Search PubMed⌕ Search

Biomedical subjects

P James

Publications and source records attributed to P James.

At least 73 records · Page 4Linked to original sources
PubMed1997

External quality assessment of the serum bactericidal test: results of a methodology/interpretation questionnaire.

Two hundred microbiology laboratories in the UK took part in two separate experimental external quality assessment distributions related to the serum bactericidal test (SBT). In the first, Staphylococcus aureus NCTC 6571 (vancomycin MIC 1 mg/L), was tested against a human serum containing vancomycin 38 mg/L plus gentamicin 0.5 mg/L. In the second, Streptococcus oralis PAJ 112/4183 (penicillin MBC < or = 0.03 mg/L) and Streptococcus sanguis PAJ 107/4184 (penicillin MBC = 128 mg/L) were tested against human serum containing penicillin 15 mg/L. Respondents returned their laboratory results and a questionnaire on clinical interpretation and technical aspects. Most laboratories (194/199, 97.5%) recommend the use of the SBT in the management of infective endocarditis but only 48 (25.2%) often or always change therapy on the basis of the result. A wide range of interpretative criteria, definitions of bactericidal endpoints and methodologies are used. Performance in the first distribution was acceptable for 75% of laboratories but in the second only 34% could identify penicillin tolerance; 34 respondents reported an SBT result of < or = 2 for the tolerant strain, 81 laboratories reported one of > or = 16. Technical factors related to acceptable performance were: sonication of broth before counting the inoculum; knowing the inoculum size in cfu/mL; use of a 4-8 h broth culture to make the inoculum; incubation of recovery plates for > 36 h; use of a calibrated pipette to sample for surviving bacteria; use of measured volumes to add the inoculum. Use of uncalibrated pipettes or standard loops to recover survivors was related to poor performance. Microbiology departments in the UK should review the clinical need to perform the SBT in the light of their local circumstances and if they elect to continue to offer this test, revise their methodologies which could be producing misleading results when testing alpha-haemolytic streptococci.

Bacteriological Techniques↗
PubMed1997

Using routine data to evaluate quality of care in British hospitals.

OBJECTIVES: As part of the process of developing an international comparative study of the outcome of hospital care it has been necessary to understand what data on hospital activity are available and how they compare with those in other countries. The authors describe the administrative data collected in British National Health Service Hospitals, with emphasis on how they differ from that available in the United States. METHODS: A description of the content of administrative data and a selective review of published literature related to their uses. RESULTS: Administrative data in the United Kingdom resembles that available in the United States, but it also has some important differences. These include a different system of procedure classification, the use of International Classification of Diseases, 10th Revision (rather than International Classification of Diseases, Ninth Revision, Clinical Modification), and the use of a different denominator ("finished consultant episodes," rather than admission spells). There also are important differences in the completeness and precision of the data that are collected. CONCLUSIONS: British administrative data can be used to describe patterns of care and have the potential to identify possible differences in outcome that can then be subjected to more detailed scrutiny. They are substantially less detailed than US data but are also somewhat less costly to collect.

Data Collection↗
PubMed1996

beta-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange.

Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C. When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured. Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C. Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence. We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL. Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement. Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.

Chaperonin 60↗
PubMed1996

The spleen protein-tyrosine kinase TPK-IIB is highly similar to the catalytic domain of p72syk.

TPK-IIB is a protein kinase that is predominant in the cytosol of spleen and is characterized by a high specific activity toward acidic peptide substrates and a low auto-phosphorylation activity. A prominent 52-kDa component purifies with the kinase [Marin, O., Donella-Deana, A., Brunati, A. M., Fischer, S. & Pinna, L. A. (1991) J. Biol. Chem. 266, 17798-17803]. Here we demonstrate that the 52-kDa protein displays sequence identity with the Miller-Dieker lissencephaly protein (LIS 1). The protein is not related to any known protein kinase and lacks an ATP-binding motif. The ATP binding and phosphotransferase activities of TPK-IIB can be fully accounted for by a minor 38-kDa protein band (p38/TPK-IIB) which can be separated from the 52-kDa protein by Mono-Q/FPLC in the presence of EDTA. Sequence analysis of p38/TPK-IIB reveals a high level of similarity, if not identity, with the catalytic domain of p72syk, a protein-tyrosine kinase implicated in the activation of hematopoietic cells. Antibodies raised against the catalytic domain of p72syk, but not antibodies raised against its N-terminal segment, cross-react with p38/TPK-IIB. The peptide substrate specificity of p72syk is almost identical to that of p38/TPK-IIB, which also supports the classification of TPK-IIB as a close relative (possibly a proteolytic or alternative spliced form) of p72syk. p38/TPK-IIB, however, exhibits a specific activity which is sixfold higher than that of p72syk and appears to be 50-fold more sensitive to inhibition by heparin. Thus, the observation that Ca(2+)-dependent degradation of p72syk by particulate fraction of rat spleen is accompanied by increased catalytic activity and increased sensitivity to heparin would be consistent with the possibility that hyperactive p38/TPK-IIB might be proteolytically generated from p72syk in response to an increase of intracellular Ca2+.

