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Biomedical subjects

R James

Publications and source records attributed to R James.

At least 127 records · Page 7Linked to original sources
PubMed1997

Translationally controlled tumor protein: a protein identified in several nontumoral cells including erythrocytes.

The translationally controlled tumor protein (TCTP) is a growth-related protein which is regulated at the translational level. It is present in mammals, higher plants and Saccharomyces cerevisiae. This study was undertaken to localize and further characterize the TCTP in human cell lysates using two-dimensional gel electrophoresis, monoclonal antibodies, and 45Ca-gel overlay. TCTP was found in several healthy and tumoral cells including erythrocytes, hepatocytes, macrophages, platelets, keratinocytes, erythroleukemia cells, gliomas, melanomas, hepatoblastomas, and lymphomas. It could not be detected in kidney and renal cell carcinoma (RCC). A monoclonal antibody raised against TCTP detected three isoforms likely due to post-translational modifications. A calcium binding property was found as well as heat stability and cytoplasmic localization. The high degree of homology from plants to man and its expression in many tissues suggests that TCTP most likely has a cell housekeeping function.

Amino Acid Sequence↗
PubMed1997

Expression of superoxide dismutase following axotomy.

Oxidative injury has been implicated in the pathophysiology of neuronal injury and neurodegenerative disease. Antioxidant proteins provide an endogenous defense against such oxidative injury and may yield important clues to mechanisms of cytoprotection and neuronal recovery. Axotomy is the simplest model of neuronal injury and lesioning the sciatic nerve allows concurrent study of both motor (spinal cord) and sensory (dorsal root ganglia, DRG) neurons affected by the same injury. This study was designed to examine the expression of superoxide dismutase (SOD), an essential antioxidant protein, in motor and sensory neurons following complete axotomy of peripheral nerve. Immunocytochemical, quantitative immunoblot, and enzymatic activity assay techniques are used. By 12 days after axotomy, immunocytochemical expression of Mn-SOD is markedly increased in affected DRG and spinal cord. A similar increase in Cu/Zn-SOD is not seen in DRG or spinal cord. This immunocytochemical staining is associated with a significant increase in specific activity and Mn-SOD protein content as measured on quantitative immunoblots. This report suggests, for the first time, that Mn-SOD and not Cu/Zn-SOD increases in sensory neurons of the DRG and motor neurons of the spinal cord following distal axotomy of the sciatic nerve. Quantitative measurements of Mn-SOD following axotomy reveals that the increase in immunocytochemical reactivity is associated with an approximately 30% increase in specific activity when comparing lesioned and contralateral spinal cord samples. These data suggest that Mn-SOD may have a more significant role in the pathophysiology of neuronal injury than Cu/Zn-SOD.

Animals↗
PubMed1997

Turner syndrome: a cytogenetic and molecular study.

Two hundred and eleven patients with a clinical diagnosis of Turner syndrome were studied. We report (i) the cytogenetic results, (ii) the frequency of cryptic mosaicism and (iii) the parental age and the parental origin of the abnormality. We scored 100 cells from blood cultures and found 97 patients to have a 45,X constitution, 15 to be 45,X/46,XX or 45,X/47,XXX mosaics, 86 to have a structurally abnormal X and 13 to have a structurally abnormal Y chromosome. Molecular methods were used to look for cryptic X and Y chromosome mosaicism in patients with a 45,X constitution. Two cryptic X but no cryptic Y mosaics were detected. In 74% of the 45,X patients the X was maternal in origin. The i(Xq)s were approximately equally likely to involve the paternal or maternal chromosome, while the majority of deletions and rings and virtually all the abnormal Y chromosomes were paternal in origin. We suggest that the preponderance of paternal errors in Turner syndrome may result from the absence of pairing along the greater part of the XY bivalent during paternal mei I, which may make the sex chromosomes particularly susceptible to both structural and non-disjunctional errors during male gametogenesis.

Adolescent↗
PubMed1997

An analysis of Michigan and California CO remote sensing measurements.

