Biomedical subjects

S Clarke

Publications and source records attributed to S Clarke.

At least 91 records · Page 5Linked to original sources

PubMed2000

A novel post-translational modification of yeast elongation factor 1A. Methylesterification at the C terminus.

Protein methylation reactions can play important roles in cell physiology. After labeling intact Saccharomyces cerevisiae cells with S-adenosyl-l-[methyl-(3)H]methionine, we identified a major methylated 49-kDa polypeptide containing [(3)H]methyl groups in two distinct types of linkages. Peptide sequence analysis of the purified methylated protein revealed that it is eukaryotic elongation factor 1A (eEF1A, formerly EF-1alpha), the protein that forms a complex with GTP and aminoacyl-tRNAs for binding to the ribosomal A site during protein translation. Previous studies have shown that eEF1A is methylated on several internal lysine residues to give mono-, di-, and tri-N-epsilon-methyl-lysine derivatives. We confirm this finding but also detect methylation that is released as volatile methyl groups after base hydrolysis, characteristic of ester linkages. In cycloheximide-treated cells, methyl esterified eEF1A was detected largely in the ribosome and polysome fractions; little or no methylated protein was found in the soluble fraction. Because the base-labile, volatile [methyl-(3)H]radioactivity of eEF1A could be released by trypsin treatment but not by carboxypeptidase Y or chymotrypsin treatment, we suggest that the methyl ester is present on the alpha-carboxyl group of its C-terminal lysine residue. From the results of pulse-chase experiments using radiolabeled intact yeast cells, we find that the N-methylated lysine residues of eEF1A are stable over 4 h, whereas the eEF1A carboxyl methyl ester has a half-life of less than 10 min. The rapid turnover of the methyl ester suggests that the methylation/demethylation of eEF1A at the C-terminal carboxyl group may represent a novel mode of regulation of the activity of this protein in yeast.

Catalysis↗

PubMed2000

PRMT3 is a distinct member of the protein arginine N-methyltransferase family. Conferral of substrate specificity by a zinc-finger domain.

S-Adenosyl-l-methionine-dependent protein arginine N-methyltransferases (PRMTs) catalyze the methylation of arginine residues within a variety of proteins. At least four distinct mammalian family members have now been described, including PRMT1, PRMT3, CARM1/PRMT4, and JBP1/PRMT5. To more fully define the physiological role of PRMT3, we characterized its unique putative zinc-finger domain and how it can affect its enzymatic activity. Here we show that PRMT3 does contain a single zinc-finger domain in its amino terminus. Although the zinc-liganded form of this domain is not required for methylation of an artificial substrate such as the glutathione S-transferase-fibrillarin amino-terminal fusion protein (GST-GAR), it is required for the enzyme to recognize RNA-associated substrates in RAT1 cell extracts. The recombinant form of PRMT3 is inhibited by high concentrations of ZnCl(2) as well as N-ethylmaleimide, reagents that can modify cysteine sulfhydryl groups. We found that we could distinguish PRMT family members by their sensitivity to these reagents; JBP1/PRMT5 and Hsl7 methyltransferases were inhibited in a similar manner as PRMT3, whereas Rmt1, PRMT1, and CARM1/PRMT4 were not affected. We were also able to define differences in these enzymes by their sensitivity to inhibition by Tris and free arginine. Finally, we found that the treatment of RAT1 cell extracts with N-ethylmaleimide leads to a loss of the major PRMT1-associated activity that was immune to inhibition under the same conditions as a GST fusion protein. These results suggest that native forms of PRMTs can have different properties than their GST-catalytic chain fusion protein counterparts, which may lack associated noncatalytic subunits.

Animals↗

PubMed2000

Amyloid beta -peptide-binding alcohol dehydrogenase is a component of the cellular response to nutritional stress.

Amyloid beta-peptide-binding alcohol dehydrogenase (ABAD) is a member of the family of short chain dehydrogenase/reductases whose distinctive properties include the capacity to bind amyloid beta-peptide and enzymatic activity toward a broad array of substrates including n-isopropanol and beta-estradiol. In view of the wide substrate specificity of ABAD and its high activity on l-beta-hydroxyacyl-CoA derivatives, we asked whether it might also catalyze the oxidation of the ketone body d-3-hydroxybutyrate. This was indeed the case, and oxidation proceeded with K(m) of approximately 4.5 mm and V(max) of approximately 4 nmol/min/mg protein. When placed in medium with d-beta-hydroxybutyrate as the principal energy substrate, COS cells stably transfected to overexpress wild-type ABAD (COS/wtABAD) better maintained 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, cellular energy charge, and morphologic phenotype compared with COS/vector cells. Using a severe model of metabolic perturbation, transgenic mice with targeted neuronal expression of ABAD subjected to transient middle cerebral artery occlusion showed strokes of smaller volume and lower neurologic deficit scores in parallel with increased brain ATP and decreased lactate, compared with nontransgenic controls. These data suggest that ABAD contributes to the protective response to metabolic stress, especially in the setting of ischemia.

