Biomedical subjects

J B Jackson

Publications and source records attributed to J B Jackson.

At least 91 records · Page 5Linked to original sources

PubMed1996

Prognostic value of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels in patients with advanced HIV-1 disease and with little or no prior zidovudine therapy. AIDS Clinical Trials Group Protocol 116A/116B/117 Team.

The association of plasma human immunodeficiency virus type 1 (HIV-1) RNA level at study entry and over time with clinical progression was evaluated in 187 patients from AIDS Clinical Trials Group protocol 116A who had little or no prior zidovudine treatment. Three-fold-higher HIV-1 RNA levels at study entry and 3-fold increases by week 8 were associated with progression (relative hazard [RH], 1.67; 95% confidence limits [CL], 1.20, 2.32; and RH, 1.45; CL, 1.02, 2.05, respectively). Having 3-fold-higher CD4 cell count at entry was independently associated with a 52% reduction in risk for progression (adjusted RH, 0.48; CL, 0.33, 0.70). When stratified by length of prior zidovudine therapy, RNA level was predictive in drug-naive patients (adjusted RH, 1.87; CL, 1.23, 2.85) but not predictive in patients with up to 16 weeks of prior therapy (adjusted RH, 1.11; CL, 0.70, 1.76). Analysis suggests that the acquisition of mutations at HIV-1 reverse transcriptase codons 215 and 74 is associated with subsequent increases in HIV-1 RNA level (relative risk, 7.00; CL, 0.86, 56.90).

Acquired Immunodeficiency Syndrome↗

PubMed1996

Association of plasma human immunodeficiency virus type 1 RNA level with risk of clinical progression in patients with advanced infection. AIDS Clinical Trials Group (ACTG) 116B/117 Study Team. ACTG Virology Committee Resistance and HIV-1 RNA Working Groups.

Human immunodeficiency virus (HIV)-1 RNA level in plasma was evaluated as a surrogate marker for disease progression in a clinical trial of advanced HIV-1 infection. Baseline HIV-1 RNA level was an independent predictor of disease progression (relative hazard [RH] for each doubling of HIV-1 RNA level, 1.26; 95% confidence interval [CI], 1.03-1.54; P = .02), after adjusting for the week 4 change in HIV-1 RNA level, baseline CD4 cell count, syncytium-inducing phenotype, clinical status at study entry, and therapy randomization. A 50% reduction in HIV-1 RNA level was associated with a 27% decrease in the adjusted risk of disease progression during the study (RH, 0.73; 95% CI, 0.52-1.02; P = .07). The partial validation of HIV-1 RNA as a predictor for clinical end points has implications for the use of HIV-1 RNA in clinical trials and practice.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Pentoxifylline therapy in human immunodeficiency virus-seropositive persons with tuberculosis: a randomized, controlled trial.

Macrophage activation and tumor necrosis factor-alpha (TNF-alpha) production are critical in tuberculosis immunity but may result in increased human immunodeficiency virus (HIV) expression and accelerated HIV disease progression in HIV-infected persons. Pentoxifylline inhibits expression of TNF-alpha and HIV. A double-blind, placebo-controlled study of adjunctive therapy with pentoxifylline (1800 mg/day) as a timed-release formulation was done in Ugandan HIV-infected patients with pulmonary tuberculosis. Subjects had early HIV disease (mean CD4 cell count, 380/microL) and did not receive other antiretroviral drugs. Pentoxifylline resulted in decreased plasma HIV RNA and serum beta 2-microglobulin and, in a subset of moderately anemic patients, improved blood hemoglobin levels. Trends were noted toward reduced TNF-alpha production in vitro and improved performance scores, but these did not reach statistical significance. No effect was noted on body mass, CD4 cell count, or survival. Additional studies of more potent TNF-alpha inhibitors in HIV-positive subjects with tuberculosis are warranted.

Adult↗

PubMed1996

Maternal HIV-1 RNA serum levels at delivery and vertical transmission in Uganda.

