Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.     

Structural analysis of zinc substitutions in the active site of thermolysin.

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.     

Structure and origin of a novel class of defective interfering particle of vesicular stomatitis virus

The genome structure and terminal sequences of a 'copyback' defective interfering (DI) particle ST1, and a novel complexly rearranged 'snapback' DI particle ST2 of vesicular stomatitis virus have been determined. The ST1 DI genome RNA possesses 54 base long inverted complementary termini, the 5' end of which is homologous to the standard virus genome 5' end. Following this region of inverted complementarity the DI RNA 5' end continues to be homologous to standard virus RNA 5' sequences, whereas the 3' end diverges into sequences within the virus L gene internal sequences. ST2 DI genome RNA does not contain colinear covalently linked plus and minus sense RNA copies of the standard infectious virus RNA 5' terminus as predicted from the prototype snapback DI structure, but instead appears to be a hairpin copy of the ST1 DI RNA genome. This is the first evidence suggesting that DI particles may be generated from RNA templates other than the standard virus RNA. Generation models and the implications of these findings for RNA virus evolution are discussed.     

Metabolic control of diabetes in general practice clinics: comparison with a hospital clinic.

An assessment was made of the degree of metabolic control achieved in diabetic patients attending mini clinics run by general practitioners compared with that in matched diabetic patients attending a hospital clinic. Patients were grouped according to whether they were being treated with diet alone, an oral hypoglycaemic, insulin once daily, or insulin twice daily. Statistical analysis showed no significant difference between patients attending mini clinics and those attending the hospital clinic in retrospective mean blood glucose, retrospective mean glycosylated haemoglobin (HbA1), or prospective HbA1 concentrations. General practitioners providing diabetic care on an organised basis can achieve a degree of glycaemic control in diabetic patients equal to that reached by a hospital clinic.     

Species turnover in deciduous forest vegetation

Summary Permanent quadrats were marked out in two areas of hardwood forest vegetation in 1969, and listings of their vascular plant species were taken on several occasions over the snow-free seasons of 1969, 1970, 1971 and 1976. Over the period of study, mean numbers of species per m² remained virtually constant, but variations in the species compositions of individual quadrats were such that mean turnover ratios of 0.115 and 0.085, respectively, were computed for the two stands. Between 1969 and 1976 averages of 20% and 14%, respectively, of the species found in individual quadrats were replaced. This was not accomplished by qualitative changes in the floras of the two stands. Rather, it reflects the operation of a system of continuous rearrangements of species in the small quadrats of both sample areas.Taxonomical nomenclature and life-form system used in this study are according to Gleason & Cronquist (1963).William Phillips, Ian Sutherland and Sheila Thompson helped in the field; Professor Keith Wade commented on the material; Abal Sen drafted the diagram; and the research is part of that funded by the National Research Council of Canada.     

Mineralocorticoid radioreceptor assay: application to adrenal-regeneration hypertension.

Identification of unknown hormones has traditionally involved utilizing a bioassay to initially detect the hormone and to follow its purification. However, radioreceptor assays may be more useful for this purpose by offering greater sensitivity and precision. A mineralocorticoid radioreceptor assay has been developed for use in conjunction with chromatographic separation of a urinary extract to detect the presence of unknown urinary mineralocorticoids. This assay utilizes competition of the unknown steroid and aldosterone for rat renal cytoplasmic mineralocorticoid receptors to enable mineralocorticoid quantitation in aldosterone equivalents. This assay provides 100 fold increase in sensitivity and a significant increase in precision over the commonly used adrenalectomized rat bioassay. The mineralocorticoid radioreceptor assay has been utilized to assay mineralocorticoid activity in chromatographic fractions of a urinary extract from rats with regenerating adrenals. A large area of mineralocorticoid radioreceptor activity has been identified which possibly represents an unknown mineralocorticoid contributing to the etiology of adrenal regeneration hypertension. This assay is applicable to other syndromes of postulated unknown mineralocorticoid excess, such as human low renin essential hypertension. In addition, similar radioreceptor assays are applicable for the initial detection of any type of hormone activity and for the subsequent purification and identification of this hormone.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.     

Electrically mediated fast polyspermy block in eggs of the marine worm, Urechis caupo

Previous work has established that the polyspermy block in Urechis acts at the level of sperm-egg membrane fusion. (J. Exp. Zool. 196:105). Present results indicate that during the first 5--10 min after insemination the block is mediated by a positive shift in membrane potential (the fertilization potential) elicited by the penetrating sperm, since holding the membrane potential of the unfertilized egg positive by passing current reduces the probability of sperm entry, while progressively reducing the amplitude of the fertilization potential by decreasing external Na+ progressively enhances multiple sperm penetrations. Also, a normal fertilization potential is correlated with a polyspermy block even under conditions (pH 7) in which eggs do not develop. We have investigated the mechanism of the electrical polyspermy block by quantifying the relationship between sperm incorporation, membrane potential and ion fluxes. Results indicate that the polyspermy block is mediated by the electrial change per se and not by the associated fluxes of Na+, Ca++, and H+.