Search PubMed⌕ Search

Biomedical subjects

P James

Publications and source records attributed to P James.

At least 55 records · Page 3Linked to original sources
PubMed1998

Identification of the binding surface on beta-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry.

Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin. Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence. From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements. This explains how hydrophobic surface on the substrate protein can become accessible while scarcely disrupting the hydrogen bond network of the native structure. An analysis of the GroEL-bound peptides bound after digestion of the beta-lactamase showed no obvious sequence motifs, indicating that binding is provided by hydrophobic patches in the three-dimensional structure.

Binding Sites↗
PubMed1998

Phosphorylation of calmodulin alters its potency as an activator of target enzymes.

Previous work has shown that calmodulin (CaM) is constitutively phosphorylated in rat liver, probably by casein kinase II [Quadroni, M., James, P., and Carafoli, E. (1994) J. Biol. Chem. 269, 16116-16122]. A procedure is now described for the isolation of the phosphorylated forms of calmodulin (PCaM) free from CaM, since in vitro phosphorylation experiments yield a 50:50 mixture of 3-4 times phosphorylated CaM and native CaM. The activation of six target enzymes by PCaM was tested: myosin light chain kinase, 3',5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase, and CaM-kinase II. In general, the phosphorylation of CaM caused a decrease in enzyme binding affinity, increasing the Kact by 2-4-fold for MLCK, PDE, PM Ca2+-ATPase, and calcineurin. The Vmax at saturating concentrations of PCaM was less affected, with the exception of CaM-kinase II, which was only minimally activated by PCaM and NOS whose Vmax was increased 2.6 times by PCaM with respect to CaM. Phosphorylation of calmodulin had very little effect on the binding of calcium to the enzyme despite the fact that Ser 101 which is phosphorylated is located in the third calcium binding loop. CD measurements performed on CaM and PCaM indicated that phosphorylation causes a marked decrease in the alpha-helical content of the protein. Phosphorylated CaM is very prone to dephosphorylation and was thus tested as a substrate for several phosphatases. It was unaffected by calcineurin (PP2B), but was a reasonable substrate for the pleiotropic phosphatases PP1gamma and PP2A.

Animals↗
PubMed1998

Sample handling for proteome analysis.

The main factor limiting the sensitivity range for the identification of proteins isolated by two-dimensional (2-D) electrophoresis is sample handling: protein detection limits on the gel, losses during extraction and digestion, as well as interference of gel contaminants and detergents with the mass spectrometry (MS) detection increasing background noise. At the one hundred picomole level, losses are fairly negligible but when the amounts drop below 1 picomole (and subfemtomole peptide detection limits have been reported recently by MS), the losses become a critical point. In order to extend proteome analysis to include very low copy number proteins, methods must be developed to minimize losses and handling steps, maximize digestion and extraction yields, as well as to lower chemical noise. We present several methods that we have developed in our laboratory to: (i) increase the amount of material available in a sodium dodecyl sulfate (SDS)-free form which does not require staining, (ii) increase protein extraction and digestion yields and lower the contamination by autoproteolytic products, and (iii) allow direct modification of the peptide mixture to generate sequence tags.

Bacterial Proteins↗
PubMed1998

An algorithm for the identification of proteins using peptides with ragged N- or C-termini generated by sequential endo- and exopeptidase digestions.

We have developed an algorithm (MassDynSearch) for identifying proteins using a combination of peptide masses with small associated sequences (tags). Unlike the approach developed by Matthias Mann, 'Tag searching', in which the sequence tags are generated by gas phase fragmentation of peptides in a mass spectrometer, 'Rag Tag' searching uses peptide tags which are generated enzymatically or chemically. The protein is digested either chemically or with an endopeptidase and the resultant mixture is then subjected to partial exopeptidase degradation. The mixture is analyzed by matrix assisted laser desorption and ionization time of flight mass spectrometry and a list of intact peptide masses is generated, each associated with a set of degradation product masses which serve as unique tags. These 'tagged masses' are used as the input to an algorithm we have written, MassDynSearch, which searches protein and DNA databases for proteins which contain similar tagged motifs. The method is simple, rapid and can be fully automated. The main advantage of this approach is that the specificity of the initial digestion is unimportant since multiple peptides with tags are used to search the database. This is especially useful for proteins like membrane, cytoskeletal, and other proteins where specific endopeptidases are less efficient and lower specificity proteases such as chymotrypsin, pepsin, and elastase must be used.

Algorithms↗
PubMed1998

Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli.

Cytochrome c552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli. In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haem-containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low mid-point potential redox centres. The best fit to the experimental data is for three n=1 components with mid-point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), -90 mV (36% of the total) and -210mV (43% of the total). Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf deletion mutant to Nrf+ or to restore formate-dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.

Anaerobiosis↗
PubMed1998

Cerebral oxygenation and the recoverable brain.