OBJECTIVE: To evaluate the clinical utility of maternal HIV-1 RNA serum levels at delivery in predicting the rate of HIV-1 vertical transmission. DESIGN AND METHODS: HIV-1 RNA levels were determined by the Roche Amplicor Monitor assay in serum specimens collected at the time of delivery from 94 transmitting and 107 nontransmitting infected mothers and 12 seronegative mothers in Uganda. Nonparametric Wilcoxon-Rank sum tests were used to identify significant differences in medians and RNA level distributions by transmission status. RESULTS: Mean HIV-1 RNA copies/mL for transmitters was 3419 +/- 7489 copies/mL versus 2483 +/- 8954 copies/mL for nontransmitters. There was a significant difference in medians and HIV-1 RNA serum level distributions between transmitting and nontransmitting mothers (p = 0.0039). However, the predictive value for any given HIV-1 RNA level for HIV-1 vertical transmission was poor. CONCLUSION: Maternal HIV-1 RNA serum levels at delivery are significantly higher in transmitting mothers versus nontransmitting mothers, but appear to be of limited value in predicting HIV-1 vertical transmission using the Roche Amplicor Monitor assay in Uganda.

Case-Control Studies↗

PubMed1996

Detection of human immunodeficiency virus type 1 (HIV-1) DNA and p24 antigen in breast milk of HIV-1-infected Ugandan women and vertical transmission.

OBJECTIVE: To determine the correlation between the detection of human immunodeficiency virus type 1 (HIV-1) in breast milk, the duration of breastfeeding, and vertical transmission of HIV-1 infection in Ugandan women. METHODS: A prospective study of HIV-1 infection in pregnant Ugandan women and their infants has been ongoing since 1990 with follow-up of mother-infant pairs for at least 2 years. Expressed breast milk specimens were collected from 201 HIV-1-seropositive and 86 HIV-1-seronegative Ugandan women approximately 6 weeks after delivery. The presence of HIV-1 DNA in the cellular fraction of the breast milk was detected by polymerase chain reaction (PCR), and HIV-1 p24 antigen was detected in the cell-free breast milk supernatant using p24 antigen enzyme immunoassay (EIA) after immune complex dissociation (ICD). The duration of breastfeeding and the clinical status of the mothers and their children were recorded. HIV-1 EIA, Western blot, PCR, or p24 antigen detection were used for the determination of the HIV-1 infection status of the children. RESULTS: Of the 201 HIV-1-infected women studied, 47 had HIV-1-infected children, 143 had children who seroreverted, and 11 had children of indeterminate status. Breast milk supernatants were available for ICD p24 antigen testing from 188 of the HIV-1-infected women and none had detectable p24 antigen. Breast milk cell pellets were available and contained amplifiable DNA in 125 of the HIV-1-infected women (20 transmitters, 104 nontransmitters, 1 indeterminate). HIV-1 DNA was detected by PCR in 72% (75/104) of nontransmitters and 80% (16/20) of the transmitters. The duration of breastfeeding by transmitter mothers (15.8 months) was not significantly different from nontransmitter mothers (14.4 months). CONCLUSIONS: No correlation was found between the detection of HIV-1 in breast milk or the duration of breastfeeding and transmission of HIV-1 infection in this study of Ugandan women.

Breast Feeding↗

PubMed1995

Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef-heart mitochondria; implications for the mechanism of proton translocation.

Transhydrogenase from beef-heart mitochondria was solubilised with Triton X-100 and purified by column chromatography. The detergent-dispersed enzyme catalysed the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH, but only in the presence of NADP+. Experiments showed that this reaction was cyclic; NADP(H), whilst remaining bound to the enzyme, was alternately reduced by NADH and oxidised by AcPdAD+. A period of incubation of the enzyme with NADPH at pH 6.0 led to inhibition of the simple transhydrogenation reaction between AcPdAD+ and NADPH. However, after such treatment, transhydrogenase acquired the ability to catalyse the (NADPH-dependent) reduction of AcPdAD+ by NADH. It is suggested that this is a similar cycle to the one described above. Evidently, the binding affinity for NADP+ increases as a consequence of the inhibition process resulting from prolonged incubation with NADPH. The pH dependences of simple and cyclic transhydrogenation reactions are described. Though more complex than those in Escherichia coli transhydrogenase, they are consistent with the view [Hutton, M., Day, J.M., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051] that, also in the mitochondrial enzyme, binding and release of NADP+ and NADPH are accompanied by binding and release of a proton. The enzyme was successfully reconstituted into liposomes by a cholate dilution procedure. The proteoliposomes catalysed cyclic NADPH-dependent reduction of AcPdAD+ by NADH only when they were tightly coupled. However, they catalysed cyclic NADP(+)-dependent reduction of AcPdAD+ by NADH only when they were uncoupled eg. by addition of carbonylcyanide-p-trifluoromethoxyphenyl hydrazone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Conformational dynamics of a mobile loop in the NAD(H)-binding subunit of proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli.

