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Biomedical subjects

E Hunter

Publications and source records attributed to E Hunter.

At least 127 records · Page 7Linked to original sources
PubMed1991

Australian Aborigines and alcohol: A review.

Following a summary of the relevant historical and legislative background, this article reviews the literature on: (i) rates of alcohol usage and problem drinking in Aboriginal populations; (ii) adverse effects of drinking; (iii) suggested causes of problem drinking among Aborigines; and (iv) treatment and preventive initiatives. The need to examine Aboriginal alcohol use in the wider context of socio-economic deprivation and rapid social change is emphasized. Key issues warranting further research attention are identified and, while recognizing the difficulties inherent in doing so, suggestions are made as to how the quality of research in the area might be raised. It is concluded that, without long-term planning and commitment of the necessary resources by government, little progress will be made in reducing the problems associated with Aboriginal use of alcohol.

Journal Article↗
PubMed1991

Amino acids encoded downstream of gag are not required by Rous sarcoma virus protease during gag-mediated assembly.

Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the active protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.

Amino Acid Sequence↗
PubMed1990

A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus.

Two different morphogenic processes of retroviral capsid assembly have been observed: the capsid is either assembled at the plasma membrane during the budding process (type C), or preassembled within the cytoplasm (types B and D). We describe here a gag mutant of Mason-Pfizer monkey virus, a type D retrovirus, in which a tryptophan substituted for an arginine in the matrix protein results in efficient assembly of capsids at the plasma membrane through a morphogenic process similar to that of type C retroviruses. We conclude that a type D retrovirus Gag polyprotein contains an additional, dominant signal that prevents immediate transport of precursors from the site of biosynthesis to the plasma membrane. Instead, they are directed to and retained at a cytoplasmic site where a concentration sufficient for self-assembly into capsids occurs. Thus, capsid assembly processes for different retroviruses appear to differ only in the intracellular site to which capsid precursors are directed.

Amino Acid Sequence↗
PubMed1990

Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane.

The capsids of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus, are preassembled in the infected cell cytoplasm and are then transported to the plasma membrane, where they are enveloped in a virus glycoprotein-containing lipid bilayer. The role of viral glycoprotein in intracellular transport of M-PMV capsids was investigated with a spontaneous mutant (5A) of M-PMV, which we show here to be defective in envelope glycoprotein biosynthesis. DNA sequence analysis of the env gene of mutant 5A reveals a single nucleotide deletion in the middle of the gene, which results in the synthesis of a truncated form of the envelope glycoprotein. Evidence is presented showing that the mutant glycoprotein is not expressed at the cell surface but is retained in the endoplasmic reticulum. Normal levels of gag-pro-pol precursor polyproteins are made and processed in mutant genome-transfected cells, and high levels of noninfectious particles lacking viral glycoprotein are released with normal kinetics into the culture medium. No intracisternal budding of capsids is observed. We conclude that viral glycoprotein is required neither for targeting preassembled capsids of M-PMV to the plasma membrane for final maturation nor for the budding process. Since the presence or absence of M-PMV glycoprotein at the site of budding does not affect the efficiency or kinetics of the targeting process, the preassembled capsid of M-PMV, in contrast to those of intracisternal type A particles, appears to have an intrinsic signal for intracellular transport to the plasma membrane.

Animals↗
PubMed1990

Structural role of the matrix protein of type D retroviruses in gag polyprotein stability and capsid assembly.

