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S Clarke

Publications and source records attributed to S Clarke.

At least 253 records · Page 14Linked to original sources
PubMed1992

Distribution of an L-isoaspartyl protein methyltransferase in eubacteria.

A protein carboxyl methyltransferase (EC 2.1.1.77) that recognizes age-damaged proteins for potential repair or degradation reactions has been found in all vertebrate tissues and cells examined to date. This enzyme catalyzes the transfer of methyl groups from S-adenosylmethionine to the carboxyl groups of D-aspartyl or L-isoaspartyl residues that are formed spontaneously from normal L-aspartyl and L-asparaginyl residues. A similar methyltransferase has been found in two bacterial species, Escherichia coli and Salmonella typhimurium, suggesting that this enzyme performs an essential function in all cells. In this study, we show that this enzyme is present in cytosolic extracts of six additional members of the alpha and gamma subdivisions of the purple bacteria: Pseudomonas aeruginosa (gamma), Rhodobacter sphaeroides (alpha), and the gamma enteric species Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Serratia marcescens. DNA probes from the E. coli methyltransferase gene hybridized only to the chromosomal DNA of the enteric species. Interestingly, no activity was found in the plant pathogen Erwinia chrysanthemi, a member of the enteric family, nor in Rhizobium meliloti or Rhodopseudomonas palustris, two members of the alpha subdivision. Additionally, we could not detect activity in the four gram-positive species Bacillus subtilis, B. stearothermophilus, Lactobacillus casei, and Streptomyces griseus. The absence of enzyme activity was not due to the presence of inhibitors in the extracts. These results suggest that many cells may not have the enzymatic machinery to recognize abnormal aspartyl residues by methylation reactions. Since the nonenzymatic degradation reactions that generate these residues occur in all cells, other pathways may be present in nature to ensure that these types of altered proteins do not accumulate and interfere with normal cellular physiology.

Amino Acid Sequence↗
PubMed1992

Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5'-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.

Adenosine↗
PubMed1992

Impairment of lung mucociliary clearance in pigeon fanciers.

Lung mucociliary clearance was measured in 15 pigeon fanciers. The study group was subdivided into two: a precipitin-positive group (n = 10; mean +/- SEM age 45 +/- 5 years) with circulating blood precipitins and a precipitin-negative group (n = 5; mean +/- SEM age 40 +/- 3 years) without. Clearance was measured using an objective, noninvasive radioaerosol technique. The data for both groups were compared with those of matched control groups of healthy subjects. The mean +/- SEM area under the tracheobronchial retention curves (AUC) over the 6-h observation period was 257 +/- 27 %h for the precipitin-positive group compared with 177 +/- 16 %h for its control group (p = 0.02)--a high AUC value denoting slow clearance. That for the precipitin-negative group was 282 +/- 34 %h compared with 150 +/- 15 %h for its control group (p = 0.02). Our study illustrates in pigeon fanciers involvement of the conducting airways in that a major defense mechanism of the airways--namely, mucociliary clearance--is substantially compromised. The presence or absence of precipitins appears not to be related to the degree of mucociliary clearance impairment.

Bird Fancier's Lung↗
PubMed1992

Effects of a videotape feedback package on the peer interactions of children with serious behavioral and emotional challenges.

Peer interactions are among the greatest challenges experienced by children who have severe emotional and behavioral problems. This study evaluated an intervention package designed to increase the ratio of these children's desirable to undesirable interactions. The package included three principal components: (a) observation of videotapes following regularly scheduled peer activity sessions; (b) self-evaluation of the children's peer interactions observed on the videotapes; and (c) delayed feedback and reinforcement for desirable peer interactions. Five students from two elementary schools participated. Multiple baseline designs and one reversal were used to evaluate the effects of the intervention package. The results showed that the intervention produced lower levels of undesirable peer interactions and higher ratios of desirable to undesirable interactions for all participants. The results are discussed in regard to their conceptual and applied implications and in terms of specific directions for future research.

Affective Symptoms↗
PubMed1992

Role of the antrum in the gastric emptying of a non-nutrient liquid in the rat.