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Ths) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Ths in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Ths specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the one-dimensional 1H-NMR spectra of Ths before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Ths (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Ths with NAD+ provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Ths. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

Alanine Dehydrogenase↗

PubMed1995

The involvement of NADP(H) binding and release in energy transduction by proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli.

Proton-translocating transhydrogenase was solubilised and purified from membranes of Escherichia coli. Consistent with recent evidence [Hutton, M., Day, J., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051], at low pH and salt concentration, the enzyme catalysed rapid reduction of the NAD+ analogue AcPdAD+ by a combination of NADH and NADPH. At saturating concentrations of NADPH, the dependence of the steady-state rate on the concentrations of NADH and AcPdAD+ indicated that, with respect to these two nucleotides, the reaction proceeds by a ping-pong mechanism. High concentrations of either NADH or AcPdAD+ led to substrate inhibition. These observations support the view that, in this reaction, NADP(H) remains bound to the enzyme: AcPdAD+ is reduced by enzyme-bound NADPH, and NADH is oxidised by enzyme-bound NADP+, in a cyclic process. When this reaction was carried out with [4A-2H]NADH replacing [4A-1H]NADH, the rate was decreased by 46%, suggesting that the H- transfer steps are rate-limiting. In simple 'reverse' transhydrogenation, the reduction of AcPdAD+ was slower with [4B-2H]NADPH than with [4B-1H]NADPH when the reaction was performed at pH 8.0, but there was no deuterium isotope effect at pH 6.0. This indicates that H- transfer is rate-limiting at pH 8.0 and supports our earlier suggestion that NADP+ release from the enzyme is rate-limiting at low pH. The lack of a deuterium isotope effect in the reduction of thio-NADP+ by NADH at low pH is also consistent with the view that NADPH release from the enzyme is slow under these conditions. A steady-state rate equation is derived for the reduction of AcPdAD+ by NADPH plus NADH, assuming operation of the cyclic pathway. It adequately accounts for the pH dependence of the enzyme, for the features described above and for kinetic characteristics of E. coli transhydrogenase described in the literature.

Escherichia coli↗

PubMed1995

Properties of the soluble polypeptide of the proton-translocating transhydrogenase from Rhodospirillum rubrum obtained by expression in Escherichia coli.