Remote sensing measurements of CO emissions from onroad vehicles were made in California in 1991 and in Michigan in 1992. It was determined that both fleets had a small linear increase in the high emitter frequency (vehicles emitting more than 4% CO) as a function of vehicle age for 1986 and newer model vehicles. Although high emitting vehicles were only a small minority of the fleet, they had a dominant impact on the mean CO and total CO emitted by the fleet. In Michigan, the highest emitting 5% of passenger cars generated 45% of the CO from cars. In California, the highest emitting 5% of passenger cars generated 38% of the CO from cars. There was a high correlation between the mean CO emitted by each model year of vehicle and the frequency of high emitting vehicles within the model year for both the Michigan and California fleets. The frequency of high emitters within any model year had no obvious relation to that model year's certification standards. The high emitter frequencies for vehicles less than nine years old were very similar for the California and Michigan fleets. An increase in the high emitter frequency in the ten-year-old and older Michigan passenger car fleet (relative to the California passenger car fleet), suggests, but does not conclusively demonstrate, that the rate of high emitters in Michigan and California is reduced by the inspection and maintenance (I/M) programs.

Automobiles↗
PubMed1997

Phase I trial of docetaxel and cisplatin in previously untreated patients with advanced non-small-cell lung cancer.

PURPOSE: To determine the maximum-tolerated doses (MTDs), principal toxicities, and pharmacokinetics of the combination of docetaxel and cisplatin administered every 3 weeks to patients with advanced non-small-cell lung cancer (NSCLC) who have not received prior chemotherapy and to recommend a dose for phase II studies. PATIENTS AND METHODS: Patients with advanced NSCLC and performance status 0 to 2 who had not received prior chemotherapy received docetaxel over 1 hour followed by cisplatin over 1 hour with hydration. Dose levels studied were (docetaxel/cisplatin) 50/75, 75/75, 75/100, and 100/75 mg/m2 repeated every 3 weeks. Colony-stimulating factor (CSF) support was not used. Pharmacokinetics of docetaxel and cisplatin were studied in the first cycle of therapy. Most patients (79%) had metastatic disease or intrathoracic recurrence after prior radiation and/or surgery. RESULTS: Of 24 patients entered, all were assessable for toxicity and 18 for response. The MTD schedules were docetaxel 75 mg/m2 with cisplatin 100 mg/m2 (dose-limiting toxicities [DLTs] in five of six patients), and docetaxel 100 mg/m2 with cisplatin 75 mg/m2 (DLTs in two of two patients, including one fatal toxicity). Limiting toxicities were febrile neutropenia and nonhematologic, principally diarrhea and renal. Two patients had neutropenic enterocolitis. Pharmacokinetics of both drugs were consistent with results from single-agent studies, which suggests no major pharmacokinetic interaction. Neutropenia was related to docetaxel area under the plasma concentration-versus-time curve (AUC). An alternative schedule was investigated, with cisplatin being administered over 3 hours commencing 3 hours after docetaxel, but toxicity did not appear to be less. Independently reviewed responses occurred in eight of 18 patients (44%; 95% confidence interval, 22% to 69%), most following 75 mg/m2 of both drugs. CONCLUSION: Docetaxel 75 mg/m2 over 1 hour followed by cisplatin 75 mg/m2 over 1 hour is recommended for phase II studies. The responses seen in this phase I study suggest a high degree of activity of this combination in previously untreated advanced NSCLC.

Adult↗
PubMed1997

The health of Canadians with diabetes.

OBJECTIVES: This article focuses on the prevalence of diabetes mellitus among Canadians, the health status of those with the disease, their socioeconomic characteristics, personal health behaviours, and use of health services. DATA SOURCE: The data are from the 1994/95 National Population Health Survey. ANALYTICAL TECHNIQUES: Weighted counts were used in computing the prevalence of diabetes and the proportions of people with the characteristics and health behaviours considered. MAIN RESULTS: In 1994/95, 3% of Canadians aged 12 and older--over 722,000 people--reported having diabetes that had been diagnosed by a health care professional. The prevalence of diabetes increases with age and is associated with low income. A higher percentage of adults with diabetes were overweight compared to those without the disease.

