Dual infection with HIV-1 and HIV-2.
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HIV Infections↗
Biomedical subjects
D M Mitchell
Publications and source records attributed to D M Mitchell.
Aspects of HIV infection: current infection control policies for HIV.
A coherent infection control policy within healthcare facilities designed for patients with human immunodeficiency virus (HIV) infection requires the application of a risk management strategy. The central feature is the adoption of universal precautions whereby it is assumed that all patients could potentially be infected by HIV. The major tenets of this are the adoption of good clinical hygiene and the adoption of agreed infection control policies with a consistent approach throughout the institution. This involves a teaching and training programme, and clearly defined policies to protect individuals from HIV infection itself and infection with other pathogens, in particular tuberculosis. Special attention is required for bronchoscopy and lung function, as well as a coherent and proactive policy regarding chemoprophylaxis for HIV infection following accidental injury such as needlestick.
Case-Control Studies↗
FTIR study of conformational substates in the CO adduct of cytochrome c oxidase from Rhodobacter sphaeroides.
Fourier transform infrared (FTIR) spectroscopy of cytochrome c oxidase from Rhodobacter sphaeroides reveals multiple CO stretch bands that are associated with different conformational substates of the enzyme. Here we report the temperature dependence of the infrared bands for the CO bound to the Fea3 heme iron and to CuB. We have also studied the kinetics of ligand return from Fea3 to CuB using temperature derivative spectroscopy (TDS). Two classes of substates (alpha/beta) can be distinguished from their different properties with regard to the width of the IR band, the temperature dependence of the peak position, and the peak of the enthalpy distribution. The pronounced temperature dependence of the stretch frequencies in the beta conformation and the lack thereof in the alpha conformation implies very different dynamic behavior in the active site and reflects structural differences between the two conformations, most likely a shift of the position of CuB in response to a change in its stereochemical environment. Similar conformational changes will be necessary during the catalytic cycle of the enzyme when dioxygen is bound in the active site.
Binding Sites↗
Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.
Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.
Carbon Monoxide↗
Polar residues in helix VIII of subunit I of cytochrome c oxidase influence the activity and the structure of the active site.
The aa3-type cytochrome c oxidase from Rhodobacter sphaeroides is closely related to eukaryotic cytochrome c oxidases. Analysis of site-directed mutants identified the ligands of heme a, heme a3, and CuB [Hosler et al. (1993) J. Bioenerg. Biomembr. 25, 121-133], which have been confirmed by high-resolution structures of homologous oxidases [Iwata et al. (1995) Nature 376, 660; Tsukihara et al. (1995) Science 269, 1069; (1996) 272, 1136]. Since the protons used to form water originate from the inner side of the membrane, and the heme a3-CuB center is located near the outer surface, the protein must convey these substrate protons to the oxygen reduction site. Transmembrane helix VIII in subunit I is close to this site and contains several conserved polar residues that could function in a rate-determining proton relay system. To test this role, apolar residues were substituted for T352, T359, and K362 in helix VIII and the mutants were characterized in terms of activity and structure. Mutation of T352, near CuB, strongly decreases enzyme activity and disrupts the spectral properties of the heme a3-CuB center. Mutation of T359, below heme a3, substantially reduces oxidase activity with only minor effects on metal center structure. Two mutations of K362, approximately 15 A below the axial ligand of heme a3, are inactive, make heme a3 difficult to reduce, and cause changes in the resonance Raman signal specific for the iron-histidine bond to heme a3. The results are consistent with a key role for T352, T359, and K362 in oxidase activity and with the involvement of T359 and K362 in proton transfer through a relay system now plausibly identified in the crystal structure. However, the characteristics of the K362 mutants raise some questions about the assignment of this as the substrate proton channel.
Amino Acid Sequence↗
A pH-dependent polarity change at the binuclear center of reduced cytochrome c oxidase detected by FTIR difference spectroscopy of the CO adduct.
A pH-dependent polarity change at the heme-copper binuclear center of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been identified by low-temperature FTIR difference spectroscopy. "Light"-minus-"dark" FTIR difference spectra of the fully reduced CO-enzyme adduct were recorded at a range of pH, and the dominance of different populations of bound CO, alpha and beta, was found to vary with pH. An apparent pKa of about 7.3 for the transition was obtained. The alpha and beta forms are differentiated by different polarities at the heme-copper binuclear center of the enzyme, sensed by the stretching frequencies of CO bound either to the heme alpha 3 Fe or to CuB. Several site-directed mutants in the vicinity of the heme-copper center are shown to favor either the alpha or the beta forms of the enyzme, suggesting that what is being monitored is an equilibrium between two conformations of the reduced form of the oxidase. Recent resonance Raman evidence has been presented demonstrating that the alpha and beta forms of the R. sphaeroides oxidase exist at room temperature; therefore, the pH-dependent change in the polarity in the vicinity of the heme-copper center may be functionally significant.
