Biomedical subjects

A J Mouland

Publications and source records attributed to A J Mouland.

10 recordsLinked to original sources

PubMed2001

RNA trafficking signals in human immunodeficiency virus type 1.

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

Animals↗

PubMed2000

The double-stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation.

Human Staufen (hStau), a double-stranded RNA (dsRNA)-binding protein that is involved in mRNA transport, is incorporated in human immunodeficiency virus type 1 (HIV-1) and in other retroviruses, including HIV-2 and Moloney murine leukemia virus. Sucrose and Optiprep gradient analyses reveal cosedimentation of hStau with purified HIV-1, while subtilisin assays demonstrate that it is internalized. hStau incorporation in HIV-1 is selective, is dependent on an intact functional dsRNA-binding domain, and quantitatively correlates with levels of encapsidated HIV-1 genomic RNA. By coimmunoprecipitation and reverse transcription-PCR analyses, we demonstrate that hStau is associated with HIV-1 genomic RNA in HIV-1-expressing cells and purified virus. Overexpression of hStau enhances virion incorporation levels, and a corresponding, threefold increase in HIV-1 genomic RNA encapsidation levels. This coordinated increase in hStau and genomic RNA packaging had a significant negative effect on viral infectivity. This study is the first to describe hStau within HIV-1 particles and provides evidence that hStau binds HIV-1 genomic RNA, indicating that it may be implicated in retroviral genome selection and packaging into assembling virions.

Binding Sites↗

PubMed1998

Vpr stimulates viral expression and induces cell killing in human immunodeficiency virus type 1-infected dividing Jurkat T cells.

In this study we investigated the effects of Vpr during human immunodeficiency virus (HIV) infection of proliferating Jurkat T cells by using a vesicular stomatitis virus envelope G glycoprotein pseudotyped HIV superinfection system. We observe that the expression of Vpr results in a severe reduction in the life span of HIV type 1 (HIV-1)-infected dividing T cells in culture. In agreement with a recent report (S. A. Stewart, B. Poon, J. B. M. Jowett, and I. S. Chen, J. Virol. 71:5579-5592, 1997), we show that events characteristic of apoptotic cell death are involved in the Vpr-mediated cytopathic effects. Our results also show that infection with viruses expressing the wild-type vpr gene results in an increase in viral gene expression and production. Interestingly, the effects of Vpr on cell viability and on viral gene expression both correlate with the ability of the protein to induce a cell cycle arrest in the G2/M phase. Mutagenesis analyses show that the C terminus of Vpr is essential for these biological activities. Although the role of Vpr is currently associated with the infection of nondividing cells, our results suggest that Vpr can also directly increase viral replication in vivo in infected dividing T cells. Furthermore, these in vitro observations suggest that Vpr-mediated cytotoxic effects could contribute to the CD4+ depletion associated with AIDS progression.

Cell Cycle↗

PubMed1998

Analysis of molecular mechanisms controlling neuroendocrine cell specific transcription of the chromogranin A gene.

Chromogranin A (CgA), a member of the granin/secretogranin family of acidic glycoproteins that play multiple roles in the process of regulated secretion of peptide hormones and neurotransmitters, is specifically expressed in endocrine and neuroendocrine cells. We previously cloned and characterized the human (h) CgA gene and showed that nucleotides -55 to +32 relative to the transcriptional start site that contain a consensus cAMP element (CRE) and TATA-box motif were sufficient for neuroendocrine cell-specific expression. Here, we examined the role of the well conserved CRE in basal and cAMP-stimulated transcription in neuroendocrine cells. Transient transfection studies with hCgA gene promoter/chloroamphenical acetyl transferase (CAT) reporter constructs were conducted in a panel of neuroendocrine cell lines as well as in nonendocrine cell lines. Deletion or mutation of the CRE resulted in loss of neuroendocrine cell specific transcriptional activity. Mutation of a well conserved region (the TG-box) located between the CRE and the TATA box had no effect or resulted in only a modest decrease in activity. Mutation of the CRE in 5'-extended (-2300 to +32 and -700 to +32) constructs resulted in a 50-75% decrease in basal activity in neuroendocrine cells. This emphasized the importance of the CRE in basal transcription and also suggested that other elements between -700 and -55 may act independently of the CRE to contribute to full basal activity in some neuroendocrine cells. Dibutyryl cAMP stimulated transcriptional activity in neuroendocrine cells, and this was abolished by mutation of the CRE. In the presence of a PKA inhibitor, dibutyryl cAMP-induced activity was completely abolished and basal activity was decreased by up to 85%. Similar protein-DNA complexes were formed in gel retardation assays with a CgA-CRE oligonucleotide and nuclear extracts from both neuroendocrine and nonendocrine cells. A predominant complex that was supershifted by addition of a CREB antibody was identical in all cell types. By immunoblot analysis, levels of total CREB protein and phosphorylated (Ser 133) CREB did not differ between neuroendocrine and nonendocrine cells. Phosphorylated CREB was increased by forskolin treatment, an effect that was blocked by a PKA-inhibitor. Expression of the transcriptional cointegrator, CREB-binding protein (CBP), assessed by both RT-PCR and Western blot analysis, did not differ between neuroendocrine and nonendocrine cells. In summary, the CRE in the hCgA gene proximal promoter is critical for both basal and cAMP-induced expression in neuroendocrine cells via a PKA-mediated pathway. However, the neuroendocrine specificity of hCgA gene transcription mediated by the CRE is not a function of levels of total CREB or phosphorylated CREB or its cointegrator CBP. Specificity may be achieved by a PKA-responsive CRE-binding protein other than CREB expressed specifically in neuroendocrine cells, expression of a repressor molecule that binds CREB in nonendocrine cells, or may lie downstream of a CRE-binding protein, e.g. in the activity or amount of cointegrators other than CBP, which are required to couple transactivators to the basal transcriptional machinery.

