Emmett J. Johnson

AIDS epidemic at the beginning of the third millennium: time for a new AIDS vaccine strategy.

Current expansion of AIDS pandemic significantly accelerates AIDS vaccine research resulting in development and clinical testing of several AIDS vaccine candidates. At the same time, available experimental and clinical data demonstrate that current AIDS vaccine strategy is unsuccessful resulting in development of inefficient and harmful vaccines. This overview briefly summarizes reported results which point out the requirement for moratorium on the current clinical trials of HIV-1 gp120/160 vaccines and urgent need for development of a new, efficient and safe AIDS vaccine strategy.

Regulation of pyruvate kinase from Thiobacillus neapolitanus

Abstract The effects on pyruvate kinase activity of 23 metabolites and EDTA were studied in extracts of the chemoautotroph Thiobacillus neapolitanus. AMP and four derivatives of the reductive pentose phosphate cycle (ribose-5-phosphate, ribulose-5-phosphate, fructose-6-phosphate, and glucose-6-phosphate) were effective activators at 0.5 m m . ATP and three derivatives of the tricarboxylic acid cycle (fumarate, oxoglutarate, and succinate) were effective inhibitors at 2.5 m m . Fructose-1,6-diphosphate and cyclic-3′,5′-AMP had no effect on pyruvate kinase activity. Metabolite effects depended on the phosphoenolpyruvate concentration, but were relatively independent of the ADP concentration. Hyperbolic velocity versus concentration data were obtained with ADP as the variable substrate while sigmoid velocity versus concentration data were obtained with phosphoenolpyruvate as the variable substrate. Fumarate (1.0 m m ) increased the sigmoidicity, while ribose-5-phosphate (0.5 m m ) converted the kinetics to hyperbolic. The level of phosphoenolpyruvate resulting in half-maximal activity was 0.5 m m . This was altered to 0.1 m m in the presence of ribose-5-phosphate (0.5 m m ) or to 0.75 m m in the presence of fumarate (1.0 m m ). The fumarate concentration resulting in half the maximal effect was 1.5 m m while that for ribose-5-phosphate was 0.05 m m . It is apparent that pyruvate kinase in this autotroph is a metabolic control point intricately connected with other regulatory circuits.

Allosteric regulation of phosphoribulokinase activity.

Inhibition of ATP-dependent CO2 fixation by AMP has been reported in several autotrophic organisms (Johnson and Peck, 1965; Mayeux and Johnson, 1966;Johnson, 1966; Gale and Beck, 1966). Johnson (1966) suggested that the site of AMP inhibition is phosphoribulokinase and that inhibition may occur through allosteric modification of the enzyme. In contrast, Gale and Beck (1966) presented data indicating that AMP inhibition of ATP-dependent CO2 fixation in extracts of Thiobacillus ferrooxidans is competitive with ATP. The studies reported below suggest that in Thiobacillusthioparus phosphoribulokinase is cooperatively affected by ATP and that inhibition of the enzyme by AMP may not be solely competitive.

Identification of an active Chi recombinational hot spot within the HIV-1 envelope gene: consequences for development of AIDS vaccines

Because of a sequence similarity between the HIV-1 envelope glycoprotein gp120 and the variable region of human immunoglobulins, we have suggested that the use of this protein as a vaccine component could strongly influence the host immune system, making it more vulnerable to HIV, and in the long term, accelerate disease progression in asymptomatic HIV patients. Using a chimeric primer consisting of the nucleotide sequence derived from the HIV-1 env gene coding for the second conserved region of gp120, and the highly conserved sequence derived from the human immunoglobulin gene coding for the V(H)III domain, we have identified in sera of AIDS patients HIV-1 field isolates carrying the complete and active Chi recombinational hot spot (GCTGGTGG). We have also demonstrated in vivo recombination between the HIV-1 gene coding for the central portion of the gp120 involving the V3 loop and the bacterial gene coding for the clp protease. These results strongly support and reinforce the previous contention and the serious concern that AIDS vaccine candidates carrying the HIV-1 env gene on viral and bacterial vectors, could result in the generation of new pathogens with unpredictable effects on the immune system.

Molecular basis of the inefficacy and possible harmful effects of AIDS vaccine candidates based on HIV-1 envelope glycoprotein gp120.

Abstract Molecular bases of possible harmful effects of AIDS vaccine candidates based on HIV-1 gp120 160 , that are prepared for Phase III clinical trials in developing countries, have been considered.

HIV retrotransposon activity and the immunopathogenesis of AIDS

This article presents the new hypothesis that HIV retrotransposon insertional mutagenesis induces genomic effects that bring about immune dysfunction through disruption, deletion or rearrangement of the genome of the host. This activity may be augmented by the action of most, if not all, the cofactors of HIV-induced disease.

