Commentaries on Landau's "The prospects of a harm reduction approach among indigenous people in Canada".
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Comment↗
Biomedical subjects
E Hunter
Publications and source records attributed to E Hunter.
Genocide: a public health issue.
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Adult↗
Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.
The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly.
Animals↗
Role of calmodulin in HIV-potentiated Fas-mediated apoptosis.
The recently demonstrated extraordinary rate of turnover of T cells in human immunodeficiency virus (HIV)-1-infected patients and the apparently concomitant high rate of viral production and death are consistent with a large amount of cell death directly due to infection. Apoptosis may be one of the major forms of T cell death in HIV-1 infection. Many apoptotic pathways depend on calcium and therefore would be expected to involve calmodulin. As the HIV-1 envelope glycoprotein, gp160, contains two known calmodulin-binding domains, we investigated the possibility that the cytoplasmic domain of the HIV-1 envelope protein gp160 could enhance Fas-mediated apoptosis, the major form of apoptosis in lymphocytes. Our studies have shown that 1) transfection of H9 and MOLT-4 cells with a non-infectious HIV proviral clone, pFN, which expresses wild-type gp160, leads to enhanced Fas-mediated apoptosis, 2) transfection of MOLT-4 cells with a pFN construct pFN delta 147, which expresses a carboxyl-terminally truncated gp160 lacking the calmodulin-binding domains, produces less Fas-mediated apoptosis than transfection with pFN, and 3) the calmodulin antagonists trifluoperazine and tamoxifen completely inhibit the pFN enhancement of Fas-mediated apoptosis in MOLT-4 cells. We have replicated all of these results using the vectors pSRHS and pSRHS delta 147, which express wild-type gp160 and truncated gp160, respectively, in the absence of other viral proteins. These investigations provide a mechanism by which HIV-1 may induce apoptosis and a possible intracellular target for future therapeutics.
Amino Acid Sequence↗
Human ehrlichiosis in a liver transplant recipient.
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Animals↗
Cloning, bacterial expression, and characterization of the Mason-Pfizer monkey virus proteinase.
We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.
Amino Acid Sequence↗
Transport of a lysosomally targeted Rous sarcoma virus envelope glycoprotein involves transient expression on the cell surface.
The details of intracellular transport pathways for glycosylated proteins remain incompletely described. We previously described a mutant Rous sarcoma virus envelope glycoprotein (gp), mu 26, with an altered membrane-spanning domain that was targeted to lysosomes after traversing the trans-Golgi. This mutant protein was not detectable on the cell surface by immunofluorescence, but its pathway for degradation remained unclear. To investigate this we have employed a second env mutation, S19, that results in a protein which is defective for normal cleavage/activation by intracellular enzymes, but remains susceptible to cleavage by extracellular proteases. Cleavage/activation of the double mutant by trypsin, which could only occur if it was exposed on the cell surface, was observed, indicating that the plasma membrane is an intermediate destination in the transport of this mutant protein. To substantiate these results, cells expressing the mu 26 glycoprotein were incubated with an antibody specific for the native protein in the presence of chloroquine. The specific accumulation of this antibody/gp complex in vesicles, as detected by internal immunofluorescence, confirmed the trypsin cleavage results. We conclude that this rapidly degraded mutant protein is transported from the trans-Golgi to the cell surface, where it is only transiently exposed, and then rapidly endocytosed and lysosomally degraded. The relevance of these results to the targeting of lysosomal proteins is discussed.
Amino Acid Sequence↗
Is there a role for prevention in aboriginal mental health?
Primary prevention is consistently identified as a priority in mental health and Aboriginal health planning. In both areas it has proved to be elusive. Aboriginal mental health therefore presents substantial challenges, particularly in remote Australia. In this paper, two brief case histories are used to demonstrate the limits of conventional psychiatric engagement in such settings and explore the dimensions of social disadvantage that compound psychiatric vulnerability. There are difficulties of primary prevention in mental health generally, and access to mental health services for those most at risk, including indigenous Australians, is unacceptably poor. Proceeding to primary prevention in Aboriginal mental health will, of necessity, include rectifying that situation and addressing the social precursors of mental disorders and distress. Such initiatives will require wide and sustained commitment toward long-term goals, with a focus on children and families, and must include effective substance-abuse strategies.