Base Sequence↗

PubMed1997

Enhancement of retroviral production from packaging cell lines expressing the human immunodeficiency type 1 VPU gene.

The HIV-1 Vpu protein stimulates virus production by enhancing the release of viral particles from infected cells. Interestingly, Vpu was also shown to enhance the release of capsids produced by gag gene contructs of other retroviruses that lack a Vpu-like activity. To investigate the effect of Vpu expression on viral particle production in retroviral packaging cell line, we developed the Damp-VpuP cell line in which vpu expression is under the control of the tetracycline-responsive promoter. Retroviral production was measured by dosage of virion-associated reverse transcriptase activity, by capsid protein immuno-detection in cell-free supernatants and by evaluating the transfer of antibiotic resistance to target cells. Induction of the Damp-VpuP cell line caused a 40-fold increase in the titer of infectious virus-like particles when compared with control cell lines. This increase in viral titer was not the result of a clonal effect nor was it a consequence of high selective pressure but rather the effect of a Vpu-mediated enhancement of viral particle production. Similar results using the third generation psi CRIP packaging cell line confirmed these findings. Constitutive expression of vpu caused a 13-fold increase in viral titer in this packaging cell line. These results indicate that the expression of HIV-1 vpu in retroviral packaging cell lines can significantly improve the titers of infectious retroviral particles.

Animals↗

PubMed1995

Chromogranin A.

Chromogranin A (CgA) is the major member of the granin family of acidic secretory glycoproteins that are expressed in all endocrine and neuroendocrine cells. Granins have been proposed to play multiple roles in the secretory process. Intracellularly, granins play a role in targeting peptide hormones and neurotransmitters to granules of the regulated pathway by virtue of their ability to aggregate in the low-pH, high-calcium environment of the trans-Golgi network. Extra-cellularly, peptides formed as a result of proteolytic processing of granins regulate hormone secretion. Some conserved features of the mature CgA protein are polyglutamic acids, calcium-binding sites, and several pairs of basic amino acids. The first 2 features are important for its intracellular functions, and the latter characteristic suggested that peptides could be released from the molecule by precursor processing enzymes. Several biologically active peptides encoded within the CgA molecule, such as vasostatin, beta-granin, chromostatin, pancreastatin, and parastatin act predominantly to inhibit hormone and neurotransmitter release in an autocrine or paracrine fashion. The biosynthesis of CgA is regulated by many different factors, including steroid hormones and agents that act through a variety of signalling pathways. CgA biosynthesis and that of the resident hormone or neurotransmitter can be regulated differentially. The widespread distribution of CgA has made the measurement of circulating immunoreactive CgA a valuable tool in the diagnosis of neuroendocrine neoplasia, and CgA immunohistochemistry can help to identify the neuroendocrine nature of tumours. Recent molecular biology studies are identifying those elements in the CgA gene promoter responsible for its specific neuroendocrine cell expression.

Animals↗

PubMed1994

Human chromogranin A gene. Molecular cloning, structural analysis, and neuroendocrine cell-specific expression.

