Site-specific mutagenesis was used to introduce amino acid substitutions at the asparagine codons of four conserved potential N-linked glycosylation sites within the gp120 envelope protein of human immunodeficiency virus (HIV). One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the env gene polypeptides gp120 and gp41 or the binding of gp120 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV env genes readily induced syncytia in CD4+ HeLa cells. These results suggest that alterations involving the second conserved domain of the HIV gp120 may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. In long-term cocultures of a T4+ lymphocyte cell line and colon carcinoma cells producing the mutant virus, revertant infectious virions were detected. Molecular characterization of two revertant proviral clones revealed the presence of the original mutation as well as a compensatory amino acid change in another region of HIV gp120.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator.

An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

Base Sequence↗

PubMed1988

The amino-terminal region of the adenovirus serotype 5 E1a protein performs two separate functions when expressed in primary baby rat kidney cells.

Adenovirus serotype 5 E1a proteins immortalize primary cells and in cooperation with products of a second oncogene, such as adenovirus serotype 5 E1b or EJ ras, produce full transformation. E1a also activates transcription of specific viral and cellular promoters, represses enhancer-dependent genes, and induces cellular DNA synthesis in quiescent cells. Comparison of different adenovirus serotypes has identified three conserved regions in the E1a protein sequence. We have analyzed E1a mutants with deletions-linker insertions in or preceding the first conserved region, region 1 (amino acids 40 through 77 of adenovirus serotype 5 E1a). E1a mutants which have in-frame deletions-substitutions in region 1 or pre-region 1 sequences were reconstructed into adenovirus to yield a total of 14 mutant viruses. All the mutant viruses showed wild-type growth in HeLa cells, confirming that region 1 is nonessential in these cells. However, we show that region 1 provides two distinct functions in infected primary rodent cells. One function is essential for induction of cell DNA synthesis, and the other is essential for focus formation. In addition, our results are consistent with a requirement for the DNA induction function in focus formation.

Adenovirus Early Proteins↗

PubMed1988

Pharmacokinetics of antimony during treatment of visceral leishmaniasis with sodium stibogluconate or meglumine antimoniate.

5 patients with visceral leishmaniasis were treated with sodium stibogluconate (2 patients) or meglumine antimoniate (3 patients) given intramuscularly at a dose of 10 mg antimony (Sb) per kg body weight daily for 30 d. Blood samples were obtained at intervals during treatment and blood Sb concentrations measured by anodic stripping voltametry. The pharmacokinetics of both drugs were remarkably similar, with peak concentrations of approximately 10 mg/litre occurring 2 h after the initial dose. Most of the Sb was eliminated rapidly, but nadir Sb concentrations increased gradually during treatment from 0.04-0.08 mg/litre 24 h after the first dose to 0.19-0.33 mg/litre 24 h after the 30th dose. For both drugs, the data were best described by a two compartment, three term pharmacokinetic model representing an initial absorption phase with a mean half-life of 0.85 h, a rapid elimination phase with a mean half-life of 2.02 h, and a slow elimination phase with a mean half-life of 76 h. The slow terminal elimination phase may be related to in vivo conversion of pentavalent Sb to trivalent Sb, which could contribute to the toxicity associated with long-term high dose therapy.

Antimony↗

PubMed1988

Structural basis of muscarinic acetylcholine receptor subtype diversity.

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Animals↗

PubMed1988

Design and management of development projects to avoid health hazards.

By drawing from the examples of two dissimilar irrigation development projects it is concluded that for the success of a healthy land development project: (i) by deliberate design a suitable management organization must be set up which has the willing support of all the interested parties, which is given substance by statute and loan agreement, and which has the active support of government; (ii) the staffing should be headed by an enthusiastic person of high rank to provide historical continuity with an adequate secretariat to give effective technical and administrative support; (iii) a deterioration in health may actually threaten the continued existence of a development programme, and health services should be considered part of the development process and integrated with it at all stages from initial planning to several years after commissioning; (iv) health programmes should be responsive to the changing health needs of the settled populations which should be monitored to allow appropriate intervention and which must involve community participation; (v) although the operation of nationally accepted levels of health care facilities should be the responsibility of the national health authority, interventions attributable to the development should be part of the development cost; (vi) interventions should be based on pre-project epidemiology surveys and forecasts of its changes taking account of the disease experience of the settlement populations; (vii) to limit intervention to disease specific problems begs the basic issues. It should be possible to provide better cost-effective health facilities for populations of development projects than for their rural counterparts because they are better defined and more settled; (viii) such interventions by way of changes in fundamental design criteria for the primary engineering works are unlikely to be cost effective; (ix) adequate health facilities must be in place and capable of operation before settlement commences.

Adolescent↗

PubMed1988

Ethics in the care of AIDS patients.

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Acquired Immunodeficiency Syndrome↗

PubMed1987

Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors.