Affinity Labels↗
PubMed1996

Analysis of global responses by protein and peptide fingerprinting of proteins isolated by two-dimensional gel electrophoresis. Application to the sulfate-starvation response of Escherichia coli.

A set of 8 proteins (SSI, sulfate-starvation-induced proteins) was observed by comparative two-dimensional electrophoresis to be induced when Escherichia coli were grown using compounds other than sulfate or cysteine as the sole sulfur source. These proteins were isolated after two-dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry. The list of peptide masses served as a protein fingerprint which was used to search the databases, allowing four of the SSI proteins (SSI2, 5, 7, 8) to be identified with a high degree of confidence. To identify the other SSI proteins, and to obtain sequence information for as many of the proteins as possible, automated on-line HPLC MS/MS (fragmentation analysis using coupled mass scanning devices) data collection was performed. The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases. Genes encoding two further proteins (SSI 1 and 3) were identified in the 8.5' region of the Escherichia coli genome. N-terminal sequencing of all of the proteins confirmed the results of protein and peptide fingerprinting and in addition showed that SSI 6 shows 50% similarity to the Bacillus subtilis orfM gene product. SSI 4 was not found in the databases by any of these methods. The methods described are of general use for the rapid analysis of complex cell responses. MS data accumulation takes about 5 min/protein for protein fingerprinting and 30 min for peptide fingerprinting and requires approximately 100 fmol of material. N-terminal sequencing however, takes about 5 h/protein and approximately 1 pmol to obtain a 10 amino acid sequence for a search.

Alkanesulfonates↗
PubMed1996

Total hip replacement: a study of customized prostheses.

The majority of widely used hip prostheses offer a limited range of offset sizes, including 35, 37, 40, 44 and 45mm. These sizes are provided to match the mean population offset which has been reported as 43.0mm (Noble et al 1988). The variability of proximal femoral geometry in terms of offset is consistent with the theory that the geometry of the femur is determined by a large number of genetic and environmental factors (Ericksen 1979). This paper suggests that the range in offset sizes offered by off-the-shelf hip protheses is not sufficient to match the extremes of anatomical offset variability of the Northern Ireland sample studied and that a custom stem that matches more accurately key femoral dimensions such as offset, may be more desirable.

Clinical Nursing Research↗
PubMed1996

Isolation from spleen of a 57-kDa protein substrate of the tyrosine kinase Lyn. Identification as a protein related to protein disulfide-isomerase and localisation of the phosphorylation sites.

A 57-kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src-like tyrosine kinase Lyn purified from the same source with a Km of 0.34 microM. The tyrosine kinases c-Fgr, Fyn, C-terminal Src kinase and p72syk, as well as the Ser/Thr-specific cAMP-dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C-terminal Src kinase, which acts to down-regulate the Src-like protein-tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide-isomerase which belongs to the superfamily of Cys-Gly-His-Cys-containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C-terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti-Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested 'processive phosphorylation' model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s).

Amino Acid Sequence↗
PubMed1996

Proteolysis of human calcitonin in excised bovine nasal mucosa: elucidation of the metabolic pathway by liquid secondary ionization mass spectrometry (LSIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI).

PURPOSE: Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. METHODS: The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. RESULTS: Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. CONCLUSIONS: Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.

Amino Acid Sequence↗
PubMed1996

The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA.

SSH1, a newly identified member of the heat shock protein (hsp70) multigene family of the budding yeast Saccharomyces cerevisiae, encodes a protein localized to the mitochondrial matrix. Deletion of the SSH1 gene results in extremely slow growth at 23 degrees C or 30 degrees C, but nearly wild-type growth at 37 degrees C. The matrix of the mitochondria contains another hsp70, Ssc1, which is essential for growth and required for translocation of proteins into mitochondria. Unlike SSC1 mutants, an SSH1 mutant showed no detectable defects in import of several proteins from the cytosol to the matrix compared to wild type. Increased expression of Ssc1 partially suppressed the cold-sensitive growth defect of the SSH1 mutant, suggesting that when present in increased amounts, Ssc1 can at least partially carry out the normal functions of Ssh1. Spontaneous suppressors of the cold-sensitive phenotype of an SSH1 null mutant were obtained at a high frequency at 23 degrees C, and were all found to be respiration deficient. 15 of 16 suppressors that were analyzed lacked mitochondrial DNA, while the 16th had reduced amounts. We suggest that Ssh1 is required for normal mitochondrial DNA replication, and that disruption of this process in ssh1 cells results in a defect in mitochondrial function at low temperatures.