Adult↗
PubMed1996

Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis.

We have used random mutagenesis to identify putative active-site residues in the C-terminal cytotoxic endonuclease domain of the bacterial toxin colicin E9. Six single-site mutations in the DNase domain were isolated which destroyed the toxic action of the colicin. DNA sequencing identified the mutations as Gly460Asp, Arg544Gly, Glu548Gly, Thr571Ile, His575Tyr and His579Tyr. All six wild-type residues are highly conserved in the DNase domains of both the E group colicins and the closely related pyocins. Site-directed mutagenesis was then used to substitute the wild-type amino acid residue at each of these positions for an alanine residue in order to distinguish important from unimportant sites. Two of the six alanine-mutant colicins (Gly460Ala and His579Ala) exhibited significant in vivo activity, unlike the original mutation of these residues, and were therefore not characterised further. The Thr571Ala mutant colicin, although not inactive, was significantly less active than the control. The other three alanine mutants (Arg544Ala, Glu548Ala and His575Ala remained completely inactive in the in vivo tests. Each 15 kDa alanine-mutant DNase domain was overexpressed and purified using a tandem-expression strategy which relies on the enzyme being able to bind to the natural inhibitor, Im9. Tryptophan emission spectra of the alanine mutants showed significant alterations in the emission maxima of all but the His575Ala mutant, suggesting changes in the tertiary structure of these mutant proteins. Activity measurements, using the spectrophotometric Kunitz assay, indicated that the Thr571Ala mutant was partially active as an endonuclease but the remaining alanine mutants were all completely inactive. All four mutant proteins, however, retained their ability to bind DNA in a gel shift assay, suggesting the mutations affect catalytic rather than substrate-binding residues. Searching the sequence databases for possible homology to other DNA-binding proteins revealed a significant match between residues 464 to 487 of the E9 DNase domain and helix IV of the POU domain of eukaryotic transcription factors.

Alanine↗
PubMed1996

Three-dimensional solution structure and 13C nuclear magnetic resonance assignments of the colicin E9 immunity protein Im9.

The 86-amino acid colicin E9 immunity protein (Im9), which inhibits the DNase activity of colicin E9, has been overexpressed in Escherichia coli and isotopically enriched with 15N and 13C. Using the 3D CBCANH and CBCA(CO)NH experiments, we have almost completely assigned the backbone 13C resonances and extended previously reported 15N/1H backbone assignments [Osborne et al. (1994), Biochemistry 33, 12347-12355]. Side chain assignments for almost all residues were made using the 3D 13C HCCH-TOCSY experiment allied to previous 1H assignments. Sixty solution structures of Im9 were determined using the DIANA program on the basis of 1210 distance restraints and 56 dihedral angle restraints. The 30 lowest-energy structures were then subjected to a slow-cooling simulated annealing protocol using XPLOR and the 21 lowest-energy structures, satisfying the geometric restraints chosen for further analysis. The Im9 structure is well-defined except for the termini and two solvent-exposed loops between residues 28-32 and 57-64. The average RMSD about the average structure of residues 4-84 was 0.94 A for all heavy atoms and 0.53 A for backbone C alpha, C = O, and N atoms. The Im9 fold is novel and can be considered a distorted antiparallel four-helix bundle, in which the third helix is rather short, being terminated close to its N-terminal end by a proline at its C-terminus. The structure fits in well with available kinetic and biochemical data concerning the interaction between Im9 and its target DNase. Important residues of Im9 that govern specificity are located on the molecular surface in a region rich in negatively charged groups, consistent with the proposed electrostatically steered association [Wallis et al. (1995a), Biochemistry 34, 13743-13750].

Bacterial Proteins↗
PubMed1996

Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus.

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

Amino Acid Sequence↗
PubMed1996

Influence of GC and AT specific DNA minor groove binding drugs on intermolecular triplex formation in the human c-Ki-ras promoter.