Adolescent↗
Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria.
The capsid precursor protein (Gag) of Mason-Pfizer monkey virus, the prototype type D retrovirus, has been expressed to high levels in bacteria under the control of the phage T7 promoter. Electron microscopic studies of induced cells revealed the assembly of capsid-like structures within inclusion bodies that formed at the poles of the cells 6 h after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The inclusion bodies and enclosed capsid-like structures were solubilized completely in 8 M urea, but following renaturation, we observed assembly in vitro of capsid-like structures that demonstrated apparent icosahedral symmetry. These results demonstrate for the first time that retroviral capsid precursors have the propensity to self-assemble in vitro and point to new approaches for the analysis of retroviral assembly and structure.
Capsid↗
Secondary structure model of the Mason-Pfizer monkey virus 5' leader sequence: identification of a structural motif common to a variety of retroviruses.
A stable secondary structure model is presented for the region 3' of the primer-binding site to 130 bases into the gag sequence of the prototype type D retrovirus Mason-Pfizer monkey virus. Using biochemical probing of RNA from this region in association with free energy minimization, we have identified a stem-loop structure in the region, which from other studies has been shown to be important for genomic RNA encapsidation. The structure involves a highly stable stem of five G-C pairs terminating in a heptaloop. Comparison of the Mason-Pfizer monkey virus structure with one predicted for squirrel monkey retrovirus demonstrates an identical stem and a common ACC motif in the loop. Free energy studies of the secondary structure of the 5' regions of eight other retroviruses predict stem loops which have similar GAYC motifs. We believe this may represent a common structural and sequence motif which among other functions may be involved in genomic RNA packaging in these viruses.
Animals↗
Oligomerization of the hydrophobic heptad repeat of gp41.
The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.
Amino Acid Sequence↗
Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: requirement of precursor cleavage for glycoprotein incorporation.
Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these two potential cleavage sites, a series of mutations has been constructed in each site individually, as well as in combinations that altered both sites simultaneously. A majority of the mutations in either potential cleavage site continued to allow efficient cleavage when present alone but abrogated cleavage of the precursor when combined. Despite being transported efficiently to the cell surface, these cleavage-defective glycoproteins were unable to initiate cell-cell fusion and viruses containing them were not infectious. Viruses that contained glycoproteins with a single mutation, and that retained the ability to be processed, were capable of mediating a productive infection, although infectivity was impaired in several of these mutants. Protein analyses indicated that uncleaved glycoprotein precursors were inefficiently incorporated into virions, suggesting that cleavage of the glycoprotein may be a prerequisite to incorporation into virions. The substitution of a glutamic acid residue for a highly conserved lysine residue in the primary cleavage site (residue 510) had no effect on glycoprotein cleavage or function, even though it removed the only dibasic amino acid pair in this site. Peptide sequencing of the N terminus of gp41 produced from this mutant glycoprotein demonstrated that cleavage continued to take place at this site. These results, demonstrating that normal cleavage of the human immunodeficiency virus type 1 glycoprotein can occur when no dibasic sequence is present at the cleavage site, raise questions about the specificity of the cellular protease that mediates this cleavage and suggest that cleavage of the glycoprotein is required for efficient incorporation of the glycoprotein into virions.
Amino Acid Sequence↗
Interactions with males promote rapid changes in gonadotropin-releasing hormone immunoreactive cells.
It is well known that hormones can regulate behaviors. However, the reciprocal interaction, the effects of behavior on hormones, has received less direct experimental attention. Dramatic changes in hormones and behaviors occur at puberty and some of these changes can be triggered by modification of the social environment. Interactions with males accelerate production of pulsatile release of gonadotropins and steroid hormones which, in turn, initiate estrous cycles, ovulation, and sexual behavior in females. Ultimately all of these actions are controlled by changes in production and secretion of gonadotropin-releasing hormone (GnRH). Little is known about how behavior affects GnRH-producing neurons. In female musk shrews, the first mating initiates the onset of puberty. Musk shrews lack a behavioral estrous cycle and they become receptive within minutes after their first contact with a male. As soon as 1 h after interactions with males there is a significant increase in the numbers of GnRH-immunoreactive (GnRH-ir) neurons in specific brain regions. In the present study, we examined changes in GnRH-ir cell number during the initial mating bout. We found dynamic changes in the numbers of GnRH-containing cells, correlated with changes in behavior. Interactions with males for less than 30 minutes induced a significant increase in GnRH-ir neurons in specific olfactory-related forebrain nuclei. At the end of a mating bout, numbers of GnRH-ir neurons declined. Because behavioral interactions have rapid and pronounced effects on the neurons that produce GnRH, this model can be used to examine the behavioral regulation of neuronal plasticity.
