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PubMed2003

Peak stimulated insulin secretion is associated with specific changes in gene expression profiles in sporadic insulinomas.

BACKGROUND: The molecular pathways that are responsible for pathologic insulin secretion by insulinomas have not been characterized. We studied gene expression profiles from insulinomas and determined associations between these changes and preoperative peak serum insulin levels. METHODS: Ten patients with insulinomas underwent calcium-stimulated arteriography and surgical resection. Tumor RNA was isolated; corresponding complementary DNA was hybridized to 10K human complementary DNA arrays. Pooled human islet cell complementary DNA served as the control. Cluster analysis of gene expression and analysis of expression ratios was performed. RESULTS: Nineteen genes were up-regulated at least 3-fold in insulinomas compared with controls, which included the genes for islet amyloid polypeptide and proprotein convertase type 2. Cluster analysis revealed 2 groups of patients with insulinoma and with distinct patterns of gene expression. Mean peak serum insulin values between groups were 196 and 1100 (U/mL (P<.05), which demonstrates a significant difference in insulin response to calcium stimulation between these 2 groups. CONCLUSION: We show that genes that are relevant to the pathogenesis of hyperinsulinemia are expressed preferentially in insulinomas. In addition, patients with a distinct and common pattern of gene expression had significantly higher stimulated insulin secretion levels. The study of these genes may help to identify the biochemical pathways that are responsible for pathologic insulin secretion.

Adult↗

PubMed1983

Complementary DNA-25S ribosomal RNA hybridization: an improved method for phylogenetic studies.

In a new combination of techniques for ribosomal RNA hybridization, complementary DNA is synthesized on 25S ribosomal RNA fragments generated by mild alkali treatment, by the enzymatic addition of polyadenylic acid tails, hybridization of these tails with oligo deoxyribosylthymine, and reverse transcription in the presence of tritiated TTP. Hybridization reactions are performed in solution. Heteroduplexes are collected on diethylaminoethylcellulose filter discs after treatment with S1 nuclease. The problems presented by secondary rRNA structure are avoided by denaturation before reverse transcription and before hybridization. The high percentage of duplex formation (78-87%), the low standard deviation of relative binding (averaging +/- 1.30% relative binding), and small differences in reciprocal hybridizations (1.71-5.18% relative binding), as well as the elimination of complications resulting from differences in the number of rRNA cistrons in nuclear DNA, make this method preferable to the membrane-filter technique commonly used in phylogenetic classifications based on the homology of large rRNAs.

DNA↗

PubMed2001

Potential role of HOXD9 in synoviocyte proliferation.

OBJECTIVE: To investigate the role of HOXD9 in the proliferation activity of cultured synoviocytes as well as the mechanisms that regulate HOXD9 transcription. METHODS: Synoviocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were transfected with HOXD9 complementary DNA to establish stable transformants that overexpressed HOXD9. HOXD9 expression was detected by Western blotting with anti-HOXD9 antibody. The growth properties of the transformants were investigated by proliferation and colony formation assays. The expression of basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNFalpha), interleukin-1beta, c-Fos, and c-Myc was examined by Western blotting. Transcriptional regulation of HOXD9 was examined by transient cotransfection. RESULTS: HOXD9 protein was highly expressed in RA synoviocytes, but there was no expression in OA synoviocytes. HOXD9 transfection induced stable HOXD9 protein expression in synoviocytes and showed an increased proliferation rate under both normal and serum-starved conditions, as well as an enhanced capacity to proliferate anchorage independently to form colonies in soft agar cultures, compared with control transfectants. Higher levels of bFGF and c-Fos were detected in HOXD9 transformants than in controls. Transient cotransfection assays of NIH3T3 fibroblasts and synoviocytes showed that HOXD9 activated the luciferase reporter construct containing the highly conserved region (HCR), an autoregulatory element of HOXD9 promoter. This activation was significantly increased by bFGF, suppressed by TNFalpha, and unchanged by transforming growth factor beta in synoviocytes. Human T lymphotropic virus type I tax also activated the luciferase reporter construct containing the HCR and had a synergistic effect with HOXD9 on HCR promoter activation. CONCLUSION: Our data suggest that HOXD9 plays a potential role in synovial proliferation. In addition, they suggest that the involvement of HOXD9 in the regulation of cellular growth might be mediated, at least in part, by up-regulation of growth-related factors such as bFGF and c-Fos and/or might result from increased transcription activity by its regulators.

3T3 Cells↗

PubMed1996

Detection of autoantibodies to nucleolar transcription factor NOR 90/hUBF in sera of patients with rheumatic diseases, by recombinant autoantigen-based assays.

