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Biomedical subjects

P James

Publications and source records attributed to P James.

At least 37 records · Page 2Linked to original sources
PubMed2000

Relation between troponin T concentration and mortality in patients presenting with an acute stroke: observational study.

OBJECTIVE: To assess whether a raised serum troponin T concentration would be an independent predictor of death in patients with an acute ischaemic stroke. DESIGN: Observational study. SETTING: Auckland Hospital, Auckland, New Zealand. SUBJECTS: All 181 patients with an acute ischaemic stroke admitted over nine months in 1997-8, from a total of 8057 patients admitted to the acute medical service. MAIN OUTCOME MEASURES: Blood samples for measuring troponin T concentration were collected 12-72 hours after admission; other variables previously associated with severity of stroke were also recorded and assessed as independent predictors of inpatient mortality. RESULTS: Troponin T concentration was raised (>0.1 microgram/l) in 17% (30) of patients admitted with an acute ischaemic stroke. Thirty one patients died in hospital (12/30 (40%) patients with a raised troponin T concentration v 19/151 (13%) patients with a normal concentration (relative risk 3.2 (95% confidence 1.7 to 5. 8; P=0.0025)). Of 17 possible predictors of death, assessed in a multivariate stepwise model, only a raised troponin T concentration (P=0.0002), age (P=0.0008), and an altered level of consciousness at presentation (P=0.0074) independently predicted an adverse outcome. CONCLUSIONS: Serum troponin T concentration at hospital admission is a powerful predictor of mortality in patients admitted with an acute ischaemic stroke.

Acute Disease↗
PubMed2000

GRP94 (endoplasmin) co-purifies with and is phosphorylated by Golgi apparatus casein kinase.

A phosphorylatable protein band of about 94 kDa (as judged by SDS-PAGE) which co-purifies and co-immunoprecipitates with Golgi apparatus casein kinase (G-CK) from rat lactating mammary gland has been shown by mass spectrometric sequence analysis to be identical or very similar to the glucose-regulated protein, GRP94. GRP94 is also readily phosphorylated by G-CK (K(m)=0.2 microM) at seryl sites which are different from the sites affected by casein kinase-2 (CK2) in the same protein. A study with peptide substrates would indicate that the G-CK sites in GRP94 conform to the motif S-R/K-E-X (X being different from D and E) which is not recognized by CK2.

Amino Acid Motifs↗
PubMed2000

A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification.

We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database. The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing. The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples. We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins. We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis.

Amino Acid Sequence↗
PubMed2000

Phosphopeptide analysis.

In this chapter we review the various methods available to the experimenter to analyse phosphorylated peptides. The initial steps in such an analysis involve the isolation of the phosphopeptides for analysis, and we outline the various current methods such as immobilised metal affinity chromatography, anti-phosphoamino acid antibodies as well as HPLC (High Pressure Liquid Chromatography) and TLC (Thin Layer Chromatography). The isolated peptides can be analysed by chemical modification followed by Edman degradation or by mass spectrometry (MS). We focus on MS methods and give examples illustrating the selective detection and sequencing of phosphopeptides.

Amino Acid Sequence↗
PubMed2000

The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-313.

Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded by ssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa.

ATP-Binding Cassette Transporters↗
PubMed2000

Insertion gain measurements using two low-powered analogue hearing aids.

Differences between insertion gain measurements and coupler measurements were investigated using two different hearing aids, to assess the accuracy of hearing aid fitting methods which involve the use of real-ear-to-coupler differences (RECDs). The hearing aids used were the NHS BE19 and the Lavis X11. They were allocated alternately to 80 new adult hearing aid users, and insertion gain measurement was carried out with the allocated aid on all subjects, and with both hearing aids on 36 subjects. Coupler measurements were also carried out with both hearing aids, at two different gain settings. Comparison of the coupler measurements and the insertion gain measurements suggests that the differences in insertion gain produced by different hearing aids cannot be predicted accurately from the differences in coupler gain measurements produced by the same aids. Although the use of RECDs measured using insert earphones may accurately predict the real-ear performance of some models of hearing aid, this method may not produce equally satisfactory results with other models.

