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Biomedical subjects

P James

Publications and source records attributed to P James.

At least 91 records · Page 5Linked to original sources
PubMed1995

Identification of the border between fibronectin type III homologous repeats 2 and 3 of the neural cell adhesion molecule L1 as a neurite outgrowth promoting and signal transducing domain.

To determine the domains of the neural cell adhesion molecule L1 involved in neurite outgrowth, we have generated monoclonal antibodies against L1 and investigated their effects on neurite outgrowth of small cerebellar neurons in culture. When the 10 antibodies were coated as substrate, only antibody 557.B6, which recognizes an epitope represented by a synthetic peptide comprising amino acids 818 to 832 at the border between the fibronectin type III homologous repeats 2 and 3, was as efficacious as L1 in promoting neurite outgrowth, increasing intracellular levels of Ca2+, and stimulating the turnover of inositol phosphates. These findings suggest that neurite outgrowth and changes in these second messengers are correlated. Such a correlation was confirmed by the ability of Ca2+ channel antagonists and pertussis toxin to inhibit neurite outgrowth on L1 and antibody 557.B6. These observations indicate for the first time a distinct site on cell surface-bound L1 as a prominent signal-transducing domain through which the recognition events appear to be funneled to trigger neurite outgrowth, increase turnover of inositol phosphates, and elevate intracellular levels of Ca2+.

Amino Acid Sequence↗
PubMed1995

Experience with community academic practice: strategies for the ambulatory education of residents.

The expectation that training programs will place more of their physician graduates into primary care has caused internal medicine programs to explore different educational experiences for their residents. One proposal is to transfer the ambulatory training site for residents from the hospital clinic to a community academic practice (CAP) site. We discuss the theoretical aspects, the practical considerations, and our experience with one CAP site, including the problems that have been solved and the problems remaining.

Academic Medical Centers↗
PubMed1995

Calmodulin-binding domains: just two faced or multi-faceted?

The Ca(2+)-binding protein calmodulin binds to and activates several cellular enzymes in response to a rise in Ca2+ concentration. It binds certain basic amphiphilic helices within these enzymes, which also act as autoinhibitory domains. The modulation of the binding equilibrium of these helices between intramolecular (inhibition) and intermolecular (activation) sites forms a focal point for crosstalk between various signalling pathways.

Amino Acid Sequence↗
PubMed1995

Assessing performance-related disability in patients with knee osteoarthritis.

This investigation describes the development and validation of a test battery for evaluating physical activity restrictions (PAR) in patients with knee osteoarthritis (OA). The tasks on the final version of the PAR include (a) a 6-min walk; (b) a stair climb; (c) a lifting and carrying task; and (d) getting into and out of a car. Data from patients with knee OA revealed that the four tasks loaded highly on a single unrotated principal component yielding an alpha internal consistency reliability of 0.92. These data suggest that investigators may choose to use an aggregate score and/or responses from individual tasks. Two week test-retest reliabilities for the four tasks were all in excess of 0.85 and there was support for their concurrent and convergent validity. Specifically, performance on the tasks correlated with time on treadmill, difficulty with self-reported ADLs, and ratings of difficulty following the performance of each task. Additional research is needed on the predictive validity of the measure and its sensitivity to change.

Activities of Daily Living↗
PubMed1995

Evidence that spinach leaves express calreticulin but not calsequestrin.

The presence of either calreticulin (CR) or calsequestrin (CS-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca(2+)-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn(2+) (i.e., under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca(2+). Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca(2+)-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.

Amino Acid Sequence↗
PubMed1995

The evaluation of pain in patients with knee osteoarthritis: the knee pain scale.

OBJECTIVE: To develop and validate a knee pain scale (KPS) for use with osteoarthritis (OA) of the knee. METHODS: Patients with documented evidence of knee OA completed the KPS and a test battery including measures of physical functioning, physical performance, and depression. Analyses were conducted to confirm the 4-factor structure of the measure, determine alpha reliabilities, assess the test-retest reliability, and examine the construct validity of the KPS. RESULTS: Confirmatory factor analysis revealed that the KPS has 4 subscales, including frequency and intensity of pain experienced during both ambulation/climbing and transfer activities. All reliabilities were in excess of 0.80 and the subscales of the KPS shared expected variance with both self-reported and objective indices of dysfunction. CONCLUSION: The KPS has good psychometric properties for assessing pain experienced in conjunction with the performance of activities of daily living. Although at present it is a research tool, with further study it should prove valuable in clinical practice as well.

Activities of Daily Living↗
PubMed1994

Purification and primary structure of metallothioneins induced by cadmium in the protists Tetrahymena pigmentosa and Tetrahymena pyriformis.

Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non-toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd(2+)-metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd(2+)-isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N-terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences. The Tetrahymena metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal-binding sites. Amino acid sequences of Tetrahymena metallothioneins have some similarity with other eukaryotic metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse metallothionein-1B.

