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Biomedical subjects

J B Jackson

Publications and source records attributed to J B Jackson.

At least 109 records · Page 6Linked to original sources
PubMed1994

Quality assurance and use of PCR in clinical trials.

Qualitative and quantitative HIV-1 DNA and RNA PCR assays are proving to be useful in the diagnosis of HIV-1 infection in infants and in assessing the in vivo antiviral activity of new therapies and regimens in clinical trials. The use of these standardized commercial assays in conjunction with an external quality assurance program has ensured that results from different laboratories are comparable. In addition, real-time proficiency monitoring has the potential to detect problems immediately before patient data are compromised.

Clinical Trials as Topic↗
PubMed1994

A phase I/II trial of intravenous L-2-oxothiazolidine-4-carboxylic acid (procysteine) in asymptomatic HIV-infected subjects.

Twenty-four asymptomatic, HIV-1-seropositive subjects with CD4 cell counts of > or = 400/microliters participated in a Phase I/II, dose escalation trial of intravenous L-2-oxothiazolidine-4-carboxylic acid (OTC: Procysteine). Four groups of six subjects each were consecutively assigned to receive OTC at an initial dose of 3, 10, 30, or 100 mg/kg, followed by the same dose given twice weekly for 6 weeks. Increases in whole-blood glutathione were observed in the highest dosage group after 6 weeks of therapy. No effects on changes in CD4 cell counts, viral load, or proviral DNA frequency were observed among the four dosage groups, although a decline in beta 2-microglobulin levels was apparent in the highest dosage group. One subject withdrew due to headaches; other probable adverse events including rash, flushing, pruritus, lightheadedness, and diminished concentration were self-limited.

Adult↗
PubMed1993

The ratio of protons translocated/hydride ion equivalent transferred by nicotinamide nucleotide transhydrogenase in chromatophores from Rhodospirillum rubrum.

The reduction of acetylpyridine adenine dinucleotide (AcPdAD+, an NAD+ analogue) by NADPH, in chromatophores treated with valinomycin, was accompanied by alkalinisation of the external medium, as measured by the absorbance change of added cresol red, a simple, non-binding pH indicator. Experiments with a stopped-flow spectrophotometer showed that initial (linear) rates of alkalinisation persisted for 1-2s. From the results of experiments in which H+ uptake was driven by a series of short flashes of light, the dependence of the outward proton leak on the extent of H+ uptake was established. Thus, the proton leak was subtracted from the initial rate of alkalinisation during transhydrogenation to give the true proton-uptake rate. The correction factor was usually about 10%. The ratio of protons translocated/H- transferred from NADPH to AcPdAD+ (the H+/H- ratio) was 0.60 +/- 0.06. The transhydrogenation reaction between NAD+ and NADPH was measured in the presence of a regeneration system for NAD+ (pyruvate and lactate dehydrogenase). In addition to the accompanying proton-translocation reaction, scalar H+ consumption linked to the regeneration system was observed and permitted internal checks on the calibration of the cresol red absorbance changes. After correction for the proton leak and scalar proton uptake, an H+/H- ratio of 0.60 +/- 0.30 was calculated from the initial rates. The water-soluble polypeptide of transhydrogenase (Ths) was washed from a sample of chromatophores to inhibit transhydrogenation activity and the accompanying H+ uptake. Re-addition of purified Ths to depleted chromatophores led to recovery of transhydrogenation activity and of H+ uptake. In this reconstituted system the H+/H- was similar to that in the native membranes. These results make it unlikely that the H+/H- ratio is artefactually low because chromatophores have a population of transhydrogenase which is not coupled to proton translocation. Further evidence that the mechanistic H+/H- ratio of chromatophore transhydrogenase is less than 1 was provided by an analysis of the kinetics of alkalinisation of the medium during reduction of AcPdAD+ by NADPH. It was shown that the progress of the transhydrogenation-induced alkalinisation was fitted by the sum of H+ uptake (the rate of transhydrogenation multiplied by the H+/H- ratio) plus the H+ leak, when the ratio was 0.6 but not when it was 1.0. The results are discussed in terms of the possible mechanism of energy coupling by transhydrogenase.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial Chromatophores↗
PubMed1993

Electrochromic responses of carotenoid absorbance bands in purified light-harvesting complexes from Rhodobacter capsulatus reconstituted into liposomes.

