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Biomedical subjects

C E Jones

Publications and source records attributed to C E Jones.

At least 19 recordsLinked to original sources
PubMed2000

Strange magnetism and the anapole structure of the proton.

The violation of mirror symmetry in the weak force provides a powerful tool to study the internal structure of the proton. Experimental results have been obtained that address the role of strange quarks in generating nuclear magnetism. The measurement reported here provides an unambiguous constraint on strange quark contributions to the proton's magnetic moment through the electron-proton weak interaction. We also report evidence for the existence of a parity-violating electromagnetic effect known as the anapole moment of the proton. The proton's anapole moment is not yet well understood theoretically, but it could have important implications for precision weak interaction studies in atomic systems such as cesium.

Journal Article↗
PubMed2000

Interaction of the bacteriophage T4 gene 59 helicase loading protein and gene 41 helicase with each other and with fork, flap, and cruciform DNA.

Bacteriophage T4 gene 59 helicase loading protein accelerates the loading of T4 gene 41 DNA helicase and is required for recombination-dependent DNA replication late in T4 phage infection. The crystal structure of 59 protein revealed a two-domain alpha-helical protein, whose N-terminal domain has strong structural similarity to the DNA binding domain of high mobility group family proteins (Mueser, T. C., Jones, C. E., Nossal, N. G., and Hyde, C. C. (2000) J. Mol. Biol. 296, 597-612). We have previously shown that 59 protein binds preferentially to fork DNA. Here we show that 59 protein binds to completely duplex forks but cannot load the helicase unless there is a single-stranded gap of more than 5 nucleotides on the fork arm corresponding to the lagging strand template. Consistent with the roles of these proteins in recombination, we find that 59 protein binds to and stimulates 41 helicase activity on Holliday junction DNA, and on a substrate that resembles a strand invasion structure. 59 protein forms a stable complex with wild type 41 helicase and fork DNA in the presence of adenosine 5'-O-(thiotriphosphate). The unwinding activity of 41 helicase missing 20 C-terminal amino acids is not stimulated by 59 protein, and it does not form a complex with 59 protein on fork DNA.

Base Sequence↗
PubMed2000

Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 A resolution crystal structure reveals a novel alpha-helical two-domain fold.

The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA. We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides. The 1.45 A resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel alpha-helical bundle fold with two domains of similar size. Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules. The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A. We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of human Syk produced using a baculovirus expression system.

The cytoplasmic tyrosine kinase p72syk (Syk) plays an essential role in signaling via a variety of immune and nonimmune cell receptors. Syk is activated in response to the engagement of the appropriate cell surface receptors and can phosphorylate downstream targets and recruit additional SH2-domain-containing proteins. In order to study the characteristics of Syk in vitro, we have overexpressed untagged, full-length human Syk in a recombinant baculovirus expression system. The enzyme was purified to 95% purity using a novel two-step affinity chromatography process using reactive yellow and phosphotyrosine columns. Yields of 3-10 mg purified Syk were obtained from 1 liter of infected insect cells. Western blotting, internal protein sequencing, and the specific tyrosine phosphorylation of a Syk peptide substrate indicated authenticity of the purified protein. The enzymatic properties of Syk were in good agreement with published data for the human enzyme, as the apparent K(m) of Syk for ATP was 10 microM and the peptide substrate was 3 microM. The recombinant protein also showed similar biochemical characteristics to the native protein isolated from B-cells such as autophosphorylation. Proteolytic cleavage of purified recombinant Syk was used to generate the kinase domain by micro-calpain. We therefore describe an efficient expression system and purification methodology to produce biologically active human Syk.

Amino Acid Sequence↗
PubMed2000

Intracellular copper routing: the role of copper chaperones.

Copper is required by all living systems. Cells have a variety of mechanisms to deal with this essential, yet toxic trace element. A recently discovered facet of homeostatic mechanisms is the protein-mediated, intracellular delivery of copper to target proteins. This routing is accomplished by a novel class of proteins, the 'copper chaperones'. They are a family of conserved proteins present in prokaryotes and eukaryotes, which suggests that copper chaperones are used throughout nature for intracellular copper routing.

Animals↗
PubMed2000

Numerical densities of myonuclei and satellite cells in muscle fiber types in the aging human thyroarytenoid muscle: an immunohistochemical and stereological study using confocal laser scanning microscopy.

