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S R Gullans

Publications and source records attributed to S R Gullans.

At least 19 recordsLinked to original sources
PubMed2002

Correcting for signal saturation errors in the analysis of microarray data.

A variety of technical errors have arisen in data analysis when using cDNA or oligonucleotide microarrays. One of the most insidious problems is the saturation of the hybridization signal of high-abundant transcripts. This problem arises from the truncation of the laser fluorescence signal. When the hybridization signal on the microarray is very strong, this truncation can result in serious consequences that may not be readily apparent to the user. As an illustration of this problem, two subclasses of normal human tissue samples (six liver and six lung samples) were analyzed with GeneChip probe arrays to evaluate the patterns of expression for approximately 7000 human genes. Five of these data sets were found to suffer from signal truncation. This caused several tissues to be incorrectly classified using hierarchical clustering. To rectify this problem so that the gene expression data could be properly compared and clustered, we developed a "filtering" procedure that identifies a subset of genes least affected by the signal saturation. This filtering procedure can be obtained at www.hugeindex.org.

Aged↗
PubMed2001

A compendium of gene expression in normal human tissues.

This study creates a compendium of gene expression in normal human tissues suitable as a reference for defining basic organ systems biology. Using oligonucleotide microarrays, we analyze 59 samples representing 19 distinct tissue types. Of approximately 7,000 genes analyzed, 451 genes are expressed in all tissue types and designated as housekeeping genes. These genes display significant variation in expression levels among tissues and are sufficient for discerning tissue-specific expression signatures, indicative of fundamental differences in biochemical processes. In addition, subsets of tissue-selective genes are identified that define key biological processes characterizing each organ. This compendium highlights similarities and differences among organ systems and different individuals and also provides a publicly available resource (Human Gene Expression Index, the HuGE Index, http://www.hugeindex.org) for future studies of pathophysiology.

Cluster Analysis↗
PubMed2001

Differential gene expression profiling in human brain tumors.

Gene expression profiling of three human temporal lobe brain tissue samples (normal) and four primary glioblastoma multiforme (GBM) tumors using oligonucleotide microarrays was done. Moreover, confirmation of altered expression was performed by whole cell patch clamp, immunohistochemical staining, and RT-PCR. Our results identified several ion and solute transport-related genes, such as N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 receptors, GABA(A) receptor subunits alpha3, beta1, beta2, and beta3, the glutamate transporter, the glutamate/aspartate transporter II, the potassium channel K(V)2.1, hK(V)beta3, and the sodium/proton exchanger 1 (NHE-1), that are all downregulated in the tumors compared with the normal tissues. In contrast, aquaporin-1, possibly aquaporins-3 and -5, and GLUT-3 message appeared upregulated in the tumors. Our results also confirmed previous work showing that osteopontin, nicotinamide N-methyltransferase, murine double minute 2 (MDM2), and epithelin (granulin) are upregulated in GBMs. We also demonstrate for the first time that the cytokine and p53 binding protein, macrophage migration inhibitory factor (MIF), appears upregulated in GBMs. These results indicate that the modulation of ion and solute transport genes and heretofore unsuspected cytokines (i.e., MIF) may have profound implications for brain tumor cell biology and thus may identify potential useful therapeutic targets in GBMs.

Aquaporin 1↗
PubMed2001

Global expression changes of constitutive and hormonally regulated genes during endometrial neoplastic transformation.

OBJECTIVE: Endometrioid endometrial carcinoma is caused by a combination of mutational events and hormonal factors. We used large-scale messenger RNA expression analysis to discover genes that distinguish neoplastic transformation and examine the patterns of tumor expression of those genes which are normally regulated during the menstrual cycle. METHODS: Expression of approximately 6000 unique genes was quantified in 4 normal (2 proliferative, 2 secretory) and 10 malignant endometria using Affymetrix Hu6800 GeneChip probe arrays. Expression differences between normal and malignant tissue groups were measured by a test of statistical significance comparing the individual t statistic for each gene to the distribution of maximum t statistics among all genes following 1001 permutations of the tissue group assignments (Permax test). Hormonally responsive genes, selected by comparison of proliferative and secretory subsets of normal endometria using a combination of filters applied to the group means and t test rankings, were then examined in the tumors. RESULTS: Fifty genes with a Permax <0.50 provided excellent discrimination between normal and malignant groups and were predominantly characterized by diminished expression levels in the cancers. We found that 100 genes which are hormonally regulated in normal tissues are expressed in a disordered and heterogeneous fashion in cancers, with tumors resembling proliferative more than secretory endometrium. CONCLUSION: Neoplastic transformation is accompanied by predominant loss of activity of many genes constitutively expressed in normal source tissues and absence of expression profiles which characterize the antitumorigenic progestin response.