Amino Acid Sequence↗
PubMed1994

Isolation of phosphorylated calmodulin from rat liver and identification of the in vivo phosphorylation sites.

A procedure is described for the isolation of calmodulin (CaM) from rat liver which produces a fraction containing non-phosphorylated, mono-, di-, and triphosphocalmodulin as determined by mass spectrometric analysis. The distribution of CaM between the various phospho-species varies from preparation to preparation even though the isolation procedure is rigidly defined, suggesting that CaM phosphorylation may be a very labile phenomenon dependent on the state of the liver as it is removed from the animal. Approximately 15% of CaM in the cell is phosphorylated. The in vivo phosphorylation sites were determined by mass spectrometric analysis of a combined CNBr and trypsin digestion of the phosphocalmodulin (phospho-CaM)-containing fractions. Phosphorylated peptides were sequenced using two mass scanning devices linked together for collisionally activated fragmentation studies to determine peptide sequences, and the phosphorylation sites were determined as Thr-79, Ser-81, and Ser-101. These correspond to three of the four in vitro target sites of calmodulin phosphorylation by casein kinase II, which indicates that this may be the enzyme responsible for the phosphorylation in vivo. A preliminary study on the modulatory activity of phosphorylated calmodulin using a sample extensively phosphorylated in vitro with casein kinase II confirmed that phospho-CaM has an altered biological activity, i.e. reduced activation of the erythrocyte plasma membrane Ca2+ pump.

Amino Acid Sequence↗
PubMed1994

Regulation of c-Fgr protein kinase by c-Src kinase (CSK) and by polycationic effectors.

The protein tyrosine kinase expressed by the protooncogene c-fgr is phosphorylated and down-regulated in vitro by the c-Src kinase (CSK). CSK catalyzed phosphorylation affects Tyr-511 of c-Fgr, homologous to Tyr-527 of c-Src and it prevents the autophosphorylation normally occurring at c-Fgr Tyr-400, homologous to c-Src Tyr-416. Polylysine, histones H1 and H2A and other polycationic proteins on the other hand stimulate c-Fgr activity while promoting enhanced autophosphorylation of both Tyr-400 and Tyr-511. Once phosphorylated at Tyr-511 and down-regulated by CSK, c-Fgr is no more susceptible to polylysine stimulation. Previous autophosphorylation (at Tyr-400) reduces c-Fgr susceptibility to down-regulation by CSK, although Tyr-511 can be still phosphorylated by it. If a more exhaustive autophosphorylation (of both Tyr-400 and Tyr-511) is performed in the presence of polylysine, c-Fgr becomes totally insensitive to CSK down-regulation. These data support the concept that down-regulation of c-Fgr by Tyr-511 phosphorylation is prevented if Tyr-400 is also phosphorylated and they are consistent with an outcompetition of phospho-Tyr-511 from the Src homology 2 domain by phospho-Tyr-400, which, in c-Fgr, is surrounded by an amino acid sequence divergent from that of the other Src-related protein tyrosine kinases.

Adenosine↗
PubMed1994

Collection of peripheral blood stem cells on spectra.

The collection of peripheral blood stem cells by apheresis is a procedure used with increasing frequency in bone marrow transplant. The Spectra Apheresis System is an automated machine allowing the procedure to be tailored to every patient for optimal safety and comfort. The ability to constantly view the products allows the operator flexibility to control the results for an optimal result.

Blood Component Removal↗
PubMed1994

Protein identification in DNA databases by peptide mass fingerprinting.

Proteins can be identified using a set of peptide fragment weights produced by a specific digestion to search a protein database in which sequences have been replaced by fragment weights calculated for various cleavage methods. We present a method using multidimensional searches that greatly increases the confidence level for identification, allowing DNA sequence databases to be examined. This method provides a link between 2-dimensional gel electrophoresis protein databases and genome sequencing projects. Moreover, the increased confidence level allows unknown proteins to be matched to expressed sequence tags, potentially eliminating the need to obtain sequence information for cloning. Database searching from a mass profile is offered as a free service by an automatic server at the ETH, Zürich. For information, send an electronic message to the address cbrg/inf.ethz.ch with the line: help mass search, or help all.

Animals↗
PubMed1994

Gas chromatography/combustion/isotope-ratio mass spectrometry analysis of urinary steroids to detect misuse of testosterone in sport.

We propose a new confirmatory method for testosterone doping in sport. The present method in use, based on measuring the testosterone/epitestosterone (T/E) ratio in urine, may miss suspicious cases, or lead to reporting cases in which the high ratio is natural. Synthetic testosterone has a 13C abundance different from that of endogenous human testosterone. The connection of a gas chromatograph to an isotope-ratio mass spectrometer via a combustion interface allows the measurement of the corresponding characteristic value (delta /1000) for testosterone, its precursors, and its metabolites. To detect exogenous administration of testosterone, 30-40 mL of urine is sufficient.