Amino Acid Sequence↗
A ligand-exchange mechanism of proton pumping involving tyrosine-422 of subunit I of cytochrome oxidase is ruled out.
The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However, several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine residue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a3 during the catalytic cycle, allowing both residues to serve as part of a proton transporting system. Site-directed mutants of Y422 have been constructed in the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides to test this hypothesis (Y422A, Y422F). The results demonstrate that Y422 is not an essential residue in the electron transfer and proton pumping mechanisms of cytochrome c oxidase. However, the results support the predicted proximity of Y422 to heme a3, as now confirmed by crystal structure. In addition, it is shown that the pH-dependent reversed electron transfer between heme a and heme a3 is normal in the Y422F mutant. Hence, these data also demonstrate that Y422 is not the residue previously postulated to interact electrostatically with heme a3, nor is it responsible for the unique EPR characteristics of heme a in this bacterial oxidase.
Base Sequence↗
"Crack lung" caused by an impure preparation.
Pulmonary complications of crack cocaine have been reported mainly from American centres. Crack usage is now on the increase in the UK. Three cases of "crack lung" are reported in patients who acquired the drug from the same source. The pulmonary syndrome they developed was due to an impure form of crack.
Adult↗
Oligoclonal V gene usage by T lymphocytes in bronchoalveolar lavage fluid from sarcoidosis patients.
The T-cell antigen receptor (TCR) repertoire was examined in lymphocytes isolated from the lungs and blood of 12 sarcoidosis patients and nine control patients. This analysis, by polymerase chain reaction (PCR), examined the variable (V)-domain genes of both the alpha and beta chains of the TCR. This is the first study to examine the usage of all known V alpha gene segments in sarcoidosis. A similar degree of diversity was observed in the TCR repertoire in the lungs and blood of the sarcoidosis patients. However, 11 of the 12 sarcoidosis patients showed an increased use of particular TCR V alpha and V beta genes in lung T cells as compared with blood. The pattern of TCR V gene bias in the lung T cells was specific for each patient. The clonality of selected V genes was examined by determining the third complementarity-determining region (CDR3) length polymorphism of particular PCR products. The majority of lung T cells with biased TCR V gene segments were oligoclonal. Altogether, these results suggest oligoclonal expansion of lung T cells in response to a local antigenic stimulus, with additional nonspecific T-cell accumulation. The variability in the V gene segments used by the expanded T-cell subsets in different sarcoidosis patients may reflect different epitopes or antigens being recognized in the lung, as well as variations in major histocompatibility complex (MHC) haplotype between the patients.
Adult↗
The lung in HIV infection: can pulmonary function testing help?
Simple lung function tests have been used to evaluate respiratory symptoms in human immunodeficiency virus (HIV) infected individuals. Abnormalities of simple lung function tests, in particular decreases in the transfer factor of the lung for carbon monoxide (TL,CO) have been described in patients with acquired immune deficiency syndrome (AIDS) who have respiratory disease. Early studies showed marked reductions in TL,CO in patients with pneumocystis pneumonia but other forms of pulmonary infection or neoplasm also resulted in reductions in TL,CO values. Lung function studies in larger numbers of patients have shown reductions in all lung function measurements, particularly in TL,CO. in all categories of HIV disease. The most marked reductions in TL,CO are seen in patients with pneumocystis pneumonia but reductions also occur in pulmonary bacterial infection, tuberculosis and pulmonary Kaposi's sarcoma. TL,CO values improved following recovery from pneumocystis pneumonia but rarely returned to normal. Zidovudine therapy does not contribute to the abnormalities of lung function but smokers generally have worse lung function tests in all categories of HIV disease. The diagnostic usefulness of simple lung function tests is limited. A reduced TL,CO is a highly sensitive index for the presence of pneumonitis (pneumocystis or otherwise) in HIV-infected individuals but lacks the necessary specificity to be a satisfactory diagnostic tool. Simple lung function tests have limitations but have a value as a simple screening test to determine the presence of underlying organic disease in HIV seropositive patients with respiratory symptoms. The presence of a normal TL,CO value makes the presence of pneumonitis unlikely.
