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Biomedical subjects

R J Roberts

Publications and source records attributed to R J Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1997

Long-term cognitive sequelae of cerebral malaria in Vietnam veterans.

The brains of fatal cases of cerebral malaria exhibit capillary occlusion, punctiform hemorrhages, and focal necrosis in subcortical white matter. Some studies have suggested that the brain pathology of survivors is similar to that of fatal cases. The purpose of this study was to investigate the hypothesis that cerebral malaria survivors would exhibit neuropsychological impairment due to the residual cerebral damage sustained from the infection. Vietnam veterans reporting a history of cerebral malaria were compared with a group of veterans with a history of combat-related injuries on standard neuropsychological tasks and on dichotic listening (DL). The cerebral malaria group performed worse on memory tasks and exhibited greater clinical impairments on DL, consistent with presumed disruption of subcortical white matter tracts.

Journal Article↗
PubMed1996

A structural basis for the preferential binding of hemimethylated DNA by HhaI DNA methyltransferase.

The crystal structure of HhaI methyltransferase complexed with non-palindromic duplex DNA, containing a hemimethylated recognition sequence, and with the cofactor analog S-adenosyl-L-homocysteine (AdoHcy), has been determined. The structure provides an explanation for the stronger affinities of DNA methyltransferases for hemimethylated DNA than for unmethylated or fully methylated DNA in the presence of AdoHcy. The unmethylated target 2'-deoxycytidine flips out of the DNA helix and the CH group at position 5 makes van der Waals' contacts with the sulfur atom of AdoHcy. Selectivity/preference for hemimethylated over fully methylated DNA may thus reflect interactions among the chemical substituent (H or CH3) at the C5 position of the flipped cytosine, protein and the bound AdoHcy. The 5-methyl-2'-deoxycytidine on the complementary strand remains in the DNA helix, with the methyl group almost perpendicular to the carboxylate group of Glu239, which is part of the sequence recognition loop. Thus, selectivity/preference for hemimethylated over unmethylated DNA appears to result largely from van der Waals' contacts between the planar Glu239 carboxylate and the methyl group of the 5-methyl-2'-deoxycytidine. Furthermore, the positive electrostatic potential originating from the bound AdoHcy extends to the DNA phosphate groups flanking the flipped cytosine. The increased binding to DNA by long-range electrostatic interactions should also occur with the methyl donor S-adenosyl-L-methionine.

Amino Acid Sequence↗
PubMed1996

Enzymatic C5-cytosine methylation of DNA: mechanistic implications of new crystal structures for HhaL methyltransferase-DNA-AdoHcy complexes.

The refined crystal structures of HhaI methyltransferase complexed with cognate unmethylated or methylated DNA together with S-adenosyl-L-homocysteine, along with the previously-solved binary and covalent ternary structures, offer a detailed picture of the active site at individual stages throughout the reaction cycle. This picture supports and extends a proposed mechanism for C5-cytosine methylation that may be general for the whole family of C5-cytosine methyltransferases. The structures of the two new complexes have been refined to crystallographic R-factors of 0.189 and 0.178, respectively, at 2.7 A resolution. We observe that both unmethylated 2'-deoxycytidine and 5-methyl-2'-deoxycytidine flip out of the DNA helix and fit into the active site of the enzyme. The catalytic sulfur atom of Cys81 interacts strongly with C6. The C5 methyl group of the flipped 5-methyl-2'-deoxycytidine is bent approximately 50 degrees out of the plane of the cytosine ring and towards the sulfur atom of S-adenosyl-L-homocysteine. This unusual position is probably due to partial sp3 character at C5 and C6 and to steric effects of the conserved amino acid residues Pro80 and Cys81. Two water molecules are held near the hydrophobic edge (C5 and C6) of the flipped cytosine by two conserved amino acid residues (Gln82 and Asn304) and the phosphoryl oxygen atom of the phosphate group 3' to the flipped nucleotide, and one of them may serve as the general base for eliminating the proton from C5. Protonation of the cytosine N3 during the methylation reaction may involve Glu119, which itself might be protonated via a water-mediated interaction between the terminal carboxyl group of Glu119 and the amino group of the methionine moiety of S-adenosyl-L-methionine. The cofactor thus plays two key roles in the reaction.

Base Sequence↗
PubMed1996

Glycine activation of human homomeric alpha 1 glycine receptors is sensitive to pressure in the range of the high pressure nervous syndrome.

