Immunological and virological analyses of persons infected by human immunodeficiency virus type 1 while participating in trials of recombinant gp120 subunit vaccines

We have studied 18 participants in phase I/II clinical trials of recombinant gp120 (rgp120) subunit vaccines (MN and SF-2) who became infected with human immunodeficiency virus type 1 (HIV-1) during the course of the trials. Of the 18 individuals, 2 had received a placebo vaccine, 9 had been immunized with MN rgp120, and seven had been immunized with SF-2 rgp120. Thirteen of the 18 infected vaccinees had received three or four immunizations prior to becoming infected. Of these, two were placebo recipients, six had received MN rgp120, and five had received SF-2 rgp120. Only 1 of the 11 rgp120 recipients who had multiple immunizations failed to develop a strong immunoglobulin G antibody response to the immunogen. However, the antibody response to rgp120 was transient, typically having a half-life of 40 to 60 days. No significant neutralizing activity against the infecting strain was detected in any of the infected individuals at any time prior to infection. Antibody titers in subjects infected despite vaccination and in noninfected subjects were not significantly different. Envelope-specific cytotoxic T-lymphocyte responses measured after infection were infrequent and weak in the nine vaccinees who were tested. HIV-1 was isolated successfully from all 18 individuals. Sixteen of these strains had a non-syncytium-inducing (NSI) phenotype, while two had a syncytium-inducing (SI) phenotype. NSI strains used the CCR5 coreceptor to enter CD4+ cells, while an SI strain from one of the vaccinees also used CXCR4. Viruses isolated from the blood of rgp120 vaccinees were indistinguishable from viruses isolated from control individuals in terms of their inherent sensitivity to neutralization by specific monoclonal antibodies and their replication rates in vitro. Furthermore, genetic sequencing of the env genes of strains infecting the vaccinees did not reveal any features that clearly distinguished these viruses from contemporary clade B viruses circulating in the United States. Thus, despite rigorous genetic analyses, using various breakdowns of the data sets, we could find no evidence that rgp120 vaccination exerted selection pressure on the infecting HIV-1 strains. The viral burdens in the infected rgp120 vaccine recipients were also determined, and they were found to be not significantly different from those in cohorts of placebo-vaccinated and nonvaccinated individuals. In summary, we conclude that vaccination with rgp120 has had,to date, no obvious beneficial or adverse effects on the individuals we have studied.     

Sulfation of endogenous compounds and xenobiotics--interactions and function in health and disease

Sulfation is a major detoxication mechanism for endogenous compounds and xenobiotics performed by a family of sulfotransferase isoenzymes. Understanding the normal cellular functions of these different sulfotransferases and the way in which endogenous and exogenous factors are able to influence their activity and expression will provide us with the information necessary to develop novel therapeutic strategies for conditions where sulfation may be implicated. This concept is discussed and is illustrated by examples including adverse drug reactions, fetal development and cancer.     

Effects of environmental conditions on food consumption in female and male rats

The present experiment examined food and water consumption under different housing conditions in 20 female and 20 male Wistar rats. Food and water consumption were measured for 6 h a day following an 18-h same-sex crowded or individual housing period for each of 6 days. All subjects were individually housed during the 6-h measurement period and had access to food and water. Female rats consumed more food and water than did male rats during the 6-h period, regardless of their 18-h housing condition. In addition, previously crowded rats consumed more food and water during the 6-h period than did rats that were previously individually housed. During the 18-h period, when subjects were differentially housed, males consumed more food and water than did females; crowded rats ate less than did individually housed rats; and crowded rats drank more water than did individually housed rats. Based on plasma corticosterone data, the female and male rats were differentially affected by housing conditions. The present results are discussed with regard to housing conditions per se and sex differences in stress responses to housing.     

Multiwavelength spectrophotometric determination of acid dissociation constants of ionizable drugs

A multiwavelength spectrophotometric approach has been developed to determine acid dissociation constants (pKa values) of sparingly soluble drug compounds. UV absorption spectra of the drug solution are acquired using a versatile device based on a fiber optics dip probe, a light source and a photodiode array (PDA) detector while the PH and the ionic strength of the chemical system is manipulated precisely by means of a commercially available titrator. Target factor analysis (TFA) has been applied to deduce the pKa values from the multiwavelength UV absorption data recorded at different pH values. We have called this multiwavelength approach the WApH technique because the pKa results are determined from changes in Wavelength and Absorbance as a function of pH (WApH). The WApH technique is exemplified by using several pure drugs, namely, niflumic acid, nitrazepam, pyridoxine, quinine and terbutaline. The pKa values obtained agree well with those derived from pH-metric titrations. It has been demonstrated that the WApH technique is able to deduce pKa values with high accuracy even if the absorption spectra of the reacting species are very similar.     

Sequence and expression of DmMKLP1, a homolog of the human MKLP1 kinesin-like protein from Drosophila melanogaster

We have isolated the Drosophila gene DmMKLP1, which has a high similarity to members of the mitotic kinesin-like subfamily of kinesin proteins. DmMKLP1 has no known close relatives in the Drosophila genome and can therefore be assumed to be the ortholog of human MKLP1 and hamster CHOI kinesin-like proteins. In situ hybridization reveals a homogeneous maternal expression in the early embryo and a terminally restricted expression pattern at blastoderm stage. Later, the expression becomes increasingly restricted to the developing central nervous system, where it remains expressed at least until the end of embryogenesis.     

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.     

Synthesis and bioreductive potential of a mono N-oxide derivative of the alkylating agent chlorambucil

Chlorambucil N-oxide (CHLN-O) was synthesized and evaluated for in vitro bioreductive antitumor activity. A time-dependent hypoxic differential was observed when EMT6 cells were exposed to CHLN-O in the presence of rat liver microsomes and reducing equivalents. The cytotoxicity of the N-oxide was potentiated under hypoxia, and augmented further by a combination of low pH and hypoxia. Metabolic studies were also undertaken, which utilized previously described HPLC methodology for the analysis of CHLN-O loss from biological fluids. These demonstrated the requirement for microsomal enzymes and reducing equivalents, and also illustrated the time-dependent manner of CHLN-O loss from isolated microsomal preparations.