OBJECTIVE: We attempted to clarify the clinical characteristics of Japanese patients with autoantibodies to nucleolar transcription factor NOR 90/hUBF (anti-NOR 90) and to analyze the autoantigenic epitopes recognized by anti-NOR 90. METHODS: Ninety-one patient sera containing anti-nucleolar antibodies (ANoA) by indirect immunofluorescence were collected. Immunoblottings were performed using recombinant fusion proteins expressed from several cloned complementary DNA (cDNA) encoding the NOR 90/hUBF autoantigen. RESULTS: Anti-NOR 90 were detected in sera from 9 (9.9%) of 91 patients with ANoA. Seven of these patients were diagnosed as having Sjögren's syndrome, 4 had concomitant rheumatoid arthritis, 1 had concomitant systemic sclerosis (SSc), and 2 had SSc alone. All 9 sera were reactive with more than 2 recombinant fusion proteins from cDNA encoding separate regions on the hUBF polypeptide. CONCLUSION: The results suggest that while anti-NOR 90 antibodies are rare, they are associated with Sjögren's syndrome in Japanese patients, and that autoimmunity is targeted toward at least 2 separate regions (amino acids 89-310 and 310-633) of the hUBF polypeptide.

Aged↗

PubMed2001

Identification and characterization of a SET/NAP protein encoded by a brain-specific gene, MB20.

A new member of the NAP/SET gene family, named MB20, was isolated from a mouse brain cDNA library by virtue of its CAG trinucleotide repetitive sequence and a brain-specific gene expression pattern. The complementary DNA sequence predicted an open reading frame of 545 amino acids, with four copies of an 11-amino-acid direct repeat. The consensus sequence for these repeats, PKE-P--K-EE, is present in the largest subunit of murine neurofilament (NF-H). The MB20 protein sequence is homologous to nucleosome assembly proteins of several species, and its C-terminus is homologous to SET proteins. Immunoblot analysis revealed that MB20 protein is expressed in the brain. Transient transfection and immunofluorescence microscopy demonstrated that MB20 is distributed in the cytoplasm as well as in the nucleus. Deletion of the N-terminal end imparts the complete localization of MB20 protein to the nucleus. The ability of MB20 to bind histone proteins was analyzed by sucrose gradient sedimentation and by retention of histone proteins by immobilized MB20 protein. On the basis of its expression pattern, predicted sequence, and protein properties, we propose that MB20 plays a unique role in modulating nucleosome structure and gene expression during brain development.

Amino Acid Sequence↗

PubMed1996

Cloning of a cDNA encoding a human homologue of CDC47, a member of the MCM family.

A complementary DNA (cDNA) clone encoding a 719-amino acid (aa) protein was isolated, which has a 49% aa identity with budding yeast CDC47, a member of the MCM family. Antiserum raised against a C-terminal polypeptide bacterially produced from the cDNA clone detected a cellular protein about 80 kDa, which coincides with the size of the in vitro transcription/translation product of the cDNA. These results indicate that the cDNA covers the entire coding region of a human homologue of CDC47.

Amino Acid Sequence↗

PubMed1993

Cloning and expression of a glutamate transporter from mouse brain.

A complementary DNA clone encoding a transporter for glutamate has been isolated from mouse brain, and its functional properties have been examined in Xenopus oocytes. The transporter displays high affinity for glutamate (Km = 72 microM) and is dependent on external Na+. The cDNA sequence predicts a protein of 543 amino acids with 6 potential transmembrane domains. Northern blot analysis indicates the mRNA encoding the glutamate transporter is expressed both in the brain and in peripheral tissues.

Amino Acid Sequence↗

PubMed1994

Myocyte-specific enhancer binding factor 2C expression in human brain development.

Myocyte-specific enhancer binding factor 2C (MEF2C) activates transcription by binding to the myocyte-specific enhancer binding factor 2 (MEF2) regulatory element and has been shown previously to be expressed in muscle and in the brain. We have now studied MEF2C expression in human brain using an antiserum raised against amino acids 140-238 of MEF2C. Western blotting demonstrated that, in fetal brain, MEF2C-immunoreactive bands have the same apparent molecular weight as those in extracts of COS cells transfected with MEF2C complementary DNA. In adult brain, however, MEF2C-immunoreactive bands have a higher molecular weight. In the cerebral cortex, MEF2C immunoreactivity is present in the cortical plate, and is not found in the intermediate zone or ventricular zone. At 14 weeks of gestation, the earliest age examined, MEF2C immunoreactivity is present in cell nuclei throughout the cortical plate. Subsequently, MEF2C immunoreactivity develops a bilaminate and then a trilaminate distribution, and ultimately is expressed preferentially in layers II, IV and VI of mature neocortex. MEF2C immunoreactivity is also found in entorhinal cortex, hippocampus, claustrum, cerebellum and amygdala, and in scattered cells in the thalamus. These findings suggest a role for MEF2C in postmitotic neuronal differentiation, in particular, in the development of certain cortical layers, but also in differentiation of other neurons as well.

