Biomedical subjects

Steven R Gullans

Publications and source records attributed to Steven R Gullans.

At least 19 recordsLinked to original sources

PubMed2003

Transcriptional activation of placental growth factor by the forkhead/winged helix transcription factor FoxD1.

Stromal-epithelial interactions play an important role in renal organogenesis. Expression of the forkhead/winged helix transcription factor FoxD1 (BF-2) is restricted to stromal cells in the embryonic renal cortex, but it mediates its effects on the adjacent ureteric bud and metanephric mesenchyme, which fail to grow and differentiate in BF-2 null mice. BF-2 is therefore likely to regulate transcription of factors secreted by stromal cells that modulate the differentiation of neighboring epithelial cells. Here, we used cells with inducible expression of BF-2, combined with microarray analysis, to identify Placental Growth Factor (PlGF), a Vascular Endothelial Growth Factor (VEGF) family member previously implicated in angiogenesis, as a downstream target of BF-2. BF-2 binds to a conserved HNF3beta site in the PlGF promoter and activates transcription. PlGF is precisely coexpressed with BF-2, both temporally and spatially, within the developing renal stroma, and it is completely absent in BF-2 null kidney stroma. Addition of PlGF to in vitro kidney organ cultures stimulates branching of the ureteric bud. Our observations indicate that PlGF is a direct and physiologically relevant transcriptional target of BF-2. The contribution of PlGF toward stromal signals that regulate epithelial differentiation suggests novel functions for a growth factor previously implicated in reactive angiogenesis.

Animals↗

PubMed2003

Gene expression changes presage neurodegeneration in a Drosophila model of Parkinson's disease.

Transgenic Drosophila expressing human alpha-synuclein faithfully replicate essential features of human Parkinson's disease, including age-dependent loss of dopaminergic neurons, Lewy-body-like inclusions and locomotor impairment. To define the transcriptional program encoding molecular machinery involved in alpha-synuclein pathology, we characterized expression of the entire Drosophila genome at pre-symptomatic, early and advanced disease stages. Fifty-one signature transcripts, including lipid, energy and membrane transport mRNAs, were tightly associated with alpha-synuclein expression. Most importantly, at the pre-symptomatic stage, when the potential for neuroprotection is greatest, expression changes revealed specific pathology. In age-matched tau transgenic Drosophila, the transcription of alpha-synuclein associated genes was normal, suggesting highly distinct pathways of neurodegeneration. Temporal profiling of progressive gene expression changes in neurodegenerative disease models provides unbiased starting points for defining disease mechanisms and for identifying potential targets for neuroprotective drugs at pre-clinical stages.

Animals↗

PubMed2003

Mammalian osmolytes and S-nitrosoglutathione promote Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function.

In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed Delta F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of Delta F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the Delta F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that Delta F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact Delta F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted Delta F508 CFTR maturation and function. S-Nitrosoglutathione-corrected Delta F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting Delta F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize Delta F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to explain why Delta F508 homozygous patients do not present with kidney dysfunction.

3T3 Cells↗

PubMed2003

Using gene expression ratios to predict outcome among patients with mesothelioma.

BACKGROUND: We have recently demonstrated that simple ratios of the expression levels of selected genes in tumor samples can be used to distinguish among types of thoracic malignancies. We examined whether this technique could predict treatment-related outcome for patients with mesothelioma. METHODS: We used gene expression profiling data previously collected from 17 mesothelioma patients with different overall survival times to define two outcome-related groups of patients and to train an expression ratio-based outcome predictor model. A Student's t test was used to identify genes among the two outcome groups that had statistically significant, inversely correlated expression levels; those genes were used to form prognostic expression ratios. We used a combination of several highly accurate expression ratios and cross-validation techniques to assess the internal consistency of this predictor model, quantitative reverse transcription-polymerase chain reaction of tumor RNA to confirm the microarray data, and Kaplan-Meier survival analysis to validate the model among an independent set of 29 mesothelioma tumors. All statistical tests were two-sided. RESULTS: We developed an expression ratio-based test capable of identifying 100% (17/17) of the samples used to train the model. This test remained highly accurate (88%, 15/17) after cross-validation. A four-gene expression ratio test statistically significantly (P =.0035) predicted treatment-related patient outcome in mesothelioma independent of the histologic subtype of the tumor. CONCLUSIONS: Gene expression ratio-based analysis accurately predicts treatment-related outcome in mesothelioma samples. This technique could impact the clinical treatment of mesothelioma by allowing the preoperative identification of patients with widely divergent prognoses.