We have used DNase I footprinting and gel shift assays to characterize the interaction of DNA binding drugs mithramycin, distamycin, and berenil with an intermolecular triplex formed by the human c-Ki-ras promoter. A purine-rich triplex-forming oligonucleotide (ODN) forms a stable intermolecular triple helix (triplex) with a homopurine (PR):homopyrimidine (PY) motif in the human c-Ki-ras promoter which contains a 22bp PR:PY region (-328 to -307). This triplex structure is comprised of 15 G.G:C triplets interspersed with 7 T.A:T triplets. Mithramycin binding sites in the human c-Ki-ras promoter encompass most of the triplex target site and three G-C-rich sequences downstream of this triplex-forming region. Mithramycin binding within the c-Ki-ras promoter completely abrogates triplex formation. Furthermore, the addition of mithramycin to pre-formed triplex by c-Ki-ras promoter displaces the major groove bound ODN. Five prominent distamycin binding sites are noted within the c-Ki-ras promoter including the triplex-forming site as well as A-T-rich regions upstream and downstream of the triplex site. Berenil does not bind within the triplex target sequence, and only one berenil binding sequence downstream of the triplex motif was present within the c-Ki-ras promoter fragment. Neither distamycin nor berenil prevents triplex formation, and, furthermore, the addition of either distamycin or berenil to the pre-formed triplex structure did not displace the major-groove-bound third strand. This study demonstrates that GC-specific and AT-specific minor groove ligands differentially affect the intermolecular pur.pur:pyr triplex. A possible biological significance of mithramycin interaction with intramolecular triplex is discussed.

Base Sequence↗
PubMed1996

The effect of pre-incisional infiltration with lignocaine on postoperative pain after molar teeth extraction under general anaesthesia.

In a double-blind, placebo-controlled investigation, we studied the effects of pre-operative inferior alveolar nerve blockade and local anaesthetic intra-oral infiltration upon postoperative pain following third molar teeth extraction under general anaesthesia. Thirty six patients (12 male), mean (range) age 25.7 (18-40) years received an injection of 2% lignocaine 2 ml with 1:200,000 adrenaline around the inferior alveolar nerve and tissues adjacent to the third molar tooth on one side and of normal saline 2 ml in a similar fashion to the other side. We measured the pain on each side of the mandible on the 1st, 4th and 11th days after surgery using a visual analogue scale. The visual analogue pain score on the lignocaine and saline sides were 25/100 and 26/100 on day 1, 23/100 and 22/100 on day 4 and 0/100 and 1/100 on day 11. This study has a power of 80% to detect differences at the 5% level of significance of 11/100 on days 1 and 4 and 7/100 on day 11. We found no significant difference in the visual analogue scores between the lignocaine side and the saline side at any stage after the procedure.

Adolescent↗
PubMed1996

Electrostatic charge on a plastic spacer device influences the delivery of salbutamol.

The aim of this study was to determine whether electrostatic charge on a plastic spacer decreases the delivery of salbutamol from a pressurized metered-dose inhaler (pMDI) and, if so, to find an optimal and practical treatment to remove the charge. Ten single actuations from a salbutamol pMDI were drawn through different Volumatic spacers at a constant flow of 60 L.min-1. The efficacies of different methods of removing charge were tested, including detergent coating of the spacers. A multistage liquid impinger was used to determine the particle size distribution of the output of the pMDI through the Volumatic spacers. The electrostatic charge on the inner surface of the spacers was measured both quantitatively with an electrometer, and qualitatively by the attraction of a thin strip of cellulose membrane to the wall of the spacer. Each experiment was repeated four times. Ionic detergent coating of the spacers removed the charge for at least 24 h. This resulted in an increase of 55-70% in small particle (< 6.8 microns) delivery compared to delivery from new spacers with high charge. We have demonstrated that electrostatic charge plays a major role in the delivery of salbutamol through plastic spacers. Adequate treatment with ionic detergent removes the charge and improves drug delivery.

Albuterol↗
PubMed1996

The experimental verification of the efficacy of finite element modeling to dental implant systems.

