Cloning and characterization of the 5'' flanking sequences (promoter region) of the human GLP-1 receptor gene

We have investigated in rat brain slices the effects of the volatile anaesthetics enflurane, isoflurane and halothane on spontaneous discharge patterns and mean firing rates of cerebellar Purkinje cells. In the absence of these anaesthetics, Purkinje cells fired bursts of action potentials separated by quiescent periods lasting less than 2 s. Mean discharge rates were 10.8 (SEM 0.4) Hz at 23 +/- 1 degrees C and 25.6 (1.2) Hz at 35 +/- 1 degrees C. The agents exhibited qualitatively different effects when applied at concentrations corresponding to 1-3 MAC. Enflurane markedly lengthened burst and inter-burst durations. Isoflurane acted in a similar manner, but effects were less pronounced. In contrast with isoflurane and enflurane, halothane shortened burst durations. At concentrations corresponding to 1-1.5 MAC, halothane, isoflurane and enflurane significantly depressed action potential firing by 15-30% (P < 0.05). Enflurane 1.2 mmol litre-1 (2.0 MAC), isoflurane 0.9 mmol litre-1 (2.8 MAC) and halothane 0.9 mmol litre-1 (3.8 MAC) depressed spontaneous spike rates by 50%. The changes in discharge patterns and the concentration-dependent decrease in the firing rates were similar at 23 +/- 1 degrees C and 35 +/- 1 degrees C. In summary, we observed that neither the anaesthetic-induced alterations in spontaneous discharge patterns nor the EC50 values of the concentration-dependent depression of the mean firing rates were in accordance with the Meyer-Overton rule. However, at clinically relevant concentrations, depression of average spike rates did not differ significantly between the anaesthetics and thus followed the rule. Our results suggest that anaesthetic actions, which are in accordance with the rule, are frequently masked by several side effects.     

Cloning and sequence analysis of the gene (eprA1) encoding an extracellular protease from Aeromonas hydrophila

A gene (eprA1) encoding the extracellular protease of Aeromonas hydrophila AH1 has been cloned and sequenced. Nucleotide sequence analysis of eprA1 predicted a single open reading frame (ORF) of 1038 bp encoding a 346 amino acid (aa) polypeptide, with a potential 21-aa signal peptide. When the eprA1 gene was expressed in minicells, one major band of approx. 37 kDa was identified, while protease activity staining experiments identified a caseinolytic band of approx. 29 kDa determined by SDS-PAGE analysis of the minicells. The deduced C-terminal aa region (Arg-290 to Gly-313) showed sequence homology to partial C-terminal sequences of other zinc metalloproteases including Penicillium citrinum metalloprotease (PlnC), Aspergillus oryzae metalloprotease (NpII), Aspergillus flavus metalloprotease (MepA), and Aspergillus fumigatus metalloprotease (Mep20), particularly with respect to zinc-binding residues.     

Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis-inhibitory factor

Cri-du-chat is a human contiguous gene deletion syndrome resulting from hemizygous deletions of chromosome 5p. Here we describe the isolation from within this interval of the human Semaphorin F (SEMAF) gene, a member of a family of proteins that has been implicated in axonal pathfinding. The human SEMAF gene covers at least 10% of the deleted region and defines a new class within this large gene family characterized by the presence of seven type 1 thrombospondin repeats. Prominent expression of murine semaphorin F (Semaf) was observed in the mouse brain, consistent with a role for semaphorin F as a signaling molecule that guides axons or migrating neuronal precursors during development. The known functions of semaphorins and the interesting pattern of expression for Semaf suggest that haploinsufficiency for SEMAF may disrupt normal brain development and might lead to some of the features of Cri-du-chat.     

