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Biomedical subjects

S Clarke

Publications and source records attributed to S Clarke.

At least 145 records · Page 8Linked to original sources
PubMed1998

Mutations in the Escherichia coli surE gene increase isoaspartyl accumulation in a strain lacking the pcm repair methyltransferase but suppress stress-survival phenotypes.

The Escherichia coli surE gene is co-transcribed with pcm, encoding the L-isoaspartyl protein repair methyltransferase, and is highly conserved among both the Eubacteria and the Archaea; however, no biochemical function has yet been identified for this gene. Isoaspartyl accumulation during stationary phase was much higher in a pcm surE double mutant than in either single mutant, suggesting that the two genes may represent two parallel pathways by which E. coli can respond to protein damage. A null mutation in surE also suppressed stress-survival defects previously observed in a pcm mutant strain, providing further evidence for an interaction between the two gene products.

Acid Phosphatase↗
PubMed1998

Trace analysis of SN-38 in human plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method with fluorescence detection was developed and validated for the quantitation of SN-38, the active metabolite of irinotecan (CPT-11), a new anticancer drug. This method uses solid-phase extraction with a C18 column for sample clean-up and concentration following acidification of human plasma with two volumes of 0.1 M HCl. Using blank plasma spiked with SN-38, we found the assay to be linear over the concentration range of 10-500 pM (3.9-195 pg/ml) with acceptable total and within-day imprecision. The recovery of SN-38 ranged from 48.3% (10 pM) to 91.5% (500 pM) whereas that of the internal standard, 20-(S)-camptothecin, was 96.9% (500 pM). This method represents a sizeable increase in sensitivity over other published methods and is shown to be suitable for the measurement of 'trough' concentrations of SN-38 during the treatment of patients with a weekly regimen of irinotecan.

Antineoplastic Agents, Phytogenic↗
PubMed1998

PRMT 3, a type I protein arginine N-methyltransferase that differs from PRMT1 in its oligomerization, subcellular localization, substrate specificity, and regulation.

Methylation is one of the many post-translational modifications that modulate protein function. Although asymmetric NG,NG-dimethylation of arginine residues in glycine-arginine-rich domains of eucaryotic proteins, catalyzed by type I protein arginine N-methyltransferases (PRMT), has been known for some time, members of this enzyme class have only recently been cloned. The first example of this type of enzyme, designated PRMT1, cloned because of its ability to interact with the mammalian TIS21 immediate-early protein, was then shown to have protein arginine methyltransferase activity. We have now isolated rat and human cDNA orthologues that encode proteins with substantial sequence similarity to PRMT1. A recombinant glutathione S-transferase (GST) fusion product of this new rat protein, named PRMT3, asymmetrically dimethylates arginine residues present both in the designed substrate GST-GAR and in substrate proteins present in hypomethylated extracts of a yeast rmt1 mutant that lacks type I arginine methyltransferase activity; PRMT3 is thus a functional type I protein arginine N-methyltransferase. However, rat PRMT1 and PRMT3 glutathione S-transferase fusion proteins have distinct enzyme specificities for substrates present in both hypomethylated rmt1 yeast extract and hypomethylated RAT1 embryo cell extract. TIS21 protein modulates the enzymatic activity of recombinant GST-PRMT1 fusion protein but not the activity of GST-PRMT3. Western blot analysis of gel filtration fractions suggests that PRMT3 is present as a monomer in RAT1 cell extracts. In contrast, PRMT1 is present in an oligomeric complex. Immunofluorescence analysis localized PRMT1 predominantly to the nucleus of RAT1 cells. In contrast, PRMT3 is predominantly cytoplasmic.

Amino Acid Sequence↗
PubMed1998

Myeloid-specific gene expression.

The mononuclear phagocytes are recruited from bone marrow precursors to most tissues of the body, particularly during inflammation or immune stimulation. This combination of accessibility as stem cells and heterogenity of tissue locations makes the myeloid cell potentially important as a carrier of therapeutic agents. Understanding the regulation of transcription in myeloid cells is necessary for any future design of tissue-specific gene targeting vectors, particularly if there are inherent size limitations. Identified members of the C/EBP, Runt/PEBP2/CBF, and Ets families of transcription factors are critical for myeloid-specific gene expression and may have myeloid-restricted expression or myeloid-specific regulation in the hematopoietic system. AP-1, Sp1, and Myb appear to be important for myeloid-restricted expression in some cases. In addition, factors involved in the up-regulation of the level of gene expression when macrophages are activated by agents such as interferon-gamma and bacterial products have been identified. Some of the sequences to which these transcription factors bind in myeloid-restricted genes have been tested in cell lines and in transgenic mice and it is now possible to make an attempt to describe the characteristics of a myeloid-specific promoter.