Adult↗

PubMed2003

Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes.

BACKGROUND: Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions, ES cells can differentiate into specialized cells, including cardiac myocytes, but the efficiency is typically low and the process is incompletely understood. METHODS AND RESULTS: We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific alpha-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP), 880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid, also known as vitamin C, markedly increased the number of EGFP-positive cells, which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and alpha-actinin. Furthermore, ascorbic acid induced the expression of cardiac genes, including GATA4, alpha-MHC, and beta-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine, Tiron, or vitamin E. CONCLUSIONS: Ascorbic acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells.

Actinin↗

PubMed2003

12-lipoxygenase in opioid-induced delayed cardioprotection: gene array, mass spectrometric, and pharmacological analyses.

12-lipoxygenase (12-LO) has been shown to be a factor in acute ischemic preconditioning (IPC) in the isolated rat heart; however, no studies have been reported in delayed PC. We characterized the role of 12-LO in an intact rat model of delayed PC induced by a delta-opioid agonist SNC-121 (SNC). Rats were pretreated with SNC and allowed to recover for 24 hours. They were then treated with either baicalein or phenidone, 2 selective 12-LO inhibitors. In addition, SNC-pretreated rats had plasma samples isolated at different times after ischemia-reperfusion for liquid chromatographic-mass spectrometric analysis of the major metabolic product of 12-LO, 12-HETE. Similar studies were conducted with inhibitors. Gene array data showed a significant induction of 12-LO message (P<0.05) after opioid pretreatment. This induction in 12-LO mRNA was confirmed by real-time polymerase chain reaction, and 12-LO protein expression was enhanced by SNC pretreatment at 24 hours relative to vehicle treatment. Both baicalein and phenidone attenuated the protective effects of SNC pretreatment on infarct size (50+/-4% and 42+/-3% versus 29+/-2%, P<0.05, respectively). No significant differences were observed in 12-HETE concentrations between baseline control and SNC-treated rats. However, 12-HETE concentrations were increased significantly at both 15 minutes during ischemia and at 1 hour of reperfusion in the SNC-treated rats compared with controls. Baicalein and phenidone attenuated the increase in 12-HETE at 1 hour of reperfusion. These data suggest that SNC-121 appears to enhance message and subsequently the activity and expression of 12-LO protein during times of stress, resulting in delayed cardioprotection.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed2002

Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury.

Renal medullary cells are exposed to elevated and variable osmolarities and low oxygen tension. Despite the harsh environment, these cells are resistant to the effects of many harmful events. To test the hypothesis that this resistance is a consequence of these cells developing a stress tolerance phenotype to survive in this milieu, we created osmotically tolerant cells [hypertonic (HT) cells] by gradually adapting murine inner medullary collecting duct 3 cells to hyperosmotic medium containing NaCl and urea. HT cells have a reduced DNA synthesis rate, with the majority of cells arrested in the G(0)/G(1) phase of the cell cycle, and show constitutive expression of heat shock protein 70 that is proportional to the degree of hyperosmolarity. Unlike acute hyperosmolarity, chronic hyperosmolarity failed to activate MAPKs. Moreover, HT cells acquired protein translational tolerance to further stress treatment, suggesting that HT cells have an osmotolerant phenotype that is analogous to thermotolerance but is a permanent condition. In addition to osmotic shock, HT cells were more resistant to heat, H(2)O(2), cyclosporin, and apoptotic inducers, compared with isotonic murine inner medullary duct 3 cells, but less resistant to amphotericin B and cadmium. HT cells demonstrate that in renal medullary cells, hyperosmotic stress activates biological processes that confer cross-tolerance to other stressful conditions.

