental period long after the last exposure to this drug. Thus, during development, METH exposure in utero might cause significant long-term changes in gene expression that might constitute, in part, some of the substrates for the behavioral and cognitive anomalies reported in the literature.

Animals↗
PubMed2003

PubMatrix: a tool for multiplex literature mining.

BACKGROUND: Molecular experiments using multiplex strategies such as cDNA microarrays or proteomic approaches generate large datasets requiring biological interpretation. Text based data mining tools have recently been developed to query large biological datasets of this type of data. PubMatrix is a web-based tool that allows simple text based mining of the NCBI literature search service PubMed using any two lists of keywords terms, resulting in a frequency matrix of term co-occurrence. RESULTS: For example, a simple term selection procedure allows automatic pair-wise comparisons of approximately 1-100 search terms versus approximately 1-10 modifier terms, resulting in up to 1,000 pair wise comparisons. The matrix table of pair-wise comparisons can then be surveyed, queried individually, and archived. Lists of keywords can include any terms currently capable of being searched in PubMed. In the context of cDNA microarray studies, this may be used for the annotation of gene lists from clusters of genes that are expressed coordinately. An associated PubMatrix public archive provides previous searches using common useful lists of keyword terms. CONCLUSIONS: In this way, lists of terms, such as gene names, or functional assignments can be assigned genetic, biological, or clinical relevance in a rapid flexible systematic fashion. http://pubmatrix.grc.nia.nih.gov/

Cell Line, Tumor↗
PubMed2003

Gene expression profiling of oxidative stress on atrial fibrillation in humans.

Atrial Fibrillation (AF) is thought be caused by oxidative stress. Oxidative stress at the cellular level results from many factors, including exposure to alcohol, medications, cold, toxins or radiation. In this study we investigated gene transcriptional profiles on the human myocardial tissues from AF and oxidative stress conditions. Right atrial appendages were obtained from AF patients (n = 26) undergoing the Maze procedure, and from control patients (n = 26) who were in normal sinus rhythm and undergoing coronary artery bypass graft operation. To examine the effects of oxidative stress on AF, we used radioactive complementary DNA (cDNA) microarrays to evaluate changes in the expression of 1,152 known genes. This technology, which monitors thousands of genes simultaneously, gives us a better picture of the interactions between AF and oxidative stress. Total RNAs prepared from the retrieved tissues were used to synthesize 33P-labeled cDNAs by reverse transcription and hybridized to cDNA microarrays. Gene expression profiles showed that 30 genes were upregulated and 25 were downregulated in AF patients compared with control patients. Moreover, comparison rank analysis revealed that the expression of five genes related to reactive oxygen species (ROS)-including flavin containing monooxygenase 1, monoamine oxidase B, ubiquitin specific protease 8, tyrosinase-related protein 1, and tyrosine 3-monooxygenase-increased by more than 2.0 of the Z-ratio, and two genes related to antioxidants including glutathione peroxidase 1, and heme oxygenase 2-decreased to the Z-ratio levels of < or = -2.0. Apparently, a balanced regulation of pro- and anti-oxidation can be shifted toward pro-oxidation and can result in serious damage similar to that of human AF. Western blotting analysis confirmed the upregulation of tyrosinase-related protein 1 and tyrosine 3-monooxygenase and the downregulation of heme oxygenase 2. These results suggested that the gene expression pattern of myocardial tissues in AF patients can be associated with oxidative stress, resulting in a significant increase in ROS. Thus, the cDNA microarray technique was useful for investigating transcription profiles in AF. It showed that the intracellular mechanism of oxidative stress plays a pivotal role in the pathologic progression of AF and offers novel insight into potential treatment with antioxidants.

Atrial Appendage↗
PubMed2003

Microarray analysis reveals interleukin-6 as a novel secretory product of the hypothalamo-neurohypophyseal system.