Forty male Wistar rats underwent liquid gastric emptying analysis with a non-nutrient liquid--99mTC-tin colloid in physiologic saline. Twenty then had two-thirds resection of the glandular stomach with Billroth-II-type reconstruction. Twenty served as controls: 10 unoperated and 10 with a laparotomy alone. Emptying was studied weekly for 4 weeks, then monthly for 4 months. Emptying was unchanged in unoperated controls. Laparotomy alone caused delayed emptying for 2 months (p less than 0.05). After Billroth-II resection emptying was delayed for 4 months (p less than 0.01). This delay included both the early and late phases of gastric emptying. We conclude that a laparotomy alone delays gastric emptying. The addition of an antrectomy causes a greater emptying delay, which persists for longer than that due to laparotomy alone. Thus, in a situation in which the small bowel and gastric feedback mechanisms are inoperative, the antrum is seen to play an important role in the gastric emptying of liquids. This supports the concept in man that antral contractions are important in ensuring the normal active emptying of liquids from the stomach.

Animals↗
PubMed1991

Structural elements affecting the recognition of L-isoaspartyl residues by the L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We have synthesized a series of L-isoaspartyl-containing (isoD) peptides and characterized their interaction with the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77). Our findings indicate that this enzyme interacts with 6 residues extending from the isoD-2 to isoD+3 positions in peptide substrates. Although peptides as simple as G-isoD-G are methylated with low affinity (Km = 17.8 mM), a wide variety of L-isoaspartyl-containing sequences in larger peptides are recognized with high affinity (Km less than 20 microM), the best yet discovered being VYP-isoD-HA, with a Km of 0.29 microM. Only two sequence elements have been found that can interfere with the high affinity binding of peptides of 4 or more residues, these being a prolyl residue in the isoD+1 position and negatively charged residues in the isoD+1, isoD+2, and/or isoD+3 positions. We investigated the effect of higher order structure on binding affinity using several L-isoaspartyl-containing proteins. Although conformation did, in some cases, lower the affinity of the methyltransferase for L-isoaspartyl residues, the range of kinetic constants for the methylation of these proteins was similar to that observed with the synthetic peptides. The L-isoaspartyl/D-aspartyl methyltransferase has been proposed to function in vivo to prevent the accumulation of L-isoaspartyl residues that arise spontaneously as proteins age. To examine whether such a mechanism is feasible given the wide range of substrate Km values observed in vitro, we set up a computer simulation to model the degradation and methylation reactions in aging human erythrocytes. Our results suggest that enough methyltransferase activity exists in these cells to significantly lower the expected number of L-isoaspartyl residues, even when these residues have millimolar Km values for methylation.

Amino Acid Sequence↗
PubMed1991

Purification, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli.

Mammalian tissues contain protein carboxyl methyltransferases that catalyze the transfer of methyl groups from S-adenosylmethionine to the free carboxyl groups of D-aspartyl or L-isoaspartyl residues (EC 2.1.1.77). These enzymes have been postulated to play a role in the repair and/or degradation of spontaneously damaged proteins. We have now characterized a similar activity from Escherichia coli that recognizes L-isoaspartyl-containing peptides as well as protein substrates such as ovalbumin. The enzyme was purified by DEAE-cellulose, hydroxylapatite, Sephadex G-100, polyaspartate, and reversed-phase chromatography and was shown to consist of a single 24-kDa polypeptide chain. The sequence determined for the N-terminal 39 residues was used to design an oligonucleotide probe that allowed the precise localization of its structural gene (pcm) on the physical map of the E. coli chromosome at 59 min. Transformation of E. coli cells with a plasmid containing DNA from this region results in a 3-4-fold overproduction of enzyme activity. The nucleotide sequence determined for the pcm gene and its flanking regions was used to deduce a mature amino acid sequence of 207 residues with a calculated molecular weight of 23,128. This sequence shows 30.8% sequence identity with the human L-isoaspartyl/D-aspartyl methyltransferase and suggests that this enzyme catalyzes a fundamental reaction in both procaryotic and eucaryotic cells.

Amino Acid Sequence↗
PubMed1991

Distinct C-terminal sequences of isozymes I and II of the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase.