Adaptation, Psychological↗

PubMed2002

Discovery of molecular mechanisms of neuroprotection using cell-based bioassays and oligonucleotide arrays.

Oxidative injury and the resulting death of neurons is a major pathological factor involved in numerous neurodegenerative diseases. However, the development of drugs that target this mechanism remains limited. The goal of this study was to test a compound library of approved Food and Drug Administration drugs against a hydrogen peroxide-induced oxidant injury model in neuroblastoma cells. We identified 26 neuroprotective compounds, of which megestrol, meclizine, verapamil, methazolamide, sulindac, and retinol were examined in greater detail. Using large-scale oligonucleotide microarray analysis, we identified genes modulated by these drugs that might underlie the cytoprotection. Five key genes were either uniformly upregulated or downregulated by all six drug treatments, namely, tissue inhibitor of matrix metalloproteinase (TIMP1), ret-proto-oncogene, clusterin, galanin, and growth associated protein (GAP43). Exogenous addition of the neuropeptide galanin alone conferred survival to oxidant-stressed cells, comparable to that seen with the drugs. Our approach, which we term "interventional profiling," represents a general and powerful strategy for identifying new bioactive agents for any biological process, as well as identifying key downstream genes and pathways that are involved.

Biological Assay↗

PubMed2002

Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma.

The pathological distinction between malignant pleural mesothelioma (MPM)and adenocarcinoma (ADCA) of the lung can be cumbersome using established methods. We propose that a simple technique, based on the expression levels of a small number of genes, can be useful in the early and accurate diagnosis of MPM and lung cancer. This method is designed to accurately distinguish between genetically disparate tissues using gene expression ratios and rationally chosen thresholds. Here we have tested the fidelity of ratio-based diagnosis in differentiating between MPM and lung cancer in 181 tissue samples (31 MPM and 150 ADCA). A training set of 32 samples (16 MPM and 16 ADCA) was used to identify pairs of genes with highly significant, inversely correlated expression levels to form a total of 15 diagnostic ratios using expression profiling data. Any single ratio of the 15 examined was at least 90% accurate in predicting diagnosis for the remaining 149 samples (e.g., test set). We then examined (in the test set) the accuracy of multiple ratios combined to form a simple diagnostic tool. Using two and three expression ratios, we found that the differential diagnoses of MPM and lung ADCA were 95% and 99% accurate, respectively. We propose that using gene expression ratios is an accurate and inexpensive technique with direct clinical applicability for distinguishing between MPM and lung cancer. Furthermore, we provide evidence suggesting that this technique can be equally accurate in other clinical scenarios.

Adenocarcinoma↗

PubMed2002

Global analysis of gene expression in renal ischemia-reperfusion in the mouse.

Ischemia-induced acute renal failure (ARF) is a relatively common disorder with major morbidity and mortality. To study global gene expression during ARF, 6-week-old C57BL/6 male mice underwent 30 min of bilateral renal ischemia followed by reperfusion [I/R] or sham operation. Oligonucleotide microarrays [Affymetrix] with approximately 10,000 genes, 6,643 of which were present in mouse kidney, were used to analyze mRNA expression for up to 4 days following I/R. Fifty-two genes at day 1 and 40 at day 4 were up-regulated more than 4-fold [400%]. Seventy genes at day 1 and 30 genes at day 4 were down-regulated to under 0.25-fold from baseline [25%]. Real-time quantitative RT-PCR confirmed changes in expression for 8 genes of interest. Most of the induced transcripts are involved in cell structure, extracellular matrix, intracellular calcium binding, and cell division/differentiation. Our data identified several novel genes that may be important in renal repair after ischemia.

ADP-Ribosylation Factor 6↗

PubMed2002

HugeIndex: a database with visualization tools for high-density oligonucleotide array data from normal human tissues.