The effect of hyperbaric pressure on the inhibitory glycine receptor has been investigated in voltage-clamped Xenopus oocytes microinjected with cRNA encoding the human alpha-1 glycine receptor subunit. Heterologous expression of the human alpha-1 subunit generated functional glycine-gated channels with properties typical of native receptors. Glycine elicited a concentration-dependent inward current which reversed polarity at -25 mV and was antagonised by nanomolar concentrations of strychnine. Concentration-response curves established for the homomeric alpha-1 glycine receptor at 5, 10 and 15 MPa were progressively shifted to the right with respect to the concentration response curve established at atmospheric pressure (0.1 MPa). Pressure had no effect on the maximal response. The EC(50) values at 0.1, 5, 10 and 15 MPa were 190 mu M, 222 mu M, 338 mu M and 482 mu M, respectively. The results demonstrate that a receptor comprised solely of the human alpha-subunit is sensitive to pressure in the range that affects divers and at which the native rat spinal cord receptor is affected. This finding is discussed in the context of the postulated binding sites for glycine and the implications for the design of drugs to protect divers from the effects of pressure.

Animals↗
PubMed1996

REBASE--restriction enzymes and methylases.

REBASE is a comprehensive database of information about restriction enzymes and their associated methylases, including their recognition and cleavage sites and their commercial availability. Information from REBASE is available via monthly electronic mailings as well as via WAIS, anonymous ftp and through the World Wide Web (http://www.neb.com/rebase). Specialized files are available that can be used directly by many software packages.

Base Sequence↗
PubMed1996

Glutathione consumption and glutathione peroxidase inactivation in fibroblast cell lines by 4-hydroxy-2-nonenal.

Treatment of cultured fibroblasts, designated HA1 cells, with 4-hydroxy-2-nonenal (4HNE) in doses up to 50 nmol/10(6) cells for 3 h results in dose-dependent cytotoxicity measured by clonogenic cell survival with 50% cytotoxicity achieved at 32 nmol 4HNE/10(6) cells. 4HNE treatment also resulted in dose-dependent reduction of cellular glutathione (GSH) content and loss of glutathione peroxidase (GPx) activity at 4HNE doses greater than 15 nmol/10(6) cells. By comparison, a 95% oxygen-resistant variant of HA1 cells, designated O2R95 cells, and a hydrogen peroxide-resistant variant of HA1 cells, designated OC14 cells, were found resistant to 4HNE cytotoxicity requiring 54 nmol 4HNE/10(6) cells and 75 nmol 4HNE/10(6) cells, respectively, for 50% cytotoxicity. In O2R95 cells, dose-dependent decreases were seen in GSH levels and GPx activity. In OC14 cells, however, any reduction in cellular GSH levels required doses of 4HNE greater than 30 nmol/10(6) cells, and GPx activity remained unchanged. No changes were seen in glutathione-S-transferase activity in any of the cell lines at any dose tested. These data indicate a correlation between glutathione modification, in a manner that prevents its recycling, the ability to inactivate enzymes with active site selenocysteine residues and the cytotoxicity of alpha, beta-unsaturated aldehydes such as 4HNE.

Aldehydes↗
PubMed1996

A milk-borne campylobacter outbreak following an educational farm visit.

After a nursery school trip to a dairy farm, 20 (53%) of 38 children and 3 (23%) of 13 adult helpers developed gastrointestinal infection. Campylobacter jejuni was isolated from 15 primary cases and from 3 of 9 secondary household cases. A cohort study of the school party found illness to be associated with drinking raw milk (relative risk 5.4, 95% confidence interval 1.4-20.4, P = 0.001). There was a significant dose response relationship between amount of raw milk consumed and risk of illness (chi 2-test for linear trend 12.1, P = 0.0005) but not with incubation period, severity of symptoms or duration of illness. All 18 human campylobacter isolates were C. jejuni resistotype 02 and either biotype I (number 16) or biotype II (number 2). Campylobacter was also isolated from samples of dairy cattle and bird faeces obtained at the farm but these were of different resisto/biotypes. Educational farm visits have become increasingly popular in recent years and this outbreak illustrates the hazard of exposure to raw milk in this setting.

Adult↗
PubMed1996

Oleic acid incorporation protects cultured hamster fibroblasts from oxygen-induced cytotoxicity.

