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S Clarke

Publications and source records attributed to S Clarke.

At least 181 records · Page 10Linked to original sources
PubMed1996

A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana.

Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5'-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.

Abscisic Acid↗
PubMed1996

Nineteenth century research on naturally occurring cell death and related phenomena.

Research on naturally occurring cell death is older than current opinion gives credit. More than 100 nineteenth century publications deal with it, and we review most of these. Soon after the establishment of the cell theory by Schleiden and Schwann, Carl Vogt (1842) reported cell death in the notochord and adjacent cartilage of metamorphic toads. Subsequent landmark discoveries included the massive cell death that occurs in pupating diptera (Weismann 1864), chondrocyte death during endochondral ossification (Stieda 1872), phagocytosis associated with cell death in the muscles of metamorphic toads (Metschnikoff 1883), chromatolytic (apoptotic) cell death in ovarian follicles (Flemming 1885), the reinterpretation of "Sarkoplasten" as "Sarkolyten" in metamorphic amphibia (Mayer 1886), the programmed loss of an entire population of neurons in fish embryos (Beard 1889), the death of scattered myocytes and myofibres in mammalian muscle (Felix 1889), and the death of many motor and sensory neurons in chick embryos (Collin 1906). Other lines of nineteenth century research established concepts important for understanding cell death, notably trophic interactions between neurons and their targets, and intercellular competition.

Animals↗
PubMed1996

Non-verbal auditory recognition in normal subjects and brain-damaged patients: evidence for parallel processing.

Three different aptitudes involved in sound object recognition were tested in 60 normal subjects and 20 brain-damaged patients: (i) capacity to segregate sound objects on different cues (intensity steps, coherent temporal modulations or signal onset synchrony); (ii) asemantic recognition of sounds of real objects by judging whether two different sound samples belonged to the same object; and (iii) semantic identification of sounds of real objects as judged by means of a multiple choice response test. In 12 patients, different aptitudes involved in auditory recognition were disrupted separately and in a way which speaks in favour of parallel rather than hierarchical processing. There was no strong association between deficits in non-verbal auditory recognition and aphasia or the side of lesion.

Adult↗
PubMed1996

Lack of tolerance to contrast-reducing actions of chlordiazepoxide with repeated reward reductions.

In each of eight cycles of repeated reward reduction, the performance of rats given brief access to 32% sucrose for 3 days, and then 4% sucrose for 2 days, was compared to rats that received 4% on all 5 days. Shifted rats consumed less than unshifted rats following each shift, with little evidence of diminution of negative contrast across the eight shifts. Acute administration of chlordiazepoxide (CDP, 8 mg/kg) on the second postshift day reduced contrast on each shift with no evidence of tolerance development to these anticontrast actions (Experiment 1a). Acute administration of CDP on the first postshift day had no effect on contrast through the first four shifts, but reliably reduced contrast on the following four shifts (Experiment 2a). There was tolerance to the sedative effects of CDP, as measured in an open field (Experiments 1b and 2b). The data thus show: a) that contrast is not lost with repeated shifts; b) no tolerance develops to the anticontrast actions of CDP and, instead, CDP gains anticontrast actions, in regard to initial contrast occurrence; c) but, concurrently, tolerance does develop to the sedative effects of CDP in an open field.

Animals↗
PubMed1996

In vivo muscle protein synthesis rates in the American lobster Homarus americanus during the moult cycle and in response to 20-hydroxyecdysone

Simultaneous measurements of in vivo rates of protein synthesis (Ks) in claw, leg and abdominal muscles were made in the American lobster Homarus americanus at three stages of the moult cycle. Ks values are significantly elevated during the premoult (stage D2-D3) and fall during the intermoult (stage C4) periods in all three muscles. Postmoult (stage A/B) levels are not significantly elevated above intermoult levels. Intermoult levels are between 0.3 and 0.4 % protein synthesized per day. In the premoult animals, the ribosomal activity (milligrams protein synthesized per microgram RNA per day) of the claw, abdominal and leg muscles is elevated three- to fivefold. The claw muscle maintains an elevated ribosomal activity into the postmoult stage whereas, by this stage, that of the other muscle tissues has fallen to intermoult levels. The RNA/protein ratios of the three muscle groups from intermoult, premoult and postmoult animals do not show any significant differences. 18S ribosomal RNA levels fluctuate slightly, with no consistent pattern over the moult cycle. In vivo injection of premoult concentrations of 20-hydroxyecdysone (20-HE) into intermoult lobsters results in elevated Ks values and ribosomal activity for the muscles after 3 days. RNA/protein ratios remain constant in the muscles in response to injections of 20-HE in vivo. In vitro preparations of leg muscle treated with 20-HE did not show similar elevated rates of protein synthesis.

Journal Article↗
PubMed1996

Surgical angioplasty of the left main coronary artery: follow-up with magnetic resonance imaging.