Acetylation↗
PubMed1993

Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta.

Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced by self-assembly in vitro, the bicistronically expressed beta subunit was autophosphorylated during formation of the holoenzyme in bacteria. Electrospray ionisation mass spectrometry (ESI) revealed Ser2 (second amino acid, first serine) as the only phosphate acceptor site. Kinetic data obtained with either the phosphorylated or the unphosphorylated form of CK-2 did not differ significantly, suggesting that the autophosphorylation had no influence on the kinetic parameters Km and Vmax. In parallel, native rat liver CK-2 beta subunit was shown to incorporate 0.1 mol phosphate/mol holoenzyme, which suggests that the enzyme is already heavily phosphorylated. ESI analysis also revealed Ser2 as the only phosphorylated residue at the amino terminus. In the case of both recombinant human CK-2 and native rat liver CK-2, treatment with alkaline phosphatase readily reversed the phosphorylated form of the beta subunit to the faster migrating dephosphorylated polypeptide.

Amino Acid Sequence↗
PubMed1993

Limited proteolysis of creatine kinase. Implications for three-dimensional structure and for conformational substrates.

Proteinase K, subtilisin, pronase E, elastase, bactotrypsin, and thermolysin are all shown here to cleave native mitochondrial creatine kinase from chicken heart (Mib-CK) very specifically at a single site, either before or after Ala-323. In analogy with hen egg ovalbumin, where the same proteases all cleaved the polypeptide chain very specifically around Ala-352, Ala-323 of Mib-CK may be located in an exposed surface loop that is sensitive to protease attack. Gel permeation chromatography demonstrated that the two proteolytic fragments of Mib-CK with M(r)'s of approximately 37,000 and approximately 6000 remain associated with each other. Proteinase K cleavage did not influence the octamer to dimer ratio of Mib-CK, indicating that selective cleavage after Ala-323 has no direct effect on dimer-dimer interfaces within the octamer. However, upon addition of MgADP plus creatine and nitrate to induce a transition-state analogue complex of the enzyme, native Mib-CK dissociated much more readily into dimers than proteinase K-digested Mib-CK. Furthermore, proteinase K cleavage of Mib-CK resulted in 2-11-fold decreases in the Vmax values, as well as in 6-23-fold increases in the Km values for phosphocreatine, creatine, and MgATP, whereas the Kd values for both MgATP and creatine were unaffected. Consequently, proteinase K cleavage of Mib-CK does not affect substrate binding per se, but interferes with substrate-induced conformational changes which are essential for catalysis and which mediate the synergism in substrate binding as it is observed with the unmodified enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Protein identification by mass profile fingerprinting.

We have developed an algorithm for identifying proteins at the sub-microgram level without sequence determination by chemical degradation. The protein, usually isolated by one- or two-dimensional gel electrophoresis, is digested by enzymatic or chemical means and the masses of the resulting peptides are determined by mass spectrometry. The resulting mass profile, i.e., the list of the molecular masses of peptides produced by the digestion, serves as a fingerprint which uniquely defines a particular protein. This fingerprint may be used to search the database of known sequences to find proteins with a similar profile. If the protein is not yet sequenced the profile can serve as a unique marker. This provides a rapid and sensitive link between genomic sequences and 2D gel electrophoresis mapping of cellular proteins.

Algorithms↗
PubMed1993

Isolation and identification of two proto-oncogene products related to c-fgr and fyn in a tyrosine-protein-kinase fraction of rat spleen.

TPK-III, a tyrosine-protein-kinase fraction previously isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] has been further purified and resolved by Mono-Q FPLC into two homogeneous compounds, Q1 and Q2, with molecular mass approximately 52 kDa, exhibiting high specific activities for phosphate incorporation into the phosphoacceptor substrate poly(Glu80Tyr20) (1194 nmol.min-1 x mg-1 and 579 nmol.min-1 x mg-1, respectively). Both Q1 and Q2 appear to be scr-related enzymes since they are readily recognised by anti-SEEP serum raised against the highly conserved segment at positions 330-345 of p60c-scr. Q1, but not Q2, interacts with a specific antibody raised against the N-terminal segment of p55c-fgr. Microsequence analysis of tryptic fragments generated from Q1 revealed five peptides which exactly overlap the expected segments of p55c-fgr. Two of these peptides were not entirely conserved in any of the other src-related tyrosine protein kinases. A sixth fragment is very similar, albeit not identical, to the C-terminus of p55c-fgr. Microsequence analysis of two tryptic fragments from the other TPK-III fraction, Q2, provided the sequences FQILNSSE and LTTQETGYIPSNY, which are identical to two segments of fyn not entirely conserved in any of the other src-related tyrosine protein kinases. These results provide evidence that the spleen tyrosine-protein-kinase fraction, conventionally designated TPK-III, is composed of products from two proto-oncogenes, one of which corresponds to fyn, while the other is either identical or very closely related to c-fgr.

Amino Acid Sequence↗