Physiological activation of the hypothalamo-neurohypophyseal system (HNS) by dehydration results is a massive release of vasopressin (VP) from the posterior pituitary. This is accompanied by a functional remodeling of the HNS. In this study we used cDNA arrays in an attempt to identify genes that exhibit differential expression in the hypothalamus following dehydration. Our study revealed nine candidate genes, including interleukin-6 (IL-6) as a putative novel secretory product of HNS worthy of further analysis. In situ hybridization and immunocytochemistry confirmed that IL-6 is robustly expressed in the supraoptic (SON) and the paraventricular (PVN) nuclei of the hypothalamus. By double staining immunofluorescence we showed that IL-6 is largely co-localized with VP in the SON and PVN. In situ hybridization, immunocytochemistry, and Western blotting all revealed IL-6 up-regulation in the SON and PVN following dehydration, thus validating the array data. The same dehydration stimulus resulted in an increase in IL-6 immunoreactivity in the axons of the internal zone of the median eminence and a marked reduction in IL-6-like material in the posterior pituitary gland. We thus suggest that IL-6 takes the same secretory pathway as VP and is secreted from the posterior pituitary following a physiological stimulus.

Animals↗
PubMed2003

Increased fibronectin expression in sturge-weber syndrome fibroblasts and brain tissue.

Sturge-Weber syndrome (SWS) is a neurocutaneous disorder that presents with a facial port-wine stain and a leptomeningeal angioma. Fibronectin expression regulates angiogenesis and vasculogenesis and participates in brain tissue responses to ischemia and seizures. We therefore hypothesized that abnormal gene expression of fibronectin and other extracellular matrix genes would be found in SWS brain tissue and SWS port-wine skin fibroblasts. Fibronectin gene and protein expression from port-wine-derived fibroblasts were compared with that from normal skin-derived fibroblasts of four individuals with SWS using microarrays, reverse transcriptase-PCR, Western analysis, and immunocytochemistry. Fibronectin gene and/or protein expression from eight SWS surgical brain samples was compared with that in two surgical epilepsy brain samples and six postmortem brain samples using microarrays, reverse transcriptase-PCR, and Western analysis. The gene expression of fibronectin was significantly increased (p < 0.05) in the SWS port-wine-derived fibroblasts compared with that of fibroblasts from SWS normal skin. A trend for increased protein levels of fibronectin in port-wine fibroblasts was found by Western analysis. No difference in the pattern of fibronectin staining was detected. The gene expression of fibronectin was significantly increased (p < 0.05), and a trend for increased fibronectin protein expression was found in the SWS surgical brain samples compared with the postmortem controls. These results suggest a potential role for fibronectin in the pathogenesis of SWS and in the brain's response to chronic ischemic injury in SWS. The reproducible differences in fibronectin gene expression between the SWS port-wine-derived fibroblasts and the SWS normal skin-derived fibroblasts are consistent with the presence of a hypothesized somatic mutation underlying SWS.

Adolescent↗
PubMed2003

The transcriptional response after oxidative stress is defective in Cockayne syndrome group B cells.

Cockayne syndrome (CS) is a human hereditary disease belonging to the group of segmental progerias, and the clinical phenotype is characterized by postnatal growth failure, neurological dysfunction, cachetic dwarfism, photosensitivity, sensorineural hearing loss, and retinal degradation. CS-B cells are defective in transcription-coupled DNA repair, base excision repair, transcription, and chromatin structural organization. Using array analysis, we have examined the expression profile in CS complementation group B (CS-B) fibroblasts after exposure to oxidative stress (H2O2) before and after complete complementation with the CSB gene. The following isogenic cell lines were compared: CS-B cells (CS-B null), CS-B cells complemented with wild-type CSB (CS-B wt), and a stably transformed cell line with a point mutation in the ATPase domain of CSB (CS-B ATPase mutant). In the wt rescued cells, we detected significant induction (two-fold) of 112 genes out of the 6912 analysed. The patterns suggested an induction or upregulation of genes involved in several DNA metabolic processes including DNA repair, transcription, and signal transduction. In both CS-B mutant cell lines, we found a general deficiency in transcription after oxidative stress, suggesting that the CSB protein influenced the regulation of transcription of certain genes. Of the 6912 genes, 122 were differentially regulated by more than two-fold. Evidently, the ATPase function of CSB is biologically important as the deficiencies seen in the ATPase mutant cells are very similar to those observed in the CS-B-null cells. Some major defects are in the transcription of genes involved in DNA repair, signal transduction, and ribosomal functions.

Adenosine Triphosphatases↗
PubMed2003

RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice.

The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. We have developed an approach, antibody-positioned RNA amplification (APRA), to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, we have observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP.

Animals↗