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Biomedical subjects

S Clarke

Publications and source records attributed to S Clarke.

At least 343 records · Page 19Linked to original sources
PubMed1986

spoIID operon of Bacillus subtilis: cloning and sequence.

The locus spoIID, involved in the sporulation of Bacillus subtilis, was cloned into derivatives of the temperate phage phi 105. Two recombinant phages were obtained which contain chromosomal DNA covering 1.6 kbp. They are both able to complement mutations spo-68 and spo-298. These mutations, which were believed to be in different loci, spoIID and spoIIC respectively, were shown to be closely linked, and both map at the position assigned to spoIID on the genetic map of B. subtilis. The sequence of 1656 bp carrying the spoIID locus was determined. Only one open reading frame was found; this codes for a protein of 343 amino acids. It is preceded by a ribosome binding site and possible recognition sequences for sigma 32- and sigma 29-RNA polymerases. Studies of the locus by means of integrational plasmid vectors defined the outer limits of the transcriptional unit. These results are completely compatible with the sequence data. The combination of sequence and mapping and the information obtained by the use of integrational plasmids confirm that the spoIID locus functions as a monocistronic operon.

Bacillus subtilis↗
PubMed1986

Topical anaesthesia for venepuncture.

A topical anaesthetic cream was tested in a randomised, double blind, placebo controlled trial of 15 children. The severity of pain experienced during venepuncture was assessed, using visual analogue and verbal rating scales. The topical anaesthetic cream was found to be significantly superior to placebo using each form of assessment.

Administration, Topical↗
PubMed1986

An evaluation of the relationship between receptive speech skills and expressive signing.

We examined the effects of receptive speech on the acquisition of manual signing among three mentally retarded children. In an alternating treatments design, we compared the acquisition of expressive signs that were, versus were not, in a child's receptive vocabulary. The children were trained via total communication in which pictorial referents were named during sign training. Signs corresponding to known words were generally acquired faster and retained better than signs corresponding to unknown words. We conducted posttests to assess the stimulus control of signing and any changes in expressive and receptive signing and speech. Observed changes in performance could be accounted for by attention to aspects of the stimulus complex during training and functional equivalence of stimuli established by training.

Child↗
PubMed1986

Interchange of callosal and association projections in the developing visual cortex.

Neurons projecting transitorily into the corpus callosum from area 17 of the cat were retrogradely labeled by the fluorescent tracer Fast Blue (FB) injected into contralateral areas 17 and 18 on postnatal days 1-5. During the second postnatal month these neurons were still labeled by the early injection, although they had eliminated their callosal axon. At this time, 15-20% of these neurons could be retrogradely relabeled by injections of Diamidino Yellow (DY) into ipsilateral areas 17 and 18, but few or none by similar injections in the other areas that receive from area 17 (19, 21a, PMLS, 20a, 20b, DLS). Similarly, area 17 neurons projecting transitorily to contralateral area PMLS during the first postnatal week could be relabeled by DY injections in ipsilateral areas 17 and 18 but not in PMLS. Already around birth, many transitorily callosal neurons in area 17 send bifurcating axons both to contralateral areas 17 and 18 and ipsilateral area 18. It is probable that during postnatal development some of these neurons selectively eliminate their callosal axon collaterals and maintain the projection to ipsilateral area 18. In fact, some transitorily callosal neurons in area 17 can be double-labeled by simultaneous perinatal injections of FB in contralateral areas 17 and 18 and of a new long-lasting retrograde tracer, rhodamine-conjugated latex microspheres, in ipsilateral area 18. The same neurons can then be relabeled by reinjecting ipsilateral area 18 with DY during the second postnatal month. This finding, however, does not exclude the possibility that some transitorily callosal neurons send an axon to ipsilateral area 18 after eliminating their callosal axon. In conclusion, area 17 neurons that project transitorily through the corpus callosum later participate, probably permanently, in ipsilateral corticocortical projections but selectively to areas 17-18. The mechanism responsible for this selectivity is unknown, but it may be related to the differential radial distribution (i.e., to birth date) of area 17 neurons engaged in the various corticocortical projections. The problems raised by the use of long-lasting retrograde fluorescent tracers in neurodevelopmental studies and by the quantification of results of double- and triple-labeling paradigms are also discussed.

Action Potentials↗
PubMed1985

Specific recognition of altered polypeptides by widely distributed methyltransferases.

Protein carboxyl methyltransferase activity has been detected in extracts prepared from bacterial cells (Salmonella typhimurium), amphibian (Xenopus laevis) oocytes, and transformed mammalian cell lines. This activity appears to specifically recognize altered aspartyl residues based on the observation that the synthetic peptide L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala is a good methyl-accepting substrate for the methyltransferase activity, but that the corresponding peptide containing a normal L-aspartyl residue is not. These activities are similar to those of the previously described human erythrocyte and bovine brain enzymes which catalyze the formation of polypeptide D-aspartyl beta-methyl esters and L-isoaspartyl alpha-methyl esters. The wide distribution of these enzymatic activites suggest that the methylation of atypical proteins is an essential function in cells.