High-density oligonucleotide arrays are a powerful tool for uncovering changes in global gene expression in various disease states. To this end, it is essential to first characterize the variations of gene expression in normal physiological processes. We established the Human Gene Expression (HuGE) Index database (www.HugeIndex.org) to serve as a public repository for gene expression data on normal human tissues using high-density oligonucleotide arrays. This resource currently contains the results of 59 gene expression experiments on 19 human tissues. We provide interactive tools for researchers to query and visualize our data over the Internet. To facilitate data analysis, we cross-reference each gene on the array with its annotation in the LocusLink database at NCBI.

Computer Graphics↗

PubMed2002

Better therapeutics through microarrays.

DNA microarrays are an integral part of the process for therapeutic discovery, optimization and clinical validation. At an early stage, investigators use arrays to prioritize a few genes as potential therapeutic targets on the basis of various criteria. Subsequently, gene expression analysis assists in drug discovery and toxicology by eliminating poor compounds and optimizing the selection of promising leads. Integral to this process is the use of sophisticated statistics, mathematics and bioinformatics to define statistically valid observations and to deduce complex patterns of phenotypes and biological pathways. In short, microarrays are redefining the drug discovery process by providing greater knowledge at each step and by illuminating the complex workings of biological systems.

Computational Biology↗

PubMed2002

Monitoring changes in gene expression in renal ischemia-reperfusion in the rat.

BACKGROUND: Although acute renal failure (ARF) is a relatively common disorder with major morbidity and mortality, its molecular basis remains incompletely defined. The present study examined global gene expression in the well-characterized ischemia-reperfusion model of ARF using DNA microarray technology. METHODS: Male Wistar rats underwent bilateral renal ischemia (30 min) or sham operation, followed by reperfusion for 1, 2, 3 or 4 days. Plasma creatinine increased approximately fivefold over baseline, peaking on day 1. Renal total RNA was used to probe cDNA microarrays. RESULTS: Alterations in expression of 18 genes were identified by microarray analysis. Nine genes were up-regulated (ADAM2, HO-1, UCP-2, and thymosin beta4 in the early phase and clusterin, vanin1, fibronectin, heat-responsive protein 12 and FK506 binding protein in the established phase), whereas another nine were down-regulated (glutamine synthetase, cytochrome p450 IId6, and cyp 2d9 in the early phase and cyp 4a14, Xist gene, PPARgamma, alpha-albumin, uromodulin, and ADH B2 in the established phase). The identities of these 18 genes were sequence-verified. Changes in gene expression of ADAM2, cyp2d6, fibronectin, HO-1 and PPARgamma were confirmed by quantitative real-time polymerase chain reaction (PCR). ADAM2, cyp2d6, and PPARgamma have not previously been known to be involved in ARF. CONCLUSION: Using DNA microarray technology, we identified changes in expression of 18 genes during renal ischemia-reperfusion injury in the rat. We confirmed changes in five genes (fibronectin, ADAM2, cyp 2d6, HO-1 and PPARgamma) by quantitative real-time PCR. Several genes, not previously been identified as playing a role in ischemic ARF, may have importance in this disease.

ADAM Proteins↗

PubMed2002

Molecular chaperones in the kidney.

The normal milieu of the kidney includes hypoxia, large osmotic fluxes, and an enormous amount of fluid/solute reabsorption. Renal adaptation to these conditions requires a host of molecular chaperones that stabilize protein conformation, target nascent proteins to their final intracellular destination, and prevent protein aggregation. Under physiologic or pharmacologic stress, inducible molecular chaperones provide additional mechanisms for repairing or degrading non-native proteins and for inhibiting stress-induced apoptosis. In contrast to intracellular chaperones, chaperones present on the cell surface regulate the immune system and have cytokine-like effects. A diverse range of chaperones and chaperone functions provide the renal cell with an armamentarium of responses to improve the chances of survival.

Animals↗

PubMed2002

Global analysis of gene expression: methods, interpretation, and pitfalls.

