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Biomedical subjects

R James

Publications and source records attributed to R James.

At least 19 recordsLinked to original sources
PubMed1992

Molecular analysis of the protein-protein interaction between the E9 immunity protein and colicin E9.

The specificity-determining region of the colicin E9 immunity protein (Im9) for its interaction with its cognate E colicin has been localized to residues 16-43 of the 86-amino-acid protein by the use of gene fusions. A comparison of the alignment of residues in this region of the Im2, Im8 and Im9 proteins have identified nine candidate specificity-determining residues. Using site-directed mutagenesis, we have changed each of these residues in the Im9 protein to the residue found in the same position in the Im8 protein. The immunity phenotype conferred by the mutant immunity protein was then tested. Of the nine residues, only one (Val34 to Asp) showed any evidence of conferring immunity to colicin E8. Changing other residues in the specificity-determining region to the equivalent Im8 residue did not affect the phenotype conferred by the mutant protein, with the exception of the change of Val37 to Glu, which resulted in low-level E8 immunity. While the substitutions at positions 34 and 37 of the Im9 protein introduced immunity towards ColE8, they did not diminish the immunity towards ColE9, suggesting that the two immunity proteins may have a common specificity framework which can be modified by single mutations. In addition, we have used chemical modification of the unique cysteine residue of Im9 (Cys23) in order to probe further this specificity-determining region. Cys23 in the purified Im9 protein is accessible to modification with the thiol-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) and the stoichiometry of labelling is close to 1:1. This residue, however, cannot be labelled by 5,5'-dithiobis(2-nitrobenzoic acid) when the Im9 protein is complexed to colicin E9. This result is consistent with the Cys23 residue being buried in the complex. However, when the purified Im9 protein modified at Cys23 with a variety of reagents was used in DNase inhibition assays with colicin E9, the modified Im9 proteins still possessed anti-DNase activity but only up to a certain derivative molecular mass. These results are discussed in terms of the proximity of Cys23 to the specificity-determining region.

Amino Acid Sequence↗
PubMed1992

Granulocyte colony-stimulating factor to prevent dose-limiting neutropenia in non-Hodgkin's lymphoma: a randomized controlled trial.

The effect of granulocyte colony-stimulating factor (G-CSF) on neutropenia, infection, and cytotoxic chemotherapy administration was studied in a randomized trial in patients receiving intensive weekly chemotherapy for non-Hodgkin's lymphoma (NHL). Eighty patients (aged 16 to 71 years) with high-grade NHL (Kiel) of any stage were randomized to receive VAPEC-B chemotherapy alone (39 patients) or with G-CSF administered as a daily subcutaneous dose of 230 micrograms/m2 (41 patients). Prophylactic ketoconazole and cotrimoxazole were administered to all patients throughout treatment. The protocol specified identical dose modification and antibiotic treatment criteria bor both groups. Neutropenia (absolute neutrophil count [ANC] less than 1.0 x 10(9)/L) occurred in 15 of 41 (37%) of the G-CSF-treated patients and in 33 of 39 (85%) of the controls, giving a relative risk for control patients of 2.31 (95% confidence interval [CI], [1.51, 3.54]; P = .00001). Fever (greater than or equal to 37.5 degrees C) with neutropenia (ANC less than 1.0 x 10(9)/L) occurred in 9 of 41 (22%) of the G-CSF group and in 17 of 39 (44%) of the controls (relative risk for control, 2.26; 95% CI [1.01, 5.06]; P = .04). There were fewer treatment delays, with shorter duration (P = .01) in patients receiving G-CSF. Chemotherapy doses were reduced in 4 of 41 (10%) of the G-CSF patients and 13 of 39 (33%) of the controls (P = .01). The dose intensity of cytotoxic chemotherapy was significantly increased in patients receiving G-CSF (median of 95% in G-CSF group compared with 83% in control patients). Three vascular deaths occurred in the G-CSF group. Delays in the control group most commonly resulted from neutropenia (19 patients, compared with 2 patients in the G-CSF-treated group, P = .000007). Severe mucositis was the major dose-limiting toxicity in G-CSF-treated patients, but did not occur more frequently than in controls (15 patients in each group). Overall, patients randomized to receive G-CSF achieved a greater dose intensity than control patients, but this did not result in significant differences in drug toxicity (other than neutropenia), intravenous antibiotic usage, or hospitalization between the two groups.

Adolescent↗
PubMed1992

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.

Bacterial Proteins↗
PubMed1992

The occurrence of cervical fractures in victims of judicial hanging.

