Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding.

Both equilibrium and nonequilibrium factors influence the efficacy of pharmaceutical agents that target intermediate states of biochemical reactions. We explored the intermediate state inhibition of gp41, part of the HIV-1 envelope glycoprotein complex (Env) that promotes viral entry through membrane fusion. This process involves a series of gp41 conformational changes coordinated by Env interactions with cellular CD4 and a chemokine receptor. In a kinetic window between CD4 binding and membrane fusion, the N- and C-terminal regions of the gp41 ectodomain become transiently susceptible to inhibitors that disrupt Env structural transitions. In this study, we sought to identify kinetic parameters that influence the antiviral potency of two such gp41 inhibitors, C37 and 5-Helix. Employing a series of C37 and 5-Helix variants, we investigated the physical properties of gp41 inhibition, including the ability of inhibitor-bound gp41 to recover its fusion activity once inhibitor was removed from solution. Our results indicated that antiviral activity critically depended upon irreversible deactivation of inhibitor-bound gp41. For C37, which targets the N-terminal region of the gp41 ectodomain, deactivation was a slow process that depended on chemokine receptor binding to Env. For 5-Helix, which targets the C-terminal region of the gp41 ectodomain, deactivation occurred rapidly following inhibitor binding and was independent of chemokine receptor levels. Due to this kinetic disparity, C37 inhibition was largely reversible, while 5-Helix inhibition was functionally irreversible. The fundamental difference in deactivation mechanism points to an unappreciated asymmetry in gp41 following inhibitor binding and impacts the development of improved fusion inhibitors and HIV-1 vaccines. The results also demonstrate how the activities of intermediate state inhibitors critically depend upon the final disposition of inhibitor-bound states.

Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common membrane insertion site.

The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1-complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.

Import of ADP/ATP carrier into mitochondria: two receptors act in parallel.

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.

A new model of Pneumocystis carinii infection in mice selectively depleted of helper T lymphocytes.

Pulmonary infections with Pneumocystis carinii are an important cause of morbidity and mortality in patients with AIDS. P. carinii infections are seen in patients with decreased numbers of helper T lymphocytes, suggesting that these cells are important in preventing infection. To test this hypothesis, we sought to establish experimental infection with P. carinii in mice selectively depleted of helper T lymphocytes. Weekly injections of a monoclonal anti-CD4 antibody produced sustained depletion of helper T lymphocytes from blood and lymphoid organs. To establish pulmonary infection, lymphocyte-depleted mice were then given intratracheal inoculations of P. carinii organisms derived from the lungs of chronically infected athymic mice. Pulmonary infection with P. carinii was demonstrable in the antibody-treated mice and was centered around the conducting airways. Infection was persistent for up to 3 mo with continued antibody treatments, and yet could be cleared from the lungs if antibody treatments were discontinued. This experimental model of P. carinii infection permits the study of infection associated with a specific immune defect and implicates the helper T lymphocyte as a critical cell in host defense against this pathogen.

Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae.

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.

HIV-1 gp41 as a target for viral entry inhibition.

The recent success of the fusion inhibitor T-20 (enfuvirtide) in clinical studies has ushered in a new chapter in the development of anti-HIV-1 therapeutics. T-20 is the first FDA-approved drug that targets the viral transmembrane protein gp41. This protein, along with gp120, promotes viral entry through a coordinated cascade of conformational transitions that lead to the fusion of the HIV-1 and target cell membranes. The interaction of gp120 with CD4 and a chemokine receptor stimulates gp41 to extend and bridge the space between the virus and cell. Subsequently, gp41 collapses into a trimer-of-hairpins structure that brings the viral and cellular membranes into close proximity necessary for fusion. Enfuvirtide targets the gp41 amino-terminal region exposed in the transient extended state, blocking the ultimate collapse into the trimer-of hairpins and inhibiting membrane fusion. The vulnerability of this transient extended state has stimulated the development of new agents, ranging from small molecules to large proteins, that bind to gp41 and inhibit its structural transformations. The discovery and characterization of these inhibitors have not only led to new antiviral strategies, but have also shed light on the accessibility of gp41 epitopes that might play a role in HIV-1 vaccine development.

Modulation of lymphocyte subsets in Peyer's patches of mice treated with monoclonal antibody against helper T-cells.