OBJECTIVE: This study determines the role of changes in numerical densities of myonuclei and satellite cells in age-related remodeling of the thyroarytenoid muscle (TA). DESIGN: Changes in numerical densities (N(V)) and ratios (N(N)) of myonuclei and satellite cells were estimated for the entire TA by use of stereological techniques. RESULTS: There was no age-related change or difference between fiber types for N(V myonucleus, fiber), but N(V myonucleus, fiber) increased with decreasing fiber diameter. There was a trend toward a decrease in N(V satellite cell, fiber) and a decrease in N(N satellite cell, myonucleus). N(V satellite cell, fiber) was higher for type 1 than for type 2 fibers, and type 1 satellite cells increased disproportionately with increasing total satellite cell numerical density. CONCLUSION: Decreased satellite cell proliferation may contribute to age-related fiber loss and atrophy in the TA. SIGNIFICANCE: Therapeutic techniques based on activation of satellite cells may block age-related fiber loss and atrophy in the TA.

Adult↗
PubMed2000

Relative patterns and rates of evolution in heron nuclear and mitochondrial DNA.

Mitochondrial cytochrome b sequence data from 15 species of herons (Aves: Ardeidae), representing 13 genera, were compared with DNA hybridization data of single-copy nuclear DNA (scnDNA) from the same species in a taxonomic congruence assessment of heron phylogeny. The two data sets produced a partially resolved, completely congruent estimate of phylogeny with the following basic structure: (Tigrisoma, Cochlearius, (((Zebrilus, (Ixobrychus, Botaurus)), (((Ardea, Casmerodius), Bubulcus), ((Egretta thula, Egretta caerulea, Egretta tricolor), Syrigma), Butorides, Nycticorax, Nyctanassa)))). Because congruence indicated similar phylogenetic information in the two data sets, we used the relatively unsaturated DNA hybridization distances as surrogates of time to examine graphically the patterns and rates of change in cytochrome b distances. Cytochrome b distances were computed either from whole sequences or from partitioned sequences consisting of transitions, transversions, specific codon site positions, or specific protein-coding regions. These graphical comparisons indicated that unpartitioned cytochrome b has evolved at 5-10 times the rate of scnDNA. Third-position transversions appeared to offer the most useful sequence partition for phylogenetic analysis because of their relatively fast rate of substitution (two times that of scnDNA) and negligible saturation. We also examined lineage-based rates of evolution by comparing branch length patterns between the nuclear and cytochrome b trees. The degree of correlation in corresponding branch lengths between cytochrome b and DNA hybridization trees depended on DNA sequence partitioning. When cytochrome b sequences were not partitioned, branch lengths in the cytochrome b and DNA hybridization trees were not correlated. However, when cytochrome b sequences were reduced to third-position transversions (i.e., unsaturated, relatively fast changing data), branch lengths were correlated. This finding suggests that lineage-based rates of DNA evolution in nuclear and mitochondrial genomes are influenced by common causes.

Animals↗
PubMed1999

Copper chaperones: function, structure and copper-binding properties.

Copper is an absolute requirement for living systems and the intracellular trafficking of this metal to copper-dependent proteins is fundamental to normal cellular metabolism. The copper chaperones perform the dual functions of trafficking and the prevention of cytoplasmic exposure to copper ions in transit. Only a small number of copper chaperones have been identified at this time but their conservation across plant, bacterial and animal species suggests that the majority of living systems utilise these proteins for copper routing. The available data suggest that each copper-dependent protein in the cell is served by a specific copper chaperone. Although copper chaperones cannot be substituted for one another in a given cell type, copper chaperones that deliver to the same protein in different cell types appear to be functionally equivalent. The majority of the copper chaperones identified thus far have an "open-faced beta-sandwich" global fold with a conserved MXCXXC metal-binding motif. Specificity for a given copper-dependent protein appears to be mediated by the residues surrounding the copper-binding motif. Copper binds to such proteins as Cu(I) in a trigonal complex with three sulfur ligands. Only the copper chaperone specific for cytochrome-c-oxidase, Cox17, deviates from this design.

Amino Acid Sequence↗
PubMed1999

The kinetics of mycophenolic acid and its glucuronide metabolite in adult kidney transplant recipients.