Cervical vertebrae from 34 victims of judicial hanging were examined. In only six cases was there a fracture of the axis and only one other fracture was seen in the series. The fractures were of two types. Three were the traditional 'Hangman's fracture' whilst three were of a type previously unreported. The incidence of fracture was unassociated with drop, date age or hangman. The results of the post mortems on these subjects were reviewed and found to be grossly inaccurate with regard to fractures. The results indicate that the traditional hangman's fracture occurred in only a small proportion of cases of judicial hanging. The cause of fractures and cause of death in judicial hanging are discussed with a brief history of judicial hanging in this country.

Capital Punishment↗
PubMed1992

Suicidal hanging in Cardiff--a 15-year retrospective study.

A retrospective study of 84 cases of suicidal hanging was undertaken in which petechial haemorrhages or congestion were seen in 27%, soft tissue haemorrhage in 29% and fracture of the hyoid or laryngeal cartilages in 36%. Fractures were found to be related to increasing age and were not found when a soft ligature had been employed. Petechial haemorrhages and congestion were related to the completeness of suspension. Apart from the lack of fractures in the soft ligature group any of these findings, either singly or in combination, was found to occur without regard to age, suspension type or ligature type. No evidence was found of clustering or seasonality. The findings are compared to other series and discussed with regard to the mechanisms of causation.

Adult↗
PubMed1992

Generation and characterization of a monoclonal antibody specific for the major thiol-activated cysteine proteinase of Porphyromonas gingivalis W83.

An IgM monoclonal antibody specified for the thiol-activated proteinase of the oral pathogen Porphyromonas gingivalis W83 was generated. The antibody reacted with a single protein of approximate molecular mass 43 kDa in outer membrane preparations of P. gingivalis. Immuno-electron microscopy using the monoclonal antibody and immunogold labelling confirmed the cell surface location of the thiol-activated proteinase. The monoclonal antibody failed to detect any proteins in Western blot analysis of other closely related oral bacteria. The specificity of this monoclonal antibody to the thiol-activated proteinase of P. gingivalis should allow its use as a diagnostic tool for the rapid enumeration of P. gingivalis in clinical samples.

Antibodies, Bacterial↗
PubMed1992

Effect of metal mixtures on activity of two respiratory enzymes and their recovery in Oreochromis mossambicus (Peters).

Effects of sublethal doses of metal (Cu, Cd, Zn) mixtures on the activities of key respiratory enzymes (succinate dehydrogenase, SDH and glyceraldehyde dehydrogenase, GDH) and their recovery following withdrawal of treatments were studied in the freshwater fish O. mossambicus. On the basis of 96 hr LC50 Cu was highly toxic followed by Zn and Cd, and the trimetal combination (Cu+Zn+Cd) was extremely toxic than any other combination; combination of Zn+Cd was least toxic. A significant gradual decrease in SDH with a concomitant increase in GDH activity observed in liver, brain, muscle and gill of animals exposed to metal suggest a metabolic shift from aerobiosis to anaerobiosis due to metal action. Exposed individuals when transferred to metal impoverished water showed an improvement in SDH activity and decline in GDH activity suggesting slow reversal to aerobic metabolism. O. mossambicus needs more time for complete recovery.

Animals↗
PubMed1992

Efficacy of the FEF colorimetric end-tidal carbon dioxide detector in children.

Direct laryngoscopy and observation of endotracheal tube (ETT) passage between the vocal cords remain the criterion standard for verifying endotracheal intubation. Detection of end-tidal carbon dioxide (ETCO2) serves as an invaluable adjunct to confirm endotracheal intubation, detect inadvertent esophageal intubation, and monitor for accidental tracheal extubation. Capnography, however, is often unavailable outside the operating suite. A commercially available, disposable, colorimetric ETCO2 detector (FEF, Fenem, Inc., New York, N.Y.), in which color changes using a numerical scale semiquantitatively measure percent carbon dioxide in exhaled gases, has proved effective in confirming endotracheal intubation in adults, but has not been thoroughly investigated in children. We studied 20 otherwise healthy children, aged 6 mo to 8 yr, with simultaneous infrared and colorimetric ETCO2 measurements during elective general anesthesia to evaluate the efficacy of the colorimetric detector. Two hundred of 200 tracheally intubated positive-pressure breaths and 198 of 200 breaths under spontaneous mask ventilation demonstrated a yellow color change (color level 5 or 6), signifying an ETCO2 greater than or equal to 15 mm Hg (2.0 kPa). Repeated-measures analysis of variance revealed no significant differences in infrared ETCO2 values between the two yellow color levels throughout the study period. The associations among color level, infrared ETCO2 determinations, ETT size, and ETT "leak" pressures estimated by Spearman rank correlation analysis were significant only for higher infrared ETCO2 values with higher ETT leak pressures (P less than 0.05). No complications were observed. The Fenem disposable colorimetric ETCO2 detector effectively confirms clinical signs of endotracheal intubation in children when capnography is unavailable.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Dioxide↗
PubMed1991

The major colonic cell mitogen extractable from colonic mucosa is an N terminally extended form of basic fibroblast growth factor.

