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Biomedical subjects

S Howell

Publications and source records attributed to S Howell.

At least 91 records · Page 5Linked to original sources
PubMed1994

Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity.

The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A2 (PLA2) shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA2 was studied. Cytosolic PLA2 has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA2 was also analysed. In contrast with a previous report, no PLA2 activity was found in brain 14-3-3, nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made.

14-3-3 Proteins↗
PubMed1994

Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells.

Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart.

Amino Acid Sequence↗
PubMed1994

Phase I trial of cisplatin in combination with glutathione.

We treated 16 patients in a phase I trial of escalating doses of intravenous cisplatin in combination with the chemoprotectant glutathione given every 21 days. Forty-three of 44 cycles (98%) were evaluable, 85% of cycles were given on time, and the median number of cycles per patient was 2. Dose-limiting nephrotoxicity was reached at a dose of 175 mg/M2 of cisplatin. Other toxicities included ototoxicity in 7 patients (44%) and grade 3 to 4 nausea and vomiting in 15 evaluable cycles (34.9%). Myelosuppression was infrequent. An increase to 175% of standard cisplatin dose intensity is attainable with the administration of glutathione; however, toxicity is substantial and the number of tolerated cycles is limited. Alternatives to the single bolus dose schedule studied in the present trial should be explored in order to better define the clinical utility of glutathione in combination with high-dose cisplatin.

Adult↗
PubMed1994

Methaemoglobin production in normal adults inhaling low concentrations of nitric oxide.

OBJECTIVE: The study was performed to determine the changes in blood methaemoglobin level during the inhalation of nitric oxide. DESIGN: The study was an unblinded dose-response study. PARTICIPANTS: 5 healthy adult volunteers aged 30-36 (4 male and 1 female) were studied on 4 occasions separated by at least one week. INTERVENTION: Nitric oxide was inhaled at inspired concentrations of 32, 64, 128, and 512 volumes per million (vpm) in air. Venous blood samples were taken every 10 min for methaemoglobin determination. Inhalation continued for 3 h (32, 64 and 128 vpm) or until the methaemoglobin exceeded 5% of the total haemoglobin (512 vpm). The methaemoglobin levels were also recorded for 3 h after 512 vpm nitric oxide had been stopped. MEASUREMENTS AND RESULTS: Both the increase in methaemoglobin fraction during nitric oxide inhalation and the decay after ceasing inhalation fitted well with a first order model describing methaemoglobin elimination. The calculated time constants were between 39-91 min. The predicted mean maximum methaemoglobin levels that would be achieved during inhalation of 32, 64, 128, and 512 vpm nitric oxide were 1.04% (0.92-1.16), 1.75% (1.80-1.90%), 3.75% (3.58-4.05), 6.93% (5.70-8.16) respectively (95% confidence interval of estimate in brackets). CONCLUSIONS: In normal individuals inhalation of up to 128 vpm of nitric oxide, greater than any dose used clinically to date, does not result in clinically significant methaemoglobinaemia. Maximum methaemoglobin levels are likely to be reached in 3-5 h after inhalation begins. However, these figures may not apply to critically ill adults and infants. Nitric oxide may have other toxic effects not examined in this study.

Administration, Inhalation↗
PubMed1994

A district epilepsy service, with community-based specialist liaison nurses and guidelines for shared care.

In order to improve epilepsy services, Doncaster Hospital doctors and general practitioners agreed upon a collaborative approach. A district epilepsy service was inaugurated around specialist services based in a hospital clinic. Guidelines were produced to clarify respective roles and to assist non-specialist hospital doctors and general practitioners in epilepsy management. A novel feature of the new service was the appointment of a community-based specialist liaison nurse. The new service is perceived to be effective, and the contribution of the specialist nurse to represent an important advance in epilepsy care. It has been possible to supervise complicated changes in medication successfully at home with a reduction in the need to attend clinic or GP. Counselling and support for patients and families, previously lacking, has been a major contribution. Evaluation to determine the extent to which the service meets the needs of those in the local population with epilepsy is planned. This document describes the first 5 years of the service and the scope and content of the specialist liaison nurse's role.