Hearing Aids↗
PubMed1999

Serine/threonine phosphorylation of calmodulin modulates its interaction with the binding domains of target enzymes.

The interaction of serine/threonine-phosphorylated calmodulin with synthetic peptides corresponding to the calmodulin-binding domains of six enzymes has been studied by fluorescence spectroscopy. For five peptides, the dissociation constant of the calmodulin-peptide complex (K(d)) increased when calmodulin was phosphorylated. An increase of more than one order of magnitude was observed with peptides derived from smooth-muscle myosin light-chain kinase and cAMP phosphodiesterase. In contrast, only a slight increase in K(d) was noted with two peptides derived from the plasma membrane Ca(2+)-ATPase and for the peptide derived from nitric oxide synthase. No significant change in affinity was detected with the peptide derived from calcineurin. In contrast, a decrease in the dissociation constant was observed with the peptide derived from the Ca(2+)-calmodulin dependent kinase II. Phosphorylation also affected the peptide-calmodulin binding stoichiometry: a decrease from two to one binding sites was observed with the peptides derived from myosin light-chain kinase and phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

A nuclear export signal prevents Saccharomyces cerevisiae Hsp70 Ssb1p from stimulating nuclear localization signal-directed nuclear transport.

Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSB gene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei of xpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-Delta cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa- or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport in nup188-Delta cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.

Adenosine Triphosphatases↗
PubMed1999

Molecular features underlying the sequential phosphorylation of HS1 protein and its association with c-Fgr protein-tyrosine kinase.

The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vivo, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis. We have now identified tyrosine 222 as the HS1 residue phosphorylated by the Src family protein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-208-401) lacking the N-terminal putative DNA binding region and the C-terminal Src homology 3 (SH3) domain is still able to undergo all the steps of sequential phosphorylation as efficiently as full-length HS1. We also show that a stable association of phospho-HS1 with c-Fgr through its SH2 domain requires previous autophosphorylation of the kinase and is prevented by subsequent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncated forms previously phosphorylated by Syk and with a peptide substrate reproducing the sequence around tyrosine 222 support the view that efficient phosphorylation of HS1 by Src family protein kinases entirely relies on TyrP-SH2 domain interaction with negligible, if any, contribution of local specificity determinants. Our data indicate that the proline-rich region of HS1 bordered by tyrosyl residues affected by Syk and Src family kinases represents a functional domain designed to undergo a process of sequential phosphorylation.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Classifying symbiotic proteins from Bradyrhizobium japonicum into functional groups by proteome analysis of altered gene expression levels.

The advent of whole genome sequences has brought with it a vast number of new potential proteins whose function is unknown. We describe an approach to sorting proteins into functional groups by comparative two-dimensional (2-D) gel mapping of cells grown under different physiological conditions. Computerized image analysis selects the proteins whose expression levels change significantly for subsequent identification by mass spectrometry. The protein groupings are further subdivided by directed alteration of their expression levels (e.g., by gene inactivation) and following the changes in the expression pattern of the mutants. We have applied this approach to study the regulation of micro- and anaerobically induced genes including the genes involved in nitrogen fixation in the symbiotic bacterium Bradyrhizobium japonicum. The results obtained show that in addition to the two known regulons controlled by the transcription factors NifA and FixK2, a third set of proteins may exist in B. japonicum which are induced by anaerobic conditions and are regulated independently. The approach can be applied generally and can be used to build up functional relationship maps of genomes. Protein identification by mass spectrometry was shown to be vital to the interpretation of the expression analysis since 15% of the 2-D gel spots contained more than one protein.

Amino Acid Sequence↗
PubMed1999

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Proteome analysis of heat shock protein expression in Bradyrhizobium japonicum.