Light-Harvesting Complexes I and II (LHI and LHII) were extracted from chromatophores of Rhodobacter capsulatus, purified in Triton X-100 and reconstituted into phospholipid vesicles. Application of membrane potentials (K+ diffusion potentials) to LHII proteoliposomes led to absorbance changes in the carotenoid bands which were spectrally similar to those in chromatophores. These (electrochromic) absorbance changes were linear with the applied membrane potential between -107 mV and +105 mV. The data were consistent with the existence of two forms of carotenoid in LHII. One form, comprising 2/3 of the total and with a long wavelength absorbance maximum at 510 nm, was not significantly affected by membrane potential. The other component, comprising 1/3 of the total and with a long wavelength absorbance maximum at 516.5 nm, was shifted by approx. 1.6 nm to the red by a membrane potential of 105 mV. Reduction of the B800 bacteriochlorophyll in LHII with NaBH4 before reconstitution did not affect the absorbance spectrum of the carotenoids and it did not affect their response to applied membrane potentials in proteoliposomes. Although the electrochromically-sensitive carotenoids might be associated with B800, interactions with the bacteriochlorophyll are perhaps not the cause of the polarisation of the carotenoid that is responsible for the linearity of the response. The carotenoids in reconstituted LHI complexes were not detectably electrochromic. The electrochromic absorbance changes of carotenoids in LHII could be useful for membrane potential measurement in liposomes containing ion-translocating proteins.

Bacterial Proteins↗
PubMed1993

Electrochromic responses of bacteriochlorophyll absorbance bands in purified light-harvesting complexes of Rhodobacter capsulatus reconstituted into liposomes.

Purified light harvesting complexes I and II (LHI and LHII) from Rhodobacter capsulatus were purified and separately incorporated into liposomes. Electrochromic absorbance changes of bacteriochlorophyll bands in the proteoliposomes in response to K(+)-diffusion potentials were recorded. In LHII proteoliposomes the application of positive-inside potentials led to red shifts in the bacteriochlorophyll absorbance bands centered at 377, 801 and 858 nm. Negative-inside potentials caused blue shifts of these bands. Electrochromism of the 590 nm band was too small to detect. The band at 858 nm was considerably more electrochromic than that at 801 nm. Electrochromic absorbance changes measured at 865-850 nm were linear with the applied diffusion potential. In LHI proteoliposomes positive-inside diffusion potentials caused red shifts of the bands centred at 374 nm and 880 nm. At 880 nm the response was linear with the applied diffusion potential and was equivalent in amplitude to that of the 858 nm band in LHII proteoliposomes. If it is assumed that the permanent dipole moment differences between the ground state and excited state of B800, B850 and B870 are similar and that polarisability effects are negligible, it follows that (a) the plane of the bacteriochlorin ring of B850 is more perpendicular to the membrane plane than that of B800 and (b) the orientations of B850 and B870 relative to the membrane plane are similar.

Bacterial Proteins↗
PubMed1993

Activation and inhibition of mitochondrial transhydrogenase by metal ions.

Mitochondrial transhydrogenase has been reported previously to be inhibited by high, rather non-physiological concentrations (in the range of 2-20 mM) of divalent cations. We show that the enzyme could be activated by low (from about 1 microM to 1 mM) concentrations of Ca2+ and Mg2+, which are within physiological range. These results bring in line the effects observed with mitochondrial enzyme to the findings with bacterial transhydrogenases. The activation of transhydrogenase by divalent cations is interpreted as an increase in affinity of the NADP(H)-binding site of the enzyme-NAD(H) complex. Reported effects of the metal ions could be important for the enzyme function in vivo.