3-Hydroxybutyric Acid↗

PubMed2000

Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains.

Src homology 3 (SH3) and WW domains are known to associate with proline-rich motifs within their respective ligands. Here we demonstrate that the proposed adapter protein for Src kinases, Sam68, is a ligand whose proline-rich motifs interact with the SH3 domains of p59(fyn) and phospholipase Cgamma-1 as well as with the WW domains of FBP30 and FBP21. These proline-rich motifs, in turn, are flanked by RG repeats that represent targets for the type I protein arginine N-methyltransferase. The asymmetrical dimethylation of arginine residues within these RG repeats dramatically reduces the binding of the SH3 domains of p59(fyn) and phospholipase Cgamma-1, but has no effect on their binding to the WW domain of FBP30. These results suggest that protein arginine methylation can selectively modulate certain protein-protein interactions and that mechanisms exist for the irreversible regulation of SH3 domain-mediated interactions.

Amino Acid Sequence↗

PubMed2000

Reconstructing historical radionuclide concentrations along the east coast of Ireland using a compartmental model.

A mathematical model is presented that simulates the annually averaged transport of radionuclides, originating from the BNFL reprocessing plant at Sellafield, throughout the Irish Sea. The model, CUMBRIA77, represents the processes of radionuclide transport and dispersion in the marine environment and allows predictions of radionuclide concentration in various environmental media, including biota, to be made throughout the whole of the Irish Sea. In this paper we describe the use of the model to reconstruct the historical activity concentrations of 137Cs and 239+240Pu in a variety of environmental media in the western Irish Sea and along the Irish east coast back to 1950. This reconstruction exercise is of interest because only limited measurements of 137Cs and 239+240Pu activity are available prior to the 1980s. The predictions were compared to the available measured data to validate their accuracy. The results of the reconstruction indicate that activity concentrations of 137Cs in the western Irish Sea follow a similar, though slightly delayed and smoothed, profile to the discharges from the Sellafield site, with concentrations at the time of peak discharge (the mid-1970s) being around an order of magnitude higher than those measured in the 1980s and 1990s. By contrast, the concentrations of 239+240Pu at the time of peak discharges were similar to those presently measured. These differences reflect the distinct marine chemistries of the two nuclides, in particular the higher propensity of plutonium to bind to sediments leading to extended transport times. Despite these differences in behaviour the doses to Irish seafood consumers from 137Cs remain significantly higher than those from 239+240Pu.

Animals↗

PubMed2000

PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells.

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.

Animals↗

PubMed2000

Fibrinogen Longmont: a dysfibrinogenemia that causes prolonged clot-based test results only when using an optical detection method.

A new fibrinogen variant was discovered as a result of discrepancies found in routine laboratory screening. The patient, a healthy 37-year-old woman, had a mild bleeding history. Initial coagulation studies on the patient revealed a prolonged prothrombin time (PT) and a normal activated partial thromboplastin time (APTT). Further investigation on the patient and her mother demonstrated both had a PT with no end point using an optical detection method (ACL3000+) and a normal PT using an electromechanical detection method (ST4 Clot Detection System). The APTT for both the patient and her mother were essentially normal with both optical and mechanical detection methods. The patient and her mother also had markedly prolonged thrombin time and reptilase time results on the ACL3000+, but they were normal on the ST4. Coagulation test results on the patient's father were all normal. We believe the fibrinogen defect in this family may affect fibrin polymerization only enough to effect light scatter interpretation, while there is enough polymerization to increase plasma viscosity and yield an end point using an electromechanical analyzer. This report should alert pathologists and clinicians to possible discrepancies between mechanical and spectrophotometric clot testing methods.

Adult↗

PubMed2000

Development of an instrument to assess readiness to recover in anorexia nervosa.

