G. Chang

We have been studying a patient who acquired human immunodeficiency virus (HIV) infection via a blood transfusion 13 years ago. She has remained asymptomatic since that time. The blood donor and two other recipients have all died of AIDS. Although this patient has shown persistently strong seroreactivity to HIV type 1 (HIV-1) antigens by Western blot (immunoblot), she has been continually HIV culture negative in results from multiple laboratories over the last 6 years and has a very low viral burden. Her CD4+ T-cell count has fluctuated around a mean of 399 cells per microliters, with little change in lymphocyte subset percentages. Strong cellular immune responses to HIV-1 epitopes by this patient have been demonstrated. We now report the results of an intensive molecular genetic analysis of the HIV-1 proviral quasispecies from this patient sampled over 5 years. Long terminal repeat region sequences supported the argument for normal basal and Tat-mediated promoter activities. Sequential sequencing of the nef gene revealed a low frequency (8.3%) of defective genes and a striking lack of sequence evolution. Functional analysis of predominant nef genes by both a cell surface CD4 downregulation and a viral infectivity complementation assay showed wild-type function. In contrast, sequential analysis of an amplicon containing the vif, vpr, vpu, tat1, and rev1 genes revealed the presence of inactivating mutations in 64% of the clones. These data suggest that this patient, initially infected with a virulent swarm of HIV-1, is presently infected with a more-attenuated viral quasispecies as a result of effective host immunity.

As determined by EPR, malic enzyme from pigeon liver binds Mn2+ with a half-site stoichiometry of two tight binding sites (KD=6 to 10 mum) per enzyme tetramer and at two to four weak binding sites (KD=0.43 to 1.34 mM). The activation of malic enzyme by Mn2+ at high levels of L-malate shows biphasic kinetics yielding two activator constants for Mn2+. The dissociation constants of Mn2+ for both classes of sites are of the same order as the kinetically determined activator constants of Mn2+, indicating active site binding at both classes of binding sites. The binding of Mn2+ to the tight sites enhances the paramagnetic effect of Mn2+ on 1/T1 of water protons by a factor (epsilon) of 17, while binding at the weak sites yields a smaller epsilon of 11. The coenzymes TPN and TPNH have no effects on epsilon, while the carboxylic acid substrates L-malate and pyruvate and the inhibitors D-malate and oxalate significantly decrease epsilon. TPNH causes a 38-fold tightening of binding of the substrate L-malate to the enzyme-Mn2+ complex, consistent with the previously described highly ordered kinetic scheme, but only a 2-fold tightening of binding of the competitive inhibitor D-malate. The dissociation constant of L-malate from the quaternary E-Mn2+-TPNH-L-malate complex (32 muM) agrees with the Km of L-malate (25 muM), indicating active site binding. The dissociation constants of pyruvate from the ternary E-Mn2+-pyruvate complex (12 mM) and from the quaternary E-Mn2+-TPN-pyruvate complex (20 mM) are similar to the Km of pyruvate (5 mM), also indicating active site binding and a less highly ordered kinetic scheme for the reactions of pyruvate than for those of L-malate. Analysis of the frequency dependence of 1/T1 of water protons indicates that two fast exchanging water ligands remain coordinated to Mn2+ in the binary E-Mn2+ complex. The binding of the substrates L-malate and pyruvate and of the transition state analog oxalate to the E-Mn2+ complex decrease the number of fast exchanging water ligands on Mn2+ by approximately 1, but the binding of D-malate has no significant effect on this parameter, indicating the occlusion or replacement of a water ligand of the enzyme-bound Mn2+ by a properly oriented substituent on C-2 of the substrate. Occlusion rather than replacement of a water ligand by pyruvate is established by studies of 1/T1 of 13COO- and 13CO-enriched pyruvate which indicate second sphere Mn2+ to pyruvate distances of 4.6 A (COO-) and 4.8 A (CO) in the ternary enzyme-Mn2+-pyruvate complex. Formation of the quaternary complex with TPN increases these distances by 0.8 A, indicating the participation of a second sphere enzyme-Mn2+-(H2O)-pyruvate complex in catalysis. Thus, malic enzyme, like five other enzymes which utilize metals to polarize carbonyl groups, forms a second sphere complex with its substrate.

We have studied the sequence and function of the human immunodeficiency virus type 1 (HIV-1) nef genes from nine patients with highly divergent rates of disease progression enrolled in a longitudinal study of HIV disease. Over an average of 7.8 years of follow-up, three patients had net positive changes in CD4+ T-cell counts, three patients had net negative changes in CD4+ T cells but did not develop AIDS, and three patients progressed to AIDS. The nef gene from each of these patients was amplified and cloned, and the sequence of 8 to 10 clones was determined. Only 2 of 88 (2.3%) nef genes recovered from these nine patients were grossly defective. Moreover, there was no relationship between the phylogeny of nef sequences and the corresponding rates of disease progression from these patients. Representative nef genes from all nine patients were tested for their abilities to downregulate cell surface CD4 in a transient-transfection assay. There was no correlation found between the functions of the nef genes from these patients and their corresponding rates of disease progression. We conclude that the nef gene is not a common mediator of the rate of HIV disease progression in natural infection.

This paper presents a simplified forward and backward approach for load flow analysis in radial distribution system. The proposed method includes two phases. At Phase I (forward sweep), the KCL and KVL are used to find the calculated voltage for each bus located at upstream of each line segment or transformer. At Phase II (backward sweep), the linear proportion concept for real and imaginary decomposition is adopted to find the ratios of real and imaginary parts of specified voltage to the calculated voltage at each upstream bus. Then, the voltage at each downstream bus is updated by real and imaginary parts of initial or calculated voltage multiplying with the corresponding ratios, respectively. The solution procedure is terminated after the mismatch of calculated voltage and specified voltage of substation is less than the tolerance value. The proposed method is tested with three IEEE distribution benchmark systems. Results show that the proposed method is effective, computationally robust, and faster than conventional forward/backward sweep and the ladder iteration method.

The RNA genome of the human immunodeficiency virus type 1 (HIV-1) is established as proviral DNA in infected cells. Only some of these cells may actively produce the array of viral RNAs that support progeny virion production. In vivo expression of a subset of proviral genotypes could influence the experimental characterization of the viral quasispecies. We have explored the relationship between DNA and cDNA genotypes of the envelope gene by the molecular cloning and nucleotide sequencing of these templates from noncultivated peripheral blood mononuclear cells from an HIV-1-infected patient. Eleven proviral DNA and nine cDNA clones representing the V1-V3 region of gp120 were recovered and sequenced. The proviral group was more heterogeneous than the cDNA group by nucleotide sequence changes and V1 length polymorphisms. Deduced amino acid sequences from this data set showed that the two groups were distinct in primary structure, in the position of N-linked glycosylation sites, and in the net charge of the V3 loop. The V1-V2 region discriminated between the groups more strongly than the V3 region. The differential representation of HIV-1 envelope genotypes in the cDNA versus the proviral compartment may have important implications for the pathogenesis of disease and for the design of antiviral therapeutics.