AIDS-Related Opportunistic Infections↗
Serum type III procollagen aminopeptide for assessing liver damage in methotrexate-treated psoriatic patients.
This study was designed to establish whether measurement of a serological marker of fibrosis might reduce the need for liver biopsy in psoriatic patients receiving methotrexate (MTX). Levels of type III procollagen aminopeptide (PIIINP-O and PIIINP-B) and laminin P1 (LamP1-B) were measured in 147 serum samples taken at the time of liver biopsy in 87 patients receiving long-term MTX treatment for severe psoriasis. Biopsies were classified as: (1) normal, (2) steatosis, (3) inflammation, (4) fibrosis, or (5) cirrhosis. Groups 3-5 were considered to show clinically relevant abnormality. Compared with controls, PIIINP-O was significantly raised in the group of MTX-treated psoriatics (P < 0.001). Within this group, levels were significantly higher in patients with inflammation, fibrosis or cirrhosis compared with those with normal histology or steatosis alone (P < 0.0001). In contrast, PIIINP-B and LamP1-B did not distinguish between controls and MTX-treated patients or between histological groups. Forty-two patients had two or more biopsies with simultaneous PIIINP-O measurement. PIIINP-O levels at the time of the first biopsy were normal in six of the seven patients whose histology was initially normal and subsequently became abnormal. A single measurement of PIIINP-O thus did not predict which patients might develop abnormal histology following further MTX. In a group of 17 patients, PIIINP-O was measured 3-monthly for up to 6 years during MTX treatment. PIIINP-O was elevated at some time during follow-up in all three patients who developed abnormal histology but was consistently normal in eight of the 11 patients whose histology remained or became normal. Our findings indicate that PIIINP-O is of value in detecting liver damage and, particularly if measured serially, may reduce the need for liver biopsy in MTX-treated patients. Although the test does not detect all patients with fibrosis, it would appear that the risk of missing significant liver damage in patients with persistently normal PIIINP-O is low.
Adult↗
EPR studies of wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides: Glu286 is not a bridging ligand in the cytochrome a3-CuB center.
Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging carboxyl ligand in the bimetallic cytochrome a3-CuB site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the carboxyl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.
Base Sequence↗
Two conformations of the catalytic site in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides.
Resonance Raman spectra of the carbon monoxy derivative of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides show two distinct Fe-CO stretching modes (519 and 493 cm-1) at room temperature. The frequency of the mode at 519 cm-1 coincides with that of other terminal oxidases at neutral pH. Two C-O stretching modes, one at 1966 cm-1 and one at 1955 cm-1, are also found. The splitting of the C-O stretching mode is consistent with the FTIR spectra of cytochrome c oxidases at cryogenic temperatures in which two different conformations (alpha and beta) of the catalytic site of the enzyme are present. The splitting of both the Fe-CO and C-O stretching modes under our conditions indicates that these two forms of the enzyme are also present at room temperature, and with the additional information on the Fe-CO modes provided here, a structural origin for the two forms may be postulated. The alpha-form has the same general structure of the active site as mammalian oxidase, a structure in which the copper atom that is the part of the Fe-CuB binuclear site interacts strongly with the bound CO. We postulate that the copper atom exerts a strong polar or steric effect on the heme-bound CO, resulting in either compression of the Fe-CO bond or distortion of the Fe-CO moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
Binding Sites↗
Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni(2+)-NTA affinity chromatography.
A rapid and highly efficient method of purifying the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been developed. This method relies upon a six-histidine affinity tag fused to the C-terminus of subunit I, which confers to the oxidase a high affinity for Ni(2+)-nitrilotriacetic acid (NTA) agarose. The histidine-tagged oxidase can be purified rapidly and with high yield by one affinity chromatography step, starting with solubilized membranes. The purified oxidase is > 95% pure and possesses structural and functional characteristics of the wildtype enzyme. The six-histidine tag can be easily added to pre-constructed site-directed mutants of subunit I, increasing the availability of purified cytochrome c oxidase mutants for biophysical and biochemical studies.
Amino Acid Sequence↗
The effect of sterols and brefeldin A on protein degradation in UT-1 cells.