A set of 19 heat shock proteins (Hsp) was observed - by subtractive two-dimensional gel electrophoresis - to be induced when Bradyrhizobium japonicum, the nitrogen-fixing root-nodule symbiont of soybean, was temperature up-shifted from 28 degrees C to 43 degrees C. Up-regulated protein spots were excised from multiple two-dimensional gels. The proteins were concentrated using a funnel-gel device before being blotted onto poly(vinylidene difluoride) membranes for digestion with trypsin before MS and tandem MS analysis or for Edman sequence determination. Five proteins in the range 8-20 kDa were identified as the small Hsp (sHsp; HspB, C, D, E and H) and three others showed strong sequence similarity to the sHsp family. Two other low molecular mass proteins corresponded to GroES1 and GroES2, and five novel proteins were found. Four proteins of approximately 60 kDa were identified as GroEL2, GroEL4, and GroEL5 and DnaK. An analysis of the heat shock induction of DnaK, of four of the most strongly induced GroESL proteins and six of the sHsp revealed that the proteins could be placed into four distinct regulatory groups based on the kinetics of protein appearance.

Amino Acid Sequence↗
PubMed1999

Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation-induced response in Pseudomonas aeruginosa PAO1.

A set of proteins induced in Pseudomonas aeruginosa PAO1 during growth in the absence of sulfate was characterized by differential two-dimensional electrophoresis and MS. Thirteen proteins were found to be induced de novo or upregulated in P. aeruginosa grown in a succinate/salts medium with sodium cyclohexylsulfamate as the sole sulfur source. Protein spots excised from the two-dimensional gels were analysed by N-terminal Edman sequencing and MS sequencing (MS/MS) of internal protein fragments. The coding sequences for 11 of these proteins were unambiguously identified in the P. aeruginosa genome sequence. Expression of these genes was investigated by reverse transcription-PCR, which confirmed that repression in the presence of sulfate was acting at a transcriptional level. Three classes of sulfur-regulated proteins were found. The first class (five proteins) were high-affinity periplasmic solute-binding proteins with apparent specificity for sulfate and sulfonates. A second class included enzymes involved in sulfonate and sulfate ester metabolism (three proteins). The remaining three proteins appeared to be part of a more general stress response, and included two antioxidant proteins and a putative lipoprotein. This study demonstrates the power of the proteomics approach for direct correlation of the responses of an organism to an environmental stimulus with the genetic structures responsible for that response, and the application of reverse transcription-PCR significantly increases the conclusions that can be drawn from the proteomic study.

Amino Acid Sequence↗
PubMed1999

Innervation density of the base of the palm.

Cutaneous nerves of the palm were localized and quantitated by longitudinal axis, proximity to the distal palmar crease, and tissue layer to identify an incision that would avoid injury to the palmar branches of cutaneous nerves. Cadaveric palms (n = 10) harvested en bloc were fixed at physiologic tension and prepared using the celloidin method and hematoxylin-eosin staining. The cutaneous nerves of the palm were counted and classified by their size and location within each tissue layer and longitudinal axis as well as by proximal, middle, and distal locations within each axis. The mean number of large nerves identified within regions of the palm differed by tissue layer and longitudinal axis but not by longitudinal location within axes. The long/ring finger web space was characterized by the lowest innervation density; the index/long finger web space and ring finger axis were characterized by the greatest innervation density. An incision in the long/ring finger web space 2 cm proximal to Kaplan's cardinal line should result in injury to fewer nerves and reduce the incidence of painful neuromas during open carpal tunnel release.

Dissection↗
PubMed1999

'Failure to rescue' as a measure of quality of hospital care: the limitations of secondary diagnosis coding in English hospital data.

Although it is widely recognized that quality of care varies between hospitals, a robust and valid measure of outcome that can be used in comparisons has proven elusive. One measure that has recently been proposed by US researchers is the 'failure to rescue' (FTR) rate. This is based on the assumption that, whereas complications may reflect both patient severity and health care factors, the ability to save patients once complications arise is much more closely related to the quality of health care. We describe an evaluation of FTR in a national sample of English hospitals using hospital episode data. We found that the rate of secondary diagnosis recording in England is about one-tenth that in the United States. The FTR rate would be highly sensitive to variations in the completeness of coding of secondary diagnoses. Unless coding is of uniformly high quality, any attempt to compare severity adjusted outcomes will be potentially unreliable.

Adolescent↗