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Biomedical subjects

G Evans

Publications and source records attributed to G Evans.

At least 19 recordsLinked to original sources
PubMed1999

Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the Km for NAD (1180 microM) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 A with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione beta-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

Catalytic Domain↗
PubMed1999

A MAD experiment performed at the white line of the iridium LIII absorption edge in lysozyme.

A multiple-wavelength anomalous diffraction (MAD) experiment was performed on an iridium derivative of hen egg-white lysozyme. Diffraction data were measured at five wavelengths on the X31 EMBL beamline on the DORIS III storage ring and at two further wavelengths on the X11 beamline. Four iridium-binding sites were located from the dispersive anomalous differences between two wavelengths at the rising and falling inflection points of the Ir LIII-edge white line using direct methods. All other attempts to determine the heavy-atom positions failed. The results demonstrate an experimental method whereby systematic error in MAD data due to sample absorption can be reduced where a white line is present in the absorption spectrum of a heavy atom.

Animals↗
PubMed1999

Residential detoxification for substance abusers with HIV/AIDS. Walden House Detoxification Program.

Substance abusers with HIV/AIDS have a complex array of problems that are not easily addressed in outpatient or long-term residential programs. A new but promising treatment modality is residential detoxification for substance abusers with HIV/AIDS, such as described by this paper. The purpose of the program is profiled, as well as its general components and the aspects that address the special needs of HIV clients. Data indicating the successes and limitations of the program are presented, along with recommendations for further addressing the needs of substance abuses with HIV/AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Solvent/detergent fresh frozen plasma as primary treatment of acute thrombotic thrombocytopenic purpura.

Three patients with acute thrombotic thrombocytopenic purpura (TTP) (one associated with E. coli infection) received as primary treatment pooled fresh frozen plasma (FFP) virus inactivated by the solvent/detergent (SD) method, plus prednisolone. All three patients attained platelet counts of > 50 x 10(9)/l by days 7, 3 and 10, respectively. Serum creatinine became corrected in parallel. Neurological features were slow to resolve in one patient, and developed de novo during plasma infusion in another with rapid reversal once intensive plasma exchange with SDFFP was instituted. All patients are in stable remission > 1 years later. SDFFP was well tolerated with no febrile or other reactions, and all patients remained negative for markers of infection with HIV, hepatitis B and hepatitis C viruses. One patient was positive for antibodies for hepatitis A virus and parvovirus B19 prior to treatment. The other two patients remained antibody negative at the end of therapy.

Adult↗
PubMed1999

What are the occupational implications of thalassaemia?

The thalassaemias are a heterogeneous group of inherited haemoglobin disorders. With modern treatment, most patients with severe disease survive to adult life. If they have no complications, and their general health is good, then the condition should have little impact on their fitness for work, though exposure to agents that are toxic or suppress the bone marrow should be avoided. Most patients with thalassaemia have minor disease and are asymptomatic. Their condition generally has no effects on their fitness to work. Pre-employment screening for thalassaemia is not justified.

Adult↗
PubMed1999

Taking MAD to the extreme: ultrafast protein structure determination.

Multiwavelength anomalous diffraction data were measured in 23 min from a 16 kDa selenomethionyl substituted protein, producing experimental phases to 2.25 A resolution. The data were collected on a mosaic 3 x 3 charge-coupled device using undulator radiation from the Structural Biology Center 19ID beamline at the Argonne National Laboratory's Advanced Photon Source. The phases were independently obtained semiautomatically by two crystallographic program suites, CCP4 and CNS. The quality and speed of this data acquisition exemplify the opportunities at third-generation synchrotron sources for high-throughput protein crystal structure determination.

Base Sequence↗
PubMed1999

Relationships of cerebral MRI findings to ultrasonographic carotid atherosclerosis in older adults : the Cardiovascular Health Study. CHS Collaborative Research Group.

