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R James

Publications and source records attributed to R James.

At least 19 recordsLinked to original sources
PubMed1998

Effects of serum protein opsonization on cytokine release by titanium-alloy particles.

This study tested whether macrophages respond differently to retrieved titanium-alloy particles than they do to machined titanium-alloy particles and assessed whether pretreatment of machined titanium-alloy particles with human serum would influence macrophage activation and cytokine release in vitro. Human monocyte/macrophages were isolated from normal healthy donors and exposed to increasing concentrations of machined and retrieved titanium-alloy particles. The profile of cytokine release was determined by commercially available ELISA kits. Machined titanium-alloy particles were opsonized with human serum and added to macrophage cultures. Serum protein binding was confirmed by SDS-PAGE analysis. The results showed that machined titanium-alloy particles and retrieved titanium-alloy particles stimulate a similar level of cytokine release when tested at comparable concentrations. Opsonization of the machined particles with human serum increased the macrophage release of cytokines in the first 12 h after exposure compared to nonopsonized particles. At 24 h, the opsonized particles stimulated significantly higher levels of cytokine release, but only at the greatest particle concentrations. This study demonstrates that machined titanium alloy induces a metabolic response in macrophages similar to that of titanium-alloy particles retrieved from failed total hip arthroplasty. In addition, these data show that serum protein binding to orthopedic biomaterial debris alters the macrophage reaction to the particles.

Alloys↗
PubMed1998

Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9 from Escherichia coli.

The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structured in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was compared with the well-studied, non-specific DNase I by using a spectrophotometric assay with calf thymus DNA as the substrate. Both enzymes require divalent metal ions for activity but, unlike DNase I, the E9 DNase is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transition metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition metals showed poor activity in a plasmid-based nicking assay, yielding significant amounts of linearized plasmid, whereas Mg2+ was very active, with the main intermediate being open-circle DNA. The results suggest that, on entry into bacterial cells, the E9 DNase is likely to exhibit primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.

Bacterial Proteins↗
PubMed1998

Dual recognition and the role of specificity-determining residues in colicin E9 DNase-immunity protein interactions.

The immunity protein Im2 can bind and inhibit the noncognate endonuclease domain of the bacterial toxin colicin E9 with a Kd of 19 nM, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 with which it shares 68% sequence identity. Previous work from our laboratory has shown that the specificity differences of these four-helix immunity proteins is due almost entirely to helix II which is largely variable in sequence in the immunity protein family. From alanine scanning mutagenesis of Im9 in conjunction with high-field NMR data, a dual recognition model for colicin-immunity protein specificity has been proposed whereby the conserved residues of helix III of the immunity protein act as the anchor of the endonuclease binding site while the variable residues of helix II control the specificity of the protein-protein interaction. In this work, we identify three residues (at positions 33, 34, and 38) in helix II which define the specificity differences of Im2 and Im9 for colicin E9 and, using alanine mutagenesis of the putative endonuclease binding surface of Im2, compare the distribution of binding energies for conserved and nonconserved sites in both immunity proteins. This comparison highlights the conserved residues of both Im2 and Im9 as the major determinants of E9 DNase binding energy. Conversely, the nonconserved, specificity-determining residues only contribute to the E9 DNase binding energy in the cognate Im9 protein, while in the noncognate immunity protein Im2, they either destabilize the complex or do not contribute to the binding energy. This comparative alanine scan of two immunity proteins therefore supports the dual recognition mechanism of selectivity in colicin-immunity protein interactions and provides a basis for understanding specificity in other protein-protein interaction systems involving structurally conserved protein families.

Alanine↗
PubMed1998

A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity.

We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity.

Amino Acid Sequence↗
PubMed1998

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Arabidopsis↗
PubMed1998

Specificity in protein-protein recognition: conserved Im9 residues are the major determinants of stability in the colicin E9 DNase-Im9 complex.