To obtain a better understanding of the role of the gag gene-encoded matrix (MA) protein in the assembly and maturation of type D retroviruses, we have made five mutants with specific in-frame deletions within the p10-coding region by the use of oligonucleotide-directed mutagenesis. The changes in the Gag polyprotein made by these mutations resulted in almost identical phenotypes. In cells expressing mutant genomes, the mutant Gag polyproteins were synthesized and modified with myristic acid in a normal manner. However, they were so unstable that the bulk of the newly synthesized polyproteins was degraded within 1 h without being processed into mature structural polypeptides. In contrast, wild-type polyproteins have a processing half-life of 3.0 to 3.5 h. The mutant Gag polyproteins were assembled with very low efficiency into capsids in the cytoplasm of the mutant-infected cells. Moreover, the few capsids that formed were neither released from nor accumulated in the cells. These results suggest that the matrix protein plays an important role in guiding the correct folding of the Gag polyprotein, which is presumably crucial for both stabilizing the molecule and facilitating the intermolecular interactions that occur during assembly of immature capsids.

Amino Acid Sequence↗
PubMed1990

Using a socio-historical frame to analyse aboriginal self-destructive behaviour.

The last two decades have seen rapid changes in many facets of Aboriginal society, including morbidity and mortality. The same period has witnessed a dramatic increase in writing about and by Aborigines and this has necessitated a re-examination of the national "history" to include the indigenous people of Australia. Medical workers in Aboriginal Australia should be alert to the historical forces determining patterns of ill-health. Psychiatry in particular must develop this perspective if it is to participate with Aborigines in addressing emergent patterns of behavioural distress including suicide, parasuicide, ludic behaviour and self-mutilation. This paper demonstrates the importance of the socio-historical frame in the examination of these behaviours from one discrete region in isolated Aboriginal Australia: the Kimberley.

Adult↗
PubMed1989

T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine.

Varicella-zoster virus (VZV) specific cytotoxicity was investigated during acute primary VZV infection, in naturally immune subjects and after vaccination with the live attenuated varicella vaccine by using T cell cultures (TCC) generated by stimulating PBMC with VZV Ag and autologous VZV-superinfected lymphoblastoid cell lines as targets. Lysis of VZV-infected lymphoblastoid cell lines was observed by TCC from acutely infected subjects, naturally immune subjects, and recipients of the varicella vaccine. VZV glycoprotein I induced cytotoxic T cells but killing was less efficient than killing by TCC stimulated with VZV Ag. The TCC were primarily CD4+ (mean 86.6%) T lymphocytes with 15.2% of the cells coexpressing Leu-19. TCC were predominantly restricted by HLA class II as demonstrated by lack of any blocking using class I mAb and blocking of 15 to 71% by L243, a mAb to class II. Unrestricted killing as measured by killing of K562 cells occurred in all TCC but was minimally greater than that observed against uninfected autologous targets. Phenotypes of PBMC during acute infection had an initial increase in CD4+ cells and an overall decrease in the percentage of circulating Leu-11+ (CD16). No enhanced K562 killing was demonstrated in PBMC from subjects with acute infection compared to subjects without infection. CD4+ CTL may function as an important primary host response in acute varicella. Immunization with live attenuated varicella vaccine induced VZV-specific, memory CTL responses comparable to those of naturally immune subjects. The demonstration of their persistence long after primary VZV infection may indicate a role for CTL in restriction of viral replication during episodes of VZV reactivation from latency.

Adult↗
PubMed1989

Isolation of Chinese hamster ovary cell lines temperature conditional for the cell-surface expression of integral membrane glycoproteins.

A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell-surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation.

Animals↗
PubMed1989

Integration of ambulance staff trained in cardiopulmonary resuscitation with a medical team providing prehospital coronary care.

Ambulance staff with advanced training in cardiopulmonary resuscitation and equipped with monitor/defibrillators were used as the initial responders to collapse calls within a medically based prehospital coronary care system. During 21 months, in a population of approximately 120,000, ambulance staff successfully resuscitated six patients from ventricular fibrillation; there were four long term survivors. The median response time of emergency ambulances to collapse calls was eight minutes compared with 20 minutes for the medically manned mobile coronary care unit. None of the patients resuscitated by ambulance staff would have survived if they had been dependent on the mobile coronary care unit acting alone. Nineteen other patients with important arrhythmias were referred for earlier medical management which in some cases may have saved lives. An additional eight long term survivors of out of hospital ventricular fibrillation were resuscitated by medical staff. The integration of paramedical with medical prehospital coronary care improved survival after out of hospital cardiac arrest.

