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Biomedical subjects

S Clarke

Publications and source records attributed to S Clarke.

At least 199 records · Page 11Linked to original sources
PubMed1995

Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins.

Proteins, like DNA, are subject to various forms of damage that can render them non-functional. Conformational changes and covalent chemical alterations occur spontaneously, and the rates of these reactions can be increased by environmental stresses such as heat, oxidative agents, or changes in pH or osmotic conditions. Although affected proteins can be replaced by de novo biosynthesis, cells--especially those subjected to stress or nutrient limitation--have developed mechanisms which can either restore damaged polypeptides to an active state or remove them. Such mechanisms can spare the biosynthetic capacity of the cell and ensure that the presence of non-functional molecules does not disrupt cell physiology. Three major mechanisms, which operate in bacteria as well as eukaryotic organisms, have been described. First, chaperones not only assist in proper de novo folding of proteins but also provide an important means of restoring activity to conformationally damaged proteins. Second, enzymatic 'repair' systems exist to directly reverse certain forms of protein damage, including proline isomerization, methionine oxidation and the formation of isoaspartyl residues. Finally, proteolysis provides a 'last-resort' means of dealing with abnormal proteins which cannot be repaired. Protein maintenance and repair may be of special importance for bacteria preparing to survive extended periods in stationary phase: both constitutive and induced mechanisms are utilized to permit survival despite greatly reduced protein synthesis.

Acclimatization↗
PubMed1995

Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides.

Aspartate and asparagine residues in polypeptides are subject to nonenzymatic reactions that lead to deamidation, isomerization, peptide bond cleavage and racemization. Much of this reactivity is due to the propensity for the initial formation of a cyclic succinimide intermediate. We have been interested in determining the effect of the side chains of neighboring histidine and cysteine residues in facilitating these reactions, particularly in the possibility that they can act as general acids and bases. In this study, we found little or no effect of histidine residues preceding an asparagine residue in hexapeptides derived from the sequence of adrenocorticotropic hormone, while a histidine residue preceding an aspartic acid residue was found to increase the rate of succinimide formation 8- to 11-fold. The presence of a histidine residue following either an asparagine or aspartic acid residue did not effect the rate of succinimide formation by peptide-bond nitrogen attack, but did increase the rate of the competing side-chain nitrogen attack leading to cleavage in the asparaginyl-containing peptide. We found that the effect of a cysteine residue following an asparagine or aspartic acid residue was in general similar to that of a serine residue, although the cleavage reaction appeared to be enhanced. These results suggest that His-Asp sequences may be particularly labile to spontaneous degradation in proteins and peptides, possibly owing to the ability of the histidine residue to facilitate succinimide formation by protonating the OH- leaving group on the side chain carboxylic acid of the aspartic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1994

Hormonal and environmental responsiveness of a developmentally regulated protein repair L-isoaspartyl methyltransferase in wheat.

The L-isoaspartyl protein methyltransferase (EC 2.1.1.77) has been proposed to be involved in the repair of spontaneously damaged proteins by facilitating the conversion of abnormal L-isoaspartyl residues to normal L-aspartyl residues. Based on the abundance of this enzyme in the seeds of a variety of plants and its unique substrate specificity, it has been hypothesized that it functions to prevent the accumulation of abnormal aspartyl residues in the proteins of aging seeds that can limit the viability of the embryo or its chances for germination. In this work, we show that the expression of the L-isoaspartyl methyltransferase is under developmental regulation in the winter wheat, Triticum aestivum. Methyltransferase mRNA and active enzyme are first detected in seeds during the late stages (III-IV) of caryopsis development. As mature seeds germinate, methyltransferase mRNA levels decline and are nearly undetectable by 72 h post-imbibition. Enzyme activity remains constant for 24 h post-imbibition and then decreases rapidly following the reduction of its corresponding mRNA. Methyltransferase activity is very low or undetectable in wheat seedlings, including leaf and root tissues. We show, however, that the L-isoaspartyl methyltransferase can be induced in vegetative tissues in response to hormone treatment and environmental stress. Abscisic acid, a phytohormone involved in seed development and desiccation tolerance, induces both methyltransferase mRNA and enzyme activity in 4-day-old wheat seedlings. Dehydration and salt stress also induce its transcription and enzymatic activity in seedlings. The ability of a plant to regulate methyltransferase activity in its seeds and vegetative tissues in response to desiccation, aging, and environmental stress may allow the plant to efficiently repair protein damage associated with these physiological changes.