OBJECTIVE: The degree to which patients with anorexia nervosa demonstrate readiness to recover from their illness has received scant theoretical or empirical attention. Investigating the prevalence and degree of amotivation for recovery in anorexia nervosa, its role in outcome, and the effectiveness of interventions designed to enhance readiness to recover necessitates the existence of a reliable and valid measure of motivational issues relevant to the disorder. The present study aimed to develop and evaluate an instrument for assessing readiness to recover in anorexia nervosa, namely, the Anorexia Nervosa Stages of Change Questionnaire (ANSOCQ), a 23-item self-report questionnaire based on Prochaska and DiClemente's stages of change model. METHOD: Seventy-one inpatients with anorexia nervosa participated in the study. On several occasions during their admission, participants completed the ANSOCQ as well as questionnaires assessing readiness to recover, anorexic symptomatology, general distress, and social desirability. RESULTS: The ANSOCQ demonstrated good internal consistency (.90) and 1-week test-retest reliability (.89). Various aspects of validity were also supported, such as significant relationships with other instruments assessing readiness to recover and the prediction of weight gain during different periods of treatment. DISCUSSION: The results suggest that the ANSOCQ is a psychometrically sound instrument that may prove useful in investigating the role of readiness to recover in anorexia nervosa.

Adolescent↗

PubMed2000

Expression, purification, and characterization of the protein repair l-isoaspartyl methyltransferase from Arabidopsis thaliana.

Protein l-isoaspartate (d-aspartate) O-methyltransferase (EC 2.1.1. 77) is a repair enzyme that methylates abnormal l-isoaspartate residues in proteins which arise spontaneously as a result of aging. This enzyme initiates their conversion back into the normal l-aspartate form by a methyl esterification reaction. Previously, partial cDNAs of this enzyme were isolated from the higher plant Arabidopsis thaliana. In this study, we report the cloning and expression of a full-length cDNA of l-isoaspartyl methyltransferase from A. thaliana into Escherichia coli under the P(BAD) promoter, which offers a high level of expression under a tight regulatory control. The enzyme is found largely in the soluble fraction. We purified this recombinant enzyme to homogeneity using a series of steps involving DEAE-cellulose, gel filtration, and hydrophobic interaction chromatographies. The homogeneous enzyme was found to have maximum activity at 45 degrees C and a pH optimum from 7 to 8. The enzyme was found to have a wide range of affinities for l-isoaspartate-containing peptides and displayed relatively poor reactivity toward protein substrates. The best methyl-accepting substrates were KASA-l-isoAsp-LAKY (K(m) = 80 microM) and VYP-l-isoAsp-HA (K(m) = 310 microM). We also expressed the full-length form and a truncated version of this enzyme (lacking the N-terminal 26 amino acid residues) in E. coli under the T7 promoter. Both the full-length and the truncated forms were active, though overexpression of the truncated enzyme led to a complete loss of activity.

Amino Acid Sequence↗

PubMed2000

Interhemispheric transfer of visual motion information after a posterior callosal lesion: a neuropsychological and fMRI study.

Interhemispheric transfer of visual information was investigated behaviourally and with functional magnetic resonance imaging (fMRI) 6 months after a lesion of the posterior two-thirds of the corpus callosum. On tachistoscopical left hemifield presentation, the patient was severely impaired in reading letters, words and geographical names and moderately impaired in naming pictures and colours. In contrast, interhemispheric transfer of visual motion information, tested by verbal report of the direction of short sequences of coherent dot motion presented within the left hemifield, was preserved. The pattern of cerebral activation elicited by apparent motion stimuli was studied with fMRI and compared to that of normal subjects. In normal subjects, apparent motion stimuli, as compared to darkness, activated strongly striate and extrastriate cortex. When presented to one hemifield only, the contralateral calcarine region was activated while regions on the occipital convexity, including putative area V5, were activated bilaterally. A similar activation pattern was found in the patient with a posterior callosal lesion; unilateral left or right hemifield stimulation was accompanied by activation in the contralateral and ipsilateral occipital convexity. Ipsilateral hemifield representation in the extrastriate visual cortex is believed to depend on callosal input. Our observation suggests that this is not the case for visual motion representation and that other, probably parallel, pathways may mediate visual motion transfer after posterior callosotomy.

Adult↗

PubMed2000

Predictors of analgesia use during supervised medical abortion. The Mifepristone Clinical Trials Group.

The object of this analysis was to identify predictors of narcotic analgesic use during medical abortion. A total of 2121 women with pregnancies of </=63 days gestational age received 600 mg mifepristone followed 48 h later by 400 microg oral misoprostol in a single arm clinical trial perfomed at 17 centers in the US. We tested the effects of subject characteristics at baseline and study centers on the use of any narcotic analgesics on the day of misoprostol use. Overall, 27% of subjects received narcotic analgesics. The main determinant of narcotic analgesic use was the study center. The relative risk of using narcotic analgesics increased with gestational age; the relative risk decreased in women with previous births, and also decreased with increasing age of the woman receiving treatment. It is concluded use of narcotic analgesia during medical abortion is least likely among older, parous women at low gestational ages; however, the clinic providing care for the patient was the most important determinant of who received narcotic analgesia.