Animals↗
PubMed1993

Inhibition of proton-translocating transhydrogenase from photosynthetic bacteria by N,N'-dicyclohexylcarbodiimide.

The effects of N,N'-dicyclohexylcarbodiimide [(cHxN)2C] on the proton-translocating enzyme, NAD(P) H(+)-transhydrogenase (H(+)-Thase), from two species of phototrophic bacteria have been investigated. The polypeptides of H(+)-Thase from Rhodobacter capsulatus are membrane-associated, requiring detergent to maintain solubility. The enzyme from Rhodospirillum rubrum, however, has a water soluble polypeptide (Ths) and a membrane-associated component (Thm) which, separately, have no activity but which can be fully reconstituted to give a functional complex. Two observations suggest that (cHxN)2C inhibited H(+)-Thase from both species by modification either close to or at the NADP(H)-binding site on the enzyme: (a) the presence of NADP+ or NADPH caused increased inhibition by (cHxN)2C and (b) after treatment of the purified enzyme from Rb. capsulatus with (cHxN)2C, the release of NADP+ became rate-limiting, as evidenced by a stimulated rate of NADPH-dependent reduction of acetylpyridine adenine dinucleotide by NADH. Experiments in which Ths and Thm from R. rubrum were separately treated with (cHxN)2C then reconstituted with the complementary, untreated component revealed that the NADP(H)-enhanced modification by (cHxN)2C was confined to Thm. In contrast to some experiments with mitochondrial H(+)-Thase [Wakabayashi, S. & Hatefi, Y. (1987) Biochem. Int. 15, 667-675], there was no protective effect of either NAD+ or NADH on the inhibition by (cHxN)2C of enzyme from photosynthetic bacteria. However, amino acid sequence analysis of proteolytic fragments of Ths revealed that the NAD(H)-protectable, (cHxN)2C-reactive glutamate residue in mitochondrial H(+)-Thase might be replaced by glutamine in R. rubrum.

Amino Acid Sequence↗
PubMed1993

Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. ACTG PCR Working Group, and the ACTG PCR Virology Laboratories.

An independent quality assurance program has been established by the Virology Committee of the AIDS Clinical Trials Group in the Division of AIDS, National Institute of Allergy and Infectious Diseases, for monitoring polymerase chain reaction (PCR) assays for human immunodeficiency virus type 1 (HIV-1) DNA that are performed by 11 laboratories participating in multicenter clinical trials in the United States. To perform HIV-1 DNA PCR for patients in AIDS Clinical Trials Group protocols, each laboratory was initially certified by correctly testing a coded certification panel consisting of eight well-defined clinical whole-blood specimens and 30 cell pellets containing 0, 2, 5, 10, 20, or 50 8E5/LAV cells per 125,000 uninfected peripheral blood mononuclear cells. PCR was performed by one of two standardized commercial assays for amplification and nonisotopic detection of HIV-1 proviral DNA. For continuing certification, each laboratory must correctly test eight coded whole-blood samples per quarter and run three or four coded cell pellets and HIV-1 DNA copy standards with every PCR assay in real time. The PCR results for the coded pellets on each run are entered into an encrypted computer file, which immediately assesses the validity of the run. To date, 10 of 11 laboratories have correctly tested all HIV-1-positive and -negative samples in the initial certification panel on their first or second attempt. Subsequently, 9 of these 11 laboratories have continued to maintain their certified status. The use of commercial HIV-1 DNA PCR assays and an external quality assurance program have ensured that results from different laboratories are comparable and that problems with sensitivity and specificity are quickly identified.

Certification↗
PubMed1993

Beta 2-microglobulin, HIV-1 p24 antibody and acid-dissociated HIV-1 p24 antigen levels: predictive markers for vertical transmission of HIV-1 in pregnant Ugandan women.