Adult↗
PubMed1988

Oligomeric structure of a prototype retrovirus glycoprotein.

The structure of the Rous sarcoma virus envelope glycoprotein complex was studied by sedimentation gradient centrifugation analyses of detergent-solubilized wild-type and mutant envelope (env) gene products. These studies show that the envelope glycoprotein forms an oligomer during biosynthesis, which is most likely a trimer, and that this is the form of the complex found in virions. Our results are consistent with oligomer formation and transport out of the endoplasmic reticulum being closely linked. From analyses of mutant envelope proteins we conclude that the extracellular domain of the glycoprotein is sufficient for oligomer formation but that the transmembrane domain is required to stabilize this complex. Additional experiments suggest that interactions between external domains of the membrane-spanning, gp37 polypeptides are those most important for the formation of trimers. The significance of these observations to retroviral replication and implications for antiviral drug development are discussed.

Animals↗
PubMed1988

Immunity to whole varicella-zoster virus antigen and glycoproteins I and p170: relation to the immunizing regimen of live attenuated varicella vaccine.

Humoral and cellular immune responses to whole varicella-zoster virus (VZV) antigen and the VZV proteins glycoprotein I (gpI) and nonglycosylated protein p170 were evaluated in healthy children and adults given lyophilized live-attenuated varicella vaccine. Children received one dose of vaccine containing 950 pfu, whereas adults received two doses of 2500 pfu. After one year, the antibody titers of adult vaccinees to whole VZV and to gpI were significantly higher than those of children. Antibody titers to whole VZV, gpI, and p170 were lower among both vaccine populations than titers in naturally immune individuals, but vaccinees who seroconverted initially retained detectable VZV antibodies. Using T lymphocyte proliferation to measure cellular immunity, we found the mean (+/- SE) transformation index to whole VZV antigen to be 4.1 +/- 0.96 in children tested at one year, a mean significantly lower than the mean of 12.7 +/- 3.39 in adults and 13.0 +/- 1.67 in naturally immune subjects. These observations suggest that the vaccine dose affects vaccine-induced immunity to VZV.

Adolescent↗
PubMed1988

Molecular cloning of the wild-type phoM operon in Escherichia coli K-12.

A metastable bacterial alkaline phosphatase (Bap) phenotype is seen in phoR mutants, which alternately express a Bap-constitutive or -negative phenotype. The alteration is affected by mutations in the phoM region near 0 min. By molecular cloning of the wild-type phoM operon onto a multicopy plasmid and recombining onto the plasmid the pho-510 mutation that abolishes variation, the phoM operon, rather than some nearby gene, was shown to control variation. Complementation tests indicated that the wild-type phoM allele is dominant to the pho-510 mutation when both are in single copy, but whichever allele is present in higher copy appears as dominant when multicopy plasmids are examined. The alternating phenotypic variation of BAP synthesis was not seen in phoR+ cells with multicopy wild-type phoM plasmids, thus showing that the variation is associated with phoM-dependent Bap expression. The alternation acted at the level of phoA transcription; it was also recA independent. BAP clonal variation is phenotypically similar to Salmonella phase variation, which is controlled by a DNA rearrangement. No evidence was found for a DNA change near the phoM operon that might be responsible for the variable Bap phenotype.

Alkaline Phosphatase↗
PubMed1987

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation.

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).

Animals↗
PubMed1987

Recombinant human interferons inhibit replication of Mason-Pfizer monkey virus in primate cells.