Abscisic Acid↗
PubMed1994

Repair of spontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase.

The non-enzymatic deamidation at residues Asn-12 and Asn-38 of Escherichia coli phosphocarrier protein, HPr, and the repair of the resulting L-isoaspartyl (or beta-aspartyl) derivatives, HPr-1 and HPr-2, by recombinant human S-adenosylmethionine-dependent L-isoaspartate-(D-aspartate) O-methyltransferase (EC 2.1.1.77) were investigated. HPr is a component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is involved in the concomitant translocation and phosphorylation of many hexose sugars. The major products of the deamidation reaction, L-isoaspartyl (or beta-aspartyl) residues at positions 12 and 38, were found to be substrates for the L-isoaspartate-(D-aspartate) O-methyltransferase, an enzyme active on a wide variety of peptides and proteins containing these abnormal residues. This enzyme has been shown to catalyze the first step in a process that can convert L-isoaspartyl residues in peptides to normal L-aspartyl residues. The affinity of a recombinant human methyltransferase for HPr-1, a form deamidated at Asn-38, was relatively poor (Km = 3.6 mM), while a greater affinity was found for HPr-2, a form deamidated at both Asn-12 and Asn-38 (Km = 197 microM). When HPr-2 was incubated with S-adenosylmethionine and the methyltransferase, the bulk of the L-isoaspartyl residues at position 12 was converted to L-aspartyl residues. The major-by-product was the D-isoaspartyl form. The conversion of L-isoaspartyl residues at position 38 to L-aspartyl residues was less complete, reflecting the lower affinity of the methyltransferase for this site. The phosphohydrolysis activity of the repaired form was found to be midway between the form containing only L-aspartyl residues at positions 12 and 38 and the deamidated HPr-2 form.

Bacterial Proteins↗
PubMed1994

An enzymatic activity in bovine brain that catalyzes the reversal of the C-terminal methyl esterification of protein phosphatase 2A.

A novel protein methyltransferase has been recently described that catalyzes the esterification of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A in a variety of eucaryotic cells. This reaction can potentially modulate the phosphatase's activity, subunit interactions, or interactions with specific phosphoprotein substrates. We present evidence here that the methylation reaction is reversible and that an enzymatic activity is present in bovine brain cytosol that catalyzes the hydrolysis of the methyl ester. We show that this activity is sensitive to inhibition by the serine-hydrolase inhibitor phenylmethanesulfonyl fluoride but is not affected by the small molecule substrate analog N-acetyl-L-leucine methyl ester. These results suggest that protein methylation and demethylation reactions can be utilized in eucaryotic cells to modulate enzyme activity in a parallel fashion to protein phosphorylation and dephosphorylation reactions.

Animals↗
PubMed1994

Amino acid polymorphisms of the human L-isoaspartyl/D-aspartyl methyltransferase involved in protein repair.

We have analyzed DNA from three exons of the human protein-L-isoaspartate(D-aspartate) O-methyltransferase gene in 30 individuals. We present evidence for two polymorphisms in these regions that result in amino acid changes. At a site corresponding to amino acid position 119, we find the ATA codon for Ile at a frequency of 0.77 and the GTA codon for Val at a frequency of 0.23. At the site corresponding to amino acid position 205, we find the AAG codon for Lys at a frequency of 0.98 and the AGG codon for Arg at a frequency of 0.02. These amino acid changes may affect the ability of this enzyme to recognize and catalyze the first step in the repair of proteins spontaneously damaged in the aging process.

Adult↗
PubMed1994

Protein carboxyl methylation in Saccharomyces cerevisiae: evidence for STE14-dependent and STE14-independent pathways.