We have purified the more acidic major isozyme (II) of the human erythrocyte L-isoaspartyl/D-aspartyl methyltransferase and compared its structure to that of the previously sequenced isozyme I. These isozymes are both monomers of 25,000 molecular weight polypeptides and have similar enzymatic properties, but have isoelectric points that differ by one pH unit. Analysis of 16 tryptic peptides of isozyme II accounting for 89% of the sequence of isozyme I revealed no differences between these enzyme forms. However, analysis of a Staphylococcal V8 protease C-terminal fragment revealed that the last two residues of these proteins differed. The Trp-Lys-COOH terminus of isozyme I is replaced by a Asp-Asp-COOH terminus in isozyme II. Southern blot analysis of genomic DNA suggests that the human genome [corrected] may contain only a single gene encoding the enzyme. We propose that the distinct C-termini of isozymes I and II can arise from the generation of multiple mRNA's by alternative splicing.

Amino Acid Sequence↗
PubMed1991

Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus.

We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.

Amino Acid Sequence↗
PubMed1991

A shared kappa reciprocal fragment and a high frequency of secondary Jk5 rearrangements among influenza hemagglutinin specific B cell hybridomas.

Ig kappa-chain gene rearrangement results in the displacement or loss of the DNA immediately 5' of Jk. This retained DNA is found on a different size fragment than in the germline (a reciprocal fragment), and contains the reciprocal joint of rearranged Vk and Jk genes, the back-to-back fusion of the heptamer/nonamer recombination signals. B cells of independent origin rarely have reciprocal fragments of the same size. However, we report that 9 of 15 B cell hybridomas of independent origin have reciprocal fragments of the same size (8-kb BamHI fragments) unrelated to their productive rearrangements. An 8-kb reciprocal fragment has also occurred on about 25% of the kappa alleles of normal splenic B cells. We find that the reciprocal fragments in two of these hybridomas contain the reciprocal joints of Jk1 genes and different Vk8 genes. In addition, we find that at least 8 of the 12 Jk4 or Jk5 expressing hybridomas have undergone double recombinations on their productive kappa alleles. The implications of these findings on the high frequency of 8-kb reciprocal rearrangements and on Vk rearrangement are discussed.

Amino Acid Sequence↗
PubMed1991

The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins.

Post-translational processing of a distinct group of proteins and polypeptides, including the a-factor mating pheromone and RAS proteins of Saccharomyces cerevisiae, results in the formation of a modified C-terminal cysteine that is S-isoprenylated and alpha-methyl esterified. We have shown previously that a membrane-associated enzymatic activity in yeast can mediate in vitro methylation of an isoprenylated peptide substrate and that this methyltransferase activity is absent in ste14 mutants. We demonstrate here that STE14 is the structural gene for this enzyme by expression of its product as a fusion protein in Escherichia coli, an organism in which this activity is lacking. We also show that a-factor, RAS1 and RAS2 are physiological methyl-accepting substrates for this enzyme by demonstrating that these proteins are not methylated in a ste14 null mutant. It is notable that cells lacking STE14 methyltransferase activity exhibit no detectable impairment of RAS function or cell viability. However, we did observe a kinetic delay in the rate of RAS2 maturation and a slight decrease in the amount of membrane localized RAS2. Thus, methylation does not appear to be essential for RAS2 maturation or localization, but the lack of methylation can have subtle effects on the efficiency of these processes.

Amino Acid Sequence↗
PubMed1991

Influence of calcium administration on the short-term hemodynamic and anti-ischemic effects of nifedipine.

This prospective study investigated whether pretreatment with intravenously administered calcium would influence the effect of nifedipine on rest hemodynamics and treadmill performance in patients with ischemic heart disease. Seventeen patients were studied after undergoing a qualifying treadmill exercise test that revealed ST segment depression indicative of ischemic heart disease. Study subjects performed three additional treadmill tests as part of the protocol. One treadmill test was obtained from each patient to provide baseline measurements without a preceding intravenous infusion and in the absence of all antianginal drugs including nifedipine; two additional exercise tests were preceded by an infusion and 10 mg of bite-and-swallow nifedipine. The infusions, administered in a randomized, double-blind, crossover fashion, consisted of either 10 ml of 10% calcium chloride (13.6 mEq) in 50 ml of 5% dextrose in water or 5% dextrose in water alone. Rest systolic blood pressure (134 +/- 4.6 mm Hg) was unchanged after placebo infusion (135 +/- 4.6 mm Hg) but decreased to 124 +/- 4.1 mm Hg (p less than 0.01) 25 min after nifedipine administration. Rest systolic blood pressure increased after calcium infusion (from 139 +/- 4.3 to 148 +/- 4.8 mm Hg, p less than 0.01) and then decreased significantly 25 min after nifedipine administration to 135 +/- 4.2 mm Hg (p less than 0.01). Despite a decrease at the time of peak nifedipine effect after either infusion, systolic blood pressure was significantly lower after administration of nifedipine alone than after administration of calcium and nifedipine (124 +/- 4.1 vs. 135 +/- 4.2 mm Hg, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Pressure↗
PubMed1991

Should disease-modifying agents be used in mild rheumatoid arthritis?

