Biomedical subjects

S Clarke

Publications and source records attributed to S Clarke.

At least 235 records · Page 13Linked to original sources

PubMed1993

Left hemisphere strategies in visual recognition, topographical orientation and time planning.

Right hemisphere lesions are known to impair, in many cases, visual recognition and visuospatial orientation. We report here on the compensatory strategies used by a patient whose posterior part of the right hemisphere was either destroyed or visually deafferented. She presented a slight appreceptive agnosia, memory disorders, and severe topographical disorientation. Her strategy relied on detail-by-detail analysis of buildings for recognition and on identification of landmarks and memorization of their sequences for finding routes. Time planning was impaired; the patient was unable to use a bi-dimensional timetable, but relied on a (linear) list of assignments. The linear, speech-related strategy she used was sustained uniquely by left hemisphere networks, since no processing of visual information occurred in the right hemisphere.

Adolescent↗

PubMed1993

Modification of eukaryotic signaling proteins by C-terminal methylation reactions.

Eukaryotic polypeptides that are initially synthesized with the C-terminal sequence -Cys-Xaa-Xaa-Xaa, including a variety of signal-transducing proteins, such as small G-proteins, large G-proteins and cGMP phosphodiesterases, can be targeted for a series of sequential post-translational modifications. This processing pathway includes the isoprenylation of the cysteine residue with a farnesyl or geranylgeranyl moiety, followed by proteolysis of the three terminal residues and alpha-carboxyl methyl esterification of the cysteine residue. The potential reversibility of the last step suggests that it may be involved in modulating the function of these proteins. Firstly, methylation may play a role in the activation of cellular peptides or proteins. Secondly, this modification may aid in the membrane attachment of cytosolic precursor proteins. Thirdly, methylation may protect the polypeptide from C-terminal proteolytic degradation once the three terminal amino acid residues are removed. Finally, reversible methylation may directly regulate the function of its target proteins. Therapeutically, inhibitors of C-terminal isoprenylcysteine methylation or demethylation reactions may prove to be useful pharmacological tools as anti-cancer and anti-inflammatory agents.

Alkyl and Aryl Transferases↗

PubMed1993

Protein methylation.

Proteins can be enzymatically modified in several ways by the addition of methyl groups from S-adenosylmethionine. Reactions forming methyl esters on carboxyl groups are potentially reversible and can modulate the activity of the target protein; in the past year, advances have been made in understanding the physiological roles of four distinct systems that modify normal and abnormal carboxyl groups on proteins. On the other hand, methylation reactions occurring on nitrogen atoms in N-terminal and side-chain positions are generally irreversible. These reactions create new types of amino acid residues and can expand the repertoire of chemistry that a protein can perform.

Aging↗

PubMed1993

Low frequencies of somatic mutation in two expressed V kappa genes: unequal distribution of mutation in 5' and 3' flanking regions.

Somatic mutation of antibody variable region genes is a hallmark of secondary responses. Most often the coexpressed VH and V kappa genes of a B cell mutate at nearly equal rates. We have previously identified hybridomas from two B cell clones that exhibited > 10-fold lower frequency of mutation in their expressed V kappa 12 or V kappa 1A genes relative to their coexpressed VH genes. To gain insight into the mechanism(s) responsible for this low frequency V kappa mutation, we determined the frequency of mutation in non-coding flanking DNA from multiple members of each clone. We find a low frequency of mutation in the 5' (4/700) and 3' (1/670) non-coding regions of the expressed V kappa 1A genes consistent with the low frequency of coding region mutation. In contrast, the distribution and frequency of mutation surrounding the expressed V kappa 12.37 gene are unusual. Among the six members of this clone there are 31 mutations 3' (31/2100 bp) and no mutations 5' (0/2010 bp) of the V kappa exon. This V kappa exon has acquired mutations that are intermediate in number to its flanking regions and are significantly skewed in distribution to the 3' end. None of the 31 3' mutations are shared by two or more members of this clone, indicating that they all occurred late in clonal expansion. These results raise the possibility that some V kappa genes may lack functional cis-regulatory elements which direct V kappa coding region mutation.

Animals↗

PubMed1993

Foreign bodies in the foot.

A symptomatic foreign object lodged in the foot can be a difficult problem. The object's location and shape are the most important factors to identify before removal of the object is attempted. Many different imaging modalities have been used to locate the foreign objects in the foot. The authors briefly reviewed several common imaging modalities. Computed tomography appears to be superior to the other imaging modalities discussed in visualizing and localizing foreign objects in the foot. Magnetic resonance scanning is probably equal to computed tomography but is less readily available and more expensive and is contraindicated with ferrous metal foreign bodies.

Adult↗

PubMed1993

The relationship between marital breakdown and childbearing in England and Wales.