Abortifacient Agents, Nonsteroidal↗

PubMed2000

Auditory agnosia and auditory spatial deficits following left hemispheric lesions: evidence for distinct processing pathways.

Auditory recognition and auditory spatial functions were studied in four patients with circumscribed left hemispheric lesions. Patient FD was severely deficient in recognition of environmental sounds but normal in auditory localisation and auditory motion perception. The lesion included the left superior, middle and inferior temporal gyri and lateral auditory areas (as identified in previous anatomical studies), but spared Heschl's gyrus, the acoustic radiation and the thalamus. Patient SD had the same profile as FD, with deficient recognition of environmental sounds but normal auditory localisation and motion perception. The lesion comprised the postero-inferior part of the frontal convexity and the anterior third of the temporal lobe; data from non-human primates indicate that the latter are interconnected with lateral auditory areas. Patient MA was deficient in recognition of environmental sounds, auditory localisation and auditory motion perception, confirming that auditory spatial functions can be disturbed by left unilateral damage; the lesion involved the supratemporal region as well as the temporal, postero-inferior frontal and antero-inferior parietal convexities. Patient CZ was severely deficient in auditory motion perception and partially deficient in auditory localisation, but normal in recognition of environmental sounds; the lesion involved large parts of the parieto-frontal convexity and the supratemporal region. We propose that auditory information is processed in the human auditory cortex along two distinct pathways, one lateral devoted to auditory recognition and one medial and posterior devoted to auditory spatial functions.

Aged↗

PubMed2000

Serotypes/groups distribution and antimicrobial resistance of invasive pneumococcal isolates: implications for vaccine strategies.

Based on the invasive pneumococcal isolates referred to reference laboratories in Scotland in 1988-99, we identified the distribution of serotypes/groups and their antimicrobial resistance patterns in order to evaluate the coverage of polysaccharide and the new pneumococcal conjugate vaccines. A total of 5659 invasive isolates were included. Of these, 5124 (90.5%) were blood isolates, 308 (5.5%) were CSF isolates, 143 (2.5%) were blood and CSF and 84 (1.5%) were other normally sterile isolates. The most prevalent 11 serotypes/groups were 14, 9, 19, 6, 23, 1, 3, 4, 7, 8 and 18, in numerical order. These accounted for 84% of total serotypes/groups. The serotypes/groups included in the 23 and 14-valent polysaccharide vaccines accounted for 96% and 88% of all isolates. Both vaccines accounted for 98% of penicillin non-susceptible and 100% of erythromycin non-susceptible isolates. The 7, 9, and 11-valent conjugate vaccines covered 61, 68 and 80% of invasive isolates respectively. The coverage of these vaccines was substantially higher in youngest age group with 84, 86 and 93% of invasive isolates in children < 2 years included in the 7, 9 and 11-valent conjugate vaccines compared with 58, 64 and 77% in adults > or = 65 years of age. The serotype/group distribution of invasive isolates in Scotland varied from year to year over the period 1993-9. The coverage of the 23-valent vaccine remained above 95% in each year but the coverage of the 7, 9 and 11-valent conjugate vaccines showed more marked fluctuation with coverage as low as 53, 60 and 75% in some years. Continued surveillance of invasive pneumococcal isolates is required to inform the development of appropriate vaccine strategies to prevent pneumococcal disease in Scotland.

Adolescent↗

PubMed2000

The prothrombin Denver patient has two different prothrombin point mutations resulting in Glu-300-->Lys and Glu-309-->Lys substitutions.

Dysprothrombinaemia is a rare, congenital cause of bleeding. Fewer than 25 families who express a functional prothrombin (factor II) defect have been reported. The original patient with prothrombin Denver had a severe haemophilia-like bleeding disorder treated with weekly prophylactic factor replacement. Analysis of factor II activity and antigen in the patient showed a factor II activity of 5 units/dl and factor II antigen of 21 units/dl. Genomic DNA from the patient, mother and brother was obtained from peripheral blood white cells. Oligonucleotides were constructed, and prothrombin exons were amplified via polymerase chain reaction (PCR). The entire sequence of the thrombin portion of the molecule (exons VIII-XIV) and that of exons I-II and IV-VII was determined. This moderately severe dysprothrombinaemia was found to be associated with compound heterozygosity for two different Glu-->Lys point mutations, at amino acid positions 300 and 309. Assays of plasma from the prothrombin Denver proband suggested that the functional defect was in the activation of zymogen to enzyme.