Chromogranin A (CgA) is an acidic glycoprotein, which is widely expressed in endocrine and neuroendocrine cells. It plays multiple important roles in the process of regulated hormone secretion. The single copy human CgA gene was isolated from a human fetal liver gene library. The gene spans 15 kilobases and contains 8 exons. Exon I encodes the 5'-noncoding region and the majority of the signal peptide coding region. Exons II-V collectively encode the highly conserved amino-terminal domain (the beta-granin sequence). Exon VI encodes a variable domain within which is the chromostatin sequence, and exon VII encodes another variable domain, which contains the pancreastatin sequence. Exon VIII encodes the highly conserved carboxyl-terminal domain and the 3'-noncoding region. The human gene promoter has a consensus TATA box, cAMP response element, and Sp-I sequence. 2.3 kilobases of the upstream regulatory region of the human CgA gene directed efficient transcription of a reporter chloramphenicol acetyltransferase gene in several neuroendocrine cell lines, including human medullary thyroid C-cell tumor, mouse pituitary corticotroph, rat pituitary tumor, and rat pheochromocytoma. The promoter was virtually inactive in nonneuroendocrine cell lines. Transient transfection studies with deleted promoter constructs showed that sequences lying between -55 and +32 base pairs relative to the transcription initiation site, containing the consensus cyclic AMP response element and TATA box, were sufficient for neuroendocrine cell-specific expression.

Amino Acid Sequence↗

PubMed1992

1,25-Dihydroxycholecalciferol regulates chromogranin-A translatability in bovine parathyroid cells.

Our previous studies indicated that regulation of bovine parathyroid chromogranin-A (CgA) by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] may occur at the level of CgA turnover or mRNA translation. In the present study, immunoprecipitation of extracts of bovine parathyroid cells that had been pulse chased with [35S]methionine revealed that 1,25-(OH)2D3 had no effect on the disappearance time of intracellular CgA. Therefore, we examined the effect of 1,25-(OH)2D3 on the polyribosome profile of CgA mRNA analyzed by sucrose density gradients. In the presence of 1,25-(OH)2D3, there was a dose-dependent recruitment of CgA mRNA into the denser polyribosomal fractions by 24 h. To determine whether this increased ribosome loading represents increased or decreased efficiency of mRNA translation, ribosome transit time experiments were conducted. The average ribosome transit time and the specific PTH ribosome transit time were not altered by 1,25-(OH)2D3. However, that for CgA was doubled in the presence of 1,25-(OH)2D3. Thus, parathyroid CgA synthesis is regulated by the vitamin D sterol at the level of peptide chain elongation. These studies, therefore, explain the lack of quantitative correspondence between 1,25-(OH)2D3-induced CgA gene transcription and CgA protein levels by revealing a previously unsuspected level of regulation of mRNA translation in the parathyroid cell.

Animals↗

PubMed1991

Regulation of synthesis and secretion of chromogranin-A by calcium and 1,25-dihydroxycholecalciferol in cultured bovine parathyroid cells.

A RIA for bovine parathyroid chromogranin-A (CgA) was developed and used to study the regulation of CgA synthesis and release in cultured bovine parathyroid cells. As previously demonstrated, an elevated medium calcium concentration (2.5 mM) led to a reduced release of CgA into the medium. In contrast, the intracellular concentration of CgA was not changed by alterations in the medium calcium concentration. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] stimulated a dose-dependent increase in the release of CgA, whereas intracellular CgA levels were not markedly altered. Alterations in medium calcium concentration did not affect CgA mRNA levels, whereas 1,25-(OH)2D3 caused a time- and dose-dependent increase in CgA mRNA levels. Therefore, changes in CgA release occurred in response to calcium and 1,25-(OH)2D3, with little change in the intracellular CgA concentration. This could arise by a change in the synthetic rate to match the alteration in the secretion rate and/or a change in the degradation rate. Calcium does not appear to affect CgA synthesis at a pretranslational level, whereas 1,25-(OH)2D3 stimulated a marked increase in CgA mRNA levels via an effect on CgA gene transcription. Furthermore, 1,25-(OH)2D3 had no effect on CgA mRNA stability. The relationship between steady state levels of CgA mRNA and total CgA levels (intracellular and extracellular) is not simple and possibly indicates that an important regulatory step for CgA synthesis occurs at the level of mRNA translation. The effect of 1,25-(OH)2D3 on CgA mRNA levels was not modulated by medium calcium concentrations. In addition, the effect of 1,25-(OH)2D3 in reducing PTH mRNA levels was not affected by medium calcium concentrations, with 1,25-(OH)2D3 and calcium acting independently to reduce PTH mRNA levels.

Amino Acid Isomerases↗

PubMed1990

Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function.

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