"The paper uses data on women from the General Household Surveys for 1986-89 [for England and Wales] to form combined multiple decrement tables on the transitions from first marriage through childbearing, divorce and remarriage. It shows that ultimately women who experience one marital breakdown tend to have around the same number of children...as those who remain married. However, their time to complete childbearing is rather longer with this extra time being concentrated largely in the birth interval in which the marital breakdown takes place. The group of women who experience more than one marital breakdown tend to have more children and consequently shorter birth intervals."

Birth Intervals↗

PubMed1993

The relationship between serial measures of disease activity and outcome in rheumatoid arthritis.

Disease activity was measured annually over a median period of 7 years (range 5-9) in a cohort of 127 patients with rheumatoid arthritis. The measurements were plotted, and the area under the resultant curve measured. The relationship of serial measures of disease activity (area under the curve) to outcome (measured radiologically, functionally and by global assessment) was investigated. A significant correlation was found between persistent disease activity and radiographic deterioration. Similar results were found for functional outcome, as measured by Steinbrocker grade, health assessment questionnaire score or global assessment (by analogue score). Single measures of disease activity did not predict outcome. Although imprecise, current methods of measuring disease activity in RA, if measured serially, are valuable in predicting outcome over a 5-10 year period.

Adrenal Cortex Hormones↗

PubMed1992

A protein methyltransferase specific for altered aspartyl residues is important in Escherichia coli stationary-phase survival and heat-shock resistance.

Proteins are subject to spontaneous degradation reactions including the deamidation, isomerization, and racemization of asparaginyl and aspartyl residues. A major product of these reactions, the L-isoaspartyl residue, is recognized with high affinity by the protein-L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77). This enzyme catalyzes the methyl esterification of the L-isoaspartyl residue in a reaction that can initiate its conversion to the normal aspartyl configuration. To directly study the physiological role of this methyltransferase, especially with respect to the potential repair of isomerized aspartyl residues in aging proteins, we examined the ability of the bacterium Escherichia coli to survive in the absence of its activity. We utilized gene disruption techniques to replace the chromosomal copy of the pcm gene that encodes the methyltransferase with a kanamycin-resistance cassette to produce mutants that have no detectable L-isoaspartyl methyltransferase activity. Although no changes in exponential-phase growth were observed, pcm- mutants did not survive well upon extended culture into stationary phase or upon heat challenge at 55 degrees C. These results provide genetic evidence for a role of the L-isoaspartyl methyltransferase in the metabolism of altered proteins that can accumulate in aging cells and limit their viability.

Aspartic Acid↗

PubMed1992

Characterization of a rat liver protein carboxyl methyltransferase involved in the maturation of proteins with the -CXXX C-terminal sequence motif.

We have utilized S-farnesyl-Leu-Ala-Arg-Tyr-Lys-Cys as a methyl-accepting substrate to characterize a membrane-bound C-terminal protein methyltransferase from rat liver. We have localized the activity to the microsomal fraction and show that the bulk of the enzyme fractionates by density gradient centrifugation with glucose-6-phosphatase, a marker of the endoplasmic reticulum, and not with 5'-nucleotidase, a marker of the plasma membrane, or galactosyl:N-acetylglucosamine transferase, a marker of the Golgi apparatus. This methyltransferase appears to form an integral part of the membrane structure. Its activity is markedly affected by a variety of detergents used to solubilize membrane proteins in their native form. All activity is lost when membranes are treated with seven different detergents at a concentration of 1% (w/v). The activity is inhibited by N-ethylmaleimide, although it can be protected against inactivation with its substrate S-adenosyl-L-methionine, or its product S-adenosyl-L-homocysteine. Finally, we find that 5'-methylthioadenosine, a substrate analogue reported to be an inhibitor of this activity in other studies, is not an effective inhibitor in vitro.

5'-Nucleotidase↗

PubMed1992

Alternative splicing of the human isoaspartyl protein carboxyl methyltransferase RNA leads to the generation of a C-terminal -RDEL sequence in isozyme II.

We have isolated two cDNA clones that correspond to the mRNAs for two isozymes of the human L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). The DNA sequence of one of these encodes the amino acid sequence of the C-terminal half of the human erythrocyte isozyme I. The other cDNA clone includes the complete coding region of the more acidic isozyme II. With the exception of potential polymorphic sites at amino acid residues 119 and 205, the deduced amino acid sequences differ only at the C-terminus, where the -RWK sequence of isozyme I is replaced by a -RDEL sequence in isozyme II. The latter sequence is identical to a mammalian endoplasmic reticulum retention signal. With the previous evidence for only a single gene for the L-isoaspartyl/D-aspartyl methyltransferase in humans, and with evidence for consensus sites for alternative splicing in corresponding mouse genomic clones, we suggest that alternative splicing reactions can generate the major isozymes previously identified in human erythrocytes. The presence of alternative splicing leads us to predict the existence of a third isozyme with a -R C-terminus. The calculated isoelectric point of this third form is similar to that of a previously detected but uncharacterized minor methyltransferase activity.