Oxygenation is the most critical function of blood flow and a sudden reduction in oxygen availability is an inevitable consequence of severe ischemia. The resulting cascade of events may result in the failure of membrane integrity of some cells and necrosis, but in the surrounding zone of tissue, less affected by hypoxia, cells survive to form the ischemic penumbra. The timing of these events is uncertain, but sufficient oxygen is available to these cells to maintain membrane ion pump mechanisms, but not enough for them to generate action potentials and therefore function as neurons. The existence of such areas has been suspected for some time based upon the nature of clinical recovery, but has now been demonstrated by SPECT imaging with a high plasma oxygen concentration under hyperbaric conditions as a tracer. A course of hyperbaric oxygen therapy frequently results in a permanent improvement in both flow and metabolism. These changes apparently represent a reversal of the changes that render neurones dormant and the activity of cells, previously undetectable by standard electrophysiological methods, can now be demonstrated. Three patients are presented in whom recoverable brain tissue has been identified using SPECT imaging and increased cerebral oxygenation under hyperbaric conditions. Improved perfusion from reoxygenation has correlated with clinical evidence of benefit especially with continued therapy.

Adolescent↗
PubMed1998

Phytochrome B affects the levels of a graft-transmissible signal involved in tuberization

Grafting experiments between phytochrome B antisense and wild-type potato (Solanum tuberosum L. subsp. andigena [line 7540]) plants provide evidence that phytochrome B is involved in the production of a graft-transmissible inhibitor of tuberization, the level of which is reduced in the antisense plants, allowing them to tuberize in noninducing photoperiods.

Journal Article↗
PubMed1998

Examination of the preclinical visit in a bachelor of nursing program.

When nursing entered the tertiary sector in 1985 the preclinical visit was introduced and became accepted practice. The initial purpose of the preclinical visit was to inform and prepare Clinical Facilitators and Nursing Unit Managers for undergraduate nursing students' clinical experience. The preclinical visit was perceived as a significant factor which potentially impacts on the quality of student learning within the clinical environment. Initial planning of the preclinical visit shapes and determines the outcomes of the clinical experience for all participants. Students personal and professional development often depend upon how well they are received in the ward area. Despite the emphasis placed on the significance of the preclinical visit, its effectiveness has not been evaluated. Ten years after the introduction of nursing to tertiary education, the preclinical visit was evaluated by a group of academics and clinicians who perceived that the preclinical visit was not achieving the desired outcomes. A survey using a convenience sample of Nursing Unit Managers and Clinical Facilitators was conducted at a major metropolitan teaching hospital in New South Wales. This paper reviews the findings of the study evaluating the preclinical visit. From the results of the study it is evident that a structured, clear format is required for the preclinical visit to be effective.

Attitude of Health Personnel↗
PubMed1997

Studies on the cytostatic and cytotoxic effects and mode of action of 8-nitro-7-hydroxycoumarin.

A derivative of coumarin, 8-nitro-7-hydroxycoumarin (8-NO2-7-OHC), was synthesised, purified and characterised. The cytostatic and cytotoxic nature of this compound was determined using both human and animal cell lines grown in vitro for 96 h in the presence of drug (0-500 microM, equivalent to 0-104 microg/ml). 8-NO2-7-OHC was shown to be cytotoxic to three cell lines, but cytostatic to all cell lines tested. With K562 and HL-60 cells, cell death was found to occur by apoptosis. This cytotoxic effect was found to be irreversible, with cell death continuing to occur following a 96 h recovery period. The cytostatic effects were found to be irreversible in four of the five cell lines tested. 8-NO2-7-OHC demonstrated its cytostatic effects within 24 or 48 h, while its cytotoxic effects appeared more gradually. The IC50 of 8-NO2-7-OHC was 475-880 microM, depending on the cell line tested. It was shown to exert its cytostatic effect through an alteration of cell cycle. It also inhibited DNA synthesis. The toxicity of 8-NO2-7-OHC does not appear to be mediated through the multi-drug resistance (MDR) protein since it caused significant cytostatic and cytotoxic effects to CHrC5 cells, which have an increased expression of this protein. This compound was shown to be non-mutagenic in a standard Ames test, both with or without a mammalian enzyme activation system. The applications and mode of actions of coumarins are discussed.

Animals↗
PubMed1997

The complete mature bovine prion protein highly expressed in Escherichia coli: biochemical and structural studies.

According to the 'protein only' hypothesis, modification of the 3-dimensional fold of the constituent cellular protein, PrP(C), into the disease-associated isoform, PrP(Sc), is the cause of neurodegenerative diseases in animals and humans. Here we describe the high-level synthesis in Escherichia coli, and purification in the monomeric form, of a histidine-tagged full-length mature PrP (25-249) of bovine brain, termed His-PrP. Based on biochemical and spectroscopic data, His-PrP displays characteristics expected for the PrP(C) isoform. The reported expression system should allow the production of quantities of bovine PrP(C) sufficient to permit 3-dimensional structure determinations.

Amino Acid Sequence↗
PubMed1997

Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels.

