PubMed Health⌕ Search

Biomedical subjects

D A Mann

Publications and source records attributed to D A Mann.

53 records · Page 3Linked to original sources
PubMed1996

The human immunodeficiency virus type 1 regulatory protein Tat inhibits interferon-induced iNos activity in a murine macrophage cell line.

Human immunodeficiency virus type 1 (HIV-1) infection is frequently associated with concurrent infection by opportunistic pathogens, against which production of nitric oxide by host macrophages provides a first line of defence. We have investigated whether regulatory HIV-1 proteins, such as Tat, can modulate the activity of the inducible nitric oxide synthase (iNos) gene when expressed in stable transfectant lines of RAW264.7 cells. A bioassay for Tat, based on transactivation of an HIV-1 LTR-CAT reporter gene, allowed selection of Tat-expressing cells. Parental and Tat-expressing macrophages accumulated identical levels of nitrite following lipopolysaccharide (LPS) stimulation. Interferon gamma (IFN-gamma) stimulation however, resulted in reduced levels of nitrite accumulation as a direct consequence of Tat expression. Conditioned media from Tat-expressing cells reduced the level of nitrite accumulation in parental cells following IFN-gamma stimulation but not stimulation with LPS. These results implicate HIV-1 Tat as a modulator of the IFN-gamma-specific signal transduction pathways leading to iNos expression.

Animals↗
PubMed1994

A molecular rheostat. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression.

The complete biologically active human immunodeficiency virus type-1 (HIV-1) rev-response element (RRE) RNA is 351 nucleotides (nt) in length, and includes an extra 58 nt on the 5' end and 59 nt on the 3' end beyond the sites proposed in the original models for the RRE secondary structure. The extra sequences are able to form a duplex structure which extends Stem I. The presence of an elongated Stem I structure in the RRE RNA was confirmed by nuclease mapping experiments. Nuclease protection experiments have shown that rev binds to restricted regions of the RRE, including the high affinity site located at the base of Stem IIb and along the length of the Stem I region. The three large stem-loop structures which protrude from Stem I and Stem IIb (Stems IIc, III+IV and V) remain accessible to nucleases even in the presence of a large excess of protein. Gel-retardation experiments show that the truncations of Stem I reduced the total number of rev molecules that can bind co-operatively and with high affinity to the RRE RNA. To test whether the elongated Stem I structure is required for maximal rev activity, a series of truncations which progressively reduced the length of Stem I was introduced into an HIV-1 derived reporter plasmid. In the presence of rev and a functional RRE, there is an increase in the levels of gag and env mRNA in the cytoplasm and a decrease in levels of tat and rev mRNAs. Each of the truncations in Stem I reduced the rev responses, with the longest truncations producing the greatest losses of activity. The data suggest that the RRE acts as a "molecular rheostat" designed to detect rev levels during the early stages of the HIV growth cycle.

Base Sequence↗
PubMed1993

HIV gene regulatory proteins tat and rev and their interactions with synthetic RNA.

Synthetic oligoribonucleotides have been prepared and annealed to form model RNA duplexes that mimic the high affinity RNA recognition sites for the HIV-1 tat and rev proteins. The contributions of individual functional groups on the model RNAs to the specificity of binding by their respective proteins were studied by use of oligoribonucleotides containing site-specifically modified nucleotides. Both tat and rev appear to recognise specifically a limited number of functional groups in the major groove of an RNA double helix distorted by virtue of unpaired or non-Watson-Crick paired nucleotides.

Base Sequence↗
PubMed1992

Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.

The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure.

Bacteriophage T7↗
PubMed1992

Alternative splicing of the cytoplasmic domain of neural cell adhesion molecule alters its ability to act as a substrate for neurite outgrowth.

A full-length cDNA encoding 180-kDa neural cell adhesion molecule (NCAM 180) has been transfected into mouse NIH-3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipitated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.

Animals↗
PubMed1991

Endocytosis and targeting of exogenous HIV-1 Tat protein.

The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.

Amino Acid Sequence↗
PubMed1991

Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs.

Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6-E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the beta 1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition "subthreshold" levels of N-CAM expression, which do not normally support neurite outgrowth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.

Animals↗
PubMed1990

Characterization of the human N-CAM promoter.

In contrast with the complex series of splicing choices that generate the various membrane-associated isoforms of the neural cell-adhesion molecule alternative splicing of 5' exons does not contribute to additional molecular diversity. A single regulatory unit in genomic DNA, mapping to a 5 kb restriction-endonuclease-HindIII fragment, controls the expression of all major RNA size classes. DNA sequence analysis of a 2 kb fragment spanning the two major identified transcriptional initiation sites (194 and 188 bp from the ATG codon) and translation start codon indicates that the regulatory unit does not possess classical TATA or CCAAT motifs. The region of the putative promoter exhibits a GC-rich content and a high frequency of the dinucleotide CpG, both characteristics of a HTF(HpaII tiny fragments)-island. Introduction of deletion-mutant chimaeric-gene constructs into human and rodent N-CAM-expressing cell lines defines an active promoter region of 467 bp (-144 to -611 bp from the ATG codon). This region of genomic DNA contains consensus sites for the interaction of known transcriptional factors.