Adenylate kinase from Thiobacillus Neapolitanus: Unique properties, possibly designed to serve a unique metabolic function

Abstract Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from Thiobacillus neapolitanus was purified to near homogeneity as evidenced by the observance of only 2 protein bands in the final preparation after disc gel electrophoresis at pH 8.3. This purified preparation was investigated to determine if the enzymes unique role in this organism might be reflected in unusual properties or sensitivities. Mol. wt 23 500; pI, pH 4.2; Km(ADP), 5.5 · 10−4M; Km(ATP), 1.1 · 10−4 M; Km(AMP), 1.3 · 10−4 M; optimal pH (forward), pH 10.0; optimal pH (reverse), pH 8.0; optimal Mg2+] (forward) = 1 2 [ADP], and reverse = 1 2 [ATP]; were determined. When compared with the properties of highly purified enzymes from other sources the forward optimal pH, optimal Mg2+:ATP ratio and a protective association of the enzyme with endogenous cythochrome c were found to be unusual. The highly alkaline pH optimum for the conversion of ADP → ATP parallels that of 9.5 for cytochrome c-compled APS reductase, another key enzume of the sulfur oxidation pathway. It is suggested that the inhibition of the reverse reaction by a high Mg2+; ATP ratio may insure the continued operation of the forward reaction and the sulfur oxidation pathway when the free ATP concentration is low and the Mg2+ concentration high. The enzyme appears to respond sharply to changes in pH, Mg2+ concentration and the presence of endogenous cytochrome c. Futute investigation of other adenylate kinases which operate far from equilibrium may reveal comparable sensitivities.

Regulation in the Chemolithotroph Thiobacillus Neapolitanus: Fructose-1,6-Diphosphatase

SummaryPartially purified fructose diphosphatase from the obligate chemolithotroph,Thiobacillus neapolitanus has been characterized, and some of its regulatory properties described. The enzyme had a high effinity for its substrate, but was inhibited by substrate at concentrations above 1 mM. The enzyme had an absolute requirement for a divalent cation. In the absence of EDTA there was a single pH optimum in the alkaline range between 8.5 and 9.5; in the presence of EDTA there was considerable was activity at both neutral and alkaline pH. This diphosphatase was inhibited by AMP at 10−4 M or greater-, the lower the pH, the greater the AMP inhibition. Treatment of the enzyme with 5×10−5 Mpara hydroxy mercuribenzoate allowed retention of full catalytic activity while abolishing considerable AMP inhibition. Exposure of the enzyme to several concentrations of urea had no effect on the AMP inhibition. Homocystine (0.06 mM) and coenzyme A (0.1 mM) had no effect. At 1 mM, PEP caused 60% inhibition, 2, 3-diphosphoglyceric acid produced 26% inhibition, and pyruvate had no effect.

A comparison of the NADH oxidase electron transport systems of two obligately chemolithotrophic bacteria.

Abstract 1. The NADH oxidase electron transport systems of two obligate autotrophs were investigated. Cytochromes c547, c550, c552, b or c554, b558 and a were found in Thiobacillus neapolitanus, and cytochromes c549, 551, c or b555, a1, and a or a3 in Thiobacillus thioparus. A soluble cytochrome c552 not present in the particulate fractions was detected in T. neopolitanus. Low potential c-type cytochromes were found in both organisms. NADH reduced both cytochromes c547 and c550 in the large particle fraction of T. neapolitanus, but only c550 in the small particle fraction. 2. Both organisms contained the ubiquinone, Q-8. The levels of flavin, quinone, and cytochrome c were comparable to those of heterotrophic bacteria. No naphthoquinone was detected. 3. The levels of NADH and ascorbate oxidases were similar to those of heterotrophic bacteria, while NADH dehydrogenase and ascorbate: N,N,N′,N′-tetramethyl-p-phenylenediamine·.2HC1 (TMPD) oxidase levels were higher. In T. Thioparus, NADH oxidase activity was located exclusively in the large-particle fraction, and in T. neapolitanus in both the large- and small-particle fractions. 4. The NADH oxidase activities of both organisms were sensitive to inhibitors usually employed in studies of electron transport. NADH oxidase of T. thioparus was completely inhibited by KCN, while that of T. neapolitanus was never inhibited by more than 80%. Ascorbate and ascorbate: TMPD oxidases were sensitive to KCN but insensitive to 2-heptyl-4-hydroxyquinoline-N-moxide. 5. Electron transport pathways are proposed for both organisms.

Anatomical locus of the common enterobacterial antigen.

Cell walls, membrane vesicles and the soluble cytoplasmic components have been isolated from Escherichia coli 0:14 and Salmonella typhimurium and examined by electron microscopy as well as hemagglutination inhibition with heterologous antisera for enterobacterial common antigen (ECA). The outer and cytoplasmic membranes also were isolated and examined for common antigen. Although common antigen was present in the isolated surface structures it was not localized exclusively in these fractions. Additional experiments revealed that, whatever the nature of the integration of ECA of Kunin into the cell envelope, it is a much more stable association in E. coli 0:14 than in S. typhimurium.