The endonuclease group of E colicins are a family of bacterial toxins whose cytotoxic activity in a producing host is inactivated by a specific immunity protein. The DNase of colicin E9 can be bound and inhibited by both cognate and noncognate immunity proteins, the dissociation constants for which span a range of 12-orders of magnitude. DNase binding specificity of the immunity proteins is governed primarily by helix II, the sequence of which is variable in this family of proteins. Heteronuclear NMR experiments have identified helix III along with helix II as the likely DNase binding site, although other regions of Im9 also showed perturbations on binding the E9 DNase. In the present work, we have used the NMR experiments as a guide for alanine scanning mutagenesis of Im9. Our data show that helices II and III of Im9 are indeed the DNase binding site and in addition quantitate the relative binding energy associated with each helix. We find that the conserved residues of helix III make the largest relative contribution toward E9 DNase binding. In conjunction with previous studies, the data suggest that specificity in the colicin-immunity system is governed by a dual recognition mechanism in which highly stabilizing interactions emanating from the conserved regions of an immunity protein act as the binding site anchor and these are modulated by interactions from neighboring, nonconserved amino acid residues. This modulation is likely to take the form of both favorable and unfavorable interactions, the balance of which define the specificity of the protein-protein interaction. The generality of such a dual recognition mechanism in other systems is also discussed.

Alanine↗
PubMed1998

NMR detection of slow conformational dynamics in an endonuclease toxin.

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid 15N- and 13C/15N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro65-Asn72, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D 1H-1H-15N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with C alpha and C beta chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp22 or Trp58, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D 1H-1H-15N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, NA and NB, which interchange at 15 degrees C with a forward rate constant of 1.61 +/- 0.5 s-1 and a backward rate constant of 1.05 +/- 0.5 s-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743-13750 and 13751-13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the NA and NB conformers of the DNase and with binding only to the NB conformer detected because the rate of dissociation of the complex of Im9 and the NA conformer, NAI. is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the NBI form. The possibility that cis-trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the NA [symbol: see text] NB interconversion to be identified, Pro65 and Pro73. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form.

Amino Acid Sequence↗
PubMed1998

Matrix metalloproteinase-2 and matrix metalloproteinase-9 in cholesteatoma and deep meatal skin.

A qualitative and quantitative study of the presence of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9) in cholesteatoma was performed. Ten cholesteatoma and four deep meatal skin specimens were analysed for gelatinase activity at molecular weights corresponding to MMP-2 and MMP-9 using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) Zymography. Gelatinase activity at 72 kDa and 92 kDa was investigated. Western blotting was employed using primary monoclonal antibodies to provide a qualitative assessment of MMP-2 and MMP-9. Non-parametric data analysis using the Mann-Whitney U test did not show a significant difference in expression of MMP-2 (P = 0.51) or MMP-9 (P = 0.14) between the two tissue types. Western blotting showed the presence of both MMP-2 and MMP-9 in the majority of specimens, both cholesteatoma and deep meatal skin.

Blotting, Western↗
PubMed1998

Immunity proteins and their specificity for endonuclease colicins: telling right from wrong in protein-protein recognition.

Immunity proteins inhibit colicins, protein toxins released by bacteria during times of environmental stress, by binding and inactivating their cytotoxic domains. This protects the producing organism as it attempts to kill off competing bacteria. The cytotoxic domains of related colicins share a high degree of sequence identity, as do their corresponding immunity proteins, yet specificity and affinity are also high, with little non-cognate biological cross-protection evident under physiological conditions. We review recent work on DNase-specific immunity proteins, which shows that, although both cognate and non-cognate proteins can bind a single toxin, their affinities can differ by as much as 12 orders of magnitude. We have termed this mode of binding dual recognition, because the DNase-binding surface of an immunity protein is made up of two components, one conserved and the other variable. The strength of the binding interaction is dominated by the conserved residues, while neighbouring variable residues control specificity. Similar dual recognition systems may exist in other biological contexts, particularly where a protein must discriminate the right binding partner from numerous, structurally homologous alternatives.

Bacterial Proteins↗
PubMed1998

Farm and tractor-related fatalities in children in South Australia.