Cerebral magnetic resonance imaging (MRI) has demonstrated a high prevalence of infarct-like lesions, white matter hyperintensities, and evidence of cerebral atrophy in older adults. While these findings are generally believed to be related to ischemia and atherosclerosis, their relationship to atherosclerosis in the carotid arteries remains to be explored. Study subjects were part of the multicenter Cardiovascular Health Study, a cross-sectional study of 3502 women and men >/=65 years of age undergoing cranial MRI and carotid ultrasonography. MRI infarcts were detected in 1068 participants (29.3%) and measurable carotid plaque in 2745 (75.3%). MRI infarcts, ventricular and sulcal widening, and white matter score were strongly associated with carotid intimal-medial thickness (IMT) and stenosis degree after adjustment for age and sex (all P<0. 01). Associations with plaque characteristics were less strong and less consistent; MRI infarcts were weakly associated only with surface irregularity, and ventricular size was weakly associated only with lesion density (both P<0.04). In contrast, sulcal widening was strongly related to plaque characteristics, with scores being higher in those with heterogeneous and irregular plaque (both P<0. 009). Adjustment for other risk factors, and for carotid IMT/stenosis, removed associations of MRI findings with plaque characteristics except for weak relationships remaining between MRI infarcts and surface irregularity and between sulcal score and heterogeneous plaque (both P<0.03). MRI abnormalities show strong and consistent relationships with increasing carotid IMT and stenosis degree but less strong associations with plaque characteristics, especially after adjusting for IMT and stenosis.

Aged↗
PubMed1999

Mainstream and sidestream cigarette smoke condensates suppress macrophage responsiveness to interferon gamma.

Sidestream smoke evolves from the smoldering end of a cigarette while the smoker is not puffing, and contributes substantially to environmental tobacco smoke (ETS). In contrast, main stream smoke emerges from the butt end of the cigarette and is mainly inhaled by the smoker. This study was performed to compare the effects of short-term exposure to cigarette smoke condensates prepared from sidestream (CSC-SS) and mainstream cigarette smoke (CSC-MS) on macrophage basal metabolism and responsiveness to two different stimuli, bacterial lipopolysaccharide (LPS) and interferon gamma (IFNgamma). Despite their generation at different temperatures and their different chemical composition, CSC - SS and CSC - MS had similar effects on macrophages. Both enhanced macrophage basal metabolism and responsiveness to LPS. Macrophage responsiveness to IFNgamma, assessed by their expression of four functional capacities, was suppressed by both CSC-SS and CSC-MS. The four assessed IFNgamma-inducible functional capacities were: enhanced phagocytosis of immuoglobulin-opsonized sheep red blood cells, TPA-induced peroxide production, class II major histocompatibility complex expression, and nitric oxide synthesis with LPS co-stimulation. The effects of CSC - SS and CSC - MS were similar qualitatively; they differ quantitatively in some cases, with CSC-MS generally effective at lower concentrations (expressed as cigarette-equivalents) than CSC-SS. Considering dilution of sidestream smoke in room air and loss during passage through the respiratory system, we expect to deliver the maximal dose to lung macrophages in situ only in rooms dense with smokers. However, only a fraction of the maximal dose can partially suppress induction of some functions, such as nitric oxide production and MHC expression. Macrophages play critical roles in tissue modeling during development. Of particular concern are neonates, whose organs are still undergoing growth and development, and are therefore susceptible to impaired development. If involuntary exposure to ETS hinders induction of macrophage functional capacities by cytokines, then development of the lungs and perhaps other organs would be impaired. In general, since macrophages are potent effectors and regulators of immunity, impairment of their responsiveness to cytokine must disrupt the proper functioning of the immune system.

Animals↗
PubMed1999

Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show biochemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification of these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.

Amino Acid Sequence↗
PubMed1998

HIV-1 envelope gp120 inhibits the monocyte response to chemokines through CD4 signal-dependent chemokine receptor down-regulation.

Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.

CD4 Antigens↗
PubMed1998

HIV-1 envelope gp41 is a potent inhibitor of chemoattractant receptor expression and function in monocytes.

