Incyte Genomics Research Articles

Research status and collaboration analysis based on big data mining: an empirical study of Alzheimer's disease

ABSTRACT This paper employs text mining techniques that aimed to facilitate technology information. First, this paper used patent data to monitor technological development trends systematically to show the technology research status from perspectives of country, institution, technology fields, and subjects. Secondly, this study explores the cooperation network institutions and inventors by applying the data mining approaches, social network analysis,. Additionally, the sequence analysis is applied to reveal a more comprehensive and objective appearance of cooperative relationships, partners, and centrality. The empirical findings reveal four significant observations. (1) The R&D centres have been mainly influenced by the United States and other developed countries. (2) All technological fields in both B IPC and Derwent manual codes are concentrated around pharmaceutical activities. (3) 1-6c alkyl, pharmaceutical composition, and central nervous system et al. are traditional research and core subjects. 2-6c alkenyl, amino acid sequence, and 1-3c alkoxy et al. are the hot subjects. (4) The influential institutions are HOFFMANN LA ROCHE & CO AG F (degree centrality is 0.0872), ASTRAZENECA AB, MERCK SHARP & DOHME CORP, PFIZER INC and UNIV CALIFORNIA, INCYTE GENOMICS. (5) The influential inventors are WANG Y, BACHER G, and PETERS D.

Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Who owns your genes?

The attempt to break the hold that Myriad has on key patents for cancer genes has reopened the debate on who can own rights to biology. At the press conference held to announce success in transplanting a synthetic genome into a living cell, scientist and cofounder of Synthetic Genomics J Craig Venter claimed: 'I have never been quite comfortable claiming patents on discoveries.' Yet Venter's name was prominent on the bundle of patents filed during his work on the Human Genome Project. In his autobiography, Venter claims he became the scapegoat for the patents filed in the 1990s patents on fragments of DNA uncovered during the sequencing work as part of a plan by the National Institute of Health to secure rights over the DNA before private researchers. But the company he founded and later left, Celera Genomics, sought patents on hundreds of potentially significant human genes despite not knowing what most of the DNA did. Even after Celera, Venter went on to help put together a wide-ranging patent to support his synthetic biology based on subsets of a natural bacterium. Celera was not alone. Incyte Genomics and Human Genome Sciences did the same. But it was Monsanto, already widely criticised for its approach to intellectual property (IP) in the biosciences, who triggered a lawsuit that changed the way that agencies such as the US Patent and Trademark Office viewed DNA patents in the mid-2000s. After that case, the USPTO decided that only genes with a known function could be patented, not just fragments of DNA that seem to come from individual genes found during mass sequencing efforts. Even that view could change again if another biotechnology company, Myriad Genetics, fails in its appeal to put its name against sequences of DNA that have existed in humans for millennia. It is the latest battle in the war to gain control over chunks of the genome, most of which is taking place in the United States of America but which has seen skirmishes in Europe and, more recently, in Australia.

Regulation of Hypocretin (Orexin) Expression in Embryonic Zebrafish

Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.

Myospryn Is a Direct Transcriptional Target for MEF2A That Encodes a Striated Muscle, α-Actinin-interacting, Costamere-localized Protein

The full repertoire of proteins that comprise the striated muscle Z-disc and peripheral structures, such as the costamere, have yet to be discovered. Recent studies suggest that this elaborate protein network, which acts as a structural and signaling center for striated muscle, harbors factors that function as mechanosensors to ensure coordinated contractile activity. Mutations in genes whose products reside in this region often result in skeletal and cardio myopathies, demonstrating the importance of this macromolecular complex in muscle structure and function. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the costamere in striated muscle. This gene, called myospryn, was identified by microarray analysis as a transcript down-regulated in MEF2A knock-out mice. MEF2A knock-out mice develop cardiac failure during the perinatal period with mutant hearts exhibiting several cardiac abnormalities including myofibrillar disarray. Myospryn is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy-associated gene 5 (CMYA5). Myospryn is expressed as a single, large transcript of approximately 12 kilobases in adult heart and skeletal muscle with an open reading frame of 3739 amino acids. This protein, belonging to the tripartite motif superfamily of proteins, contains a B-box coiled-coil (BBC), two fibronectin type III (FN3) repeats, and SPRY domains and interacts with the sarcomeric Z-disc protein, alpha-actinin-2. Our findings demonstrate that myospryn functions directly downstream of MEF2A at the costamere in striated muscle potentially playing a role in myofibrillogenesis.