Animals↗
PubMed1998

Colour constancy impairments in patients with lesions of the prestriate cortex.

Colour matching and colour constancy were studied in seven patients and 46 control subjects. Subjects were required to match Munsell Colour Chips presented under either identical or different illumination. Three of the patients had deficits in colour constancy, i.e. failure to compensate for the change in the wavelength composition of the illumination. Two of the patients with defective constancy had suffered bilateral cortical damage to the posterior lingual and fusiform gyri, and one patient had a lesion restricted to the same regions of the right hemisphere. Our observations indicate that these cortical areas, which include part of putative human area V4, play an important role in colour constancy.

Adult↗
PubMed1998

RNA and protein interactions modulated by protein arginine methylation.

This review summarizes the current status of protein arginine N-methylation reactions. These covalent modifications of proteins are now recognized in a number of eukaryotic proteins and their functional significance is beginning to be understood. Genes that encode those methyltransferases specific for catalyzing the formation of asymmetric dimethylarginine have been identified. The enzyme modifies a number of generally nuclear or nucleolar proteins that interact with nucleic acids, particularly RNA. Postulated roles for these reactions include signal transduction, nuclear transport, or a direct modulation of nucleic acid interactions. A second methyltransferase activity that symmetrically dimethylates an arginine residue in myelin basic protein, a major component of the axon sheath, has also been characterized. However, a gene encoding this activity has not been identified to date and the cellular function for this methylation reaction has not been clearly established. From the analysis of the sequences surrounding known arginine methylation sites, we have determined consensus methyl-accepting sequences that may be useful in identifying novel substrates for these enzymes and may shed further light on their physiological role.

Amino Acid Sequence↗
PubMed1998

Compartments within human primary auditory cortex: evidence from cytochrome oxidase and acetylcholinesterase staining.

The human primary auditory area (AI) corresponds to granular cortex located on Heschl's gyrus. We studied its pattern of cytochrome oxidase and acetylcholinesterase activity in 10 normal human hemispheres. In cytochrome-oxidase-stained coronal sections layer IV was prominent by its dark staining. The overall staining intensity varied along the medio-lateral extent of Al; a 2.0-2.5-mm-wide antero-posterior dark band was present at mid-AI. In acetylcholinesterase-stained coronal sections a dark antero-posterior band appeared at the same location, corresponding to the highly granular part of Al. In cytochrome-oxidase-stained tangential sections of flattened Al, approximately 500-microm thick alternating dark and light cytochrome oxidase stripes were present in layers III and IV. These stripes were perpendicular to the dark band. Comparison with tonotopic maps of human Al obtained by activation studies suggests that the cytochrome oxidase and acetylcholinesterase dark band is most likely parallel to isofrequency lines and may correspond to the representation of frequencies critical for speech comprehension. The narrow stripes may be related to particular binaural or ampliotopic domains, whose presence is suggested by evidence from electrophysiological recordings in cat Al and from magnetoencephalographic studies in humans.

Acetylcholinesterase↗
PubMed1998

The L-isoaspartyl protein repair methyltransferase enhances survival of aging Escherichia coli subjected to secondary environmental stresses.

Like its homologs throughout the biological world, the L-isoaspartyl protein repair methyltransferase of Escherichia coli, encoded by the pcm gene, can convert abnormal L-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885-9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation in rpoS, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158-4163, 1997). We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new pcm mutant lacks these phenotypes. Interestingly, the newly constructed pcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42 degrees C. The pcm mutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functional pcm gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E. coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.

Bacterial Proteins↗
PubMed1998

Simulating hospital patient flows.

This case study development has benefited by the use of clear on-screen logic which has facilitated useful discussions between the analyst and end-users. The screen displays enable a realistic depiction of a system to be developed. A simple first model was gradually made more realistic in a step-by-step process involving both analyst and end-users. The investigation is continuing and the model is being further refined. Data collection is underway, and system data will be used to provide the model's input parameters of arrival and service rates. Times and lengths of queues of patients and total time in the system will be compared with their model equivalents. The resulting model is likely to be acceptable to the end-users because of their involvement in its development. This involvement has been facilitated by a simulation environment which enables on-screen iconic representations of system components to be created by analyst and end-user together.

Ambulatory Care Facilities↗
PubMed1998

Quantified EEG activity in adolescent attention deficit hyperactivity disorder.