BACKGROUND: Mycophenolic acid kinetics have been reported to vary after renal transplantation, and mycophenolic acid area under the concentration-time curve (AUC) is the best predictor of suppression of graft rejection. METHODS: To determine whether mycophenolic acid kinetics vary after renal transplantation and to examine the potential role of enterohepatic recirculation, we investigated the kinetics of mycophenolic acid and mycophenolic acid glucuronide on days 2, 5, and 28 after transplantation in 10 kidney transplant recipients (male/female ratio, 1.5; mean age, 41.7 +/- 5.0 years) given 1 g mycophenolate mofetil twice a day. To facilitate therapeutic drug monitoring, we examined a limited sampling strategy for estimating 12-hour mycophenolic acid [AUC(0-12)]. RESULTS: The mean +/- SE AUC(0-12) for mycophenolic acid on day 28 was 38.5 +/- 1.6 mg x h/L, with a secondary peak 4 to 8 hours after dosing that was attributable to enterohepatic recirculation. Marked variability was shown in the kinetic profile of mycophenolic acid among patients across the three sampling days. Mycophenolic acid AUC(0-12) was positively predicted by both serum creatinine (P = .01) and serum albumin (P = .03) but not by time after transplantation, body weight, or trough concentration. Limited sampling (at 0, 1, 3, and 6 hours) accounted for 84.1% of the variability in the mycophenolic acid AUC(0-12) data and predicted the AUC(0-12) closely (r2 = 0.954) when evaluated in 10 different kidney transplant recipients. CONCLUSIONS: Mycophenolic acid AUC(0-12) is predicted by serum albumin and creatinine after kidney transplantation, and the AUC(0-12) may be determined during the early posttransplant period while the patient remains hospitalized with use of a limited sampling strategy to facilitate therapeutic drug monitoring.

Adult↗
PubMed1998

Characterization, crystallization and preliminary X-ray investigation of glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus.

Recombinant Sulfolobus solfataricus glyceraldehyde-3-phosphate dehydrogenase has been purified and found to be a tetramer of 148 kDa. The enzyme shows dual cofactor specificity and uses NADP+ in preference to NAD+. The sequence has been compared with other GAPDH proteins including those from other archaeal sources. The purified protein has been crystallized from ammonium sulfate to produce crystals that diffract to 2.4 A with a space group of P43212 or P41212. A native data set has been collected to 2.4 A using synchrotron radiation and cryocooling.

Amino Acid Sequence↗
PubMed1998

High-performance liquid chromatography determination of mycophenolic acid and its glucuronide metabolite in human plasma.

Two HPLC-UV assays are reported here: one is a rapid assay for mycophenolic acid (MPA) and the other is a simultaneous assay for MPA and its metabolite mycophenolic acid glucuronide (MPAG). For both methods, plasma samples (500 microl) with added internal standard were acidified and extracted using C18 solid-phase extraction cartridges. Chromatographic separation was achieved on a C18 Novapak column using a mobile phase consisting of methanol-0.05% orthophosphoric acid (40:60, v/v) for the rapid MPA assay and 30:70 for the simultaneous MPA and MPAG assay. The assays were linear over the ranges 0.1 to 50.0 mg/l for MPA and 2.8 to 225.8 mg/l for MPAG. Mean absolute recovery for all analytes was >99%. These methods are suitable for therapeutic drug monitoring and pharmacokinetic studies.

Chromatography, High Pressure Liquid↗
PubMed1998

Microscale high-performance liquid chromatography-electrospray tandem mass spectrometry assay for cyclosporin A in blood.

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC-electrospray tandem mass spectrometry assay. Blood samples (100 microl) were prepared by acetonitrile precipitation and C18 solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5-1000 microg/l (r> or =0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 microg/l and 2.8% at 800 microg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x + 10.0, r=0.996). This HPLC-ESI-MS-MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.

Chromatography, High Pressure Liquid↗
PubMed1998

The Kidney's role in glucose balance following partial hepatectomy.

It has long been believed that the liver is the major contributor to glucose balance during fasting and stressful situations. Recently, investigators have implicated the kidney as having a significant contribution to systemic glucose appearance. We studied the relative contributions of the kidney and liver to glucose homeostasis in fasted nonoperated, sham-operated, and 70% hepatectomized rats. Systemic glucose appearance, renal glucose release and uptake, and hepatic glucose release were determined by glucose balance and isotopic dilution techniques. Systemic glucose appearance remained unchanged following hepatectomy. There was a significant output of glucose by the kidney in all groups, accounting for >50% of total glucose appearance. Despite the kidney's appreciable contribution to circulating glucose in the postabsorptive state, renal glucose release was not increased in the hepatectomized rats compared to controls. Total glucose appearance was maintained following hepatectomy by an increase in hepatic glucogenesis. There was a significant increase in the rate of hepatic glucose release from resected rats when normalized to gram of remaining liver (P < 0.001). Despite the substantial amount of renal glucose output in the postabsorptive state, preservation of glucose balance following 70% hepatectomy is accomplished by adaptation in hepatic glucose output.

Animals↗
PubMed1998

Production of human tissue factor using the Pichia pastoris expression system.