UT-1 cells, a mutant Chinese hamster ovary (CHO) cell line induced to produce an abundance of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), were used to determine the effects of sterols and brefeldin A on the degradation of this enzyme. Brefeldin A has been shown to cause retention of proteins in and relocation of proteins to the endoplasmic reticulum (ER). UT-1 cells were incubated with (a) sterols only (12 micrograms/ml cholesterol and 0.2 microgram/ml 25-hydroxycholesterol), (b) sterols and brefeldin A (0.5 microgram/ml), and (c) brefeldin A only. Western blot analysis showed that incubation with sterols and brefeldin A decreased HMGR levels more slowly than incubation with sterols alone over the first 24-36 h of incubation; however, the rates were not significantly different. By 48 h of incubation, HMGR had decreased to a level comparable to that found when cells were incubated in sterols only. Incubation with brefeldin A alone did not cause a decrease in HMGR over the same 48-h time period. HMGR was undetectable in parental CHO cells under all of the conditions described. Indirect immunofluorescence microscopy revealed a pattern of tight, perinuclear staining with sterol incubation. After 48 h in sterols, HMGR staining was uniformly decreased throughout the cytoplasm. This change in staining pattern is also observed during incubation of UT-1 cells with sterols and brefeldin A. Incubation for 48 h with brefeldin A alone had no effect on the tight perinuclear pattern originally observed. Diffuse, faint staining of CHO cells under all conditions served as a negative control. The results of these experiments indicated that brefeldin A, and therefore retention of proteins in the ER, does not interfere with the degradation of HMG CoA reductase. Despite the presence of brefeldin A, sterol-mediated dispersal and degradation of the crystalloid ER (CER) continued in UT-1 cells. Lack of brefeldin A sensitivity implied that the mechanism for CER dissolution was distinct from previously described mechanisms for ER to Golgi transport.
Animals↗
Evolution of pulmonary HIV-1 infection in a patient with Pneumocystis carinii pneumonia.
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Acquired Immunodeficiency Syndrome↗
Abnormalities of serum type III procollagen aminoterminal peptide in methotrexate-treated psoriatic patients with normal liver histology do not correlate with hepatic ultrastructural changes.
In a previous study, it was shown that the serum levels of type III procollagen aminoterminal peptide (P3NP) were significantly greater in patients receiving methotrexate (MTX) treatment for psoriasis than in untreated control patients with psoriasis. Although levels were highest in patients with hepatic fibrosis and cirrhosis, serum P3NP concentrations in those patients with normal liver histology on light microscopy were also shown to be significantly higher than in controls. In the present study, liver biopsies from 22 such 'normal' patients were examined by electron microscopy, in order to determine whether P3NP levels correlated with ultrastructurally demonstrable fibrosis. Fibrosis in the perisinusoidal space of Disse was present in as many as 82% of biopsies. Although the prevalence of such fibrosis in psoriasis patients who have not received MTX is unknown, the high prevalence of Disse space fibrosis and of raised P3NP in MTX-treated patients suggests that MTX causes subtle liver damage in a majority of treated patients. However, we were unable to show a statistical correlation between P3NP and the degree of Disse space fibrosis.
Adult↗
Sequential liver biopsies during long-term methotrexate treatment for psoriasis: a reappraisal.
One hundred and eighty-two liver biopsies were performed over a 10-year period on patients receiving long-term, low-dose, once weekly oral methotrexate (MTX) for severe psoriasis. Forty-nine patients had two or more biopsies during continued treatment and formed the study population for our analysis. The first and last biopsies were compared to determine progression of any histological abnormalities. Liver biopsies were assessed without knowledge of the MTX dose and allocated to one of five groups according to the severity of the histological abnormalities. These were defined as: (1) normal; (2) steatosis alone; (3) inflammation without fibrosis; (4) fibrosis; and (5) cirrhosis. The mean cumulative dose of MTX at the time of the first biopsy was 2743 mg (range 315-10,024), given over 275 weeks (range 26-738). In the interval between the first and last biopsies, patients received, on average, a further 2362 mg (range 390-7155) over 225 weeks (range 60-460). There was improvement in the histological assessment in 12 patients, no change in 28 patients, and deterioration in nine patients. None developed cirrhosis. Liver biopsy findings prompted discontinuation of MTX in four of the 49 patients on long-term treatment. This has to be weighed against the cost and morbidity of the 124 biopsies performed in these patients. Our results suggest that, with careful follow-up, the risk of development or progression of liver disease in patients receiving long-term, low-dose, once weekly oral MTX for psoriasis is modest, and that the requirement for performing routine liver biopsies in these patients needs to be reconsidered.
Biopsy↗