OBJECTIVE: To analyse the circumstances surrounding farm and tractor-related childhood fatalities in South Australia, to identify potentially dangerous situations and to delineate prevention strategies. METHODS: A retrospective search of files was conducted at the Women's and Children's Hospital Department of Histopathology from 1981 to 1996, and the State Coroner's Office from 1988 to 1996. RESULTS: Fifteen fatalities were identified comprising 11 boys and four girls (age range, 2 years 11 months to 13 years; average, 6 years). Activities prior to death included riding on a tractor/trailer (n=8); playing near a field grain storage bin (n=2); playing near machinery (n=2); sleeping in a car near a burn off (n=1); walking around a dam (n=1) and riding as a passenger on a motorbike (n=1). Causes of death included multiple skull fractures and cerebral damage (n=11), asphyxia (n=2), drowning (n=1) and incineration (n=1). CONCLUSIONS: The most dangerous activity involved children riding unrestrained on tractors; falls resulted in extensive injuries from tractor rear wheels or towed machinery. Allowing children on farms to ride on tractors or machinery only if there is a safe seat with a restraining harness would substantially reduce the number of fatal farm-related childhood injuries in South Australia.

Accidents↗
PubMed1998

Widespread axonal injury in gunshot wounds to the head using amyloid precursor protein as a marker.

In order to determine whether axonal injury (AI) is a factor in cases of penetrating head injury, the brains of 14 patients who died shortly after sustaining a fatal gunshot wound (GSW) to the head were examined, and the presence of AI determined using immunohistochemical staining for amyloid precursor protein (APP). The distribution of AI was mapped throughout the cerebral hemispheres and brain stem. AI was present in all cases in a diffuse distribution distant to the missle track with severe involvement of the brain stem in all cases. There was no axonal APP immunoreactivity in the direct region of the missle track at the point of primary axotomy. The APP-positive AI in these cases is likely to be a mixture of primary and secondary AI as APP immunostaining is unable to distinguish primary AI due to mechanical deformation from AI secondary to hypoxic-ischemic damage.

Adolescent↗
PubMed1998

Amphetamine derivative fatalities in South Australia--is "Ecstasy" the culprit?

OBJECTIVE: To analyze features of a series of fatalities caused by amphetamine-derivative designer drugs marketed as "Ecstasy" in South Australia, and to identify reasons for the recent marked increase in number of these deaths. MATERIALS AND METHODS: Following the death of a 26-year-old woman after alleged ingestion of Ecstasy tablets, a retrospective search of files at State Forensic Science, Adelaide and the South Australian State Coroner's Department was undertaken from February 1992 to January 1997 to identify similar cases. RESULTS: Six fatalities were found, all of which have occurred since September 1995 (M:F ratio, 1:1; age range, 22 to 36 years; average age, 27.7 years). All individuals had histories of recent ingestion of illegal drugs thought to be Ecstasy (methylenedioxymethamphetamine, MDMA) at the time of purchase. Delay occurred in seeking medical attention, despite severe symptoms. Causes of death involved documented hyperthermia in 3 cases (temperatures of 41.5-46.1 degrees C), with features of hyperthermia in one other case, and intracranial hemorrhage in another. Drugs in toxic/lethal amounts identified at postmortem included paramethoxyamphetamine (PMA) in all cases, amphetamine/methamphetamine in 4 cases, and methylenedioxymethamphetamine (MDMA or Ecstasy) in only 2 cases. Interaction with a prescription medication (fluoxetine) may have occurred in 1 case. CONCLUSIONS: The number of deaths due to amphetamine derivatives apparently due to substitution of PMA for MDMA (Ecstasy) have recently increased markedly in Adelaide. Potential users should be warned that PMA has been associated with a much higher rate of lethal complications than other designer drugs, and that no guarantee can be made that tablets sold as Ecstasy are not PMA.

Adult↗
PubMed1998

Do shorter-acting neuromuscular blocking drugs or opioids associate with reduced intensive care unit or hospital lengths of stay after coronary artery bypass grafting? CABG Clinical Benchmarking Data Base Participants.