Cloning of cDNAs encoding the human BAG1 protein and localization of the human BAG1 gene to chromosome 9p12

cDNAs encoding the human homolog of BAG1, a Bcl-2-binding protein with anti-apoptotic function, were cloned. DNA sequence analysis of human BAG1 cDNAs predicts a protein with an additional 55 amino acids at its NH2-terminus compared to the mouse protein. Immunoblot assays using monoclonal antibodies raised against bacterially produced h-BAG1 protein confirmed the larger size of the human protein (approximately 34 kDa) compared to mouse. PCR analysis of DNA from human x rodent somatic cell hybrids using human BAG1-specific primers localized the gene to human chromosome 9. Cosmid clones of h-BAG1 were obtained and used for fluorescence in situ hybridization analysis of normal metaphase chromosomes, thus localizing h-BAG1 to 9p12, a region associated with hereditary disorders that may involve developmental dysregulation of programmed cell death.     

Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F)

Blocking CD28-B7 T cell costimulatory activation by the fusion protein CTLA4Ig prevents rejection and induces long-term graft acceptance in various experimental transplant models. There are reported differences in the efficacy of CTLA4Ig in renal and cardiac rodent allograft models, but it is not clear whether these are due to the strain or species differences investigated in the different studies reported. This study investigates the effect of blocking CD28-B7 T cell costimulation with murine CTLA4Ig in rat models of acute renal and cardiac allograft rejection models, using the same complete major histocompatibility complex-incompatible strain combination. A single injection of murine CTLA4Ig 2 d after engraftment was able to induce long-term graft acceptance (> 100 d) in 54% of Lewis rat recipients of Wistar-Furth kidneys. Transferring this protocol into the acute Wistar-Furth to Lewis heart allograft model resulted in a mean graft survival time of 24.7+/-16.9 d, and all grafts were ultimately rejected. Only concomitant injection of donor cells (4 x 10(7) splenocytes) plus a single injection of CTLA4Ig on the day of transplant could induce long-term graft acceptance in 50% of animals. In both the cardiac and renal transplant models, the thymus and spleen were required for induction of tolerance. The maintenance phase of tolerance, however, did not require an intact thymus but did require the presence of a spleen. These data have important clinical applicability because human studies with T cell costimulatory blockade are being planned.     

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.     

The structure and organization of the human erythroid anion exchanger (AE1) gene

The AE1 (anion exchanger, band 3) protein is expressed in erythrocytes and in the A-type intercalated cells of the kidney distal collecting tubule. In both cell types it mediates the electroneutral transport of chloride and bicarbonate ions across the lipid bilayer, and, in erythrocytes, it also serves as the critical attachment site of the peripheral membrane skeleton. We have characterized the human AE1 gene using overlapping clones isolated from a phage library of human genomic DNA. The gene spans approximately 20 kb and consists of 20 exons separated by 19 introns. The structure of the human AE1 gene corresponds closely with that of the previously characterized mouse AE1 gene, with a high degree of conservation of exon/intron junctions, as well as exon and intron nucleotide sequences. The putative upstream and internal promoter sequences of the human AE1 gene used in erythroid and kidney cells, respectively, are described. We also report the nucleotide sequence of the entire 3' noncoding region of exon 20, which was lacking in the published cDNA sequences. In addition, we have characterized 9 Alu repeat elements found within the body of the human AE1 gene that are members of 4 related subfamilies that appear to have entered the genome at different times during primate evolution.     

The teratogenicity of N-methyl-D-aspartate (NMDA) receptor antagonists

While it is generally accepted that y-aminobutyric acid type A (GABA(A))-receptor agonists decrease anxiety by facilitating the neuronal influx of chloride, the site of action within the brain is not clearly delineated. To gain further insight into the locus of anxiolytic action of GABA in the brain, we measured the distribution of GABA-stimulated chloride influx in seven regions of the brain from high- and low-anxiety rats. Our results show a significant increase in GABA-gated chloride influx in the hypothalamus of rats exhibiting low anxiety. The role of the hypothalamus in the regulation of anxiety is briefly discussed.     