Transhydrogenase, which catalyses the reduction of NADP+ by NADH coupled to proton translocation across a membrane, may be unique in the photosynthetic bacterium Rhodospirillum rubrum. Unlike the homologous enzyme from animal mitochondria and other bacterial sources, it has a water-soluble polypeptide, which exists as a dimer (Ths), that can be reversibly dissociated from the membrane component [Williams, R., Cotton, N. P. J., Thomas, C. M. & Jackson, J. B. (1994) Microbiology, 140, 1595-1604]. We have expressed the gene for Ths in cells of Escherichia coli under control of the tac promoter and a strong ribosome binding site. The protein, purified by column chromatography, fully reconstituted transhydrogenation activity to everted membrane vesicles of Rhs. rubrum that had been washed to remove Ths. The purified expressed protein was prepared in quantities over 100-fold greater than were obtained from wild-type Rhs. rubrum. The fluorescence spectrum of purified expressed Ths had an intense and unusually short wavelength emission maximum at 310 nm with shoulders at 298 and 322 nm. Time-resolved measurements indicated that the fluorescence decay was almost monoexponential with a lifetime of 5.2 ns. On denaturation with 4 M guanidine hydrochloride, the emission band shifted to 352 nm and decreased in intensity. In the native protein, the fluorophore was relatively inaccessible to quenching solutes, such as iodide ions and acrylamide. It is concluded that the fluorescence emission arises mainly from the single tryptophan residue of Ths (Trp72), which is locked into a rigid conformation and is located in highly non-polar environment. The 310-nm fluorescence of Ths was quenched by NADH, maximally to 46%. The apparent binding constant was 18 microM. The fluorescence of Ths-bound NADH was enhanced relative to the nucleotide in free solution and its emission maximum was shifted to a shorter wavelength (440 nm). These data support previous indications that the NADH binding site is located in domain I of proton-translocating transhydrogenase. Excitation of Ths at 280 nm did not lead to sensitized emission at 440 nm from bound NADH. This indicates that the quenching of fluorescence of Ths by NADH does not result from resonance energy transfer from Trp72 to the bound nucleotide. NAD+, NADP+ and NADPH had little effect on the protein fluorescence. The kinetics of quenching of Ths fluorescence by NADH were examined after mixing in a stopped-flow device. The 'on' rate constant for nucleotide binding was approximately 8 x 10(6) M-1 s-1 and the 'off' constant approximately 150 s-1.

Amino Acid Sequence↗

PubMed1995

Long-term follow-up of blood donors with indeterminate human immunodeficiency virus type 1 results on Western blot.

BACKGROUND: At present, tens of thousands of United States blood donors who are at low risk for human immunodeficiency virus type 1 (HIV-1) infection are indefinitely deferred. These persons are repeatably reactive for HIV-1 antibody in enzyme immunoassay (EIA) and are indeterminate in Western blot. STUDY DESIGN AND METHODS: To determine the significance and persistence of anti-HIV-1 reactivity in plasma from volunteer blood donors with HIV-1-indeterminate Western blots, 66 donors were retested for HIV-1 antibody by the same manufacturers' EIA and Western blot 5 to 7 years after the initial Western blot. In addition, donors' peripheral blood mononuclear cells were tested by polymerase chain reaction (PCR) for HIV-1 DNA gag sequences. RESULTS: Thirty-five (53%) of 66 donors were still repeatedly reactive for HIV-1 on EIA and indeterminate on Western blot, 23 (35%) were negative on EIA and indeterminate on Western blot, 7 (11%) were negative in EIA and Western blot, and 1 (2%) was repeatedly reactive on EIA and negative on Western blot. Donors with persistently indeterminate Western blots had a band pattern nearly identical to that on the original Western blot. No donor was positive in Western blot, p24 antigen, or PCR testing. No donor had signs or symptoms of HIV-1 infection. CONCLUSION: Long-term follow-up of Western blot-indeterminate blood donors does not reveal evidence of HIV-infection. A mechanism to return these donors to the donor pool should be considered.

Base Sequence↗

PubMed1995

Human immunodeficiency virus (HIV) type 1 infection status and in vitro susceptibility to HIV infection among high-risk HIV-1-seronegative hemophiliacs.

Blood samples were obtained from 16 hemophiliacs who had a 50%-94% defined risk of human immunodeficiency virus (HIV) type 1 infection on the basis of treatment history and from 14 controls not at risk for HIV infection. HIV-1 was not detected in any of 12 patient samples by cocultivation nor in 14 patient samples by the polymerase chain reaction. Peripheral blood cells from 7 seronegative hemophiliacs at highest risk of seroconversion (94%) were less susceptible to HIV-1 infection in vitro than were cells from healthy controls (P < .025, one-tailed Wilcoxon rank sum test). In contrast, the susceptibility to HIV-1 infection of lymphocytes from 6 seronegative hemophiliacs at moderate risk (50%-56%) of seroconversion did not differ from that of cells from controls or from high-risk hemophiliacs. Therefore, prolonged periods of seronegative HIV-1 infection are not common in this high-risk population. In addition, among hemophiliacs there may exist heterogeneity in susceptibility to HIV-1 infection in vitro and in vivo.

Adult↗

PubMed1995

Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes.