Adolescent↗
PubMed1993

Antibodies against the major brain isoforms of 14-3-3 protein. An antibody specific for the N-acetylated amino-terminus of a protein.

14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. They have been implicated in the regulation of a wide range of biological processes [reviewed in Aitken et al. (1992) Trends Biochem. Sci. 17, 498-501]. We have raised specific antibodies against each mammalian brain isoform of 14-3-3 employing peptides synthesised from the amino-terminal regions. The peptides were, like the proteins from mammalian brain, N-acetylated. The antiserum specific for the epsilon isoform did not recognise the recombinant form of this protein (lacking the N-acetyl co-translational modification) expressed in E. coli until it was chemically acetylated.

14-3-3 Proteins↗
PubMed1993

Toxicity of paracetamol in cultured chick hepatocytes treated with methotrexate.

Cultured chick hepatocytes were used to investigate the hepatotoxicity of methotrexate alone and in combination with paracetamol. Treatment with methotrexate alone at concentrations as high as 1 mg/ml resulted in no toxicity in cultured chick hepatocytes, as indicated by no detachment of cells and no effect on protein synthesis or on release of the intracellular enzyme lactate dehydrogenase. However, treatment with methotrexate alone resulted in a 30% decrease in reduced glutathione levels. Combined treatment with methotrexate and paracetamol was toxic, but only in cells preinduced for cytochrome P450 1A by treatment with beta-naphthoflavone. Under these conditions, methotrexate lowered the threshold concentration of paracetamol at which toxicity was observed. This methotrexate-mediated increase in paracetamol toxicity was associated with decreased formation of the glucuronide, sulfate and thiol metabolites of paracetamol and with increased covalent binding of radiolabeled paracetamol to macromolecules. In cells pretreated with beta-naphthoflavone, additional treatment with either methotrexate or buthionine sulfoximine, an inhibitor of glutathione synthesis, together with paracetamol, was associated with decreased restoration of glutathione levels. These results suggest that methotrexate increased paracetamol toxicity by decreasing the amount of glutathione available for conjugation with reactive metabolites of paracetamol.

Acetaminophen↗
PubMed1993

Membrane peptidases on human osteoblast-like cells in culture: hydrolysis of calcitonin and hormonal regulation of endopeptidase-24.11.

Five membrane peptidase activities have been identified on cultured human osteoblast-like cells. These consisted of the four exopeptidases aminopeptidase-A, aminopeptidase-N, aminopeptidase-W and carboxypeptidase-M, and the endopeptidase, endopeptidase-24.11. The presence of endopeptidase-24.11 was confirmed immunochemically by immunofluorescent staining and by enzyme-linked immunosorbent assay. The inclusion of phosphoramidon partially inhibited the hydrolysis of human calcitonin by a membrane fraction prepared from osteoblast-like cell membranes, thus implicating endopeptidase-24.11 in its inactivation. Another metallopeptidase also contributed substantially to calcitonin hydrolysis. Purified porcine endopeptidase-24.11 (1 microgram) was shown to hydrolyse calcitonin with a half-life of 23 min, which compared to a half-life of 0.5 min for substance P under similar conditions. Sequence data revealed that the initial site of hydrolysis of calcitonin was between residues Lys18 and Phe19. The expression of endopeptidase-24.11 by cultured osteoblast-like cells was shown to be modified by various agents: expression was decreased by phorbol 12-myristate-13-acetate (160 nM for 48 h) and increased in the presence of calcitonin (1.5 nM for 48 h) and 1,25-dihydroxyvitamin D3 (0.01-1 microM for 72 h).

Amino Acid Sequence↗
PubMed1993

Mosaic expression of membrane peptidases by confluent cultures of Caco-2 cells.