Adult↗
PubMed2000

A novel, sensitive detection system for high-density microarrays using dendrimer technology.

To improve signal detection on cDNA microarrays, we adapted a fluorescent oligonucleotide dendrimeric signal amplification system to microarray technology. This signal detection method requires 16-fold less RNA for probe synthesis, does not depend on the incorporation of fluorescent dNTPs into a reverse transcription reaction, generates a high signal-to-background ratio, and can be used to allow for multichannel detection on a single chip. Furthermore, since the dendrimers can be detected individually, it may be possible, by employing dendrimer-binding standards, to calculate the numbers of bound cDNAs can be estimated. These features make the dendrimer signal detection reagent ideal for high-throughput functional genomics research.

Biotechnology↗
PubMed2000

Prospective use of DNA microarrays for evaluating renal function and disease.

At the forefront of the revolution in human genomics is DNA microarray technology, which evaluates expression levels or genotypes of thousands of genes simultaneously, by means of miniaturization and parallel processing. Furthermore, advances in bioinformatics will result in the creation of large databases, which will require complex software programming for structural analysis. Over the next decade, DNA microarrays, combined with sophisticated informatics and genomic databases, will provide molecular fingerprints of disease processes and prognoses. This review provides an update on DNA microarray technology and its application to renal diseases.

DNA↗
PubMed1999

Genomic organization of the gene coding for TIRC7, a novel membrane protein essential for T cell activation.

A novel human membrane protein, TIRC7, was recently identified and demonstrated to be essential in T cell activation. Here we report on the genomic organization of the TIRC7 gene, which is composed of 15 exons and spans 7.9 kb. The seven predicted transmembrane-spanning domains of the TIRC7 protein coincide well with exon-intron boundaries. TIRC7 and OC116, a recently described putative subunit of the vacuolar proton pump that was demonstrated to be expressed in an osteoclastoma tumor as well as in a human pancreatic adenocarcinoma cell line, are demonstrated to be alternative transcripts of the same gene. OC116 consists of 20 exons with the last 14 introns and exons being identical with those of TIRC7. The chromosomal locus for both transcripts was identified on chromosome 11q13.4-q13.5. In human alloactivated T lymphocytes, mRNA expression of TIRC7, but not OC116, is demonstrated, indicating that OC116 is not involved in regular T cell proliferation.

Amino Acid Sequence↗
PubMed1999

The human homolog of Drosophila cornichon protein is differentially expressed in alloactivated T-cells.

To identify novel genes induced in the early stage of T-cell activation, mRNA expression in alloactivated human lymphocytes was examined. Differential display-reverse transcription PCR analysis revealed a 207-bp cDNA fragment which was upregulated 24 h after allostimulation of a human T-cell line. The corresponding complete 1396 bp cDNA, named TGAM77, encodes a predicted 134 amino acid protein which shares 63% homology with the cornichon (cni) protein of Drosophila melanogaster. Upregulation of TGAM77 mRNA in the early phase of T-cell activation was confirmed by Northern blot and RT-PCR analysis of activated human lymphocytes. TGAM77 mRNA is expressed in a variety of human tissues with various expression levels. In analogy to cni which is involved in an epidermal growth factor-like signaling pathway inducing cellular asymmetry in Drosophila oogenesis, TGAM77 might function in similar signaling establishing vectorial re-localization and concentration of signaling events in T-cell activation.

Amino Acid Sequence↗
PubMed1999

Cloning and expression of a murine histone deacetylase 3 (mHdac3) cDNA and mapping to a region of conserved synteny between murine chromosome 18 and human chromosome 5.

Histone deacetylases (HDACs) are enzymes that play a pivotal role in transcription, differentiation, and cell cycle progression. We previously cloned human HDAC3 cDNA and showed that its transfection into THP-1 cells results in G2/M cell cycle accumulation. Using bioinformatic screening and PCR, we have now cloned the murine Hdac3 cDNA, which encodes a 428-amino-acid protein with near complete identity to its human ortholog. To establish a link to a potential disease locus, we performed PCR-based chromosomal mapping for the mHdac3 gene and chromosomal fluorescence in situ hybridization (FISH) for the human gene. mHdac3 localizes to chromosome 18 and human HDAC3 gene localizes to a syntenic region in chromosome 5 at band q31.3-q32 telomeric to the cytokine gene cluster. Transfection of mHdac3 into HeLa cells led to accumulation in G2/M. Our results suggest a cell cycle function for murine Hdac3, reflecting the complex regulatory roles of this gene family.

Amino Acid Sequence↗
PubMed1998

Differential expression of human histone deacetylase mRNAs in response to immune cell apoptosis induction by trichostatin A and butyrate.