The aim of this study was to explore elements of the maturational and cortical hypoarousal models in adolescent ADHD, by examining EEG activity in a rest eyes open condition, in 26 adolescent unmedicated ADHD males and 26 age and sex matched normal controls. ADHD adolescents were found to have increased anterior EEG absolute theta activity and reduced posterior relative beta activity compared with controls. These results lend some support to the continuation of a maturational lag and reduced cortical arousal in adolescent ADHD. These measures need to be further explored using concomitant EEG with electrodermal measures of arousal.

Adolescent↗
PubMed1997

Targeted gene disruption of the Caenorhabditis elegans L-isoaspartyl protein repair methyltransferase impairs survival of dauer stage nematodes.

The methylation of abnormal L-isoaspartyl residues by protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) can lead to their conversion to L-aspartyl residues. For polypeptides damaged by spontaneous reactions that generate L-isoaspartyl residues, these steps represent a protein repair pathway that can limit the accumulation of potentially detrimental proteins in the aging process. We report here the construction and the characterization of an animal model deficient in this methyltransferase. We utilized Tc1-transposon-mediated mutagenesis in the nematode Caenorhabditis elegans to construct a homozygous excision mutant lacking exons 2-5 of the pcm-1 gene encoding this enzyme. Nematodes carrying this deletion exhibited no detectable L-isoaspartyl methyltransferase activity. These worms demonstrated normal morphology and behavior and adult mutant nematodes exhibited a normal lifespan. However, the survival of dauer-phase mutants was diminished by 3.5-fold relative to wild-type dauers after 50 days in the dauer phase. The fitness of the pcm-1 deletion nematodes was reduced by about 16% relative to that of wild-type nematodes as measured by the ability of these mutants to compete reproductively against a wild-type population. We found that the absence of the functional methyltransferase gene leads to a modest accumulation of altered protein substrates in aged dauer worms. However, in the viable fraction of these dauer worms, no differences were seen in the levels of altered substrate proteins in the parent and methyltransferase-deficient worms, suggesting that the enzyme in wild-type cells does not efficiently catalyze the repair of spontaneously damaged proteins.

Alleles↗
PubMed1997

Single trial variability within the P300 (250-500 ms) processing window in adolescents with attention deficit hyperactivity disorder.

The traditional averaging process used to derive event related potential components (ERPs) is a soundly based method of determining the underlying ERP. Averaging, however, ignores the variability due to the single-trial ERPs that constitute the traditional average ERP. This variability may reflect complementary functional information to the average measure. Our group applied a simple procedure, the response variance curve (RVC), which measures single-trial ERP variability relative to their average. In this study, the average ERP and RVC measures (generated from the same single-trial task-relevant target ERPs) were assessed in an auditory oddball paradigm, in 17 unmedicated male adolescents with attention deficit hyperactivity disorder (ADHD) and in 17 age- and sex-matched normal controls. P300 amplitude, latency and point of maximum variability of the RVC were measured within the P300 processing window (250-500 ms post-stimulus). There were no significant differences in P300 amplitude or latency between the groups. Unmedicated ADHD patients, however, showed significantly increased single-trial variability within the P300 window compared with controls. This variability was significantly reduced with stimulant medication.

Acoustic Stimulation↗
PubMed1997

Human erythrocyte protein L-isoaspartyl methyltransferase: heritability of basal activity and genetic polymorphism for thermal stability.

Protein L-isoaspartyl methyltransferase (PIMT) is believed to play an important role in the disposition of age-damaged proteins by catalyzing the repair of abnormal isoaspartyl linkages resulting from the spontaneous deamidation of asparaginyl residues or isomerization of aspartyl residues. As a step toward testing the hypothesis that human disease- or age-related pathology might be associated with a deficiency in PIMT, we investigated basal activity and thermal stability of PIMT in erythrocyte lysates from 299 U.S. family members. Thermal stability was measured because it is a sensitive measure of variation in amino acid sequence. Basal activity was normally distributed with a mean+/-SD of 558+/-43 units/ml erythrocytes. Statistical analysis of the data revealed that basal PIMT activity exhibited a high degree of heritability. Enzyme thermal stability showed a skewed bimodal frequency distribution, and segregation analysis of family member pedigrees was consistent with Mendelian inheritance of two major alleles. No DNA was available from the family samples, so we tested two additional population samples for a known Ile/Val polymorphism at codon 119 and for PIMT activity and thermal stability, using blood donated by 25 Norwegians and by 20 Koreans. Single-stranded conformational polymorphism analysis using polymerase chain reaction revealed a 100% correlation between thermal stability grouping and this polymorphism. The high thermal stability samples were all homozygous Ile, the low thermal stability samples were all homozygous Val, and the intermediate thermal stability samples were all heterozygous. Furthermore, this polymorphism was responsible, in part, for the variation observed in basal erythrocyte PIMT activity. These results will help provide a foundation for future studies aimed at correlating levels of PIMT activity, or other properties of this enzyme, with human disease.