Gene Expression in Normal Urothelium Depends on Location within the Bladder: A Possible Link to Bladder Carcinogenesis

ObjectivesClinical studies have shown that more than 70% of primary bladder tumours arise in the area around the ureteric orifice. In this study a genomic approach was taken to explore the molecular mechanisms that may influence this phenomenon. MethodsRNA was isolated from each individual normal ureteric orifice and the dome biopsy from 33 male patients. Equal amounts of the pooled ureteric orifice and dome mRNAs were labelled with Cy3 and Cy5, respectively before hybridising to the gene chip (UniGEM 2.0, Incyte Genomics Inc., Wilmington, Delaware, USA). Results: Significant changes (more than a twofold difference) in gene expression were observed in 3.1% (312) of the 10,176 gene array: 211 genes upregulated and 101 downregulated. Analysis of Cdc25B, TK1, PKM, and PDGFra with RT-PCR supported the reliability of the microarray result. Seladin-1 was the most upregulated gene in the ureteric orifice: 8.3-fold on the microarray and 11.4-fold by real time PCR. ConclusionsOverall, this study suggests significant altered gene expression between these two anatomically distinct areas of the normal human bladder. Of particular note is Seladin-1, whose significance in cancer is yet to be clarified. Further studies of the genes discovered by this work will help clarify which of these differences influence primary bladder carcinogenesis.

Thymosin β4 Is a Determinant of the Transformed Phenotype and Invasiveness of S-Adenosylmethionine Decarboxylase–Transfected Fibroblasts

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the synthesis of polyamines essential for cell growth and proliferation. Its overexpression induces the transformation of murine fibroblasts in both sense and antisense orientations, yielding highly invasive tumors in nude mice. These cell lines hence provide a good model to study cell invasion. Here, the gene expression profiles of these cells were compared with their normal counterpart by microarray analyses (Incyte Genomics, Palo Alto, CA, and Affymetrix, Santa Clara, CA). Up-regulation of the actin sequestering molecule thymosin beta4 was the most prominent change in both cell lines. Tetracycline-inducible expression of thymosin beta4 antisense RNA caused a partial reversal of the transformed phenotype. Further, reversal of transformation by dominant-negative mutant of c-Jun (TAM67) caused reduction in thymosin beta4 mRNA. Interestingly, a sponge toxin, latrunculin A, which inhibits the binding of thymosin beta4 to actin, was found to profoundly affect the morphology and proliferation of the AdoMetDC transformants and to block their invasion in three-dimensional Matrigel. Thus, thymosin beta4 is a determinant of AdoMetDC-induced transformed phenotype and invasiveness. Up-regulation of thymosin beta4 was also found in ras-transformed fibroblasts and metastatic human melanoma cells. These data encourage testing latrunculin A-like and other agents interfering with thymosin beta4 for treatment of thymosin beta4-overexpressing tumors with high invasive and metastatic potential.

NF90 Regulates Cell Cycle Exit and Terminal Myogenic Differentiation by Direct Binding to the 3′-Untranslated Region of MyoD and p21WAF1/CIP1 mRNAs

NF90 and splice variant NF110/ILF3/NFAR are double-stranded RNA-binding proteins that regulate gene expression. Mice with targeted disruption of NF90 were engineered. NF90(-/-) mice were born small and weak and succumbed to perinatal death within 12 h because of neuromuscular respiratory failure. Lung inflation and morphology were normal in NF90(-/-) mice. The diaphragm and other skeletal muscles in NF90(-/-) mice demonstrated disorganized arrangement and paucity of myofibers, evidence of myocyte degeneration and increased apoptosis. The expression of myogenic regulators, MyoD, myogenin, and p21WAF1/CIP1, was severely decreased in NF90(-/-) mice. These myogenic transcription factors and cell cycle inhibitors are regulated in part through post-transcriptional mRNA stabilization. Northwestern blotting revealed that NF90 is the principal and specific p21WAF1/CIP1 and MyoD 3'-untranslated region RNA-binding protein in developing skeletal muscles. NF90 regulates transcription factors and a cell cycle inhibitor essential for skeletal muscle differentiation and for survival.

Gene expression of fibroblasts following light irradiation

Gene expression of fibroblasts following light irradiation

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis: Atsushi Tanaka, Asahi Life Fdn, Tokyo Japan; Patrick S Leung, Thomas P Kenny, University of California, Davis, CA; Janice Au-Young, Incyte Genomics, Inc, Palo Alto, CA; Thomas Prindiville, University of California, Davis, CA; Ross L Coppel, Monash University, Victoria Australia; Aftab A

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis: Atsushi Tanaka, Asahi Life Fdn, Tokyo Japan; Patrick S Leung, Thomas P Kenny, University of California, Davis, CA; Janice Au-Young, Incyte Genomics, Inc, Palo Alto, CA; Thomas Prindiville, University of California, Davis, CA; Ross L Coppel, Monash University, Victoria Australia; Aftab A