Treatment of mice with anti-L3T4, a monoclonal antibody directed against helper T-cells, impairs clearance of intestinal Giardia muris infection. The present study examined the effect of anti-L3T4 treatment on mouse Peyer's patch cytoarchitecture and on the distribution of T-cell subsets within microenvironments of the follicle. Female BALB/c mice, aged 8 weeks, were given 4-7 weekly injections of either anti-L3T4 (1 mg/wk) or PBS (control group), and Peyer's patches were examined by immunohistochemistry or flow cytometry. In anti-L3T4-treated mice, Peyer's patch follicles (B-cell areas) were about two thirds the size of follicles in controls, and virtually all the size difference occurred in germinal centers. Peyer's patches were depleted of L3T4+ cells, yet the proportion of Thy-1.2+ (all T) cells was not decreased correspondingly, and the distribution of Thy-1.2+ cells in the patches was similar to that in control mice. In anti-L3T4-treated mice, Thy-1.2+ cells consisted of (a) Ly-2+ (cytotoxic/suppressor T) cells, and (b) a population of Thy-1.2+ cells that were neither L3T4+ nor Ly-2+. After treatment, Ly-2+ cells accounted for most of the T-cells in interfollicular areas and were also scattered in follicles, in germinal centers, and below the dome epithelium--in areas where L3T4+ cells predominated in control mice. Thy-1.2+ cells that were L3T4- and Ly-2- were mainly localized below the dome epithelium. These shifts indicate complex interrelationships among different lymphocyte subsets in Peyer's patches.

Generation of L3T4-/Ly-2- T cells in Peyer's patches of mice treated with monoclonal antibody against helper T cells.

The effect of in vivo administration of anti-L3T4 monoclonal antibody on the generation of an L3T4-/Ly-2- (CD4-/CD8-) population of Thy-1.2+ cells was examined in Peyer's patches of mice by two-color flow cytometry. Female BALB/c mice aged 8 wk were given 4-6 weekly injections of either anti-L3T4 (1 mg/wk) or phosphate-buffered saline (control group), and dispersed Peyer's patch cells analyzed for the presence and absence of L3T4 and Ly-2 on Thy-1.2+ cells. In anti-L3T4 treated mice, L3T4-/Ly-2- T cells accounted for 25-30% of the Thy-1.2+ cells, whereas in control mice these cells represented only 3-4% of the T cells. The remaining 70-75% of the Thy-1.2+ cells after anti-L3T4 treatment were Ly-2+ and not L3T4+. The L3T4-/Ly-2- population of Thy-1.2+ cells is a novel subset which has not been previously found in Peyer's patches and is amplified when helper T cells are depleted.

Intrathymic changes in murine CD3, CD5 and CD8 expression after in vivo administration of anti-CD4.

Intrathymic development of murine cortical and medullary thymocyte subpopulations was examined after in vivo administration of anti-CD4 mAb. Four days after mice received 1 mg anti-CD4, expression of CD4 was reduced to about 25% the levels of controls. On cortical CD8+/PNAhigh thymocytes, CD5 and CD8 expression both decreased, but not in parallel, whereas CD3 expression increased almost 2-fold. Partial shifts in CD3 expression were seen 3 and 6 h after injection, but modulation of CD4 preceded the increase in CD3 expression. On medullary CD8-/PNAlow thymocytes, both CD3 and CD5 were down regulated. On nontargeted CD8+/PNAlow medullary thymocytes, expression of these molecules was decreased less than 20%, but the decrease in CD8 expression was primarily due to the appearance of a CD8low subpopulation. The results indicate that anti-CD4 mAb differentially affects the intrathymic development of T cell populations.

Changes in melatonin secretion with age and pubescence.

At the turn of the century, reports on a coincidence of pineal tumors and precocious puberty in man initiated a host of animal experiments. Results of these experiments ultimately revealed a modulating effect on sexual maturation and on gonadal activity in several species, brought about by the pineal hormone melatonin (aMT) and particularly its circadian secretion pattern. In addition, it renewed interest in a possible interaction of the pineal and human sexual function. The introduction of specific and precise aMT assays provided exact evaluation of the circadian aMT secretion pattern in adults. Despite considerable efforts, however, a precise characterisation of this pattern has not yet been achieved in children. In a first step we examined single day- and nighttime serum samples from 280 subjects of all ages. aMT levels were low during early infancy, increased to their highest values around 1 to 3 years and dropped progressively by 75 percent until young adulthood, when they remained fairly stable. Thus, the pattern of aMT levels is the reverse of that of gonadotropin levels or gonadal activity in humans. Whether there is a causal relationship between the activity of the pineal gland and the hypothalamo-pituitary-gonadal axis in humans or whether the described negative correlation in the activity of both glands is purely coincidental remains to be clarified.

Oxidation of sulphide minerals--I: determination of ferrous and ferric iron in samples of pyrrhotite, pyrite and chalcopyrite.