In vitro experimental data of angular displacement vs. applied force were obtained on a fully osseointegrated pair of endosseous implants in a mandible of a macaca mulatta monkey. These data are compared with calculated displacement data for a given force from a three-dimensional finite element model reproducing the geometric structure of a portion of the mandible holding the implants. The displacements show agreement over a range of forces in the elastic region within 3%. The FE model yields a full description of the bending of the implant and the distribution of the stress in the bone surrounding the implants.

Alveolar Process↗
PubMed1995

Mutagenesis of active site residues in type I dehydroquinase from Escherichia coli. Stalled catalysis in a histidine to alanine mutant.

Chemical modification experiments have previously implicated four amino acid residues in the mechanism of type I dehydroquinase from Escherichia coli. To further test their importance, these residues were mutated, and the resulting mutants were expressed, purified, and characterized. When the highly conserved, Schiff base-forming lysine residue was mutated (K170A) the resulting enzyme showed a approximately 10(6)-fold reduction in catalytic activity, but was still able to bind both substrate and product, as shown by a novel fluorescence-based ligand-binding assay. This is consistent with Lys-170 playing a central role in catalysis and shows that, although forming a covalent bond with the substrate, it is not essential for ground state binding of substrate or product. Conversely, substituting leucine for the conserved, iodoacetate-reactive methionine residue (M205L) had little effect on kcat or Km. Diethylpyrocarbonate experiments had previously implicated either His-143 or His-146 as the putative active site general base. Substituting alanine for each shows that H146A retains full catalytic activity while H143A shows a 10(6)-fold loss of activity. As with the K170A mutant, H143A can bind ligand, and in addition to the predicted role of this residue as the proton-abstracting general base, our data suggest that it is also involved in the formation and breakdown of Schiff base intermediates. Isoelectric focusing, electrospray ionization mass spectrometry, and fluorescence spectroscopy show that the H143A mutant preferentially stabilizes the formation of the product Schiff base, and that this results in burst kinetics reminiscent of p-nitrophenyl acetate hydrolysis by chymotrypsin. The most striking illustration of this stabilization is the fact that the H143A mutant is isolated from overexpressing cells with a significant proportion of the enzyme monomers covalently bound to the product, 3-dehydroshikimate, via a Schiff base linkage. Our data suggest that the H143A mutant is able to slowly transform substrate to product but that the hydrolytic release of the product is stalled. The proposed dual role of His-143 in the mechanism of type I dehydroquinase may explain why the elimination reaction catalyzed by the enzyme proceeds with syn stereochemistry.

Alanine↗
PubMed1995

Protein-protein interactions in colicin E9 DNase-immunity protein complexes. 1. Diffusion-controlled association and femtomolar binding for the cognate complex.

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a nonspecific DNase housed in the C-terminus of the protein. A kinetic and thermodynamic analysis of complex formation for both the holotoxin and the isolated DNase domain with the cytoplasmic inhibitor of this enzyme, the immunity protein Im9, is presented. The dissociation constant for each complex was calculated from the ratio of the association and dissociation rate constants. Association was monitored by stopped-flow fluorescence and comprises at least two steps for both complexes, an initial fluorescence enhancement followed by a fluorescence quench. The data are consistent with a two-step binding mechanism in which the rate of formation of an encounter complex (k1) is rate determining and essentially diffusion controlled (4.0 x 10(9) M-1 s-1 for colicin E9) in buffer of low ionic strength. This encounter complex then rearranges to the final stable complex. Sequential stopped-flow experiments using 5-hydroxy-L-tryptophan labeled DNase domain support the two-step mechanism and further show that the rate of encounter complex rearrangement is significantly faster than its dissociation. The overall rate of dissociation of the colicin E9-Im9 complex (k(off)) was determined by radioactive subunit exchange to be 3.7 x 10(-7) s-1. Thus, the Kd for the complex (k(off)/k1) is 9.3 x 10(-17) M, which corresponds to a change in free energy on binding of -21.9 kcal mol-1 at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