Animals↗
PubMed1985

Demethylation of protein carboxyl methyl esters: a nonenzymatic process in human erythrocytes?

We have compared the demethylation rate of protein carboxyl methyl esters from isolated human erythrocyte membranes with the corresponding rate of metabolic turnover of these same methyl groups in the intact erythrocyte. Surprisingly, the apparent spontaneous demethylation of these membrane protein methyl esters was significantly faster at physiological pH than the corresponding rate determined by pulse-chase analysis of intact cells incubated with L-[methyl-3H]methionine. Readdition of erythrocyte lysate to purified membranes did not increase the rate of demethylation, as might be expected if there were cytosolic or membrane-bound protein methylesterase activity, but resulted instead in an apparent stabilization of these methyl esters. Thus, the metabolic lability of these protein methyl esters in intact cells may be quantitatively explained by spontaneous, rather than enzymatic, demethylation reactions. A model is presented in which a rapid but nonenzymatic intramolecular demethylation reaction results in the formation of a polypeptide imide or anhydride intermediate. The metabolic fate of these hypothetical intermediates is unknown but may lead to the repair or degradation of protein D-aspartyl and L-isoaspartyl residues, which appear to be the substrates for the initial transmethylation reaction.

Erythrocyte Membrane↗
PubMed1985

Ornithine transcarbamylase from Neurospora crassa: purification and properties.

Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and Km's of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Km's and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms.

Amino Acids↗
PubMed1985

The modulating effects of flurbiprofen on adriamycin plus vincristine or vindesine in the treatment of advanced breast cancer.

To assess the modulating effects of a non-steroidal anti-inflammatory drug on chemotherapeutic agents, 183 patients with advanced breast cancer have been treated in a randomised study with flurbiprofen or placebo and adriamycin plus a vinca alkaloid. To assess the efficacy of the new vinca alkaloid, vindesine, in breast cancer, patients were further randomised to receive vindesine or vincristine. The overall response rate in evaluable patients was 57%, and the median duration of response in the different treatment groups varied from 6 to 10 months. Response rates and toxicity in vindesine- and vincristine-treated patients were similar, although with vindesine neurotoxicity was slightly lower. Flurbiprofen did not improve the response rate or reduce the toxicity of adriamycin plus vinca alkaloid.

Adult↗
PubMed1985

Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions.

A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with L-[methyl-3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.

Aspartic Acid↗
PubMed1985

Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages.

Ordinarily packaged in DNA, adenine deoxyribonucleotides are preferentially concentrated in erythrocyte and lymphocyte cytosol in adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. A spectrum of cytosol enzyme activities are defined in terms of reaction velocities, K0.5s, and nucleotide partition after incubation with ribo- and deoxyribonucleotides. AMP and dAMP were dephosphorylated, but only AMP was deaminated in vitro. Although nucleotidase activity is much stronger in lymphocytes, AMP deaminase was the dominant degradative reaction in all erythrocyte and lymphocyte lysates under the conditions specified. For most cytosolic enzymes, ribonucleotides were preferred cofactors, implying that dADP and dATP often may be bystanders at metabolic events. The adenylate kinase-mediated partition of approximately equimolar ribo- and deoxyribonucleotide substrates yielded a very large preponderance of AMP in the monophosphate compartment, the monophosphates alone being directly vulnerable to degradative loss. The adenylate kinase(s) of lymphocytes differed strikingly from those of erythrocytes in reaction velocities with nucleotide cofactors, K0.5s, and in susceptibility to substrate inhibition.

AMP Deaminase↗
PubMed1984

The organization of immature callosal connections.