Gene expression profiling of bovine macrophages in response to Escherichia coli O157:H7 lipopolysaccharide

The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St. Louis, MO). Of these target sequences, 44 were differentially expressed upon exposure to LPS, including 18 genes not previously reported to exist in cattle. These included a pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and inhibitors of apoptosis. Using the human microarray, cDNA from bovine LPS-treated and control macrophages consistently hybridized to targets known to be expressed constitutively by macrophages, as expected given the predicted cDNA sequence homology. That this human system was accurately estimating levels of bovine transcripts was further verified by real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-PCR) using bovine-specific primers. This first report of bovine–human cross-species expression profiling by microarray hybridization demonstrates the utility of this technique in bovine gene expression and discovery.

Downregulation of gene expression in the ageing lens: a possible contributory factor in senile cataract.

To study the molecular characteristics of lens epithelial cells from patients with senile cataract by cDNA microarray technique. Lens epithelial cells adhering to anterior capsules taken during cataract surgery collected from 108 patients, aged 56-92 years (senile cataract group), were pooled. Pooled epithelial cells of normal, noncataractous lenses from one patient with ocular trauma, one patient with lens subluxation, and 25 cadaveric eyes, all under the age of 55 years, served as a control. Total RNA was extracted by conventional methods from the two groups of cells, and a fluorescent probe was prepared for each group. The probes were hybridized on 9700 known human cDNA clones. Hybridized clones were analysed using a scanning laser and the results were processed by GEMTools (Incyte Genomics) software. A total of 1827 clones hybridized with the two probes. Of these, 400 showed differences of more than two-fold in gene expression between the two probes. Relative to controls, gene expression in the senile cataract lenses was upregulated in 318 clones and downregulated in 82. Three genes-filensin, inwardly rectifying potassium channel (IRPC), and pigment epithelium-derived factor (PEDF) were strongly downregulated (by 41.3-, 6.8-, and 5.9-fold, respectively) in senile cataract. Cataractogenesis is associated with numerous changes in the genetic profile of the lens epithelial cells. Since filensin, IRPC, and PEDF genes are known to have important roles in the physiology and morphology of the transparent lens, substantial downregulation of their expression might contribute to the formation of senile cataract.

Catalytic Activity of ADAM8, ADAM15, and MDC-L (ADAM28) on Synthetic Peptide Substrates and in Ectodomain Cleavage of CD23

The ADAM family of disintegrin metalloproteases plays important roles in "ectodomain shedding," the process by which biologically active, soluble forms of cytokines, growth factors, and their receptors are released from membrane-bound precursors. Whereas ADAM8, ADAM15, and MDC-L (ADAM28) are expressed in specific cell types and tissues, their in vivo functions and substrates are not known. By screening a library of synthetic peptides as potential substrates, we show that soluble recombinant forms of these enzymes have similar proteolytic substrate specificity, clearly distinct from that of ADAM17 (TNFalpha-converting enzyme). A number of tumor necrosis factor (TNF) family proteins and CD23 were screened as potential substrates for ectodomain cleavage. We found that ADAM8, ADAM15, and MDC-L, but not ADAM17, catalyzed ectodomain shedding of CD23, the low affinity IgE receptor. ADAM8-dependent, soluble CD23 release required proteolytically active ADAM8, and a physical association of ADAM8 was observed with the membrane-bound form of CD23. The ADAM8-dependent release of sCD23 and the endogenous release from B cell lines could be similarly inhibited by a hydroxamic acid, metalloprotease inhibitor compound. We conclude that ADAM8 could contribute to ectodomain shedding of CD23 and may thus be a potential target for therapeutic intervention in allergy and inflammation.

Microarray analysis of changes in bone cell gene expression early after cadmium gavage in mice