We incubated yeast cells (Saccharomyces cerevisiae) with the methyl donor S-adenosyl-L-[methyl-3H]methionine and then fractionated their cellular components by gel electrophoresis in sodium dodecyl sulfate. By analyzing gel slices for [3H]methyl esters by a vapor-phase diffusion assay, we detect major methyl-esterified species that migrate at apparent polypeptide sizes of 24 and 22 kDa and minor species of 49, 38, 35, 33, 31, and 26 kDa. Incubation of extracts from labeled cells with ribonuclease A or proteinase K revealed that the 24- and 22-kDa species represent methyl-esterified RNAs, whereas the other species are methyl-esterified polypeptides. The 38-, 33-, 31-, and 26-kDa polypeptides were not methyl-esterified in an isogenic yeast strain lacking the STE14 gene encoding a C-terminal isoprenylcysteine methyltransferase, suggesting that they are substrates for the STE14 methyltransferase. On the other hand, the amount of the methylated 49-kDa polypeptide is reduced in the ste14 mutant, indicating that at least two methylated polypeptides are present--one a substrate of the STE14 methyltransferase and one a substrate of a STE14-independent methyltransferase. The 35-kDa polypeptide also appears to be methylated by a STE14-independent methyltransferase. When cells were incubated in the presence of the protein synthesis inhibitor cycloheximide, little or no methylation of the STE14-dependent species was detected while the methylation of the STE14-independent substrates was unaffected. Pulse-chase studies revealed significant turnover of all of the methylated species in a 4-h period, with the exception of the 38-kDa polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Cycloheximide↗
PubMed1994

Association and intrinsic connections of human extrastriate visual cortex.

Human extrastriate visual cortex contains several functionally defined visual areas of which four (V2, VP, V4 and V5) can be identified using cyto- and myeloarchitectonic criteria. A case with a small lesion in the superolateral part of cytoarchitectonic area 19, outside the previously defined visual areas, has been studied with the Nauta method for anterogradely degenerating axons. The infarcted part of cortex formed dense intrahemispheric connections in some visual areas (V1, V2, V3 and probably a part of V4) and weaker connections in others (VP, V5). Connections to V1 and to V2 were mainly directed to the representation of the lower paramedian part of the visual field. Intrinsic connections, visualized by the degenerating axon segments in the cortex around the lesion, had a patchy distribution. In conclusion, intrahemispheric corticocortical connections link preferentially some of the visual areas and within these areas certain parts.

Afferent Pathways↗
PubMed1994

Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes.

Three regions of sequence similarity have been reported in several protein and small-molecule S-adenosylmethionine-dependent methyltransferases. Using multiple alignments, we have now identified these three regions in a much broader group of methyltransferases and have used these data to define a consensus for each region. Of the 84 non-DNA methyltransferase sequences in the GenBank, NBRF PIR, and Swissprot databases comprising 37 distinct enzymes, we have found 69 sequences possessing motif I. This motif is similar to a conserved region previously described in DNA adenine and cytosine methyltransferases. Motif II is found in 46 sequences, while motif III is found in 61 sequences. All three regions are found in 45 of these enzymes, and an additional 15 have motifs I and III. The motifs are always found in the same order on the polypeptide chain and are separated by comparable intervals. We suggest that these conserved regions contribute to the binding of the substrate S-adenosylmethionine and/or the product S-adenosylhomocysteine. These motifs can also be identified in certain nonmethyltransferases that utilize either S-adenosylmethionine or S-adenosylhomocysteine, including S-adenosylmethionine decarboxylase, S-adenosylmethionine synthetase, and S-adenosylhomocysteine hydrolase. In the latter two types of enzymes, motif I is similar to the conserved nucleotide binding motif of protein kinases and other nucleotide binding proteins. These motifs may be of use in predicting methyltransferases and related enzymes from the open reading frames generated by genomic sequencing projects.

Amino Acid Sequence↗
PubMed1994

Modular organization of human extrastriate visual cortex: evidence from cytochrome oxidase pattern in normal and macular degeneration cases.

The human extrastriate occipital cortex contains several visual areas that are probably analogues of macaque areas V2, V3, VP, V4 and V5. Tracing of callosal connections has led to the anatomical identification of these areas and to the characterization of some of them by cyto- and myeloarchitecture (Clarke and Miklossy, 1990). The pattern of cytochrome oxidase activity in these visual areas is now described in a normal case and in a case of age-related bilateral macular degeneration. In normal cortex, the laminar distribution of cytochrome oxidase activity was similar in V2, V3, VP, V4 and V5; a prominent dark band covered most of layers III and IV, and its upper and lower limits were gradual. In V2, V3, V4 and V5 but not VP, layer II tended to be darker than the infragranular layers. The overall intensity of the staining varied between areas: VP was very light, V2, V3 and V4 were darker, and V5 was very dark. A different, two-band pattern of cytochrome oxidase activity was found in a restricted region of the posterosuperior precuneus. The bilateral age-related macular degeneration had led to a great loss of ganglion cells in the central, but not in the peripheral retinae. The central representation in the lateral geniculate nuclei showed abnormally weak staining for cytochrome oxidase, particularly in the parvocellular layers. In the cortex, the contrast between lightly and darkly stained regions was greater than in the normal case. In particular, V5 was very heavily stained, and in V1 and V2 there were two different types of dark stripes that may represent compartments driven predominantly by the magnocellular system.