In newborn kittens, the anterograde transport of horseradish peroxidase, alone or bound to wheat-germ agglutinin, indicates that callosal axons have entered selectively the restricted portions of the neocortical gray matter (e.g., the area 17/18 border) which receive callosal afferents in adults. The callosal axons do also reach regions where they lack in the adult, but there they seem not to penetrate far into the gray matter. Neonatal injections of retrograde fluorescent tracers restricted to the gray matter in areas 17, 18, and posteromedial lateral suprasylvian area (PMLS) label neurons in the contralateral hemisphere only when the tracers were directed into regions known to receive callosal axons. In particular, injections near the 17/18 border label neurons in the contralateral hemisphere at the homologous site and at restricted, retinotopically corresponding locations in other visual areas: a pattern similar to the adult one. In contrast, an injection reaching the white matter of areas 17 or 18 labels a wider, continuous territory extending mediolaterally over most visual areas from 17 to posterolateral lateral suprasylvian area (PLLS) and including regions which later become acallosal; in addition, labeled neurons are found in the limbic cortex medial to area 17 and in the auditory cortex lateral to PLLS, none of which is known to project to either 17 or 18 in the adult. In flattened reconstructions of the cortex, the shape of the territory labeled by each of these injections is characteristically, although somewhat irregularly, crescent shaped; its rostrocaudal position varies with that of the injection. An injection extending into the white matter of more lateral visual areas (19, 21a, PMLS) labels callosal neurons over a similar territory, which extensively overlaps that labeled by the 17/18 border injections and likewise includes regions which are acallosal in the adult. In spite of the overlapping distribution of labeling obtained from separate injection sites, as in adults, each cytoarchitectonically (or retinotopically) defined area seems to receive from a different set of neurons, although a few neurons send bifurcating axons to more than one area. In conclusion, injections restricted to the cortical gray matter reveal a topographic organization of juvenile callosal connections similar to that of the adult. In contrast, injections extending into the white matter and adequate to reach the transitory callosal axons which appear to be confined there reveal what appears to be an earlier organization. These two organizations probably reflect different morphogenetic factors.

Animals↗
PubMed1984

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues.

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.

Amino Acid Sequence↗
PubMed1984

Membrane protein carboxyl methylation does not appear to be involved in the response of erythrocytes to cytoskeletal stress.

We have investigated the effect of changes of human erythrocyte cell shape on the degree of covalent modification by carboxyl methylation of membrane cytoskeletal proteins. The results indicate that the cell probably does not utilize carboxyl methylation to respond to cytoskeletal perturbations caused by such agents as A23187, 2,4-dinitrophenol, and chlorpromazine, all of which are known to cause large changes in cell shape. Protein carboxyl methylation also remained unchanged in the presence of cytochalasin B, which prevents such changes in cell shape. These results are not consistent with a cytoskeletal regulatory role for protein methylation reactions in the intact erythrocyte.

2,4-Dinitrophenol↗
PubMed1984

Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences.

S-Adenosyl-L-homocysteine was used to inhibit the methylation of carboxylic acid residues of membrane proteins in intact human erythrocytes. Incubation of erythrocytes for 24 h with 5 mM each of adenosine and L-homocysteine resulted in the intracellular accumulation of S-adenosyl-L-homocysteine and substantially inhibited membrane protein carboxyl methylation. From the degree of inhibition and from the observed turnover of methylated proteins, we estimate that the number of protein methyl esters in cells incubated with adenosine and L-homocysteine for 20 h is less than 20% that of cells incubated without these inhibitors. No significant differences in the physical deformability properties of the membrane of these hypomethylated cells were detected. However, there was a small but significant (p less than 0.001) increase in the amount of membrane protein D-aspartyl residues in these cells compared to control cells. These observations are consistent with the hypothesis that methylation of membrane proteins at D-aspartyl residues may result in the selective removal or repair of these uncommon residues.

Adenosine↗
PubMed1984

Carboxyl methylation of cytosolic proteins in intact human erythrocytes. Identification of numerous methyl-accepting proteins including hemoglobin and carbonic anhydrase.

Intact human erythrocytes incubated with L-[methyl-3H]methionine incorporated radioactivity into base-labile linkages with membrane and cytosolic proteins which are characteristic of protein methyl esters. Kinetic analysis of the methylation reactions in intact cells shows that individual erythrocytes contain approximately 38,000 and 115,000 protein methyl esters with biological half-lives of 150 min or less in the membrane and cytosolic protein fractions, respectively. Fractionation of the methylated cytosolic species by gel filtration chromatography at pH 6.5 followed by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 reveals that many different cytosolic proteins serve as methyl acceptors and that the degree of modification varies widely for individual proteins. For example, hemoglobin is modified to the extent of 3 methyl groups/10(6) polypeptide chains, while carbonic anhydrase contains 1 methyl group/approximately 16,500 polypeptide chains at steady state. Aspartic acid beta-[3H]methyl ester (Asp beta-[3H]Me) can be isolated from carboxypeptidase Y digests of cytosol proteins. By synthesizing and separating diastereomeric L-Leu-L-Asp beta Me and L-Leu-D-Asp beta Me dipeptides, we show that all of the Asp beta-[3H]Me recovered from cytosolic proteins is in the D-stereoconfiguration. Based on these data and on previous observations that erythrocytes contain a single methyltransferase which also methylates red cell membrane proteins at D-aspartyl residues both in vivo (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464) and in vitro (O'Connor, C. M., and Clarke, S. (1983) J. Biol. Chem. 258, 8485-8492), we propose that protein carboxyl methylation is part of a generalized mechanism for metabolizing damaged proteins. The infrequent and spontaneous occurrence of D-aspartyl residues in proteins adequately explains the broad substrate specificity and limited stoichiometries of protein carboxyl methylation reactions.

Blood Proteins↗