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Biomedical subjects

Kevin G Becker

Publications and source records attributed to Kevin G Becker.

16 recordsLinked to original sources
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↗
PubMed2003

Analysis of microarray data using Z score transformation.

High-throughput cDNA microarray technology allows for the simultaneous analysis of gene expression levels for thousands of genes and as such, rapid, relatively simple methods are needed to store, analyze, and cross-compare basic microarray data. The application of a classical method of data normalization, Z score transformation, provides a way of standardizing data across a wide range of experiments and allows the comparison of microarray data independent of the original hybridization intensities. Data normalized by Z score transformation can be used directly in the calculation of significant changes in gene expression between different samples and conditions. We used Z scores to compare several different methods for predicting significant changes in gene expression including fold changes, Z ratios, Z and t statistical tests. We conclude that the Z score transformation normalization method accompanied by either Z ratios or Z tests for significance estimates offers a useful method for the basic analysis of microarray data. The results provided by these methods can be as rigorous and are no more arbitrary than other test methods, and, in addition, they have the advantage that they can be easily adapted to standard spreadsheet programs.

Cluster Analysis↗
PubMed2002

A genomic-scale view of the cAMP response element-enhancer decoy: a tumor target-based genetic tool.

Enhancer DNA decoy oligodeoxynucleotides (ODNs) inhibit transcription by competing for transcription factors. A decoy ODN composed of the cAMP response element (CRE) inhibits CRE-directed gene transcription and tumor growth without affecting normal cell growth. Here, we use DNA microarrays to analyze the global effects of the CRE-decoy ODN in cancer cell lines and in tumors grown in nude mice. The CRE-decoy up-regulates the AP-2beta transcription factor gene in tumors but not in the livers of host animals. The up-regulated expression of AP-2beta is clustered with the up-regulation of other genes involved in development and cell differentiation. Concomitantly, another cluster of genes involved in cell proliferation and transformation is down-regulated. The observed alterations indicate that CRE-directed transcription favors tumor growth. The CRE-decoy ODN, therefore, may serve as a target-based genetic tool to treat cancer and other diseases in which CRE-directed transcription is abnormally used.

Adenocarcinoma↗
PubMed2002

Microarray analysis of gene expression in the prefrontal cortex in schizophrenia: a preliminary study.

Microarray studies can be used to examine expression levels for large numbers of genes simultaneously and may be applied to identify genes involved in schizophrenia. A microarray with 1127 brain-relevant genes was used to screen relative gene expression in the dorsolateral prefrontal cortex (DLPFC) from three pools of patients with schizophrenia (n = 15) and three matched control pools (n = 15). Pooling of tissue samples was employed as a strategy to detect changes in gene expression that are consistently found across individual cases of schizophrenia. Differences in gene expression were examined by z-ratios in addition to traditional normalized ratios. Three genes that showed consistently decreased expression in schizophrenia by both z-ratio differences and decreased normalized numerical ratios were identified. These were histidine triad nucleotide-binding protein (HINT), ubiquitin conjugating enzyme E2N (UBE2N) and glutamate receptor, ionotropic, AMPA 2 (GRIA2). Moreover, HINT gene expression was decreased to a similar degree in a prior study. In addition, a decrease in AMPA receptor expression is consistent with a decrease in glutamate synaptic function. These results are subject to limitations based on variations inherent to human subjects and tissue samples, possible effects of neuroleptic treatment, and the requirement for verification using independent techniques.

Adult↗
PubMed2002

Double round hybridization of membrane based cDNA arrays: improved background reduction and data replication.

Microarray analysis provides the ability to obtain gene expression data on thousands of gene targets in parallel and is being applied to an increasing variety of neurobiological samples. Nylon filter based cDNA arrays often provide an alternative to oligo-based or glass-based gene expression analysis systems. cDNA arrays regardless of the platform, are usually quite expensive to purchase and equally expensive to produce. As such, obtaining both high quality and replicated data, as well as the ability to recover from an apparently failed hybridization experiment are important factors in microarray experimental design. Using a collection of 1152 neuronally relevant cDNAs, this report describes simple modifications in experimental design of radioactive microarray experiments that, (1) improves reproducibility; (2) allows recovery from a failed experiment and; (3) provides a type of replicate data set with no requirement for additional arrays, no additional probe labelings, and little extra cost.

DNA, Complementary↗
PubMed2002

Global analysis of stress-regulated mRNA turnover by using cDNA arrays.

cDNA array technology has proven to be a powerful way to monitor global changes in gene expression patterns. Here, we present an approach that extends the current utility of cDNA arrays to allow the evaluation of the relative roles of transcription and mRNA turnover in governing gene expression on a global basis, compared with current individual gene-by-gene analyses. This method, which involves comparison of large-scale hybridization patterns generated with steady-state mRNA versus newly transcribed (nuclear run-on) RNA, was used to demonstrate the importance of mRNA turnover in regulating gene expression following several conditions of stress.

Carcinoma, Non-Small-Cell Lung↗
PubMed2002

Development of a highly specialized cDNA array for the study and diagnosis of epithelial ovarian cancer.