BACKGROUND: Ostial stenosis of the left main coronary artery is a serious condition with a dismal prognosis. The treatment is surgical, with the two viable options being coronary artery bypass grafting and surgical angioplasty of the left main coronary artery. METHODS: We describe the use of surgical angioplasty to treat 3 patients (2 women and 1 man) with left main ostial stenosis using the posterior approach. Patency of the angioplasty was demonstrated subsequently with magnetic resonance imaging. RESULTS: All 3 patients were free of angina 12, 18, and 24 months after operation. Magnetic resonance imaging scans in all 3 patients demonstrated the widely patent left main coronary artery. CONCLUSIONS: Surgical angioplasty is an effective alternative to coronary artery bypass grafting in patients with left main ostial stenosis. Magnetic resonance imaging is an excellent noninvasive method for monitoring the patency of the left main coronary artery.

Adult↗
PubMed1996

Iso- or antiperistaltic anastomosis: does it matter?

To assess the importance of gastrojejunal anastomoses being performed in an isoperistaltic manner, 25 rats underwent solid and liquid gastric emptying studies before and at 1, 2, 3, 4, 8, 12 and 16 weeks after a Polya-type gastrectomy. No differences between the two anastomotic directions were evident, either in the deaths associated with post-operative gastric dilatation, or in the rates of solid and liquid gastric emptying following surgery.

Animals↗
PubMed1995

Protein L-isoaspartyl methyltransferase from the nematode Caenorhabditis elegans: genomic structure and substrate specificity.

We identified a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) in the nematode worm Caenorhabditis elegans. The methylation of abnormal L-isoaspartyl residues by this enzyme can lead to their conversion to L-aspartyl residues and represents a protein repair step for polypeptides damaged by spontaneous reactions during the aging process. We show that the levels of this enzyme increase 2-fold in C. elegans in the dauer larval form, a developmental stage where the organism can survive for extended periods of time. Utilizing degenerate oligonucleotide primers derived from conserved amino acid sequences of mammalian, plant, and bacterial L-isoaspartyl methyltransferases and PCR amplification, we made DNA probes that allowed us to obtain cDNA and genomic DNA clones encoding this enzyme in the nematode. The deduced amino acid sequence is 53% identical to the human enzyme and 29% identical to the Escherichia coli enzyme. Overexpression of the cDNA for the C. elegans enzyme in E. coli gave an active product with micromolar Km values for L-isoaspartyl-containing peptide substrates and for the methyl donor S-adenosyl-L-methionine. No methylation of D-aspartyl-containing peptides was detected under conditions where the human enzyme catalyzed this reaction, suggesting that the ability to methylate D-aspartyl residues in addition to L-isoaspartyl residues was a later evolutionary adaptation of this enzyme. The C. elegans gene for the methyltransferase, designated pcm-1, was mapped to a single site in a 31 kb region in the central portion of chromosome V. The gene is 3.2 kb in length and includes six introns. Although much smaller, its genomic organization is similar to that of the corresponding mouse gene, with identically positioned intron--exon splice junctions at five of seven sites. We propose that this gene plays an important role in facilitating the long term survival of this organisms.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli.

We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells.

Amino Acid Sequence↗
PubMed1995

Expression and purification of a human recombinant methyltransferase that repairs damaged proteins.

We report the construction of a plasmid (pDM2x) containing the coding sequence of the more acidic isozyme II of the human protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) and the overexpression and purification of the recombinant protein. This intracellular enzyme is present in all tissues and can catalyze the first step of a repair reaction where proteins containing abnormal L-isoaspartyl (or D-aspartyl) residues can be converted to forms containing normal L-aspartyl residues. When the methyl-transferase cDNA is expressed in Escherichia coli strain BL21 (DE3) under the T7 phage promoter, we find that active enzyme is produced in amounts up to 20% of the total soluble protein. We have developed a rapid and efficient purification method utilizing a one column-step nonaffinity fractionation that allows for the preparation of 10.2 mg of homogeneous enzyme from 2.6 liters of Luria-Bertani broth culture in less than 24 h. The product is soluble and fully active (10,000 pmol of methyl groups transferred to ovalbumin/mg enzyme/min from S-adenosyl-L-methionine at 37 degrees C). Conditions have been developed to concentrate this enzyme to 30 mg/ml. Analyses of the purified enzyme by N-terminal Edman sequencing and electrospray mass spectroscopy reveal that it is identical to the human isozyme II with the exception that the N-terminal alanine residue is not acetylated.

Amino Acid Sequence↗
PubMed1995

The auditory pathway in cat corpus callosum.

The cortical auditory fields of the two hemispheres are interconnected via the corpus callosum. We have investigated the topographical arrangement of auditory callosal axons in the cat. Following circumscribed biocytin injections in the primary (AI), secondary (AII), anterior (AAF) and posterior (PAF) auditory fields, labelled axons have been found in the posterior two-thirds of the corpus callosum. Callosal axons labelled by small individual cortical injections did not form a tight bundle at the callosal midsagittal plane but spread over as much as one-third of the corpus callosum. Axons originating from different auditory fields were roughly topographically ordered, reflecting to some extent the rostro-caudal position of the field of origin. Axons from AAF crossed on average more rostrally than axons from AI; the latter crossed more rostrally than axons from PAF and AII. Callosal axons originating in a discrete part of the cortex travelled first in a relatively tight bundle to the telo-diencephalic junction and then dispersed progressively. In conclusion, the cat corpus callosum does not contain a sector reserved for auditory axons, nor a strictly topographically ordered auditory pathway. This observation is of relevance to neuropsychological and neuropathological observations in man.