A method has been developed for determining small amounts of both ferrous and ferric iron in oxidized samples of pyrrhotite, pyrite and chalcopyrite. The oxidized iron is selectively dissolved in 10M phosphoric acid under reflux and can be determined with the accuracy generally accepted in chemical phase analysis.

Oxidation of sulphide minerals--II: determination of metal in the oxidation products of galena, sphalerite and chalcocite.

A 15% ammonium acetate and 3% acetic acid solution has been shown to be useful in the determination of the principal metal in small amounts of the oxidation products of galena, sphalerite and chalcocite. Its use in the determination of the extent of oxidation in concentrates or complex ores of these minerals is implied.

Determination of tin as stannite-kesterite and cassiterite in ores.

Tin is stannite-kesterite and as cassiterite in a tin-bearing ore can be determined by the selective decomposition of the stannite-kesterite phase with a mixture of sodium nitrate and acetic acid.

Chemical phase-analysis of ores and rocks: a review of methods.

The methods employed in the chemical phase-analysis of rocks, ores and their treatment products are reviewed. An attempt has been made to be critical, selective and thorough in the choice of methods available in the literature, in order to provide a convenient manual for workers interested in this aspect of analytical chemistry.

Determination of the elemental sulphur content of minerals and ores.

A new technique has been developed to determine trace quantities of elemental sulphur in minerals and ores. After separation from the sample by sublimation, the sulphur is determined spectrophotometrically in ethanol. Sulphide, sulphite, sulphate and thiosulphate do not interfere. The effect of the presence of xanthate or dixanthogen is discussed briefly.

The stability of the certified reference ores MP-1 KC-1 and SU-1 towards air oxidation.

The stability of three certified reference sulphide ores, MP-1, KC-1 and SU-1, towards air oxidation has been measured at 50 degrees and 40, 62 and 82% relative humidity, and at 62% relative humidity and 34 degrees and 67 degrees . Both the relative humidity and temperature affect the rate of oxidation but their relative importance depends on the mineralogical composition of the ore. Changes in the water-extractable metals and elemental sulphur content on oxidation have been determined. It is concluded that these ores may be stored safely in sealed bottles under normal laboratory conditions.

Oxidation of sulphide minerals-VI Ferrous and ferric iron in the water-soluble oxidation products of iron sulphide minerals.

A pseudo-kinetic method has been developed for determining the ferrous and ferric iron in the water-soluble oxidation products of pyrrhotite, pyrite and chalcopyrite, and ores and concentrates containing them. Two determinations are required for each material. In one, the total iron is determined with 1,10-phenanthroline after reduction to Fe(II). In the other, the reduction of Fe(III) is retarded by complexation with fluoride. The difference in the amount of ferrous phenanthranoline complex produced in these two determinations is a function of the original FE(III) concentration and of time.

Effect of EDTA/NaF solutions on the orion Cu(II) ion-selective electrode.

The titration of ethylenediaminetetra-acetic acid in fluoride medium with Cu(II) solution with the Orion Cu(II) ion-selective electrode as indicator, results (after several titrations) in a broad end-point which is unsatisfactory for exact analytical purposes. This effect has been found to arise from an enhanced rate of response to changes in EDTA concentration when the electrode has been exposed to an EDTA/NaF medium for prolonged periods.

Titrimetric determination of aluminium in zinc-aluminium alloys, with edta and a cu(II)-selective electrode.

The end-point for the titration of EDTA with Cu(II), as measured by a Cu(II)-selective electrode, varies with pH and temperature. Moreover, the effect of pH and temperature on the behaviour of this electrode differs according to whether fluoride is present. As a consequence, the determination of aluminium in zinc-aluminium alloys by the Freegarde and Allen method with use of a Cu(II)-selective electrode must be performed with close control of pH and temperature to maximize accuracy and repeatability.

Oxidation of sulphide minerals-III determination of sulphate and thiosulphate in oxidised sulphide minerals.

A method has been developed to determine sulphate and thiosulphate in small amounts of the oxidation products of sulphide minerals. The sample is treated with ammonium sulphide solution to promote ion-exchange between sulphide ion and the sulphur-bearing anions of the oxidation products. Sulphate is determined alone and then all other sulphoxy anions are oxidized to sulphate and determined as such. The non-sulphate anions are reported as thiosulphate. The relative error is about 10% or less for 2 mg or more of sulphoxy anion. Although this method does not yield exact results with respect to sulphite or polythionates, a clearer understanding of the oxidation of sulphide minerals is now available.