Animals↗
PubMed1990

Detection of acute cholecystitis in children.

Hepatobiliary imaging has an established diagnostic role in adults with acute cholecystitis but is underutilized in children. The authors report two children with acute cholecystitis, whose condition was diagnosed with the aid of a 99m-technetium-labeled iminodiacetic acid (IDA) derivative. The diagnosis was confirmed surgically.

Acute Disease↗
PubMed1989

Increased intracellular cyclic AMP differentially modulates nerve growth factor induction of three neuronal recognition molecules involved in neurite outgrowth.

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.

Antigens, Surface↗
PubMed1988

Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-1 and N-CAM in PC12 cell cultures.

The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture. Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression. Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM. Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures. Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1. Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin. Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM. Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response.

Antigens, Surface↗
PubMed1987

Cholera toxin and dibutyryl cyclic AMP inhibit the expression of neurofilament protein induced by nerve growth factor in cultures of naive and primed PC12 cells.

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neurofilament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme-linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30-fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B-cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.

Adrenal Gland Neoplasms↗
PubMed1987

Cell-cell interactions modulate the responsiveness of PC12 cells to nerve growth factor.

The growth of PC12 cells on a collagen substratum or on monolayers of several non-neuronal cell types was studied by measuring nerve growth factor (NGF)-dependent increases in the expression of a 150 X 10(3) (Mr) neurofilament protein subunit and the membrane glycoprotein Thy-1. Both responses were found to be greatly suppressed in cultures of fibroblasts as compared to the C2 and G8-1 muscle cell lines and the C6 glioma cell line. This suppression was associated with an inhibition of NGF-dependent neuritic outgrowth from PC12 cells grown on fibroblast monolayers. There was no evidence that fibroblasts secrete soluble molecules that directly inhibit these responses or neutralize NGF. In addition, there was no difference in the neurofilament protein response from PC12 cells that had been treated with NGF prior to coculture, and the now primed PC12 cells readily extended axons over fibroblast monolayers. These data demonstrate that cell-cell and/or cell-matrix interactions can modulate biochemical responses to NGF and suggest that responsiveness of neuronal cells to environmental cues is not immutable. Control of the latter may be at the level of expression of receptor molecules for cell-surface- or matrix-associated macromolecules and a similar mechanism operating during development could play a role in growth cone guidance.

Cell Communication↗
PubMed1985

Rapid transfer of DNA from agarose gels to nylon membranes.

The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.

Animals↗
PubMedDate not supplied

Analysis of the Th1/Th2 paradigm in transplantation: interferon-gamma deficiency converts Th1-type proislet allograft rejection to a Th2-type xenograft-like response.

The rejection mechanisms for fetal proislet allografts and pig proislet xenografts in mice are characterized by different intragraft cytokine mRNA profiles and cellular responses. Allograft rejection is predominantly CD8 T-cell-dependent and is associated with a Th1-type cytokine pattern (i.e., IFN-gamma, IL-2 but no IL-4 or IL-5 mRNA). In contrast, xenograft rejection is CD4 T-cell-dependent and is accompanied by a strong Th2-type response (i.e., enhanced expression of IL-4 and IL-5 mRNA) and by marked eosinophil accumulation at the graft site. We have now examined and compared the regulatory role of IFN-gamma in both proislet allograft and xenograft rejection processes. The histopathology and intragraft cytokine mRNA profile of BALB/c (H-2d) proislet allografts were examined in IFN-gamma-deficient and wild-type C57BL/6J recipient mice. The survival of pig proislet xenografts was also assessed in IFN-gamma -/- and wild-type hosts. Both proislet allografts and xenografts were acutely rejected in IFN-gamma -/- and wild-type mice. Unlike the conventional allograft reaction, which lacks eosinophil infiltration, the rejection of proislet allografts in IFN-gamma-deficient hosts correlated with intragraft expression of IL-4 and IL-5 mRNA (i.e., a Th2-type response) and eosinophil recruitment. The rejection of proislet allografts and xenografts can therefore occur by IFN-gamma-independent pathways; IFN-gamma, however, regulates the pathology of the allograft reaction but not the xenograft response. The immune destruction of proislet allografts is not prevented by Th2 cytokine gene expression; instead, the latter correlated with the recruitment of unconventional inflammatory cells (eosinophils), which may play an accessory role in effecting graft injury. Significantly, the Th1-to-Th2-like switch resulted in the novel conversion of an allograft rejection reaction into a xenograft-like rejection process.

Acute Disease↗