Amino Acid Sequence↗

PubMed1992

Maturation of isoprenylated proteins in Saccharomyces cerevisiae. Multiple activities catalyze the cleavage of the three carboxyl-terminal amino acids from farnesylated substrates in vitro.

Eukaryotic polypeptides containing COOH-terminal-CXXX sequences can be posttranslationally modified by isoprenylation of the cysteine residue via a thioether linkage, proteolytic removal of the three terminal amino acids, and alpha-carboxyl methylation of the cysteine residue. Through the development of an indirect coupled assay, we have identified three in vitro activities in the yeast Saccharomyces cerevisiae that can catalyze the proteolytic cleavage of the three COOH-terminal amino acids of the synthetic peptide substrate N-acetyl-KSKTK[S-farnesyl-Cys]VIM. One of these is the vacuolar protease carboxypeptidase Y. Using a mutant strain deficient in this enzyme, we find evidence for an additional soluble activity as well as for a membrane-associated activity. These latter activities are candidates for roles in the physiological processing of isoprenylated protein precursors. They are both insensitive to inhibitors of serine and aspartyl proteinases but are sensitive to sulfhydryl reagents and 0.5 mM ZnCl2. The soluble activity appears to be a metalloenzyme, inhibitable by 2 mM o-phenanthroline but not by 1 mM N-ethylmaleimide, whereas the membrane-associated enzyme is inhibitable by 1 mM N-ethylmaleimide but not 2 mM o-phenanthroline. We show that the membrane-bound protease is not an activity of the membrane-bound methyltransferase, because protease activity is observed in membrane preparations that lack the STE14-encoded methyltransferase. The soluble activity appears to be a novel carboxypeptidase of approximately 110 kDa that catalyzes a processive removal of amino acids from the COOH terminus from both the farnesylated and non-farnesylated substrate, but not from three other unrelated peptides. Finally, we find no evidence for non-vacuolar membrane or soluble activities that catalyze the ester hydrolysis of N-acetyl-S-farnesyl-L-cysteine methyl ester.

Alkyl and Aryl Transferases↗

PubMed1992

VH12 rearrangements in adult peritoneal B cells.

About half of the phosphatidylcholine (PtC)-binding peritoneal B cells of B10.H-2aH-4bp/Wts mice express the VH12 gene. In these cells the D-region gene segments are restricted in length and sequence and are always rearranged to JH1, suggesting that PtC-specific B cells are clonally selected. To assess the extent to which this VH gene is used by peritoneal B cells to encode antibodies of other specificities, we have analyzed the length and sequence of D-region gene segments of VH12-D-JH1 rearrangements in peritoneal B cells independent of antigen specificity by PCR. We find that all 34 randomly chosen VH12-D-JH1 rearrangements analyzed are productive and have D regions that are restricted identically to those of PtC-specific B cells. These data suggest that essentially the entire repertoire of VH12-D-JH1 rearrangements are used by B cells that bind PtC, further illustrating the degree to which this repertoire is shaped by antigen selection.

Animals↗

PubMed1992

Use of thermometers in general practice.

OBJECTIVE: To identify the attitudes of general practitioners towards the use of thermometers in general practice. DESIGN: Postal questionnaire survey. SETTING: All general practitioners in the catchment area of Frimley Park Hospital, Surrey. SUBJECTS: 145 general practitioners. MAIN OUTCOME MEASURES: Answers to questions covering a variety of aspects concerning the use of thermometers in general practice. RESULTS: 116 (80%) doctors replied. Seven doctors did not have any method of taking a patient's temperature; up to 12 more doctors did not use their thermometers and 56 doctors used them infrequently, less than once a fortnight. Mercury glass thermometers were most commonly used (80 doctors; 69%), but only 8% of doctors used them correctly. Six doctors failed to clean their thermometers between patients. The study failed to identify the roles of axillary and rectal temperature readings. CONCLUSION: There is a wide variation in attitudes towards the use of thermometers in general practice.