The cell-surface expression of endopeptidase-24.11 (EC 3.4.24.11) on Caco-2 cells cultured to confluency is markedly heterogeneous unlike that of dipeptidylpeptidase IV (EC 3.4.14.5). Here we have investigated the cell-surface expression of three other ectopeptidases: angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase N (EC 3.4.11.2) and aminopeptidase W (EC 3.4.11.16). We show by indirect immunofluorescent staining that these three enzymes are present on the surface of some cells but not on others. However, these enzymes were detected in the majority of detergent-permeabilised Caco-2 cells indicating the presence of intracellular pools of these enzymes. This suggests that there may either be differential regulation of apical transport for these peptidases or that they recycle at different rates.

Aminopeptidases↗
PubMed1993

Dose-response relationship for inhaled nitric oxide in experimental pulmonary hypertension in sheep.

We have examined the effect of inhaled nitric oxide 4-512 p.p.m. in six sheep with pulmonary hypertension induced first with hypoxia and then with 6 micrograms kg-1 of E. coli endotoxin. A similar dose-dependent reduction in pulmonary artery pressure occurred in pulmonary hypertension induced by hypoxia or endotoxin, with a maximum effect of 25-30% decrease with nitric oxide 64 p.p.m. Increasing the dose to 512 p.p.m. had no further effect. The ED50 for inhaled nitric oxide was 39 p.p.m. for pulmonary hypertension induced by hypoxia and 48 p.p.m. for endotoxin-induced pulmonary hypertension. A dose-dependent increase in arterial oxygenation, which reached a maximum with nitric oxide 64 p.p.m., was seen with inhaled nitric oxide after endotoxin infusion. If the toxicity of inhaled nitric oxide can be determined, it may prove useful in the treatment of pulmonary hypertension secondary to the adult respiratory distress syndrome.

Animals↗
PubMed1993

Evaluating the competence of physicians in practice: from peer review to performance assessment.

Evaluation of physician competence has traditionally been defined by the medical profession largely through standardized tests and board certifying examinations. However, a level of physician evaluation that takes place outside academic medical centers and certifying boards is rapidly developing. This article describes three programs for such physician evaluation: (1) the program of US Healthcare, a national managed health care company; (2) the DEMPAQ--Developing and Evaluating Methods to Promote Ambulatory Care Quality--project, a joint research effort between Harvard University (Cambridge, Massachusetts) and the State of Maryland's (and District of Columbia's) Peer Review Organization (PRO); and (3) a project initiated by the American College of Physicians (ACP) in Philadelphia, Pennsylvania, to assess both medical competence and technical performance in the hospital setting. The authors argue for the need to inform physicians-in-training about the types of evaluation to which they will be subjected over the courses of their careers. In order to further this goal, the authors advocate increased collaboration between leaders in the academic setting and those spearheading these new programs for assessment of physician performance.

Ambulatory Care↗
PubMed1993

Chondroid syringoma with hyaline cell change.

Four cases of chondroid syringoma containing large numbers of hyaline or plasmacytoid cells are described. Three cases occurred in the hand and one in the foot. Hyaline cells are commonly seen in mixed tumours and myoepitheliomas of salivary glands and rarely in chondroid syringomas. The hyaline-cell change in three of the cases initially caused diagnostic difficulties and the possibility of sarcoma was raised in two cases. In addition to the characteristic hyaline cells, the presence of tubulo-glandulo-ductal structures, benign squamous epithelium and myxochondroid stroma aided diagnosis. Immunohistochemically, the hyaline cells exhibited positivity for vimentin, cytokeratin, S-100 protein, carcino-embryonic antigen, focal glial fibrillary acidic protein (3 cases), neuron-specific enolase (3 cases) and focal alpha-smooth muscle actin (2 cases). Occasional cells were Ber EP4 positive (2 cases). In some cells, a striking peripheral ring-like positivity for cytokeratin and S-100 protein was noted. Ultrastructurally, desmosomes, varying numbers of tonofibrils and non-bundling intermediate filaments were seen. Scanty fine filaments with vague focal densities were detected in some cells. Our studies suggest that the hyaline cells represent modified epithelial as well as myoepithelial cells. One of our cases also exhibited collagenous spherulosis.

Actins↗
PubMed1993

Orthodontic treatment needs in Westmead Hospital Dental Clinical School.