BACKGROUND: The authors hypothesized that shorter-acting opioid and neuromuscular blocking drugs would be associated with reductions in duration of intubation, length of stay (LOS) in the intensive care unit (ICU) after tracheal extubation, or postoperative (exclusive of ICU) LOS, and that shorter durations of intubation would be associated with reduced ICU LOS after extubation and postoperative (exclusive of ICU) LOS. METHODS: One-thousand ninety-four patients undergoing primary coronary artery bypass graft surgery at 40 academic health centers were studied. Multiple patient-related factors were included in multivariate models for hypothesis testing. RESULTS: The duration of tracheal intubation, ICU LOS after extubation, and postoperative (exclusive of ICU) LOS all varied significantly by site. There was no difference between vecuronium and pancuronium in duration of intubation, ICU LOS after extubation, or postoperative (exclusive of ICU) LOS. Use of sufentanil rather than fentanyl was associated with a significant (P=0.045) reduction of 1.9 h (95% CI, 0.04 to 4.1 h) in duration of tracheal intubation but had no significant effect on ICU LOS after extubation, total ICU LOS, postoperative (exclusive of ICU) LOS, or total postoperative LOS. The authors' best model predicts a complex association between increasing duration of intubation and both ICU LOS after tracheal extubation and postoperative (exclusive of ICU) LOS, which was associated with an increase in those measures when duration of intubation exceeded 7.3 or 3 h, respectively. CONCLUSIONS: The LOS measures varied considerably among the institutions. Use of shorter-acting opioid and neuromuscular blocking drugs had no association with ICU LOS after tracheal extubation or with postoperative (exclusive of ICU) LOS. Only when the duration of intubation exceeded threshold values was it associated with increased LOS measures.

Aged↗
PubMed1998

Three patients with a 45,X/46,X,psu dic(Xp) karyotype.

Few cases of isochromosomes for the short arm of the X have been reported and all are dicentric with variable portions of the long arms interposed between the two centromeres. This paper reports three cases of complete short arm duplication of one X chromosome in unrelated female patients. All patients also have a 45,X cell line and present with some characteristic features of Turner syndrome. We used conventional cytogenetics, in situ hybridisation, and molecular genetics to describe all three structurally abnormal chromosomes and the parental origin of two of them. We briefly discuss the "inactivation enhancement" theory; however, any genotype-phenotype correlation is complicated by the presence of the 45,X cell line.

Adolescent↗
PubMed1998

Phase II study of docetaxel and cisplatin in advanced non-small-cell lung cancer.

PURPOSE: Docetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, France) and cisplatin are two of the most active single agents used in the treatment of non-small-cell lung cancer (NSCLC). A recently reported phase I study of the combination of docetaxel and cisplatin recommended a dose of 75 mg/m2 of both drugs every 3 weeks for subsequent phase II study. PATIENTS AND METHODS: Eligible patients were aged 18 to 75 years with a World Health Organization (WHO) performance status < or = 2 and life expectancy > or = 12 weeks, with metastatic and/or locally advanced NSCLC proven histologically or cytologically. Patients were not permitted to have received prior chemotherapy, extensive radiotherapy, or any radiotherapy to the target lesion and must have had measurable disease. Concurrent treatment with colony-stimulating factors (CSFs) or prophylactic antibiotics was not permitted. Docetaxel (75 mg/m2) in 250 mL 5% dextrose was given intravenously (i.v.) over 1 hour immediately before cisplatin (75 mg/m2) in 500 mL normal saline given i.v. over 1 hour in 3-week cycles. Premedication included ondansetron, dexamethasone, promethazine, and standard hyperhydration with magnesium supplementation. RESULTS: A total of 47 patients, two thirds of whom had metastatic disease, were entered onto this phase II study. The majority of patients were male (72%) and of good (WHO 0 to 1) performance status (85%). All 47 patients were assessable for toxicity and 36 were for response. Three patients were ineligible and eight (17%) discontinued treatment because of significant toxicity. In assessable patients, the overall objective response rate was 38.9% (95% confidence limits [CL], 23.1% to 56.5%), 36.1% had stable disease, and 25% progressive disease. On an intention-to-treat analysis, the objective response rate was 29.8%. Median survival was 9.6 months and estimated 1-year survival was 33%. Significant (grade 3/4) toxicities included nausea (26%), hypotension (15%), diarrhea (13%), and dyspnea mainly related to chest infection (13%). One patient experienced National Cancer Institute (NCI) grade 3 neurosensory toxicity after eight cycles. Grade 3/4 neutropenia was common and occurred in 87% of patients, but thrombocytopenia > or = grade 3 was rare (one patient). Significant (grade 3/4) abnormalities of magnesium levels were common (24%). Febrile neutropenia occurred in 13% of patients and neutropenic infection in 11%, contributing to two treatment-related deaths. No neutropenic enterocolitis or severe fluid retention was reported. CONCLUSION: Compared with other active regimens used in this setting, the combination of docetaxel and cisplatin in advanced NSCLC is an active regimen with a similar toxicity profile to other combination regimens.