Localization of the human NMDAR2D receptor subunit gene (GRIN2D) to 19q13.1-qter, the NMDAR2A subunit gene to 16p13.2 (GRIN2A), and the NMDAR2C subunit gene (GRIN2C) to 17q24-q25 using somatic cell hybrid and radiation hybrid mapping panels

1. Three hundred 4-week-old commercial Hibrow broiler chicks were fed ad libitum, 85% and 70% of ad libitum or ad libitum for 5 d per week with 3rd and 5th days of food withdrawal for 28 d to examine the effects of food restriction on liveweight, growth rate, food conversion efficiency, dressing percentage, abdominal fat and mortality. 2. Quantitative food reduction to 85% and 70% of ad libitum, and reduction in feeding time by 2 d/week significantly reduced final body weight and rate of growth. 3. Food restriction (to 70% ad libitum) resulted in lower abdominal fat than ad libitum feeding, 15% food restriction or reduction in feeding time by 2 d/week. 4. There were no significant differences between the effects of 15% food restriction and 2-d reduction in feeding time per week on final body weight, growth rate and abdominal fat. 5. Reduction in feeding time by 2 d/week resulted in the same food efficiency as ad libitum feeding and quantitative food restriction by 15% and a significantly better food efficiency than 30% quantitative food reduction. 6. Reducing feeding time by 2 d/week seemed to have less severe effects on the birds than quantitatively reducing food supply by 30%.     

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons

The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.     

Cloning and nucleotide sequence of the gene encoding dinitrogenase reductase (nifH) from the cyanobacterium Nostoc 6720

The nucleotide sequence of the 3' end of the nifU coding sequence, the complete coding sequence of nifH and a substantial part of the 5' end of nifD coding sequence from Nostoc 6720 is presented. The coding sequences are highly conserved with those of Anabaena 7120 and Anabaena sp. L31. However the intergenic region between nifU and nifH contains two segments of short tandemly repetitive repeat sequences (STRRs) that differ from the STRR that is common to both Anabaena7120 and Anabaenasp. L31. Various sequence structures that are common to Nostoc 6720, the Anabaena strains and Plectonema boryanum are discussed.     

Structure of the gene encoding the alpha subunit of the human interleukin 3 receptor

Interleukin 3 is a cytokine that stimulates proliferation and differentiation of hematopoietic progenitor cells. Its receptor consists of two subunits, an interleukin 3-specific alpha subunit and a beta subunit shared by garanulocyte-macrophage colony stimulating factor and interleukin 5 receptors. In this paper, we determined the genomic structure of the alpha subunit of the human interleukin 3 receptor, which spans approximately 40 kb and has 12 exons. We found that the genomic structures of the alpha subunits of the human interleukin 3 and granulocyte-macrophage colony stimulating factor receptors are very similar. They possess a unique additional intron in the 'C domain', which is absent in the alpha subunit of the interleukin 5 receptor. These results suggest a shared evolutionary pathway of these two genes.     

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.     

Cloning, sequencing and analysis of cDNA encoding bovine intercellular adhesion molecule-1 (ICAM-1)

Intercellular adhesion molecule-1 (ICAM-1) is an inducible glycoprotein that interacts with the leukocyte beta 2-integrins, LFA-1 and Mac-1. We have isolated and analyzed a cDNA clone coding for the putative bovine ICAM-1 gene and compared it with known comparative sequences from other species as well as bovine ICAM-3. The 3398-bp bovine ICAM-1 cDNA sequence codes for 535 amino acids and shows 57% homology with human ICAM-1 and 47% homology with bovine ICAM-3 at the amino acid levels. The predicted number and positions of cysteine residues in bovine ICAM-1 are all conserved among species including bovine ICAM-3. It has two arginine-glycine-aspartate (RGD) sites in the extracellular region and a serine residue in the cytoplasmic tail. Northern blot results show that the bovine ICAM-1 gene is expressed in stimulated leukocytes whereas bovine ICAM-3 is expressed predominantly in resting neutrophils.