OBJECTIVES: To evaluate the clinical utility of plasma beta 2-microglobulin (beta 2M) levels, acid-dissociated HIV-1 p24 antigen, and HIV-1 p24-antibody titers in predicting HIV-1 vertical transmission in 227 HIV-1-infected Ugandan pregnant women. DESIGN: Plasma beta 2M levels, acid-dissociated HIV-1 p24-antigen positivity, and HIV-1 p24-antibody titers were determined using commercial enzyme immunoassays (EIA) in a Ugandan cohort of 52 HIV-1-seropositive transmitting mothers, 175 HIV-1-seropositive non-transmitting mothers, and 52 seronegative mothers within 6 weeks prior to delivery. RESULTS: Transmitter mothers had significantly higher plasma concentrations of beta 2M (1.80 +/- 1.13 mg/l) than non-transmitter seropositive mothers (1.32 +/- 0.81 mg/l; P = 0.0013). Similarly, a significantly higher proportion of transmitter mothers had detectable p24 antigen than non-transmitter mothers [six out of 51 (11.8%) versus six out of 173 (3.5%); P = 0.03]. Compared with the vertical transmission rate of 23% in the seropositive group, the positive predictive values of a beta 2M level > 1.5 mg/l or detectable HIV-1 p24 antigen for vertical transmission were 34 and 50%, respectively. Five of six (83.3%) seropositive mothers with both a beta 2M level > 1.5 mg/l and detectable p24 antigenemia transmitted HIV-1 infection to their infants compared with 25 of 124 (20.2%) seropositive mothers with values below the cut-off values for both tests (P = 0.00249). However, beta 2M was not found to be a significant independent predictor of vertical transmission when analyzed in a multivariate model with p24 antigenemia. There was no significant difference in HIV-1 p24-antibody titers in transmitter mothers versus non-transmitter mothers (P = 0.299). CONCLUSION: beta 2M levels and acid-dissociated HIV-1 p24-antigen assays may be used to predict which HIV-1-infected pregnant women are at greatest risk for vertical transmission. However, only the p24-antigen test was independently predictive of vertical transmission and its clinical utility is limited.

Adolescent↗
PubMed1993

Detection and quantitation of human immunodeficiency virus type 1 using molecular DNA/RNA technology.

The direct detection and quantitation of human immunodeficiency virus type 1 are becoming increasingly important for diagnosis and viral load determinations in human immunodeficiency virus type 1 infection. Traditional molecular-based assays such as Southern, Northern, and in situ hybridization have had limited application in this area due to the relatively low number of proviral copies per cell, the low percentage of infected cells, and transcriptional dormancy in many of the infected cells. Recent technological advances in polymerase chain reaction assays promise a number of useful applications in the clinical research and clinical laboratory setting.

Acquired Immunodeficiency Syndrome↗
PubMed1992

The relation between the soluble factor associated with H(+)-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli.

Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H(+)-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Ths) and a membrane-bound component are together required for activity. Ths was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH4)2SO4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an Mr of approx. 74,000 under non-denaturing conditions but analysis of the pure protein by SDS-PAGE revealed a single polypeptide, Mr 43,000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatophores and decreased the capacity of Ths to restore activity to depleted membranes. They reacted with a polypeptide of Mr 43,000 in crude cell extract, chromatophore membranes and chromatophore washings but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43,000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the alpha subunit of the E. coli protein. The break between the alpha and beta polypeptides of E. coli transhydrogenase is such that both components are membrane-associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Ths has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Ths and the reported sequence of alanine dehydrogenase from Bacillus but Ths did not have any alanine dehydrogenase activity.

Amino Acid Sequence↗
PubMed1992

Nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus; the H+/H- ratio and the activation state of the enzyme during reduction of acetyl pyridine adenine dinucleotide.