The reversible acetylation of histones by histone deacetylases (HDACs) and acetyltransferases (HATs) plays a fundamental role in gene transcription. We previously showed that HDAC mRNA is upregulated in immune cells upon PHA-induced activation. Little is known, however, about the differential regulation of HDAC mRNAs by the HDAC inhibitors Trichostatin A (TSA) and butyrate, agents known to block proliferation and induce apoptosis. We report that apoptosis-inducing concentrations of TSA and butyrate upregulate the expression of HDAC mRNAs in a differential manner and act synergistically with PHA to induce HDAC expression, suggesting the presence of independent HDAC regulatory mechanisms. Moreover, we show that HDAC inhibitor-induced apoptosis is associated with early abrogation of gamma-IFN production by Th1 lymphocytes and with p53 mRNA downregulation. Our findings highlight the dynamic interplay of cell cycle-, activation- and apoptosis-related proteins in association with time-dependent expression of HDACs and are suggestive of different specific roles for these enzymes.

Acetyltransferases↗
PubMed1998

Identification of a novel gene--SSK1--in human endothelial cells exposed to shear stress.

To identify transcriptionally regulated genes potentially involved in the effect of shear stress on endothelial gene expression, we performed a differential display analysis of mRNAs from human umbilical vein endothelial cell (HUVEC) exposed to laminar shear stress (12 dynes/cm2) in comparison to HUVEC maintained in static condition. We identified a cDNA fragment overexpressed by laminar shear stress. The full-length, SSK1, was 3653 long and encoded for a novel protein of 1050 amino acids. Northern blot demonstrates that SSK1 mRNA is expressed at high levels also in placenta, a weak transcript was present in heart, skeletal muscle, kidney and pancreas. Homology searches of the protein databases showed that SSK1 is related to numerous serine-threonine kinases. The highest homology was found with a very recently described gene, BUBR1, an analogue of BUB1, which is a kinase involved in the regulation of cell cycle. The most conserved residues in catalytic domains II, III, VIb, VII, VIII and IX of serine-threonine protein kinases were found in the C terminal region of SSK1 which further supports the kinase nature of the new protein. The putative serine-threonine kinase SSK1 may represent a tool by which mechanical forces regulates phosphorylation events within endothelial cells.

Amino Acid Sequence↗
PubMed1998

Differential display cloning of a novel human histone deacetylase (HDAC3) cDNA from PHA-activated immune cells.

The nucleosomal histones can be modified through reversible acetylation by histone acetyltransferases (HATs) and deacetylases (HDACs). HATs induce nucleosomal relaxation and allow DNA-binding by transcriptional activators. HDACs from corepressor complexes which negatively regulate cell growth. However, the HDAC inhibitors butyrate and Trichostatin A block T cell proliferation, suggesting that not all effects of HDACs lead to repression. Using mRNA differential display and 5'RACE we isolated human HDAC3, a novel gene that is upregulated in PHA-activated T cell clones. HDAC3 is homologous to other human HDACs and yeast RPD3. In peripheral blood mononuclear cells (PBMCs), activation by PHA, PMA and alpha-CD3 increased HDAC mRNA but no effect was seen with IFN-gamma, LPS, or IL-4. In contrast, GMCSF downregulated PBMC levels of HDAC3 mRNA. All HDACs were found to be ubiquitously expressed in immune and non-immune tissues. In human myeloid leukemia THP-1 cells, HDAC3 transfection resulted in increased size, aberrant nuclear morphology and cell cycle G2/M cell accumulation. Functional activity of the expressed HDAC3 protein was confirmed in alpha-HDAC3 antibody immunoprecipitates by a histone deacetylase assay. Our study suggests the participation of HDACs in cell cycle progression and activation.

Amino Acid Sequence↗
PubMed1998

Prevention of acute allograft rejection by antibody targeting of TIRC7, a novel T cell membrane protein.

A novel 75 kDa membrane protein, TIRC7, is described that exhibits a central role in T cell activation in vitro and in vivo. Modulation of TIRC7-mediated signals with specific anti-TIRC7 antibodies in vitro efficiently prevents human T cell proliferation and IL-2 secretion. Moreover, anti-TIRC7 antibodies specifically inhibit type 1 subset specific IFN-gamma expression but spare the type 2 cytokine IL-4. Diminished proliferation but not IFN-gamma secretion is reversible by exogenous rIL-2. An anti-TIRC7 antibody that cross-reacts with the 75 kDa rat homolog exhibits inhibition of rat alloimmune response in vitro and significantly prolongs kidney allograft survival in vivo. Targeting of TIRC7 may provide a novel therapeutic approach for modulation of the immune response.