Over the past 15 years, global analysis of mRNA expression has emerged as a powerful strategy for biological discovery. Using the power of parallel processing, robotics, and computer-based informatics, a number of high-throughput methods have been devised. These include DNA microarrays, serial analysis of gene expression, quantitative RT-PCR, differential-display RT-PCR, and massively parallel signature sequencing. Each of these methods has inherent advantages and disadvantages, often related to expense, technical difficulty, specificity, and reliability. Further, the ability to generate large data sets of gene expression has led to new challenges in bioinformatics. Nonetheless, this technological revolution is transforming disease classification, gene discovery, and our understanding of regulatory gene networks.

Computational Biology↗

PubMed2001

DNA microarray analysis of complex biologic processes.

DNA microarrays, or gene chips, allow surveys of gene expression, (i.e., mRNA expression) in a highly parallel and comprehensive manner. The pattern of gene expression produced, known as the expression profile, depicts the subset of gene transcripts expressed in a cell or tissue. At its most fundamental level, the expression profile can address qualitatively which genes are expressed in disease states. However, with the aid of bioinformatics tools such as cluster analysis, self-organizing maps, and principle component analysis, more sophisticated questions can be answered. Microarrays can be used to characterize the functions of novel genes, identify genes in a biologic pathway, analyze genetic variation, and identify therapeutic drug targets. Moreover, the expression profile can be used as a tissue or disease "fingerprint." This review details the fabrication of arrays, data management tools, and applications of microarrays to the field of renal research and the future of clinical practice.

Drug Design↗

PubMed2001

Identification of the gamma-aminobutyric acid receptor beta(2) and beta(3) subunits in rat, rabbit, and human kidneys.

The properties and functions of gamma-aminobutyric acid (GABA(A)) receptors in the mammalian central nervous system are well studied. However, the presence and significance of GABA(A) receptors in nonneural tissue is less clear. The goal of this study was to examine the expression and localization of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney. Reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and cerebellum and rabbit S(2) segments were amplified by use of PCR and GABA(A) beta(2) and beta(3) subunit-specific primers. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(2) subunit were identical in nucleotide composition. The rabbit kidney and rabbit neuronal GABA(A) receptor beta(2) subunit were 99% identical in nucleotide composition. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(3) subunits were 93% and 95% identical in nucleotide and amino acid composition, and rabbit kidney cortex and rabbit neuronal GABA(A) receptor beta(3) subunits were 95% and 98% identical in nucleotide and amino acid composition, respectively. PCR screening of a human kidney cDNA library and sequencing revealed that the human kidney cortex and neuronal beta(3) subunits were identical in nucleotide composition. Immunoblot analysis of rat kidney cortex and brain identified immunoreactive proteins in the 55 to 57 kD region, corresponding to the GABA(A) receptor beta(2) and beta(3) subunits. Immunohistochemistry revealed cytosolic and basolateral staining of the proximal convoluted and straight tubule. These results provide compelling evidence for the expression of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney of multiple species and the localization of the beta(2)/beta(3) subunits to the renal proximal tubule.

Amino Acid Sequence↗

PubMedDate not supplied

Alzheimer's disease drug discovery targeted to the APP mRNA 5'untranslated region.

We performed a screen for drugs that specifically interact with the 5' untranslated region of the mRNA coding for the Alzheimer's Amyloid Precursor Protein (APP). Using a transfection based assay, in which APP 5'UTR sequences drive the translation of a downstream luciferase reporter gene, we have been screening for new therapeutic compounds that already have FDA approval and are pharmacologically and clinically well-characterized. Several classes of FDA-pre-approved drugs (16 hits) reduced APP 5'UTR-directed luciferase expression (> 95% inhibition of translation). The classes of drugs include known blockers of receptor ligand interactions, bacterial antibiotics, drugs involved in lipid metabolism, and metal chelators. These APP 5'UTR directed drugs exemplify a new strategy to identify RNA-directed agents to lower APP translation and A beta peptide output for Alzheimer's disease therapeutics.

5' Untranslated Regions↗