Chromatophores from Rhodobacter capsulatus were incubated in the dark with NADPH and acetylpyridineadenine dinucleotide (AcPdAD+) in the presence of different concentrations of myxothiazol. The transhydrogenase activity was monitored until an appropriate mass action ratio, [AcPdAD+][NADPH]/[AcPdADH][NADP+], was reached. The sample was then illuminated and the initial rate of either AcPdAD+ reduction by NADPH or AcPdADH oxidation by NADP+ was recorded. The ratio of H+ translocated per H- equivalent transferred by transhydrogenase was calculated from the value of the membrane potential (delta pH = 0) at which illumination caused no net reaction in either direction. The mean value for the H+/H- ratio was 0.55. At greater values of [AcPdAD+][NADPH]/[AcPdADH][NADP+] than were employed in the above experiments and over a wider range of concentrations of myxothiazol, it was found that incremental increases in the membrane potential always gave rise to a decrease, never an increase in the rate of AcPdAD+ reduction. In contrast to the H(+)-ATP synthase, there is no evidence of any activation/deactivation of H(+)-transhydrogenase by the protonmotive force.

Membrane Potentials↗
PubMed1992

New testing approaches in transfusion medicine.

A revolution has occurred in the availability of molecular diagnostic technology in the last decade. We are just now beginning to bridge the gap between research and the availability of rapidly licensed clinical assays. Many of these new technologies will be useful in the general practice of transfusion medicine. Some of these techniques may be discarded as impractical. However, many of these methods should prove to be useful in clinical transfusion research and diagnosis.

Blood Transfusion↗
PubMed1991

Cytochrome c2 is essential for electron transfer to nitrous oxide reductase from physiological substrates in Rhodobacter capsulatus and can act as an electron donor to the reductase in vitro. Correlation with photoinhibition studies.

1. Addition of nitrous oxide to a periplasmic fraction released from Rhodobacter capsulatus strains MT1131, N22DNAR+ or AD2 caused oxidation of c-type cytochrome, as judged by the decrease in absorbance at 550 nm. The periplasmic fraction catalysed reduction of nitrous oxide in the presence of either isoascorbate plus phenazine ethosulphate or reduced methyl viologen. The rates with these two electron donors were similar and were comparable to the activity observed with a quantity of cells equivalent to those from which the periplasm sample had been derived. Activity in the periplasm could not be observed with ascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine although this reductant was effective with intact cells treated with myxothiazol to block the activity of the cytochrome-bc1 complex. 2. Cells of R. capsulatus MTG4/S4, a mutant from which the gene for cytochrome c2 has been specifically deleted, did not catalyse detectable rates of nitrous-oxide reduction. A nitrous-oxide reductase activity was present, as shown by activity of both cells and a periplasmic fraction with isoascorbate plus phenazine ethosulphate as reductant. The rates in cells and the periplasmic fraction were similar to those observed in the corresponding wild-type strain (MT1131). In contrast to wild-type cells, 2,3,5,6-tetramethyl-p-phenylenediamine and N,N,N',N'-tetramethyl-p-phenylenediamine [Ph(NMe2)2] were ineffective as mediators of electrons from isoascorbate. Visible absorption spectra showed that no detectable cytochromes in either the periplasm or intact cells of the MTG4/S4 mutant were oxidised by nitrous oxide. 3. Purified ferroycytochrome c2 from R. capsulatus was oxidised by nitrous oxide in the presence of periplasm from R. capsulatus MTG4/S4. The rate of oxidation was proportional to the amount of periplasm added, but was considerably lower than the rate of nitrous-oxide reduction observed with the same periplasmic fraction when either ascorbate plus phenazine ethosulphate or reduced methyl viologen were used as substrates. The oxidation of cytochrome c2 was inhibited by acetylene and by low concentrations of NaCl. 4. Oxidation of ferrocytochrome c2 by nitrous oxide was observed when the purified cytochrome was mixed with a preparation of nitrous-oxide reductase. However, oxidation of ferrocytochrome c' by nitrous oxide was not observed in the presence of the reductase. The observations with the mutant MTG4/S4 suggest that cytochrome c2 is the only periplasmic cytochrome involved in nitrous-oxide reduction. 5. Nitrous-oxide-dependent oxidation of a c-type cytochrome was observed in a periplasmic fraction from Paracoccus denitrificans, provided the fraction was first reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome c Group↗