One hundred and fifty-four adolescents, 76 female and 78 male consented to take part in this survey. The two authors examined and assessed them for dental aesthetics, malocclusion related features and TMJ related signs. The patients were also questioned about their opinion of dental aesthetics, their interest in having orthodontic treatment and their TMJ symptoms. Only 63 per cent of the adolescents who were considered suitable for orthodontic treatment expressed an interest in wanting treatment. Twenty-seven per cent of patients had signs and/or symptoms of TMJ disturbance. No association was found between individual malocclusion problems and TMJ signs and symptoms. The authors considered 56.5 per cent of the total group for orthodontic treatment, the majority for aesthetic reasons, and placed them into high, medium and low priority groups. Fixed appliance therapy for both arches was the recommended type of treatment for most patients.

Adolescent↗
PubMed1992

A survey of membrane peptidases in two human colonic cell lines, Caco-2 and HT-29.

The expression of cell-surface peptidases was examined in two human colon carcinoma cell lines, Caco-2 and HT-29. Enzymic assays revealed the presence of eight cell-surface peptidases on a Caco-2 cell line (passage number 82-88), namely aminopeptidase N, dipeptidyl peptidase IV, peptidyl dipeptidase A (angiotension-converting enzyme), aminopeptidase P, aminopeptidase W, endopeptidase-24.11, gamma-glutamyl transpeptidase and membrane dipeptidase. The presence of dipeptidyl peptidase IV and endopeptidase-24.11 was also confirmed immunochemically. After 15 days culture, the activities of aminopeptidase P, peptidyl dipeptidase A and alkaline phosphatase activities on Caco-2 cells reached a plateau, and that of membrane dipeptidase began to decline. In contrast, aminopeptidase N, dipeptidyl peptidase IV and endopeptidase-24.11 activities were still rising after 26 days in culture. Caco-2 cells of passage number 181-183 were found to lack endopeptidase-24.11, but maintained dipeptidyl peptidase IV expression. Two populations of HT-29 cells were surveyed. Both the standard, undifferentiated population and a differentiated population expressed only three peptidases: dipeptidyl peptidase IV, aminopeptidase W and carboxypeptidase M. In the differentiated HT-29 cells the activity of dipeptidyl peptidase IV after 14-21 days was beginning to plateau whereas aminopeptidase W activity was still rising and that of carboxypeptidase M had begun to decline. These differences in activity profiles observed among this group of cell-surface peptidases indicate that these cell lines, especially Caco-2, are useful models to study the regulation of their expression.

Aminopeptidases↗
PubMed1992

The complete sequences of trout (Salmo gairdneri) thymosin beta 11 and its homologue thymosin beta 12.

Two forms of beta-thymosins, designated thymosin beta 11 and thymosin beta 12, were isolated from trout (Salmo gairdneri) spleen. This suggests that the presence of two beta-thymosins, previously thought to be a property of mammalian tissues only, is a more general phenomenon in vertebrate species. Both trout beta-thymosins were found to be N-terminally blocked by a group identified as acetyl by m.s. Automated protein sequencing of tryptic, thermolytic and Staphylococcus aureus in 41-residue V8 proteinase fragments revealed that one of the two beta-thymosins corresponds to the previously reported 41-residue-long sequence of thymosin beta 11 with two substitutions at positions 5 and 7, i.e. Asn instead of Asp, and Glu instead of Gln, whereas the other beta-thymosin, designated thymosin beta 12, was found to be a 42-residue polypeptide closely similar in sequence to thymosin beta 11, with five substitutions (i.e. at positions 5, 7, 10, 11 and 41, with Asp, Ala, Ser, Asn and Thr instead of Asn, Glu, Ala, Ser and Ser respectively) and one addition at position 42 (Ala). Comparison of the known six sequences of beta-thymosins together with the sequences reported here showed that the sequence similarity of the two beta-thymosins in trout (86%) is greater than that of the two beta-thymosins in mammalian species (74%) and that residues at 28 positions are identical in all beta-thymosins, the longer conserved segments located at positions 16-26 and 31-38.

Amino Acid Sequence↗