The effects of oleic, linoleic and arachidonic acid on oxygen toxicity were evaluated in cultured hamster fibroblasts. Each fatty acid was incorporated separately using a protocol that resulted in dose-dependent increases in the respective cellular fatty acid content to as much as 20-fold greater than unsupplemented controls. Linoleic acid produced no changes in cell survival after 48 h treatment with 95% oxygen, regardless of fatty acid content of the cells. Oleic acid incorporation resulted in a dose-dependent increase in cell survival at 48 h in 95% oxygen, whereas arachidonic acid incorporation resulted in a dose-dependent decrease in cell survival at 48 h in 95% oxygen. No significant differences in amounts of linoleic or arachidonic acids were detected in control, oleic acid- enriched or linoleic acid-enriched cells during oxygen exposure. In cells enriched with arachidonic acid, exposure to oxygen significantly reduced the amounts of linoleic and arachidonic acid to 79 and 84%, respectively, of the amounts found in air-exposed cells. The results indicate that oleic acid incorporation into cells provides protection against 95% oxygen-induced cytotoxicity. In contrast, arachidonic acid incorporation led to sensitization of cells to 95% oxygen-induced cytotoxicity that was accompanied by a loss of polyunsaturated fatty acids. As a result, it would appear that in situations of increased oxidative stress, high monounsaturated fatty acid diets that increase cellular oleic acid content may provide a protective environment compared with high polyunsaturated fatty acid diets that increase cellular arachidonic acid content.

Animals↗
PubMed1996

Determination of site-specific modifications of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal using matrix assisted laser desorption time-of-flight mass spectrometry.

Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications. Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide. The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme. Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence. One modified segment contains Lysine-182 that has been found to be part of the enzyme active site. Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence. The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1,4-addition of 4HNE. These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1,4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions.

Aldehydes↗
PubMed1996

Design fluency among normals and patients with closed head injury.

A Design Fluency task was administered to 86 patients who had suffered closed head injury (CHI) with loss of consciousness and 87 normal control subjects. Subjects were asked to draw as many novel designs as they could in 5 minutes without scribbling, drawing a nameable object, or repeating a design that had been drawn previously. The mean performance of the CHI group was significantly poorer than that of the control group, with 47% of head injured performing defectively (below the 5th percentile of controls There was no significant relationship between Design Fluency and prorated IQ, psychomotor speed, or Word Fluency. The findings demonstrate that a standardized version of the "free condition" of Design Fluency is likely to be useful in the evaluation of patients with closed head injury.

Journal Article↗
PubMed1995

Reasons for non-uptake of measles, mumps, and rubella catch up immunisation in a measles epidemic and side effects of the vaccine.

OBJECTIVE: To investigate the reasons for poor uptake of immunisation (non-immunisation) and the possible side effects of measles, mumps, and rubella vaccine in a catch up immunisation campaign during a community outbreak of measles. DESIGN: Descriptive study of reasons for non-immunisation and retrospective cohort study of side effects of the vaccine. SETTING: Secondary schools in South Glamorgan. SUBJECTS: Random cluster sample of the parents of 500 children targeted but not immunised and a randomised sample of 2866 of the children targeted. MAIN OUTCOME MEASURES: Reasons for non-immunisation; symptoms among immunised and non-immunised children. RESULTS: Immunisation coverage of the campaign was only 43.4% (7633/17,595). The practical problems experienced included non-return of consent forms (6698/17,595), refusal of immunisation (2061/10,897 forms returned), and absence from school on day of immunisation (1203/8836 children with consent for immunisation). The most common reasons cited for non-immunisation were previous measles infection (145/232), previous immunisation against measles (78/232), and concern about side effects (55/232). Symptoms were equally common among immunised and non-immunised subjects. However, significantly more immunised boys than non-immunised boys reported fever (relative risk 2.31 (95% confidence interval 1.36 to 3.93)), rash (2.00 (1.10 to 3.64), joint symptoms (1.58; 1.05 to 2.38), and headache (1.31 (1.04 to 1.65)). CONCLUSIONS: Many of the objections raised by parents could be overcome by emphasising that primary immunisation does not necessarily confer immunity and that diagnosis of measles is unreliable. Measles, mumps, and rubella vaccine is safe in children aged 11-15.

Adolescent↗
PubMed1995

Disruption of the target G-C base-pair by the HhaI methyltransferase.

HhaI MTase binds DNA duplexes containing mismatches at the target base position with higher affinity than that observed for the canonical substrate. The stability of these MTase-DNA complexes inversely correlates with the strength of the base pair that is disrupted upon interaction. This finding may offer a general tool to detect other enzymes that flip bases out of the DNA helix.

Base Composition↗
PubMed1995

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.

5-Methylcytosine↗
PubMed1995

Functional analysis of Gln-237 mutants of HhaI methyltransferase.