Ovarian cancer is a major cause of cancer death in women. Unfortunately, the molecular pathways underlying ovarian cancer progression are poorly understood, making the development of novel diagnostic and therapeutic strategies difficult. On the basis of our previous observations obtained from serial analysis of gene expression, we have constructed a specialized cDNA array for the study of ovarian cancer. Small, specialized arrays have several practical advantages and can reveal information that is lost in the "noise" generated by irrelevant genes present in larger arrays. The array, which we named Ovachip, contains 516 cDNAs chosen from our serial analysis of gene expression and cDNA array studies for their relevance to ovarian cancer. The gene expression patterns revealed with the Ovachip are highly reproducible and extremely consistent among the different ovarian specimens tested. This array was extremely sensitive at differentiating ovarian cancer from colon cancer based on expression profiles. The Ovachip revealed clusters of coordinately expressed genes in ovarian cancer. One such cluster, the IGF2 cluster, is particularly striking and includes the insulin-like growth factor II, the cisplatin resistance-associated protein, the checkpoint suppressor 1, the cyclin-dependent kinase 6, and a protein tyrosine phosphatase receptor. We also identified a cluster of down-regulated genes that included the cyclin-dependent kinase 7 and cyclin H. Thus, the Ovachip allowed us to identify previously unidentified clusters of differentially expressed genes that may provide new paradigms for molecular pathways important in ovarian malignancies. Because of the relevance of the arrayed genes, the Ovachip may become a powerful tool for investigators in the field of ovarian cancer and may facilitate progress in understanding the etiology of this disease and in its clinical management.

Carcinoma, Papillary↗
PubMed2002

Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity.

The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2002

AF5, a CNS cell line immortalized with an N-terminal fragment of SV40 large T: growth, differentiation, genetic stability, and gene expression.

Central nervous system progenitor cells that are self-renewing in culture and also differentiate under controlled conditions are potentially useful for developmental studies and for cell-based therapies. We characterized growth and plasticity properties and gene expression in a rat mesencephalic cell line, AF5, that was immortalized with an N-terminal fragment of SV40 large T (T155g). For over 150 population doublings in culture, the growth rate of AF5 cells remained steady, the cells remained responsive to bFGF, and telomerase activity and telomere lengths were unchanged. While karyotype analyses revealed some chromosomal abnormalities, these were also unchanged over time; additionally, no mutations in p53 gene sequences were found, and wild-type p53 activation was normal. AF5 cells produced PDGF, TGFbeta1, TGFbeta2, and bFGF. Similar to primary progenitor cells, AF5 cells retained their plasticity in culture; they could be propagated in an undifferentiated state as "neurospheres" in serum-free media or as adherent cultures in serum-containing media, and they differentiated when allowed to become confluent. Adherent subconfluent actively growing cultures expressed a marker for immature neurons, nestin, while few cells expressed the mature neuronal cell marker betaIII-tubulin. Confluent cultures ceased growing, developed differentiated morphologies, contained few or no nestin-expressing cells, and acquired betaIII-tubulin expression. Global gene expression was examined using a 15,000 gene microarray, comparing exponential growth with and without bFGF stimulation, and the differentiated state. The AF5 cell line exhibited stable genetic and growth properties over extended periods of time, while retaining the ability to differentiate in vitro. These data suggest that the AF5 cell line may be useful as an in vitro model system for studies of neural differentiation.

Animals↗
PubMed2002

Dissecting the circuitry of protein kinase A and cAMP signaling in cancer genesis: antisense, microarray, gene overexpression, and transcription factor decoy.

Expression of the RI alpha subunit of the cAMP-dependent protein kinase type I (PKA-I) is enhanced in human cancer cell lines, in primary tumors, in transformed cells, and in cells upon stimulation of growth. Signaling via the cAMP pathway may be complex, and the biological effects of the pathway in normal cells may depend upon the physiological state of the cells. However, results of different experimental approaches such as antisense exposure, 8-Cl-cAMP treatment, and gene overexpression have shown that the inhibition of RI alpha/PKA-I exerts antitumor activity in a wide variety of tumor-derived cell lines examined in vitro and in vivo. cDNA microarrays have further shown that in a sequence-specific manner, RI alpha antisense induces alterations in the gene expression profile of cancer cells and tumors. The cluster of genes that define the "proliferation-transformation" signature are down-regulated, and those that define the "differentiation-reverse transformation" signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers, exhibiting the molecular portrait of the reverted (flat) phenotype of tumor cells. These results reveal a remarkable cellular regulation, elicited by the antisense RI alpha, superimposed on the regulation arising from the Watson-Crick base-pairing mechanism of action. Importantly, the blockade of both the PKA and PKC signaling pathways achieved with the CRE-transcription factor decoy inhibits tumor cell growth without harming normal cell growth. Thus, a complex circuitry of cAMP signaling comprises cAMP growth regulatory function, and deregulation of the effector molecule by this circuitry may underlie cancer genesis and tumor progression.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Microarray technology and its application on nicotine research.

Since its development, microarray technique has revolutionized almost all fields of biomedical research by enabling high-throughput gene expression profiling. Using cDNA microarrays, thousands of genes from various organisms have been examined with respect to differentiation/development, disease diagnosis, and drug discovery Nevertheless, research on nicotine using cDNA microarrays has been rather limited. Therefore, it is our intention in this article to report the findings of our cDNA microarray study on nicotine. We first present an overview of the microarray technology, particularly focusing on the factors related to microarray design and analysis. Second, we provide a detailed description of several newly identified biological pathways in our laboratory, such as phosphatidylinositol signaling and calcium homeostasis, which are involved in response to chronic nicotine administration. Additionally, we illustrate how comparisons between microarray studies help identify candidate genes that potentially may explain the observed inverse association between smoking and schizophrenia. Lastly, given the early stage of microarray research on nicotine, we elaborate on the need for an efficient analysis of genetic networks to further enhance our understanding of the mechanisms involved in nicotine abuse and addiction.

Animals↗