Aged↗
PubMed1994

Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain.

We have recently described a novel protein carboxyl methylation system that results in the reversible modification of a 36-kDa polypeptide component of a 178-kDa protein in the cytosol of a variety of eucaryotic cells. This reaction, catalyzed by a cytosolic 40-kDa methyl-transferase, results in the methyl esterification of the alpha-carboxyl group of the C-terminal leucine residue. We have now purified the major methylated 36-kDa polypeptide from bovine brain. N-terminal sequence analysis of a tryptic fragment of this polypeptide revealed identity to the catalytic subunit of protein phosphatase 2A. This enzyme exists in the cell predominantly as a trimeric 151-kDa native species containing the 36-kDa catalytic polypeptide that terminates in a leucine residue. We then fractionated bovine brain cytosolic extracts to separate the major phosphatase isoforms 2A1 and 2A2 and found that both could be methylated by a partially purified preparation of the methyltransferase. A synthetic C-terminal octapeptide based on the sequence of the 36-kDa catalytic subunit is neither a substrate nor an inhibitor of this methyltransferase, suggesting that this enzyme recognizes aspects of the tertiary and/or quaternary structure of the native phosphatase. Because this modification reaction is readily reversible in extracts, it may represent a novel strategy of the cell to modulate the function of this protein phosphatase.

Animals↗
PubMed1994

Primary small cell carcinoma of the prostate: unusual modes of presentation.

Primary small cell carcinoma of the prostate is an uncommon condition. Between August 1989 and July 1991 seven patients with this pathology presented to the Repatriation General Hospital, Melbourne. Four of these patients presented by means not previously described. Included in this group is the first reported case of this tumour localized to the prostate gland and apparently cured by radical prostatectomy. The generally poor prognosis and lack of hormonal response associated with this condition warrant a greater awareness of the diagnosis among urologists.

Acid Phosphatase↗
PubMed1994

Personal constructs of survivors of childhood sexual abuse receiving cognitive analytic therapy.

Increasing numbers of survivors of childhood sexual abuse (CSA) are now seeking psychological therapy; yet little evidence currently exists concerning optimum treatment formats, especially when re-victimization has occurred in adulthood. This study reports an exploratory investigation of re-victimization, and of the use of cognitive analytic therapy (CAT) with seven CSA survivor women, six of whom had experienced re-victimization and five of whom were self-abusive. Results of single element and dyad grids administered before and after therapy were consistent with the exploratory hypothesis that abuse forms a central component of the women's relationships, hence making recurrence of abuse a real possibility. Outcome of therapy using CAT was positive, although the considerable and significant symptomatic changes observed were accompanied by significant change in only a relatively small number of the women's constructs, suggesting the persistence of the centrality of abuse despite therapy. For two women, levels of disturbance remained high after termination, and some evidence of relapse was also evident at three-month follow-up.

Adolescent↗
PubMed1994

A new gene involved in stationary-phase survival located at 59 minutes on the Escherichia coli chromosome.

We determined the DNA sequence of a 2,232-bp region immediately upstream of the pcm gene at 59 min on the Escherichia coli chromosome that encodes an L-isoaspartyl protein methyltransferase with an important role in stationary-phase survival. Two open reading frames of 477 and 1,524 bp were found oriented in the same direction as that of the pcm gene. The latter open reading frame overlapped the 5' end of the pcm gene by 4 bp. Coupled in vitro transcription-translation analysis of DNA containing the 1,524-bp open reading frame directly demonstrated the production of a 37,000-Da polypeptide corresponding to a RNA species generated from a promoter within the open reading frame. The deduced amino acid sequence showed no similarity to known protein sequences. To test the function of this gene product, we constructed a mutant strain in which a kanamycin resistance element was inserted at a BstEII site in the middle of its coding region in an orientation that does not result in reduction of Pcm methyltransferase activity. These cells were found to survive poorly in stationary phase, at elevated temperatures, and in high-salt media compared with parent cells containing the intact gene, and we thus designate this gene surE (survival). surE appears to be the first gene of a bicistronic operon also containing the pcm gene. The phenotypes of mutations in either gene are very similar and indicate that both gene products are important for the viability of E. coli cells under stressful conditions.

Acid Phosphatase↗