This study compared the levels of human immunodeficiency virus (HIV) virion RNA in plasma from whole blood collected in VACUTAINER CPT (cell preparation tube), VACUTAINER PPT (plasma preparation tube), VACUTAINER SST (serum separation tube), and standard VACUTAINER tubes with sodium heparin, acid citrate dextrose, sodium citrate, and potassium EDTA used as anticoagulants. Quantitative plasma HIV RNA levels were measured by branched-DNA signal amplification. Blood from all tubes was either processed within 1 to 3 h after collection or stored at room temperature or at 4 degrees C for analysis at 6 to 8 and 30 h postdraw. Immediately separated plasma from sodium citrate CPT tubes held at 4 degrees C maintained better stability of HIV RNA equivalents than whole blood held at room temperature or 4 degrees C. The highest number of HIV RNA equivalents was seen with EDTA VACUTAINER tubes. HIV RNA equivalents in all types of plasma were significantly higher than in SST tubes. Although a decline in HIV RNA equivalents was seen in all collection devices after 30 h, a significantly greater decline in plasma HIV RNA equivalents occurred in acid citrate dextrose VACUTAINER tubes than in citrate CPT, PPT, and standard EDTA VACUTAINER tubes. In order to minimize the variability of quantitative HIV RNA test results, our data suggest that samples collected for a particular assay should be processed at the same time postdraw using a particular tube type throughout a given study.

Anticoagulants↗

PubMed1995

Acute decrease in HIV-1 viral load after initiation of zidovudine therapy: implications for interrupting vertical transmission.

Use of zidovudine (AZT) in pregnant women has recently been reported to reduce the rate of vertical transmission of human immunodeficiency virus type 1 (HIV-1) possibly through a reduction in maternal viral load. To determine how quickly AZT is able to reduce viral load, infectious virus and plasma HIV-1 RNA were sequentially measured in an HIV-1-infected patient at short intervals (hours) after initiation of oral AZT. Peripheral blood samples were collected at baseline, 1, 2, 4, 8, 12, 24, 48 hours, and 1 week after initiation of AZT therapy (500 mg/day) for quantitative plasma HIV-1 RNA levels, p24 antigen levels, and AZT levels. Quantitative HIV-1 peripheral blood mononuclear cell and plasma cultures, CD4 cell counts, and MT-2 cell assays for syncytium-inducing phenotype were performed at baseline, 1, 2 days, and 1 week after initiation of AZT therapy. A significant drop in viral load did not occur until after 24-48 hours. AZT should probably be administered at least 2 days prior to anticipated delivery, if a reduced maternal viral load is responsible for AZT's efficacy in preventing vertical transmission.

CD4 Lymphocyte Count↗

PubMed1995

A critical analysis of the use of radiation inactivation to measure the mass of protein.

Measurements are presented of the radiation inactivation of four enzymes exposed to a 6 MeV proton beam. It has long been thought that the measurement of the susceptibility of an enzyme to ionizing radiation can be used to determine its molecular mass. Results are frequently interpreted using the empirical analysis of Kempner and Macey (Biochim. Biophys. Acta 163, 188-203, 1963). We examine this analysis and discuss the validity and limitations of the assumptions on which it is based. Our results indicate that the specific biochemical properties of each enzyme make a significant contribution to its radiation sensitivity.

Animals↗

PubMed1994

Proton-translocating transhydrogenase and NAD- and NADP-linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria.