Amino Acid Sequence↗
PubMed1996

Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

Base Sequence↗
PubMed1996

ZD0870 treatment of murine candidiasis caused by fluconazole resistant isolates of Candida albicans.

Mice were infected intravenously with three fluconazole susceptible and ten fluconazole resistant isolates of Candida albicans then treated with escalating doses of 0.25, 0.5, 1, 2.5, 10 and 40 mg/kg/day of the new antifungal triazole, ZD0870, for 10 days. A minimum protective dose of < 0.25 mg/kg was determined for infections introduced by the three fluconazole susceptible C. albicans and one of the fluconazole resistant isolates whereas doses ranging from 2.5 to 10 mg/kg/day were required for infections induced by seven of the resistant isolates and > or = 40 mg/kg/day for the remainder. Thus, infections caused by fluconazole resistant C. albicans may be successfully treated with ZD0870, though higher doses than those used to treat infections due to susceptible yeast may be required.

Animals↗
PubMed1996

SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum.

The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p. Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment. We report the characterization of a novel Hsp70 of the ER, Ssi1p. Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions. However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese. The localization of Ssi1p to the ER, suggested by the presence of a conserved S. cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence. The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2. Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER. Unlike KAR2, however, SSI1 is not induced by heat shock. Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue. Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process.

Amino Acid Sequence↗
PubMed1995

The secondary structure of phospholamban: a two-dimensional NMR study.

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids which regulates the Ca2+ pump of the sarcoplasmic reticulum of heart, slow-twitch and smooth muscle (SR): it is normally assumed to exist in the membrane as a homopentamer. A monomeric analogue of phospholamban PLN(C41F), in which Cys41 was replaced by a Phe, was synthesized and its conformation studied by 1H NMR spectroscopy in a 1:1 mixture of chloroform/methanol. Most of the resonances in the 1H NMR spectra were assigned. The work has shown that the C-terminal hydrophobic portion forms a very stable alpha-helix. The hydrophilic N-terminal part adopts an alpha-helix configuration which is much less stable except for the stretch containing the phosphorylation sites.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of the arylsulfatase synthesized by Pseudomonas aeruginosa PAO during growth in sulfate-free medium and cloning of the arylsulfatase gene (atsA).

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PAO1 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDS/PAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57 degrees C and pH 8.9, and a Km of 105 microM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in P. aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5-751 mutant of P. aeruginosa PAO1. Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30-40% and 25-30% identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the P. aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the P. aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

Amino Acid Sequence↗
PubMed1995

Hierarchical phosphorylation of a 50-kDa protein by protein tyrosine kinases TPK-IIB and C-Fgr, and its identification as HS1 hematopoietic-lineage cell-specific protein.

TPK-IIB is an acidophilic protein tyrosine kinase devoid of autophosphorylation activity and unrelated to the Src family kinases [Marin, O., Donella-Deana, A., Brunati, A.M., Fischer, S. & Pinna, L. A. (1991) J. Biol. Chem. 266, 17798-17803]. Here, we describe the purification to homogeneity of a 50-kDa prominent substrate (p50) of TPK-IIB from rat spleen homogenates. Microsequence analysis of fragments from purified p50 discloses its homology to HS1, a hematopoietic-lineage cell-specific protein implicated in B-cell-antigen receptor-mediated signalling [Yamanashi, Y., Okada, M., Semba, T., Yamori, T., Umemori, H., Tsunasawa, S., Toyoshima, K., Kitamura, D., Watanabe, T. & Yamamoto, T. (1993) Proc. Natl Acad. Sci. USA 90, 3631-3635]. p50 is an excellent substrate for TPK-IIB, exhibiting very favourable kinetic constants (Km = 0.07 microM, kcat = 1.5) and incorporating up to 4 mol P/mol protein. p50 is, however, a weak substrate for the Src-related protein kinases Lyn and c-Fgr. Once phosphorylated by TPK-IIB, however, p50 is converted into a good substrate for c-Fgr and, to a lesser extent, for Lyn. p50 phosphorylated by TPK-IIB associates with c-Fgr and Lyn, as judged by co-immunoprecipitation with anti-Fgr IgG and anti-Lyn IgG, respectively. These data suggest the involvement of TPK-IIB in B-cell-antigen receptor-mediated signalling, and support the idea that phosphorylation by TPK-IIB might be a prerequisite for the recruitment of certain protein substrates by Src-related protein tyrosine kinases.

Adaptor Proteins, Signal Transducing↗