We have developed a gel electrophoresis system that can concentrate proteins from spots cut out of up to 50 two-dimensional electrophoresis gels. During protein concentration, SDS is substituted with a non-ionic detergent (octyl beta-glucopyranoside) which allows digestion and MS analysis of the protein directly extracted from the gel without fixation or staining. The system avoids the problems associated with the digestion of dilute protein in multiple bands by (a) greatly reducing the gel volume for digestion and thus the amount of protease required, hence lowering contamination by autodigestion products, (b) reducing the volume of solvent required for extraction of protein from the gel, thus minimising loss of material to container surfaces, and (c) removing SDS which interferes with subsequent MS or HPLC analysis. The efficiency of protein recovery ranges between an average of 80% for proteins from silver stained two-dimensional gels to 90% for fluorescence and Coomassie-blue-stained gels. The method is compatible with MS analysis of very low amounts of protein from any staining system, but appears not to be useful for Edman sequencing of silver-stained or fluorescent-stained proteins since the amount of N-terminal blockage appears to increase as the amount of protein isolated from the two-dimensional gel decreases.

Amino Acid Sequence↗
PubMed1997

Subcellular and tissue distribution, partial purification, and sequencing of calmodulin-stimulated Ca2+-transporting ATPases from barley (Hordeum vulgare L.) and tobacco (Nicotiana tabacum).

The subcellular distribution of plasma-membrane-type Ca2+-transporting ATPases was studied in barley leaves (Hordeum vulgare L.). A highly enriched plasma membrane (PM) fraction was analysed for Ca2+ pumps and compared with several inner-membrane preparations, including the tonoplast, the envelope of the chloroplast, and an endoplasmic reticulum (ER)-enriched fraction. The enzymes were identified and characterised with regard to the phosphointermediate formation, their nucleotide specificity and their binding to calmodulin. A Ca2+-transporting ATPase (molecular mass approximately 130 kDa), which showed high specificity for ATP and high affinity for calmodulin, was localised in the PM. A 116-kDa Ca2+-transporting ATPase, probably located in the ER, showed lower nucleotide specificity and weaker affinity for calmodulin. A comparison of the distribution of the pumps in leaves and roots indicated that the ratio of expression of the two enzymes changed in a tissue-specific manner: the PM pump was dominant in leaves, while the inner-membrane pump was expressed at a higher level in the roots. For the purification of calmodulin-binding proteins (Ca2+ pumps), microsomes isolated from tobacco cell cultures were used. Two active Ca2+ pumps were identified, and the one at 116 kDa was partially sequenced.

Adenosine Triphosphate↗
PubMed1997

Of genomes and proteomes.

The era of complete genome sequences has arrived and with it vast amounts of data which must be annotated, cross referenced, and placed within the regulatory networks which define the physiology of an organism. One eucaryotic and three procaryotic genomes have been completed and the data made available and another 50 sequences are expected to be completed by the end of the decade. One of the first steps in the new post genome era will be to decipher the functions of the huge numbers of new open reading frames. Various approaches to investigate what unknown genes do and how genes interact together within an organism are being undertaken, including (1) the simultaneous measurement of the expression levels of all genes in a cell and (2) the mapping and quantitation of all proteins expressed within a cell. The idea of systematically mapping and identifying the total protein complement of the genome (the "proteome") arose over 20 years ago when the separation of proteins from total cell extracts by two dimensional (2D) gel electrophoresis was developed. This review will focus on the use of 2D gel electrophoresis as the basis for constructing proteome maps and on the rapid advances in mass spectrometry which will allow the large-scale, automated identification of proteins which is necessary for the creation of such databases.

Animals↗
PubMed1997

Functional specificity among Hsp70 molecular chaperones.

Molecular chaperones of the 70-kilodalton heat shock protein (Hsp70) class bind to partially unfolded polypeptide substrates and participate in a wide variety of cellular processes. Differences in peptide-binding specificity among Hsp70s have led to the hypothesis that peptide binding determines specific Hsp70 functions. Protein domains were identified that were required for two separate functions of a yeast Hsp70 family. The peptide-binding domain was not required for either of these specific Hsp70 functions, which suggests that peptide-binding specificity plays little or no role in determining Hsp70 functions in vivo.

Adenosine Triphosphatases↗
PubMed1997

Cisplatin-induced renal effects and thromboxane A2 receptor blockade.

The aim of this study was to evaluate the renal protective effect of linotroban, a thromboxane A2 receptor antagonist, in 25 patients with malignant tumours scheduled for cisplatin therapy. Cisplatin was administered 1 h after the start of a 24-h continuous infusion of linotroban or placebo. Glomerular filtration rate and effective renal plasma flow were measured. Infusions of cisplatin decreased glomerular filtration rate by 17 +/- 25 mL min-1 (P = 0.049 vs. baseline) and effective renal plasma flow by 94 +/- 150 mL min-1 (P = 0.049 vs. baseline) in the placebo group. In the linotroban group a decrease in glomerular filtration rate by 11 +/- 18 mL min-1 (P = 0.050 vs. baseline) and in effective renal plasma flow by 26 +/- 63 mL min-1 (P = 0.2 vs. baseline) was noted. However, no difference was noted between groups in response to treatment. Our findings indicate that linotroban may not be useful for prevention of cisplatin's acute nephrotoxic effects.

Adult↗