H(+)-transhydrogenase (H(+)-Thase) and NADP-linked isocitrate dehydrogenase (NADP-ICDH) are very active in animal mitochondria but their physiological function is only poorly understood. This is especially so in the case of the heart and muscle, where there are no major consumers of NADPH. We propose here that H(+)-Thase and NADP-ICDH have a combined function in the fine regulation of the activity of the tricarboxylic acid (TCA) cycle, providing enhanced sensitivity to changes in energy demand. This is achieved through cycling of substrates by NAD-linked ICDH, NADP-linked ICDH and H(+)-Thase. It is proposed that NAD-ICDH operates in the forward direction of the TCA cycle, but NADP-ICDH is driven in reverse by elevated levels of NADPH resulting from the action of the transmembrane proton electrochemical potential gradient (delta p) on H(+)-Thase. This has the effect of increasing the sensitivity to allosteric modifiers of NAD-ICDH (NADH, ADP, ATP, Ca2+ etc), potentially giving rise to large changes in the net flux from iso-citrate to alpha-ketoglutarate. Furthermore, changes in the level of delta p resulting from changes in the demand for ATP would, via H(+)-Thase, shift the redox state of the NADP pool and this, in turn, would lead to a change in the rate of the reaction catalysed by NADP-ICDH and hence to an additional and complementary effect on the net metabolic flux from isocitrate to alpha-ketoglutarate. Other consequences of this substrate cycle are, (i) the production of heat at the expense of delta p, which may contribute to thermoregulation in the animal, and (ii) an increased rate of dissipation of delta p (leak).

Animals↗

PubMed1994

Kinetic resolution of the reaction catalysed by proton-translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates.

The mechanism, by which transhydrogenase couples transfer of H- equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichia coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP+ or NADPH. This indicates that the rates of release of NADP+ and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP+ and NADPH are accompanied by the release of H+ from the enzyme and (b) increased ionic strength decreases the value of the pKa of the group responsible for H+ release. Modification of the enzyme with N,N1-dicyclohexylcarbodiimide led to inhibition of the rate of release of NADP+ and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD+, NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates. It is considered that the binding and release of H+, which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane-bound state.

Binding Sites↗

PubMed1994

Use of semiquantitative human immunodeficiency virus type 1 culture to guide therapy in a premature infant.

Semiquantitative cultures for human immunodeficiency virus type 1 were used to initiate and guide therapy in an asymptomatic, heavily infected premature infant. The cultures were performed on fivefold serial dilutions of peripheral blood mononuclear cells, and viral growth was detected by the appearance of p24 antigen in the supernatant. While the patient was receiving low-dose zidovudine, her titer diminished from 206 infectious units per 10(6) cells to undetectable levels over 20 weeks, and she remained asymptomatic throughout her 1st year of life. This case suggests that semiquantitative HIV-1 culture should be further evaluated for its ability to guide practical therapeutic decisions in patients who do not fulfill currently established criteria for treatment.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Cloning and sequencing of the genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and the implications for the domain structure of the enzyme.

The genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum have been cloned using a probe constructed with the polymerase chain reaction, genomic DNA as target and oligonucleotide primers corresponding to amino acid sequence obtained from the purified soluble subunit. There is a cluster of three genes, designated pntAA, pntAB and pntB, whose translation products indicate polypeptides of 384, 139 and 464 amino acids, respectively. This contrasts with the situation in the enzymes from Escherichia coli (two polypeptides) and bovine mitochondria (one polypeptide) but there is close similarity between the sequences. PntAA is the soluble subunit of the enzyme from R. rubrum, equivalent to the relatively hydrophilic domain I that forms the N-terminal part of the alpha polypeptide of E. coli transhydrogenase and which probably contains the NAD(H)-binding site. PntAB corresponds to the strongly hydrophobic domain IIa at the C-terminus of the alpha polypeptide of the E. coli transhydrogenase. PntB corresponds to the E. coli beta polypeptide, which comprises the strongly hydrophobic domain IIb and the relatively hydrophilic domain III, thought to contain the NADP(H)-binding site. The peptide bond between PntAA-Lys237 and -Glu238 of both the denatured and the native soluble subunit is very sensitive to proteolysis by trypsin and the neighbouring peptide bond Lys227-Thr228 to cleavage by the endoproteinase Lys-C. Related sites have been reported to be sensitive to trypsin in the E. coli and bovine mitochondrial enzymes. The two tryptic fragments from the native R. rubrum soluble subunit are unable to reconstitute transhydrogenase activity to membranes depleted of the soluble subunit but they can block reconstitution by intact soluble subunit. It is suggested that this protease-sensitive region separates two subdomains and that, after trypsinolysis, at least one retains structural integrity and can dock with domains II and/or III.

Amino Acid Sequence↗