Tissue factor plays an important role in the initiation of the blood coagulation cascade resulting in the formation of a fibrin clot. The extracellular domain of human tissue factor has been expressed in the protease-deficient strain of the methylotrophic yeast Pichia pastoris, SMD1168. Tissue factor was expressed with a human influenza hemagglutinin tag fused at the C-terminus under control of the regulatory sequences from the Pichia AOX1 gene. Expressed protein was secreted in a soluble form at levels of up to 10 mg L-1 and correct processing of the PHO1 signal sequence was confirmed by N-terminal amino acid sequence analysis. Tissue factor was produced in Pichia as three discrete forms which appeared as three bands in the range 37-45 kDa by SDS-PAGE. These were all recognized by an anti-tissue factor monoclonal antibody. Deglycosylation studies using Endo H showed that the three forms were the result of differences in glycosylation of the protein. The low levels of secreted proteins produced by P. pastoris make this an efficient host for producing biologically active recombinant tissue factor requiring little purification.

Amino Acid Sequence↗
PubMed1998

Estrogen replacement reverses endothelial dysfunction in postmenopausal women.

PURPOSE: To measure the effect of long-term clinical hormone replacement therapy on brachial artery vasomotor responses, and to compare these responses in premenopausal and postmenopausal women. PATIENTS AND METHODS: We studied 23 postmenopausal women, including 18 who were evaluated prior to starting clinically indicated oral hormone replacement therapy. Twelve postmenopausal women received estrogen alone, the other 6 were treated with estrogen/medroxyprogesterone combinations. Eleven premenopausal volunteers served as a comparison group. Change in brachial artery diameter in response to postischemic hyperemic flow and sublingual nitroglycerin was measured by ultrasound. RESULTS: The 18 postmenopausal subjects receiving hormone replacement showed a progressive improvement in their postischemic vasodilation. Mean (+/-SD) postischemic vasodilation was 0.4%+/-7.1% prior to estrogen replacement. There were significant increases in postischemic vasodilation of 4.8%+/-6.6% after 1 month and 8.3%+/-3.4% after 6 months of estrogen replacement. The response to nitroglycerin was similar at all time points studied. Women with the most abnormal responses to hyperemic flow at baseline demonstrated the greatest improvement after 6 months of hormone replacement therapy. Premenopausal and postmenopausal subjects differed in their response to hyperemic flow, with premenopausal women showing 5.8% vasodilatation compared with a 0.6% vasodilation in postmenopausal women (P=0.046). CONCLUSIONS: Endothelial function is abnormal in many postmenopausal women compared with premenopausal women, and in some postmenopausal women it can be enhanced by estrogen replacement therapy. This effect may increase with prolonged use.

Adult↗
PubMed1998

Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay.

Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n=102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/-standard deviation [SD]) bias (EMIT-HPLC) was 1.88+/-0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.

Chromatography, High Pressure Liquid↗
PubMed1998

Plasma indomethacin assay using high-performance liquid chromatography-electrospray-tandem mass spectrometry: application to therapeutic drug monitoring and pharmacokinetic studies.

The authors report the use of high-performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) for the quantification of indomethacin (IND) in plasma with microscale sample preparation. Plasma samples (100 microL) and mefanamic acid (internal standard [IS]), buffered to pH 3.5, were prepared using solid phase extraction and chromatographed using a C8 column. The mobile phase composition was 80% methanol to 20% ammonium acetate buffer (40 mM, pH 5.1). A flow rate of 300 microL per minute was used with a 1-to-12 postcolumn split into the mass spectrometer. Selected reaction monitoring with mass transitions m/z 357.9-->139.0 and m/z 242-->209.0 were used for IND and IS, respectively. The chromatographic analysis time was 4 minutes. The assay was linear from 5 microg/L to 2000 microg/L with interday imprecision (n=5) over the analytic range (5%). At four concentrations (10 microg/L, 25 microg/L, 250 microg/L, 1500 microg/L), assay imprecision was 9% (total coefficient of variation [CV]) and accuracy ranged between 96.5% and 102.8% (n=16). The absolute recovery of IND and IS was 74% (n=8) and 95% (n=24), respectively. This method was developed and validated in less than 10 working days, had a lower limit of quantification than reported HPLC-ultraviolet (UV) methods, and uses small sample volumes. These factors illustrate the power of HPLC-ESI-MS/MS for drug analysis. Furthermore, the ability of this method to measure IND over a wide concentration range makes it suitable for therapeutic drug monitoring and pharmacokinetic studies.

Chromatography, High Pressure Liquid↗