When the HhaI (cytosine-5) methyltransferase (M.HhaI) binds DNA it causes the target cytosine to be flipped 180 degrees out of the helix. The space becomes occupied by two amino acids, Ser-87 and Gln-237, which enter the helix from opposite sides and form a hydrogen bond to each other. Gln-237 may be involved in specific sequence recognition since it forms three hydrogen bonds to the orphan guanosine, which is the partner of the target cytosine. We have prepared all 19 mutants of Gln-237 and tested their biochemical properties. We find that mutations of this residue greatly affect the stability of the M.HhaI-DNA complex without affecting the enzyme's specificity for the target sequence. Surprisingly, all mutants retain detectable levels of enzymatic activity.

Base Sequence↗
PubMed1995

Contribution of increased glutathione content to mechanisms of oxidative stress resistance in hydrogen peroxide resistant hamster fibroblasts.

An H2O2-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line, which demonstrates cross resistance to 95% O2 and a 2-fold increase in total glutathione content, was utilized to investigate mechanisms responsible for cellular resistance to H2O2- and O2-toxicity. OC14 and HA1 cells were pretreated with buthionine sulfoximine (BSO) to deplete total cellular glutathione. Following BSO pretreatment, cells were either placed in 250 microM BSO to maintain the glutathione depleted condition and challenged with 95% O2, or challenged with hydrogen peroxide in the absence of BSO. Total glutathione and the activities of CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase were evaluated immediately following the BSO pretreatment as well as following 39 to 42 hr of exposure to 250 microM BSO. BSO treatment did not cause significant decreases in any cellular antioxidant tested, except total glutathione. Glutathione depletion resulted in significant (P < 0.05) sensitization to O2-toxicity and H2O2-toxicity in both cell lines at every time point tested. However, glutathione depletion did not completely abolish the resistance to either O2- or H2O2-toxicity demonstrated by OC14 cells, relative to HA1 cells. Also, glutathione depletion did not effect the ability of OC14 cells to metabolize extracellular H2O2. These data indicate that glutathione dependent processes significantly contribute to cellular resistance to acute H2O2- and O2-toxicity, but are not the only determinants of resistance in cell lines. The contribution of aldehydes formed by lipid peroxidation in mechanisms involved with the sensitization to O2-toxicity in glutathione depleted cells was tested by measuring the lipid peroxidation byproduct, 4-hydroxy-2-nonenal (4HNE), bound in Schiff-base linkages or in its free form in cell homogenates at 49 hr of 95% O2-exposure. No significant increase in 4HNE was detected in glutathione depleted cells relative to glutathione competent cells, indicating that glutathione depletion does not sensitize these cells to O2-toxicity by altering the intracellular accumulation of free or Schiff-base bound 4HNE.

Aldehydes↗
PubMed1995

Can self-reported data accurately describe the prevalence of overweight?

Overweight is an important public health problem affecting around 50% of the population of Wales, resulting in increased risk of illness, premature disability and premature death. The aim of this study was to examine critically the accuracy of self-reported data in describing the prevalence of overweight in Wales. A sample of 1622 adults aged 18 to 64 years was taken from the Welsh Heart Health Survey 1985. In that survey weight and height data were collected on a self-completed questionnaire and by clinical measurement. Mean differences between self-reported and measured weight and height were used as indicators of bias, and the accuracy of BMI and the prevalence of overweight based on this data were analysed. Weight was reported without significant bias in men, but women under-reported their weight by an average of 1.1 kg. Height was over-reported by 1.4 cm in men, and 0.7 cm in women, on average. More than two-thirds of subjects reported to within 2.3 kg and 2.5 cm of their actual weight and height. Reporting was more biased in older and overweight groups. The calculation of body mass index resulted in amplification of bias and underestimation of the prevalence of overweight and obesity in the study sample of 4.5% in men and 6.7% in women. The results have important implications for the use of self-reported data for the scientific measurement of the prevalence of overweight, especially in longitudinal studies, and suggest that further research should be conducted into the stability of reporting bias over time.

Adult↗
PubMed1995

Significance of unilateral ear extinction on the dichotic listening test.

The association between unilateral ear extinction on the dichotic listening test and the lateralization of epileptogenic foci was examined in a sample of 49 seizure patients undergoing preoperative evaluation for epilepsy surgery. Results from patients who were left hemisphere dominant for speech indicated that right ear impairment was always predictive of a unilateral left hemisphere focus, but left ear extinction was associated with unilateral lesions of either hemisphere. In this patient sample, dichotic listening performance reflected an interaction of both lateralized foci and hemispheric preference of language processing. Implications concerning clinical use of the dichotic listening test are discussed.

Adolescent↗