Animals↗
PubMed1995

Morphology and spatial distribution of corticothalamic terminals originating from the cat auditory cortex.

In this paper we studied the morphology and spatial distribution of corticothalamic axons and terminals originating from the auditory cortical fields of the cat. The anterograde tracer biocytin was injected at electrophysiologically characterized loci in the primary (AI) (N = 2), anterior (AAF) (N = 1), posterior (PAF) (N = 1) and secondary (AII) (N = 2) auditory fields. In all cases, two different types of labeled terminals were found in the auditory thalamus: small spherical endings (1-2 microns) and giant, finger-like endings (5-10 microns). After biocytin injections in AI and AAF, the majority of anterogradely labeled axons terminated in the rostral half of the pars lateralis (LV) of the ventral division of the medial geniculate body (vMGB). In LV, the corticothalamic axons ramified profusely, giving rise to dense terminal fields forming well delineated curved stripes, with small spherical endings. Additional terminal fields formed by small endings were observed in the medial division of the medial geniculate body (mMGB). Giant endings were observed in a small area in the dorsal nucleus (D) of the dorsal division of the medial geniculate body (dMGB), near its border with the vMGB. PAF projections were located in the caudal half of vMGB and in mMGB, where only small terminals were found. Giant endings were seen in the superficial part of dMGB emerging from labeled corticothalamic axons oriented in parallel to the dorsal surface of the MGB. Projections from AII gave rise to a main terminal field of small endings in D; a second terminal field consisting of giant endings intermingled with small endings was found in the deep dorsal nucleus (DD) of dMGB. We conclude that small terminals serve the feedback projection to the thalamic nucleus from which the injected cortical field receives its main input, whereas giant terminals cross the borders between the parallel ascending auditory pathways.

Acoustic Stimulation↗
PubMed1995

Molecular immunobiology of macrophages: recent progress.

Macrophage molecules involved in adhesion, signalling, secretion and interactions with pathogens and T-helper lymphocytes have been cloned and defined using monoclonal antibodies and genetic disruption. Studies of the regulation of macrophage differentiation and activation by cytokines have yielded insights into the heterogeneity of their functions in natural and acquired immunity, including immunity to infections by mycobacteria and HIV.

Animals↗
PubMed1995

Tissue-specific targeting of cytokine unresponsiveness in transgenic mice.

The ubiquitous cellular distribution of certain cytokine receptors has hampered attempts to define the physiologically important cell-specific functions of cytokines in vivo. Herein, we report the generation of transgenic mice that express a dominant-negative IFN gamma receptor alpha chain mutant under the control of either the human lysozyme promoter or the murine lck proximal promoter, which display tissue-specific unresponsiveness in the macrophage or T cell compartments, respectively, to the pleiotropic cytokine, IFN gamma. We utilize these mice to identify previously undefined cellular targets of IFN gamma action in the development of a murine antimicrobial response and the mixed lymphocyte reaction. Moreover, we identify the macrophage as a critical responsive cell in manifesting the effects of IFN gamma in regulating CD4+ T helper subset development. These studies thus represent a novel approach to studying the cell-specific actions of an endogenously produced pleiotropic cytokine in vivo.

Animals↗
PubMed1995

Homograft replacement of fungal endocarditic pulmonary valve.

Right-sided infective endocarditis is uncommon but is increasingly seen as a reflection of the prevalence of drug abuse, chronic intravenous catheters and associated congenital malformations. Isolated pulmonary valve endocarditis has rarely been reported and there has been only one previous report of isolated pulmonary valve fungal endocarditis. This resolved with antimicrobials. We describe a case of isolated mycotic pulmonary valve endocarditis resistant to existing anti-fungal chemotherapy, which necessitated pulmonary valve resection and replacement with a homograft.

Adult↗
PubMed1995

Small joint synovitis in rheumatoid arthritis: should it be assessed separately?

In the assessment of disease activity in rheumatoid arthritis (RA) small joint synovitis is traditionally included only as a component of active, tender or swollen joint counts. By contrast, in the assessment of disease damage in RA, the X-ray score of hands and feet represents one of the most common parameters used and is regarded as a major indicator of outcome. Data presented in this study lead us to hypothesize that the small joints require separate assessment in any study of disease activity or outcome in RA: (i) there is clear evidence that small joint synovitis often occurs in the absence of an abnormal acute phase response (ESR or C-reactive protein) and (ii) measured synovitis is an individual (PIP) joint has been shown to be reliable and to be related to subsequent X-ray changes in the same joint. Our findings show that, in a study of a treatment of RA, it is quite possible for disease activity measures to appear controlled while inflammation continues in the small joints causing radiological damage. This radiological damage is reflected as an adverse outcome. Hence the paradox of improving disease activity but not outcome. We argue that small joint inflammation and damage should be recognized as one aspect of the RA disease process offering unique information and as such should be assessed independently.

Adult↗