We developed an in vivo model for cadmium-induced bone loss in which mice excrete bone mineral in feces beginning 8 h after cadmium gavage. Female mice of three strains [CF1, MTN (metallothionein-wild-type), and MT1,2KO (MT1,2-deficient)] were placed on a low-calcium diet for 2 weeks. Each mouse was gavaged with 200 μg Cd or vehicle only. Fecal calcium was monitored daily for 9 days, beginning 4 days before cadmium gavage, to document the bone response. For CF1 mice, bones were taken from four groups: +/− Cd, 2 h after Cd and +/− Cd, 4 h after Cd. MTN and MT1,2KO strains had two groups each: +/−Cd, 4 h after Cd. PolyA+ RNA preparations from marrow-free shafts of femura and tibiae of each +/− Cd pair were submitted to Incyte Genomics for microarray analysis. Fecal Ca results showed that bone calcium excreted after cadmium differed for the three mouse strains: CF1, 0.24 ± 0.08 mg; MTN, 0.92 ± 0.22 mg; and MT1,2KO, 1.7 ± 0.4 mg. Gene array results showed that nearly all arrayed genes were unaffected by cadmium. However, MT1 and MT2 had Cd+/Cd− expression ratios >1 in all four groups, while all ratios for MT3 were essentially 1, showing specificity. Both probes for MAPK 14 (p38 MAPK) had expression ratios >1, while no other MAPK responded to cadmium. Vacuolar proton pump ATPase and integrin alpha v (osteoclast genes), transferrin receptor, and src-like adaptor protein genes were stimulated by Cd; other src-related genes were unaffected. Genes for bone formation, stress response, growth factors, and signaling molecules showed little or no response to cadmium. Results support the hypothesis that Cd stimulates bone demineralization via a p38 MAPK pathway involving osteoclast activation.

Identification and Immunohistochemical Characterization of a Mucin-Like Glycoprotein Expressed in Early Stage Breast Carcinoma

In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq^® Expressed Sequence Tag database. A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types. BS106 mRNA was detected in 90% of the breast tissues examined. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media. Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer. BS106 protein was detected in the vast majority of carcinomas (70–100%) and overexpressed in approximately 30% of the 22 specimens analyzed. BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma.

ABOUT THE AUTHORS

ABOUT THE AUTHORS

GENOMICS FIRMS SHIFT OPERATIONS

GENOMICS COMPANIES HYseq Pharmaceuticals and Incyte Genomics have struck deals to merge with other small continuing a trend among gene sequencing and genomics companies to build integrated product-based businesses (C&EN, Oct. 14, page 25). Hyseq's merger with Variagenics will the development of the most promising products of both companies, Hyseq Chairman George B. Rathmann says. new company will be in a position to advance a strong development program and accelerate a significant pipeline. Hyseq will exchange about $56 million of its stock for the stock of the pharmacogenomics and molecular diagnostics firm. The new company will be based in Sunnyvale, Calif., with diagnostics development capabilities in Cambridge, Mass. It anticipates having 110 to 120 employees after the merger and enough cash to fund operations through 2004. Similarly, Incyte will pay up to $28.3 million in cash and stock, and a possible $14 million in future milestone payments, to acquire small-molecule d...

GEM? Microarrays and drug discovery

Incyte Genomics' GEM Gene Expression Microarray is a proven genomics tool used by a large number of pharmaceutical companies to speed up the drug discovery and development process. The development and integration of this technology, together with Incyte's sequence databases and clone resources, have resulted in GEM microarrays that span approximately 60,000 human genes as well as approximately 60,000 plant, rat, mouse, yeast, and bacterial genes. The technology underlying the use of these arrays and their application to the drug discovery process is highlighted.

Microarray technology GEM microarrays and drug discovery.

Incyte Genomics' GEM Gene Expression Microarray is a proven genomics tool used by a large number of pharmaceutical companies to speed up the drug discovery and development process. The development and integration of this technology, together with Incyte's sequence databases and clone resources, have resulted in GEM microarrays that span approximately 60,000 human genes as well as approximately 60,000 plant, rat, mouse, yeast, and bacterial genes. The technology underlying the use of these arrays and their application to the drug discovery process is highlighted.

Annotating the human proteome: the Human Proteome Survey Database (HumanPSD) and an in-depth target database for G protein-coupled receptors (GPCR-PD) from Incyte Genomics.

The Proteome Division of Incyte Genomics has released new volumes to the BioKnowledge Library to add human, mouse and rat protein information to its rich collection of model organism Proteome Databases. The Human Proteome Survey Database (HumanPSD) compiles the fundamental properties of more than 25 000 characterized mammalian proteins. HumanPSD includes clear, concise and current protein descriptions (Title Lines), the protein sequence, calculated physical properties, precomputed BLAST alignments, controlled-vocabulary protein properties and Gene Ontology terms, and a list of published references. Each report also contains expression data, Pfam domain information and an associated Mouse Mutant Phenotype section describing behavioral, physiological and cellular phenotypes for over 1500 mouse mutant phenotypes. GPCR-PD contains more than 3200 Protein Reports from the three mammalian species for G protein-coupled receptors, their protein ligands, associated G-proteins and their downstream signaling proteins. In addition to the features described above, each GPCR-PD Protein Report displays annotations of experimental findings from over 10 000 publications. These databases provide important new volumes of Proteome's BioKnowledge Library (http://www.incyte.com), integrating protein information from model organisms with the human proteome.