Biological variability of human immunodeficiency virus type-1 (HIV-1) is involved in the pathogenesis of acquired immunodeficiency syndrome (AIDS). Syncytium-inducing (SI) HIV-1 variants emerge in 50 percent of infected individuals during infection, preceding accelerated CD4+ T cell loss and rapid progression to AIDS. The V1 to V2 and V3 region of the viral envelope glycoprotein gp120 contained the major determinants of SI capacity. The configuration of a hypervariable locus in the V2 domain appeared to be predictive for non-SI to SI phenotype conversion. Early prediction of HIV-1 phenotype evolution may be useful for clinical monitoring and treatment of asymptomatic infection.

We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.

It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.

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Four 2,3-oxidosqualene analogs, 3, 4, 5, and 6, which are irreversible, time-dependent inhibitors of the enzyme lanosterol synthase, were found to attach covalently within the 231−236 (yeast numbering) segment (Figure ). The attachment was determined by tryptic digestion of the inactivated enzyme, separation of the tryptic cleavage products by C18 reverse phase HPLC, and fragment identification by mass spectroscopy or Edman degradation. W232 and H234 are the targets of the chemical inactivation by cations derived from analogs 3−6. 2,3-Oxidosqualene analogs 7, 8, and 9 inactivated the enzyme with covalent attachment to the 486−512 segment (Figure ), which is in a domain that is predicted to be an amphipathic α-helix. Site-directed mutagenesis of various amino acid residues (76 total) in lanosterol synthase which are conserved in five different species has revealed that residues D456, H146, and H234 are essential for catalytic activity. These and other data permit the formulation of a hypothetical working model of some aspects of the activation and binding of 2,3-oxidosqualene by lanosterol synthase. The model is depicted in Figure . In that model D456 and protonated H146 initiate cyclization, and the domains containing 231−236 and 486−512 make contact with the reacting substrate.

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis−Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C−6 methyl was replaced by H (3) or Cl (4). The measured V max/K M ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D → N mutation) and each of the nine conserved glutamic acid residues (E → Q) revealed that only one, D456, is essential for catalytic function of the enzyme. The essential D456 residue is a likely candidate for electrophilic (specifically protic) activation of the oxirane function.

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Endotoxin shock has been reported to alter endothelial structure as well as function of large arteries from in vitro experiments. Cremaster muscle arteriolar dilator reactivity of pentobarbital-anesthetized rats was determined by videomicroscopy at control and 30, 90, 150, and 210 min after intravenous infusion of Escherichia coli endotoxin (6 mg/kg, 1-h period). The dilator response was tested by intra-arterial injections of 90 ng acetylcholine (ACh). At control A1, A2, and A3 arterioles dilated 45, 21, and 34%, respectively. Postendotoxin arterial pressure decreased progressively, the A1 arterioles constricted (P < 0.05), A2 diameters were unchanged and A3 diameters increased. Postendotoxin ACh dilations averaged 28, 23, and 25%. A1 dilation was significantly (P < 0.05) less than at control. Methylene blue (2.5 mg ia) attenuated the ACh response at control, but after endotoxin an intense downstream vasoconstriction resulted in stasis and reduced survival time occurred. Hydroquinone (HQ) partially blocked the responses to ACh postendotoxin. HQ significantly increased the survival time postendotoxin. It is evident postendotoxin that the endothelia of arterioles are functional and able to release nitric oxide (NO) throughout the entire survival period. The microvascular release of NO and the dilation response to ACh were substantially attenuated by methylene blue and HQ. The latter may block the more lethal effects of the inducible NO synthase.