To investigate the molecular basis for the diversity in muscarinic cholinergic function, we have isolated the genes encoding the human M1 and M2 muscarinic receptors (mAChR) as well as two previously undiscovered mAChR subtypes, designated HM3 and HM4. The amino acid sequence of each subtype reflects a structure consisting of seven, highly conserved transmembrane segments and a large intracellular region unique to each subtype, which may constitute the ligand-binding and effector-coupling domains respectively. Significant differences in affinity for muscarinic ligands were detected in individual mAChR subtypes produced by transfection of mammalian cells. Each subtype exhibited multiple affinity states for agonists; differences among subtypes in the affinities and proportions of such sites suggest the capacity of mAChR subtypes to interact differentially with the cellular effector-coupling apparatus. Subtype-specific mRNA expression was observed in the heart, pancreas and a neuronal cell line, indicating that the regulation of mAChR gene expression contributes to the differentiation of cholinergic activity.

Amino Acid Sequence↗

PubMed1987

Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen.

The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).

Acquired Immunodeficiency Syndrome↗

PubMed1987

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor.

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.

Antibodies, Monoclonal↗

PubMed1987

Primary structure and biochemical properties of an M2 muscarinic receptor.

A partial amino acid sequence obtained for porcine atrial muscarinic acetylcholine receptor was used to isolate complementary DNA clones containing the complete receptor coding region. The deduced 466-amino acid polypeptide exhibits extensive structural and sequence homology with other receptors coupled to guanine nucleotide binding (G) proteins (for example, the beta-adrenergic receptor and rhodopsins); this similarity predicts a structure of seven membrane-spanning regions distinguished by the disposition of a large cytoplasmic domain. Stable transfection of the Chinese hamster ovary cell line with the atrial receptor complementary DNA leads to the binding of muscarinic antagonists in these cells with affinities characteristic of the M2 receptor subtype. The atrial muscarinic receptor is encoded by a unique gene consisting of a single coding exon and multiple, alternatively spliced 5' noncoding regions. The atrial receptor is distinct from the cerebral muscarinic receptor gene product, sharing only 38% overall amino acid homology and possessing a completely nonhomologous large cytoplasmic domain, suggesting a role for the latter region in differential effector coupling.

Amino Acid Sequence↗

PubMed1987

Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein.

HIV LTR-directed expression is markedly stimulated in trans by coexpression of a region of the HIV genome encoding a portion of the tat reading frame. Transient expression assay analysis reveals that trans-activation of LTR-directed expression results primarily from an increase in mRNA accumulation. Deletion analysis of the LTR indicates that upstream promoter and enhancer elements are dispensible for trans-activation, while sequences 3' of the RNA start site displaying strict orientation and position dependence are required. These sequences, contained in the 5' leader of all HIV transcripts, form a stable stem-loop structure with twofold symmetry in the cognate mRNA. Analysis of mutations in the trans-acting region demonstrates that the trans-activator is the protein product of the tat gene, identified biochemically in HIV-infected and transfected cells as an Mr 15,000 polypeptide. We discuss possible mechanisms whereby the interaction of p15tat with the dyad element promotes the accumulation of LTR-directed mRNA.

Acquired Immunodeficiency Syndrome↗

PubMed1987

Oocyte pickup by laparoscopy replaced by transvaginal aspiration in an in vitro fertilization program.

The results of laparoscopic (lap) and transvaginal (TV) oocyte pickups (OPUs) performed concurrently for in vitro fertilization in 232 consecutive treatment cycles have been reviewed. The patients were compared for age, preoperative estradiol concentration, luteal-phase support, and number of follicles aspirated and were found to be similar but were not matched for cause of infertility. The lap OPU group had more oocytes recovered per follicle aspirated (P less than 0.001), but because of a lower fertilization rate (P less than 0.01), the number of embryos transferred was similar. Nevertheless, more (P less than 0.05) pregnancies occurred in the TV OPU group. Patients were subgrouped so that comparisons of patients with the same cause of infertility, tubal disease alone, were considered. This showed that the pregnancy rate was still higher in the TV OPU group (P less than 0.05). TV OPU was less painful and not associated with increased morbidity, and since the data suggest that TV OPU was at least as successful as lap OPU, it is recommended that all oocyte pickups in the future be performed transvaginally.

Adult↗

PubMed1987

Variation in the timing of multiple ovulations following gonadotropin releasing hormone treatment and its relevance to collecting pronuclear embryos of sheep.

Timing of superovulation was examined by repeated laparoscopy in two Merino flocks treated with either pregnant mare serum gonadotropin (PMSG) plus gonadotropin releasing hormone (GnRH) or follicle stimulating hormone (FSH) plus GnRH. Observations were made in May (late breeding season), August (early anoestrous season), November (late anoestrous season), and February (midbreeding season). Data examined were time to first ovulation, time to all ovulations, and time from first to last ovulation. A GnRH-induced synchrony in the timing of superovulation occurred in Flock 1 irrespective of the month of observation. Approximately 80% of ovulations were recorded within 3 h with the median ovulation occurring 47 to 49 h after progestagen treatment. A similar synchrony was observed in Flock 2 in November and February. However, in May and August, the timing was asynchronous with some ewes superovulating as early as 10 or more hours before the median time obtained in November and February. An examination of this phenomenon indicated that 1) it also occurred when GnRH was not included in the treatment protocol, 2) it occurred irrespective of when ewes were exposed to vasectomized rams, and 3) it was more common in anovular ewes induced to superovulate than in spontaneously cyclic ewes. We concluded that treatment protocols developed for the collection of pronuclear embryos in Merino ewes during the breeding season can be less reliable when used out of season, thus increasing the possibility of collecting two- to four-cell embryos.

Journal Article↗