Pretreatment of Mason-Pfizer monkey virus-infected human embryonic kidney cells with either of the cloned human interferons, A or A/D, reduces the release of infectious virus particles to the same extent (greater than 97%) as determined by 125I-protein A binding radioimmune assay. While interferon A has no significant effect on the release of infectious viruses from a Mason-Pfizer monkey virus-infected monkey cell line consistent with the species specificity of this group of compounds, human interferon A/D can block the release of virions from the same monkey cell line. This block appears to be at the level of viral particle release from the cells since both labeling with radioactive precursors and electron microscopic observations show few extracellular particles but normal levels of intracytoplasmic A particles. The results show that cloned human interferon A/D can block the replication of an immunosuppressive retrovirus in primate cells and raise the possibility of testing the efficacy of this interferon in primate model systems.

Animals↗
PubMed1987

A charged amino acid substitution within the transmembrane anchor of the Rous sarcoma virus envelope glycoprotein affects surface expression but not intracellular transport.

Two point mutations were introduced by oligonucleotide-directed mutagenesis into the region of the Rous sarcoma virus envelope gene that encodes the hydrophobic transmembrane anchor of the receptor glycoprotein. Single-nucleotide substitutions ultimately converted a hydrophobic leucine, located centrally within the membrane-spanning domain, to either a similarly hydrophobic methionine or a positively charged arginine. The altered coding region was reinserted into an intact copy of the envelope gene, cloned into simian virus 40 late-replacement vector and expressed in primate cells. Analysis of envelope gene expression in CV-1 monkey cells revealed normal levels of synthesis of a membrane-spanning precursor for both the mutants; however, the arginine-containing mutant [mu 26(arg)] exhibited greatly reduced cell surface expression of mature protein, as determined by indirect immunofluorescence and 125I labeling of surface proteins. In experiments in which cells producing the mu 26(arg) polypeptide were pulsed with radioactive leucine and then chased for 5 h, no intracellular accumulation or extracellular secretion of mature products (gp85 and gp37) could be detected. Treatment of mu 26(arg)-infected cells with lysosomal enzyme inhibitors (chloroquine and leupeptin) resulted in the accumulation of gp85 and gp37, indicating that they were being degraded rapidly in lysosomes. The fact that terminally glycosylated and proteolytically cleaved env gene products were observed under these conditions showed that modifications associated with passage through the trans compartment of the Golgi apparatus occurred normally on the mutant polypeptide; thus insertion of a highly charged amino acid into the transmembrane hydrophobic region of gp37 results in the postGolgi transport to lysosomes. It is proposed that the insertion of this mutation into the transmembrane anchor of the envelope glycoprotein does not affect membrane association, orientation with respect to the membrane, or intracellular transport at early stages during maturation. At a step late in the transport pathway, however, the presence of the charged side chain alters the protein in such a manner that the molecules are transported to the lysosomes and degraded. It seems likely that transport of the protein from the trans-Golgi to the cell surface is either directly blocked, or that after expression on the cell surface the mature glycoprotein complex is unstable and rapidly endocytosed.

Amino Acid Sequence↗
PubMed1987

Trans-activation of long terminal repeat sequence-mediated gene expression is not a property of type D retrovirus replication.

Several retroviruses encode trans-acting factors which activate gene expression directed by long terminal repeat (LTR) sequences and play a role in the positive feedback regulation of virus replication. We have examined two Mason-Pfizer monkey virus (MPMV) strains for their ability to produce and respond to such factors. Plasmids with the LTR of either MPMV or type D retrovirus/New England (D/NE) were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of these plasmids into several different human cell lines gave rise to significant CAT activity, demonstrating the strong transcriptional promoter activity of these LTRs. However, little or no increase in CAT activity was found upon transfection of these plasmids into MPMV- or D/NE-infected cell lines as compared with uninfected cell lines. Furthermore, CAT activity was not enhanced in uninfected cells by cotransfecting either a functional MPMV DNA clone, a plasmid expressing the human T-lymphotropic retrovirus trans-activator genes, tat-1 or tat-3. These data show that the property of trans-activation of LTR-mediated gene expression is a function in the replication of only certain retroviruses.

Cell Line↗