Attitude of Health Personnel↗

PubMed1992

Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We provide here the first direct evidence that D-aspartyl residues in peptides are substrates for the L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). We do this by showing that D-aspartic acid beta-methyl ester can be isolated from carboxypeptidase Y digests of enzymatically methylated D-aspartyl-containing synthetic peptides. The specificity of this reaction is supported by the lack of methylation of L-aspartyl-containing peptides under similar conditions. Methylation of D-aspartyl residues in synthetic peptides was not observed previously because with Km values ranging from 2.5 to 4.8 mM, these peptides are recognized by the methyltransferase with 700-10,000-fold lower affinity than are their L-isoaspartyl-containing counterparts. The physiological significance of D-aspartyl methylation was investigated in two ways. First, analysis of in situ methylated human erythrocyte proteins showed that at least 22% of the methyl groups associated with the proteins ankyrin and band 4.1 are on D-aspartyl residues, suggesting that D-aspartyl methylation is an important function of the methyltransferase in vivo. Second, mathematical modeling of the protein aging and methylation reactions occurring in intact erythrocytes indicated that the accumulation of D-aspartyl residues can be reduced as much as 2-5-fold by the methyltransferase activity. Although this reduction is much less than that predicted for L-isoaspartyl residues, it may be significant in maintaining functional proteins throughout the 120-day life span of these cells.

Amino Acid Sequence↗

PubMed1992

Lactational transfer of 3,3',4,4'-tetrachloro- and 2,2',4,4',5,5'-hexachlorobiphenyl induces cytochrome P450IVA1 in neonates. Evidence for a potential synergistic mechanism.

On the first day of lactation, material rats were treated with a single low dose of 5 mg/kg body weight of 3,3',4,4'-tetrachlorobiphenyl (TCB) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) or with a combination of both congeners. Lactational transfer of these polychlorinated biphenyls (PCBs) was found in neonates and significant increases in microsomal cytochrome P450, cytochrome b5 and in glutathione-S-transferase activity were observed. Treatment with HCB did not increase neonatal ethoxyresorufin-O-de-ethylation (EROD) activities whereas a more than 26-fold increase in EROD activity was noted in response to exposure to TCB. However, EROD activities were increased more than 65-fold in response to the combined exposure to TCB and HCB. Exposure via milk to TCB caused a significant reduction in the N-demethylation of aminopyrine, but the combined exposure to TCB and HCB produced a significant reduction in the N-demethylation of dimethylnitrosamine. Lactational transfer of either TCB or HCB reduced marginally peroxisomal enzyme activities; however, exposure to a combination of TCB and HCB resulted in the highly significant reduction in KCN-insensitive palmitoyl-CoA oxidation and acetyl-CoA oxidation. Contrary to the reduction of these enzyme activities, the specific concentrations of CYP4A1 were significantly increased when neonates were exposed to either TCB or HCB. The largest induction, however, was observed in response to the combined exposure to both PCBs. Evidence is presented to suggest an induction of CYP4A1 which may be independent of the molecular substitution pattern of the two PCBs used in our studies but on a possible mode of synergistic interaction.

Animals↗

PubMed1992

In vivo differential prenylation of retinal cyclic GMP phosphodiesterase catalytic subunits.

A number of phototransducing proteins in vertebrate photoreceptors contain a carboxyl terminal -CXXX motif (where C = cysteine and X = any amino acid), known to be a signal sequence for their post-translational prenylation and carboxyl methylation. To study the roles of these modifications in the visual excitation process, we have utilized an intravitreal injection method to radiolabel the prenylated proteins of rat retinas in vivo. We showed that two of the major prenylated polypeptides in the rod outer segments are the PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase (PDE). By chromatographic analyses of the amino acid constituents generated by exhaustive proteolysis of PDE alpha and PDE beta, we further demonstrated that they are differentially prenylated by farnesylation and geranylgeranylation, respectively. While a number of proteins ending with the -CXXX sequence have already been reported to possess either a farnesyl or a geranylgeranyl group, PDE is the first enzyme shown to be modified by both types of prenyl groups. The prenyl modification of PDE most likely plays a major role in membrane attachment and in correctly positioning the PDE molecule for phototransduction.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1992

Macrophages in tissues and in vitro.

Macrophages have specialized functions in different tissue microenvironments such as lymphohaemopoietic organs and the nervous system. Recently, progress has been made in defining cellular and molecular properties of isolated and tissue macrophages in the developing and adult animal.

Animals↗

PubMed1992

The L-isoaspartyl/D-aspartyl protein methyltransferase gene (PCMT1) maps to human chromosome 6q22.3-6q24 and the syntenic region of mouse chromosome 10.

We have mapped the genes for the human and mouse L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77) using cDNA probes. We determined that the human gene is present in chromosome 6 by Southern blot analysis of DNA from a panel of mouse-human somatic cell hybrids. In situ hybridization studies allowed us to confirm this identification and further localize the human gene (PCMT1) to the 6q22.3-6q24 region. By analyzing the presence of an EcoRI polymorphism in DNA from backcrosses of C57BL/6J and Mus spretus strains of mice, we localized the mouse gene (Pcmt-1) to chromosome 10, at a position 8.2 +/- 3.5 cM proximal to the Myb locus. This region of the mouse chromosome is homologous to the human 6q24 region.

Animals↗