Adult↗

PubMed1995

Cloning of a rat cDNA encoding a novel LIM domain protein with high homology to rat RIL.

A complementary DNA (1392 bp) encoding a protein with high homology to rat reversion-induced LIM (RIL) protein was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted CLP-36, encodes a 327-amino-acid (aa) protein that contained a restrictively conserved LIM (a Cys-rich domain with consensus aa sequence C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-20-C-X2-C/H/D that was initially identified in homeodomain proteins, Lin-11 [Freyd et al., Nature 344 (1990) 876-879], Isl-1 [Karlsson et al., Nature 344 (1990) 879-882] and Mec-3 [Way et al., Cell 54 (1988) 5-16] in the C-terminal portion (aa 192-327). It shared an overall 45.1% identity to a rat LIM domain RIL protein [Kiess et al., Oncogene 10 (1995) 61-68], with 62.0% identity in the N terminus (aa 1-89) and 50.0% identity in the C terminus (aa 188-327). The rat CLP-36 mRNA is expressed most abundantly in heart, lung and liver, moderately in spleen and skeletal muscle, and at extremely low levels (if at all) in testis and brain tissues. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with a fragment of clone CLP-36 as probe, demonstrated a single band with a mobility corresponding to a size of 1.4 kb, whose level was significantly decreased during chemical hypoxia.

Amino Acid Sequence↗

PubMed2003

Swine Toll-like receptor 9(1) recognizes CpG motifs of human cell stimulant.

Complementary DNA (cDNA) encoding swine Toll-like receptor 9 (sTLR9) was isolated from Peyer's patches (Pps) of gut-associated lymphoid tissue (GALT). The complete open reading frame (ORF) of sTLR9 contains 3093 bp coding deduced 1030 amino acid residues. The amino acid sequence of sTLR9 was characterized by a signal peptide followed by multiple leucine-rich repeats, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor (TIR). The sTLR9 showed a higher amino acid identity with humans (81.8%) and felis catus (86.7%) than mice (74.9%). The HEK293T cells transfected with pCXN2.1-FLAG DNA containing the sTLR9 cDNA were expressed sTLR9 as a membrane-bound molecules, which were reactive with anti-sTLR9 rabbit polyclonal antibody. Moreover, the transfectant was responsible for the CpG oligo DNA. sTLR9 was preferentially expressed in Pps and mesenteric lymph nodes (MLNs), and its degree was approximately three times higher than a spleen but weak in the other tissues by the real-time quantitative PCR analyses. The strong expression of sTLR9 in Pps and MLNs and its recognizing CpG DNA for human cell stimulant are shown first in this study, which may help in understanding the intestinal immune system mediated by a bacterial DNA through TLR9.

Amino Acid Sequence↗

PubMed1997

Restoration of TATA-dependent transcription in a heat-inactivated extract of tobacco nuclei by recombinant TATA-binding protein (TBP) from tobacco.

We isolated a complementary DNA (cDNA) that encoded a TATA-binding protein (TBP) from a cDNA library of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2). A comparison among deduced amino acid sequences of plant TBPs revealed the presence of a long conserved region within the amino acid sequence of the TBP. Genomic Southern analysis revealed that tobacco TBP (tTBP) is encoded by only a small number of copies of a gene in the tobacco genome. Addition of recombinant tTBP to an extract of tobacco nuclei (TNE) enhanced the basal transcriptional activity in vitro. This result indicates that the level of tTBP is a rate-limiting factor for basal transcriptional activity in TNE. We subsequently succeeded in the functional complementation of TATA-dependent initiation of transcription that was associated with a plant promoter in a homologous plant system. Addition of bacterially expressed recombinant tTBP to a heat-inactivated TNE restored transcriptional activity, as did the addition of human TBP. Moreover, heating of the recombinant tTBP eliminated its ability to restore transcriptional activity. It appears that the heat inactivation of TNE was caused by the heat inactivation of tTBP in TNE.

Amino Acid Sequence↗

PubMed2003

Molecular fingerprinting of TGFbeta-treated embryonic maxillary mesenchymal cells.

The transforming growth factor-beta (TGF(beta)) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGF(beta) on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGF(beta) in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGF(beta) action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's Atlas Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGF(beta)-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGF(beta)-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImage analysis in order to determine differences in gene expression between control and TGF(beta)-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGF(beta). Affected genes could be grouped into three general functional categories: transcription factors and general DNA-binding proteins; growth factors/signaling molecules; and extracellular matrix and related proteins. The extent of hybridization of each gene was evaluated by comparison with the abundant, constitutively expressed mRNAs: ubiquitin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ornithine decarboxylase (ODC), cytoplasmic beta-actin and 40S ribosomal protein. No detectable changes were observed in the expression levels of these genes in-response to TGF(beta) treatment. Gene expression profiling results were verified by Real-Time quantitative polymerase chain reaction. Utilization of cDNA microarray technology has enabled us to delineate a preliminary transcriptional map of TGF(beta) responsiveness in embryonic maxillary mesenchymal cells. The profile of differentially expressed genes offers revealing insights into potential molecular regulatory mechanisms employed by TGF(beta) in orchestrating craniofacial ontogeny.