HIV-1 uses CD4 and chemokine receptors as cofactors for cellular entry. The viral envelope transmembrane protein gp41 is thought to participate in viral fusion with CD4(+) cells. We investigated whether gp41 interacts with chemokine receptors on human monocytes by testing its effect on the capacity of cells to respond to chemokine stimulation. Monocytes preincubated with gp41 of the MN strain showed markedly reduced binding, calcium mobilization, and chemotaxis in response to a variety of chemokines as well as to the bacterial peptide fMLP. This generalized inhibition of monocyte activation by chemoattractants required the presence of CD4, since the effect of gp41 was only observed in CD4(+) monocytes and in HEK293 cells cotransfected with chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Confocal microscopy showed that gp41 caused internalization of CXCR4 in HEK293 cells provided they were also cotransfected with intact CD4. In addition, pretreatment of monocytes with protein kinase C inhibitors partially reversed the inhibitory effect of gp41. Thus, gp41, which had not previously been implicated as interacting with HIV-1 fusion cofactors, downregulates chemoattractant receptors on monocytes by a CD4-dependent pathway.

CD4 Antigens↗
PubMed1998

Oral contraceptives and the risk of hereditary ovarian cancer. Hereditary Ovarian Cancer Clinical Study Group.

BACKGROUND: Women with mutations in either the BRCA1 or the BRCA2 gene have a high lifetime risk of ovarian cancer. Oral contraceptives protect against ovarian cancer in general, but it is not known whether they also protect against hereditary forms of ovarian cancer. METHODS: We enrolled 207 women with hereditary ovarian cancer and 161 of their sisters as controls in a case-control study. All the patients carried a pathogenic mutation in either BRCA1 (179 women) or BRCA2 (28 women). The control women were enrolled regardless of whether or not they had either mutation. Lifetime histories of oral-contraceptive use were obtained by interview or by written questionnaire and were compared between patients and control women, after adjustment for year of birth and parity. RESULTS: The adjusted odds ratio for ovarian cancer associated with any past use of oral contraceptives was 0.5 (95 percent confidence interval, 0.3 to 0.8). The risk decreased with increasing duration of use (P for trend, <0.001); use for six or more years was associated with a 60 percent reduction in risk. Oral-contraceptive use protected against ovarian cancer both for carriers of the BRCA1 mutation (odds ratio, 0.5; 95 percent confidence interval, 0.3 to 0.9) and for carriers of the BRCA2 mutation (odds ratio, 0.4; 95 percent confidence interval, 0.2 to 1.1). CONCLUSIONS: Oral-contraceptive use may reduce the risk of ovarian cancer in women with pathogenic mutations in the BRCA1 or BRCA2 gene.

Adult↗
PubMed1998

Angiotensinogen and angiotensin converting enzyme genotypes and carotid atherosclerosis: the atherosclerosis risk in communities and the NHLBI family heart studies.

BACKGROUND: Polymorphisms of the renin-angiotensin system are associated with cardiovascular pathology. Therefore, the association of the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene and the T235 (methionine to threonine substitution) polymorphism of the angiotensinogen (AGT) gene with intima-media thickness of the carotid artery was investigated. METHODS AND RESULTS: Subjects were randomly selected from two centers participating in both the Atherosclerosis Risk in Communities (ARIC) and NHLBI Family Heart Studies. Probands were 45-64 years of age who were free of cardiovascular disease and had B-mode ultrasound measured carotid intima-media thickness. Multiplex polymerase chain reaction amplification was used to evaluate the ACE I/D and AGT T235 polymorphisms: genotype information was available on 495 and 475 participants, respectively. The frequencies of the ACE D and AGT T alleles were 0.56 and 0.52, respectively; 30% were homozygous for the ACE D allele, and 29% were homozygous for the AGT T allele. After adjustment for systolic blood pressure, antihypertensive medication use, diabetes, age, sex and LDL cholesterol, the mean intima-media thickness was 0.729, 0.732 and 0.721 mm in the ACE DD, ID, and II genotypes, respectively (partial F test 1.53, P = 0.22), and 0.727, 0.732 and 0.724 mm in the AGT MM, MT, and TT genotypes, respectively (partial F test 0.91, P = 0.40). Combining the genotypes for ACE and AGT, there were also no differences in intima-media thickness across the eight joint genotypes. CONCLUSION: We found no evidence that the ACE I/D and AGT T235 polymorphisms of the renin-angiotensin system were associated with carotid intima-media thickness in this population-based sample of middle-aged adults with no history of cardiovascular disease. The lack of an association between these variants and intima-media thickness may indicate that early atherosclerosis is mediated by factors other than these RAS polymorphisms.

Angiotensinogen↗