Acute Disease↗
PubMed1998

Characterization of the Hsp110/SSE gene family response to hyperosmolality and other stresses.

Hsp110, Osp94, and Hsp70RY are members of the recently described Hsp110/SSE subfamily of (heat and osmotic) stress proteins whose members are structurally related to the Hsp70/BiP gene superfamily. To date, little is known about the response of this gene family to stresses in vitro or in vivo. In this study, an analysis of mRNA expression showed that Hsp110 and Osp94, like Hsp70, are induced in renal murine inner medullary collecting duct (mIMCD3) epithelial cells by heat shock, hyperosmotic NaCl, and cadmium, whereas low pH had a suppressive effect on Osp94. H2O2 decreased expression of Osp94 while inducing levels of Hsp110 and Hsp70 message. Tunicamycin, hypertonic urea, and tumor necrosis factor- had no effects. Hsp70RY was responsive exclusively to cadmium chloride. Moreover, enhanced expression of Hsp110 and Osp94 was subsequent to induction of Hsp70 and was suppressed by inhibition of protein synthesis by cycloheximide. RT-PCR analysis showed Hsp110, Osp94, and Hsp70RY are ubiquitously expressed in mouse tissues. In murine kidney, there was a corticomedullary gradient of expression of Hsp110, Osp94, Hsp70RY, and Hsp70 but not Hsc70 or BiP. Furthermore, dehydration increased inner medullary expression of Hsp110 and Osp94. An analysis of stress tolerance in mIMCD3 cells showed that heat shock and hyperosmotic NaCl stress are cross-tolerant stresses, suggesting hyperosmolality is a physiological correlate of heat shock in mammalian kidney. Thus Hsp110 and Osp94 behave as heat shock proteins, although they are regulated differently than Hsp70.

Animals↗
PubMed1998

A combination of NaCl and urea enhances survival of IMCD cells to hyperosmolality.

Physiological adaptation to the hyperosmolar milieu of the renal medulla involves a complex series of signaling and gene expression events in which NaCl and urea activate different cellular processes. In the present study, we evaluated the effects of NaCl and urea, individually and in combination, on the viability of murine inner medullary collecting duct (mIMCD3) cells. Exposure to hyperosmolar NaCl or urea caused comparable dose- and time-dependent decreases in cell viability, such that 700 mosmol/kgH2O killed >90% of the cells within 24 h. In both cases, cell death was an apoptotic event. For NaCl, loss of viability at 24 h paralleled decreases in RNA and protein synthesis at 4h, whereas lethal doses of urea had little or no effect on these biosynthetic processes. Cell cycle analysis showed both solutes caused a slowing of the G2/M phase. Surprisingly, cells exposed to a combination of NaCl + urea were significantly more osmotolerant such that 40% survived 900 mosmol/kgH2O. Madin-Darby canine kidney cells but not human umbilical vein endothelial cells also exhibited a similar osmotolerance response. Enhanced survival was not associated with a restoration of normal biosynthetic rates or cell cycle progression. However, the combination of NaCl + urea resulted in a shift in Hsp70 expression that appeared to correlate with survival. In conclusion, hyperosmolar NaCl and urea activate independent and complementary cellular programs that confer enhanced osmotolerance to renal medullary epithelial cells.

Animals↗
PubMed1998

Apoptosis of endothelial cells is associated with paracrine induction of adhesion molecules: evidence for an interleukin-1beta-dependent paracrine loop.

Monocytic infiltration of the vessel wall is a hallmark of injury in a variety of vascular diseases. In the present study, we explored the relationship between endothelial apoptosis and hyperadhesiveness for monocytic cells. Apoptosis of human umbilical vein endothelial cells (HUVECs) was induced by either growth factor deprivation (GFD) for 24 hours or by incubation with mitomycin C (MMC) at 0.01 mg/ml for 24 hours and confirmed by light microscopy and DNA laddering. In parallel assessments of cell-cell adhesion, GFD and MMC induced hyperadhesiveness of HUVECs for the THP-1 monocytic cell line. Hyperadhesiveness developed in association with induction of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 on HUVECs and was attenuated by monoclonal antibodies directed against these ligands. Culture medium conditioned by apoptotic HUVECs up-regulated the expression of adhesion molecules on normal HUVECs, suggesting that paracrine factors in the apoptotic milieu led to induction of adhesion molecules. Interleukin (IL)-1beta was implicated as a putative mediator in this setting because 1) exogenous IL-1beta up-regulates ICAM-1 and VCAM-1 with kinetics similar to those noted during endothelial cell apoptosis, 2) endothelial apoptosis was associated with increased expression of IL-1beta converting enzyme, and 3) the adhesion-promoting actions of GFD and MMC were attenuated by an anti-IL-1beta antibody.

Apoptosis↗