Animals↗

PubMed1994

NF-AT components define a family of transcription factors targeted in T-cell activation.

The NF-AT transcription complex is required for the expression of a group of proteins that collectively coordinate the immune response. Here we purify two proteins encoded by separate genes that represent the pre-existing (p) and cytosolic (c) components of NF-AT. Expression of the full-length complementary DNA encoding NF-ATc activates the interleukin (IL-2) promoter in non-T lymphocytes, whereas a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon T-cell activation. The other protein, NF-ATp, is highly homologous to NF-ATc over a limited domain which shows similarity to the Dorsal/Rel family, but has a wider tissue distribution. Agents that increase intracellular Ca2+ or activate protein kinase C independently modify NF-ATc, indicating that distinct signalling pathways converge on NF-ATc to regulate its function.

Amino Acid Sequence↗

PubMed1996

Functional redundancy of the muscle-specific transcription factors Myf5 and myogenin.

The myogenic basic helix-loop-helix transcription factors, Myf5, MyoD, myogenin and MRF4, play key roles in skeletal muscle development. All of them induce myogenic differentiation in cultured non-muscle cells, suggesting that they might be functionally redundant. But the genes are expressed at different times during embryogenesis and mice carrying a mutation in any of the genes have different phenotypes. A rib cage defect was observed in Myf5-deficient mice, which die perinatally. We investigated whether the rib cage defect was due to the failure of the early activation of the gene or to the unique interactions of Myf5 with specific downstream targets. For this we inserted a myogenin complementary DNA into the Myf5 locus by homologous recombination which simultaneously disrupted Myf5 function. We report here that mice homozygous for this myogenin gene knock-in (ki) developed a normal rib cage and were viable, therefore demonstrating functional redundancy of Myf5 and myogenin for rib formation.

Animals↗

PubMed1997

A family of cytokine-inducible inhibitors of signalling.

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Amino Acid Sequence↗

PubMed1997

Structure and function of a new STAT-induced STAT inhibitor.

The signalling pathway that comprises JAK kinases and STAT proteins (for signal transducer and activator of transcription) is important for relaying signals from various cytokines outside the cell to the inside. The feedback mechanism responsible for switching off the cytokine signal has not been elucidated. We now report the cloning and characterization of an inhibitor of STAT activation which we name SSI-1 (for STAT-induced STAT inhibitor-1). We found that SSI-1 messenger RNA was induced by the cytokines interleukins 4 and 6 (IL-4, IL-6), leukaemia-inhibitory factor (LIF), and granulocyte colony-stimulating factor (G-CSF). Stimulation by IL-6 or LIF of murine myeloid leukaemia cells (M1 cells) induced SSI-1 mRNA expression which was blocked by transfection of a dominant-negative mutant of Stat3, indicating that the SSI-1 gene is a target of Stat3. Forced overexpression of SSI-1 complementary DNA interfered with IL-6- and LIF-mediated apoptosis and macrophage differentiation of M1 cells, as well as IL-6 induced tyrosine-phosphorylation of a receptor glycoprotein component, gp130, and of Stat3. When SSI-1 is overexpressed in COS7 cells, it can associate with the kinases Jak2 and Tyk2. These findings indicate that SSI-1 is responsible for negative-feedback regulation of the JAK-STAT pathway induced by cytokine stimulation.

Amino Acid Sequence↗

PubMed2000

Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium.

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

Amino Acid Sequence↗

PubMed1997

cDNA cloning and genomic organization of enhancer of split groucho gene from nematode Caenorhabditis elegans.

This first genomic Enhancer of split groucho (ESG) gene and its full length complementary DNA (cDNA) from nematode C. elegans were cloned and sequenced via homology with the corresponding Drosophila groucho cDNA. The cDNA of 2.1-Kb encodes a protein of 612 amino acids, and the nematode ESG protein is the smallest and most different in structure compared to all ESG related proteins. The gene isolated is 4,246-bp in size, including 1,219-bp promoter region. A putative TATA-box at position -1166, two consensus sequence of ACTGG, characteristic of leader binding protein-1 (LBP-1) binding motifs at position -563 and -211 and nine CAAT boxes were found in the promoter region of ESG gene. The protein-coding sequence is interrupted by five introns. The length of introns 1 to 5 is 52, 252, 87, 53 and 518 bp, respectively. The overall structural relationships of the ESG-related proteins among human, mouse, rat, Xenopus, Drosophila and nematode were also analyzed.

Amino Acid Sequence↗