A 12-month double-blind controlled study comparing hydroxychloroquine 400 mg daily with placebo in 104 patients with mild RA was conducted to see whether patients with mild rheumatoid arthritis (RA) benefit from treatment with disease-modifying agents. Mild RA was defined as synovitis limited to the hands and feet, an ESR less than 30 mm/h and C-reactive protein less than 20 mg/l, a situation where accepted clinical practice is to use a non-steroidal anti-inflammatory agent alone. By 6 months, the improvement of clinical and laboratory parameters in the hydroxychloroquine treated patients was significant compared with pretreatment levels and significantly greater than the control group. This improvement was maintained at 12 months. In addition, fewer patients withdrew through lack of efficacy, eight on hydroxychloroquine versus 18 on placebo. The implications of treating this well defined group of patients is discussed.

Arthritis, Rheumatoid↗
PubMed1991

Fine needle aspiration cytodiagnosis as a pre-requisite for primary medical treatment of breast cancer.

The accurate non-surgical diagnosis of breast lumps allows assessment of breast cancer patients for conservation or neoadjuvant primary treatment before surgical intervention. We have analysed the accuracy of clinical assessment, fine needle aspiration cytology (FNAC), and mammography in over 868 women seen in a symptomatic breast clinic. Clinical examination by an experienced breast physician, together with FNAC, detected over 99% of the cancers with a 12% false positive rate. A cytological diagnosis of definite carcinoma was obtained in 69% of women with breast cancer with no false positive result from the women with benign conditions producing a 100% positive predictive value. These results indicate that it would be acceptable to give pre-surgical systemic endocrine or chemotherapy to women with positive cytology which is therefore a prerequisite for a neoadjuvant therapy programme.

Biopsy, Needle↗
PubMed1991

Spontaneous degradation and enzymatic repair of aspartyl and asparaginyl residues in aging red cell proteins analyzed by computer simulation.

The inherent instability of proteins may be a limiting factor in the longevity of an organism. Spontaneously altered forms may themselves be toxic, or their accumulation may simply crowd out normal proteins. Two of the major sites of nonenzymatic degradation are aspartyl and asparaginyl residues, which are susceptible to an intramolecular reaction that results in the deamidation of asparaginyl residues and the isomerization and racemization of both aspartyl and asparaginyl residues. In all eucaryotic cells examined so far, an enzyme is present that can recognize at least some of these damaged sites and initiate their conversion to normal forms. This enzyme, the type II protein carboxyl methyltransferase, catalyzes the methyl esterification of L-isoaspartyl and D-aspartyl residues, enabling them to spontaneously revert to their normal L-aspartyl configurations. In this study, we utilize data on the rates of spontaneous degradation and enzymatic methylation in a computer program that simulates these reactions in the intact human erythrocyte. The results show that the methyltransferase may have an important role in limiting the accumulation of proteins containing altered aspartyl and asparaginyl residues.

Asparagine↗
PubMed1991

Functional assessment, curricular revision, and severe behavior problems.

An adolescent female with multiple handicaps and a long history of severely disruptive behavior participated in a functional assessment linked directly to specific revisions in her school curriculum. During Phase 1, reversal designs were used to test hypotheses pertaining to antecedent and curricular influences on problem behavior. During Phase 2, a multiple baseline across afternoon and morning time periods demonstrated that the curricular revisions were effective in eliminating severely disruptive behavior and increasing on-task responding. Data also showed that inappropriate "psychotic" speech was reduced and appropriate social interactions were increased. Follow-up results showed that the changes were maintained throughout the school year. Questionnaire data provided social validation of the procedures and outcomes. The findings are discussed in relation to their implications for functional assessment, individualized curricula, and positive programming for students with disabilities and serious behavior problems.

Achievement↗