Adult↗
PubMed1998

Scaling of intrinsic contractile properties and myofibrillar protein composition of fast muscle in the fish myoxocephalus scorpius L

Contractile properties were determined for fast muscle fibres from short-horn sculpin (Myoxocephalus scorpius L.) ranging from 5 to 35 cm total body length (L) and from 2.0 to 758 g body mass. Maximum unloaded shortening velocity (V0) decreased with increasing body size, scaling as 19.5L-0.34, and isometric twitch activation and tetanus relaxation times became longer, scaling as 12.0L0.31 and 19. 5L0.42 respectively. Myofibrillar Mg2+/Ca2+-ATPase activity scaled as 2.51L-0.28. In order to investigate the mechanisms underlying the scaling of contractile properties, myofibrillar protein composition and I filament lengths were determined. One-dimensional SDS-PAGE and two-dimensional isoelectric focusing/non-equilibrium isoelectric focusing-PAGE revealed no differences in the myofibrillar protein isoforms of myosin light chains, actin, tropomyosin, troponin-T and troponin-C in fish of differing body size. Peptide maps of purified myosin heavy chains digested with eight different proteolytic enzymes were also similar in all fish examined. Three isoforms of troponin-I were present in fish less than 20 cm in total length with relative molecular masses of 17 (TnIf3), 22 (TnIf2) and 23 (TnIf1). The ratio of TnI isoforms varied with body length, and only TnIf3 was present in fish greater than 28 cm total length. The length of I filaments was independent of body length. Thus, although the expression of TnI isoforms changes during growth, the underlying mechanism responsible for increased shortening velocity with decreased body size remains unknown.

Journal Article↗
PubMed1998

Scaling of muscle performance during escape responses in the fish myoxocephalus scorpius L

Fast-starts associated with escape responses were studied in short-horn sculpin (Myoxocephalus scorpius L.), ranging from 5.5 to 32 cm in total length (L). Electromyography and sonomicrometry were used simultaneously to measure muscle activation and length changes, respectively, in the superficial layers of fast muscle in rostral myotomes. Escape responses consisted of a half tailbeat to bend the body into a C-shape (C-bend), another half tailbeat (contralateral contraction), followed by one or two more tailbeats and/or a gliding phase. The scaling relationships for both muscle strain and shortening duration differed between the C-bend and the contralateral contraction. As a result, relative muscle shortening velocity (V/V0) scaled as -1.18L1.06 for the C-bend and as 1.23L-0. 66 for the contralateral contraction. Therefore, the scaling relationships for muscle shortening velocity varied throughout the time course of the escape response. Muscle power output was determined by using the work-loop technique to subject isolated muscle fibres to in vivo strain and stimulation patterns. Plots of the instantaneous muscle forces and velocities achieved during the contralateral contraction were found to deviate from the steady-state force-velocity relationship. Maximum instantaneous muscle power output was independent of body size, with mean maximum values of 307 and 222 W kg-1 wet muscle mass for the C-bend and the contralateral contraction, respectively.

Journal Article↗