Animals↗
"Freedom's just another word": aboriginal youth and mental health.
OBJECTIVE: The aim of the study was to review the available material on Aboriginal youth mental health, exploring certain contemporary issues. METHOD: The review covers the psychiatric and social science literature, focusing on the recent period of major social change. RESULTS: The available material is severely limited, particular deficiencies being normal child development, the impact of social change and its consequences for the construction of Aboriginal identity. CONCLUSION: There is a need for more work in this area and for psychiatric engagement at a political level.
Adolescent↗
Conflicting constructions of aboriginal suicide: a letter in response to Robert Parker.
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Australia↗
Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex.
For a number of viruses, oligomerization is a critical component of envelope processing and surface expression. Previously, we reported that a synthetic peptide (DP-107) corresponding to the putative leucine zipper region (aa 553-590) of the transmembrane protein (gp41) of human immunodeficiency virus type 1 (HIV-1) exhibited alpha-helical secondary structure and self-associated as a coiled coil. In view of the tendency of this type of structure to mediate protein association, we speculated that this region of gp41 might play a role in HIV-1 envelope oligomerization. However, later it was shown that mutations which should disrupt the structural elements of this region of gp41 did not affect envelope processing, transport, or surface expression (assembly oligomerization). In this report we compare the effects of amino acid substitutions within this coiled-coil region on structure and function of both viral envelope proteins and the corresponding synthetic peptides. Our results establish a correlation between the destabilizing effects of amino acid substitutions on coiled-coil structure in the peptide model and phenotype of virus entry. These biological and physical biochemical studies do not support a role for the coiled-coil structure in mediating the assembly oligomerization of HIV-1 envelope but do imply that this region of gp41 plays a key role in the sequence of events associated with viral entry. We propose a functional role for the coiled-coil domain of HIV-1 gp41.
Amino Acid Sequence↗
Mutations within the transmembrane glycoprotein of Mason-Pfizer monkey virus: loss of SU-TM association and effects on infectivity.
We have previously reported that an 11-amino-acid deletion (D33) within the immunosuppressive peptide (ISP) region of the Mason-Pfizer monkey virus transmembrane (TM) glycoprotein, gp22, caused the loss of interaction between TM and the surface (SU) glycoprotein, gp70. This resulted in the secretion of large amounts of biologically active SU glycoprotein, and we postulated that the ISP might represent a point of contact between the two glycoproteins. To further define the amino acids that might be involved in this proposed region of interaction, we have made two neighboring 4-amino-acid deletions within the area defined by the D33 mutation and have carried out saturation mutagenesis on this 8-amino-acid region. We found that one of the smaller deletions (delta D), and two single point mutations (R68S and L72P), gave the same phenotype as the original D33 mutant. These results provide additional support for the hypothesis that this region of the TM glycoprotein is in contact with the SU glycoprotein and is important in maintaining the noncovalent interactions of the glycoproteins that function to hold the complex together.
Amino Acid Sequence↗
A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent.
Replication of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev protein. This protein acts in concert with the cis-acting rev-responsive element present in intron-containing RNAs to facilitate nuclear export of these RNAs. Here we show that a cis-acting 219-nucleotide sequence from an unrelated "simple" retrovirus, Mason-Pfizer monkey virus (MPMV), enables Rev-independent HIV-1 replication. This sequence is present in an untranslated region near the 3' end of the MPMV genome. The MPMV element is also able to efficiently substitute for Rev in expression of Gag/Pol and Env proteins from subgenomic constructs. We hypothesize that the MPMV element functions by interacting with a cellular factor that plays a role in mRNA transport analogous to that of the Rev protein. It might be possible to exploit this element in the development of an HIV vaccine.
Biological Transport↗