Aging↗
PubMed1997

Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice.

L-Asparaginyl and L-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing L-isoaspartyl and D-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77). The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal L-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues. To define the physiological relevance of this protein-repair pathway and to determine whether deficient protein repair would cause central nervous system dysfunction, we used gene targeting in mouse embryonic stem cells to generate protein L-isoaspartate(D-aspartate) O-methyltransferase-deficient mice. Analyses of tissues from methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain, heart, liver, and erythrocytes. The knockout mice showed significant growth retardation and succumbed to fatal seizures at an average of 42 days after birth. These results suggest that the ability of mice to repair L-isoaspartyl- and D-aspartyl-containing proteins is essential for normal growth and for normal central nervous system function.

Animals↗
PubMed1997

Cytochrome oxidase, acetylcholinesterase, and NADPH-diaphorase staining in human supratemporal and insular cortex: evidence for multiple auditory areas.

The pattern of cytochrome oxidase, acetylcholinesterase, and NADPH-diaphorase activity was studied in the supratemporal plane, the posterior part of the superior temporal gyrus, and the insula of normal human brains. Five dark cytochrome oxidase regions were found: (i) on Heschl's gyrus (area TC of von Economo and Koskinas); (ii) on the planum polare (area TC/TG); (iii) posterior to Heschl's gyrus (within area TA); (iv) on the posterior convexity of the superior temporal gyrus (within area TA); and (v) on the posterosuperior insula (area IB). More lightly stained cortex separated these regions (areas IA, TD, and part of TB). The laminar distribution of cytochrome oxidase activity varied in different areas. Acetylcholinesterase-positive fibers predominated in area TC and pyramidal neurons in areas TA and IA and in parts of TB; a mixture of fiber and neuronal staining was found in TC/TG, TD, and IB. NADPH-diaphorase positive profiles included large darkly stained nonpyramidal neurons, mostly in infragranular layers and in subcortical white matter, small faintly stained cells, and a dense array of fibers. The NADPH-diaphorase staining pattern did not vary between areas. The present results suggest that the supratemporal plane, the posterior part of the superior temporal gyrus, and the insula contain at least eight putative cortical areas. Comparison with activation studies by others suggest that, apart from the primary auditory area, six other putative areas may be auditory whereas one putative area, on posterior insula, may be vestibular.

Acetylcholinesterase↗
PubMed1997

Effects of combined exposure to noise and toxic substances--critical review of the literature.

A number of studies have been conducted in recent years investigating the potential effects on various health endpoints of the combination of noise and a variety of different industrial substances. This review indicates that the information available in both animals and humans on this subject is limited particularly with respect to assessing occupational risk. Most studies have focused on auditory effects in animals, although investigations have been performed for other toxicological endpoints. For some substances, notably toluene, the information from animals studies does suggest an interaction but these were performed only at exposure levels to both noise and chemicals which were each individually ototoxic. Single simultaneous exposure to noise and styrene did not result in any enhancement of auditory impairment above that produced by noise or styrene alone. Single simultaneous exposure to noise and carbon monoxide (CO), however, showed some evidence of enhancement of ototoxicity beyond that produced by noise or carbon monoxide alone, although only at high atmospheric concentrations of CO. When 1,3-dinitrobenzene was administered parentally at neurotoxic dose levels with continuous noise exposure, there was an increased severity of effects in the brain stem. Combined exposure to noise and lead and/or cadmium resulted in histopathological heart lesions of undefined severity, a finding which was not observed for either of those agents in isolation. Dermal exposure to dimethylformamide and noise or inhalation exposure to xylene and noise resulted in some biochemical changes in cardiac muscle which were of doubtful toxicological significance. In developing mice, there was evidence that combined exposure to cadmium sulphate and noise caused an increased incidence of external and skeletal malformations but only at dose levels of cadmium which would have induced developmental effects. Overall, for each of these chemicals and endpoints observed there is a suggestion of some interaction with noise exposure. From the data that are currently available, however, inferences cannot be drawn on whether or not interactions would have occurred at lower, more occupationally relevant, levels of exposure. A number of studies have investigated human populations exposed to both noise and industrial chemicals. Due to confounding factors, however, it was concluded that these data were inadequate for assessing the combined effects of noise and chemical exposure on hearing.

Animals↗