Adolescent↗

PubMed2000

Practice observed: is speculum examination on admission a necessary procedure in the management of all cases of antepartum haemorrhage?

The objective of the study was to determine the justification or otherwise of admission speculum examination in women presenting with antepartum haemorrhage and the outcome/ benefits of the practice on women with antepartum haemorrhage presenting to the delivery suite of a UK teaching hospital. Factors studied included gestational age at presentation, associated factors with the bleeding, grade of medical/ midwifery staff performing the admission speculum examination, findings on speculum examination and ultrasound scan and the overall pregnancy outcome. Of the 564 (4.9%) women who presented with antepartum haemorrhage; 521 had an admission speculum examination; 94.4% of these were by senior house officers (SHOs). The findings were a normal cervix (69%) and ectopy (21.3%). Of the women, 14.4% had recurrent bleeding and 8.5% went into preterm labour. The complications of the pregnancies, timing of delivery and management were independent of speculum examination. Admission speculum examination did not influence the management of pregnancies and the complications thereafter. These results suggest that such an intrusive procedure may not be justifiable in women presenting with antepartum haemorrhage.

Journal Article↗

PubMed2000

Hierarchy within human SI: supporting data from cytochrome oxidase, acetylcholinesterase and NADPH-diaphorase staining patterns.

The human primary somatosensory cortex consists of four cytoarchitectonic subdivisions (3a, 3b, 1 and 2) that are likely to contain distinct somatosensory representations. The intraareal organization of these areas as well as that of the primary motor cortex (area 4) has been analyzed using histochemical stains of cytochrome oxidase, acetylcholinesterase and NADPH-diaphorase activity in normal human brains. Cytochrome oxidase activity was revealed in individual cortical neurons and neuropil. Areas 4, 3a and 3b were on average darker than areas 1 and 2. The laminar distribution of cytochrome oxidase activity varied in different areas. A prominent dark band was present in layers IV and lower III in areas 3a and 3b and in layer III in areas 1, 2 and 4. Acetylcholinesterase staining revealed fibers and pyramidal cells in layers III and V; stained layer III pyramids were rare in areas 3a and 3b and numerous in areas 1, 2 and 4. NADPH-diaphorase positive elements included Golgi-like stained non-pyramidal neurons and Nissl-like stained pyramidal neurons; the former were found, in small numbers, in layer II of areas 4, 3a, 3b and 1, and the latter in layers III and V of areas 4 and 3a and in layer V of areas 1 and 2. The dark cytochrome oxidase staining of layer IV and the paucity of acetylcholinesterase positive pyramids in areas 3a and 3b resemble the pattern found in primary visual and auditory areas, whereas the dark cytochrome oxidase staining in layer III and abundance of acetylcholinesterase positive pyramids in areas 1 and 2 that of association areas. These results suggest that the four areas included in human SI constitute hierarchical stages of cortical processing, with 3a and 3b corresponding to primary and 1 and 2 to secondary areas.

Acetylcholinesterase↗

PubMed2000

The tolerability of efavirenz after nevirapine-related adverse events.

Eight patients who were infected with human immunodeficiency virus, and who had each sustained an adverse drug reaction while following a regimen including nevirapine, were switched to a regimen including efavirenz. None of the patients experienced adverse events identical to that which necessitated discontinuation of nevirapine. This study demonstrates that adverse events related to nevirapine are not a class-specific effect.

Adult↗

PubMed2000

Electrophysiological evidence for fast visual processing through the human koniocellular pathway when stimuli move.

There is increasing evidence from cellular recordings in primates and behavioral studies in humans that motion can be processed by other than the magnocellular (M) pathway and the cortical dorsal stream. Little is known about cortical processing of moving stimuli when the information is conveyed by the third retinogeniculocortical pathway - the so-called koniocellular (K) pathway. We addressed this issue in humans by studying the spatio-temporal dynamics of the brain electrical fields evoked by tritan (S-cone isolating) and luminance-defined moving stimuli. Tritan and luminance stimuli are presumably carried by the K and M pathways respectively. We found two time intervals where significant stimulus-specific electric fields were evoked: an early period between 40 and 75 ms after stimulus onset, and a later period between 175 and 240 ms. Some of these fields were identical for tritanand luminance-motion, suggesting that the processing of moving stimuli share common cortical substrates when mediated via K and M pathway input. However, tritan-motion stimuli also evoked unique electric fields that appeared earlier in time than the common motion-specific fields, indicating very fast activation of cortical areas specific to input through the K pathway. A distributed source localization procedure revealed simultaneous activation of striate and extrastriate areas even at the early processing stages, strongly suggesting a very fast activation of the visual cerebral network.

Adult↗