Colonic growth factors (CGFs) were extracted from porcine intestinal epithelium and mucosa. Under acidic conditions, very little mitogenic activity (as assayed using murine 3T3 fibroblasts and a human colonic cell line) was extractable. However, by extracting at neutral or slightly alkaline pH, significant mitogenic activity for both the murine fibroblasts and human colonic carcinoma cell line could be detected. CGFs are present throughout the intestine and cecum. The epithelial mucosa of the distal colorectal region appeared to contain mitogens which were more potent for the colonic cells than the 3T3 fibroblasts. Purification of CGFs from the colonic mucosa required removal of associated mucin by pH precipitation prior to chromatographic fractionation. It was then possible to develop a complete purification (390,000-fold) scheme for the major CGF, an 18-kDa protein which bound to heparin-Sepharose. N-terminal sequence analysis yielded a single sequence (Q)SPGGAMAAGSITTLPALP, i.e. an N-terminally extended form of basic fibroblast growth factor. Apart from the substitution of Gly in bovine basic fibroblast growth factor by a Ser in porcine CGF, the proteins are identical. A similar extraction procedure using purified human colonic crypt epithelial cells yielded a mitogen for the human colonic cell line with similar chromatographic properties.

Amino Acid Sequence↗
PubMed1991

The North Coast Cholesterol Check Campaign. Results of the first three years of a large-scale public screening programme.

Although cardiovascular disease (CVD) mortality has been declining, CVD is still the major cause of death in Australia and an elevated blood cholesterol level is considered a major contributor. Large-scale community-based screening programmes in other countries have demonstrated that a population approach can be effective in reducing cholesterol levels and the risk of CVD. The North Coast Cholesterol Check Campaign is the largest community-based cholesterol intervention programme in Australia. Since its inception in 1987, 13% of the Region's adult population (over 29,000 persons) have been screened. About half had elevated blood cholesterol levels (greater than or equal to 5.5 mmol/L) and were given dietary counselling to reduce fat intake. Mean blood cholesterol levels were significantly reduced between initial screening and follow-up in all three years. Reductions, after correction for regression, were 8%, 6% and 10%, respectively, in 1987, 1988 and 1989. There was also a consistent and significant 1.5% to 2% reduction in weight. All age/sex cohorts above age 35 were well represented each year although self-referral did bias both initial and follow-up samples towards women and the aged. Nevertheless, the proportion of men and men in their middle age increased during the three years. The proportion of participants with elevated cholesterol levels increased in each successive year while the proportion of participants who complied with referrals to visit their general practitioner and with requests to return for follow-up decreased. Over half of the North Coast adult population has now had a cholesterol test. The rate of increase in testing since the inception of the Campaign has been approximately four times the national rate. North Coast general practitioners have played a major role by catering for the increased community demand for cholesterol testing and by providing an effective referral service for the Campaign. Community-based screening programmes in Australia can detect and beneficially influence large numbers of persons with elevated cholesterol levels. Interventions like the North Coast Cholesterol Check Campaign, when conducted over a number of years, do attract higher risk and less compliant people.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Homeobox gene expression in the intestinal epithelium of adult mice.

Using a polymerase chain reaction-based strategy, we have detected the expression of nine different homeobox genes in adult mouse intestine. Included among these are the recently described intestine-specific Cdx-1 gene and a new, related gene, Cdx-2. Southern blot experiments show that Cdx-2 is present in a single copy in the mouse genome. Of several adult mouse tissues assayed, intestine was the only one that contained detectable levels of Cdx-2 mRNA. Expression of all nine homeobox genes in different regions of the intestine was quantitated by RNase protection analysis, which revealed a unique expression profile for each gene. These observations suggest that homeobox gene expression may play an important role in cellular differentiation in the adult intestine.

Amino Acid Sequence↗
PubMed1991

A high-resolution networked computer system for radiologic instruction of medical students.

A network of 18 Macintosh II-based workstations (Apple Computer Corp, Cupertino, CA) having high-resolution 1,024-line monitors is used for the radiologic instruction of medical students in our institution. Although this is an expensive system, it is time-shared with the Departments of Pathology, Physiology, Neuroscience, Biochemistry, Parasitology, and Microanatomy. The number of workstations available allows the entire class to access the system simultaneously by working in small groups. Because of its high resolution, medical imaging studies and text may be displayed on the same monitor side by side. We have chosen a question-and-answer format (Carousel). Forty-one radiologists in our affiliated medical institutions contributed cases. These cases in question-and-answer form supplement the lectures and film-based tutorials given as part of our mandatory third year radiology clerkship.

Computer Communication Networks↗
PubMed1991

2-(3,4-Dichlorophenyl)-N-methyl-N-[2-(1-pyrrolidinyl)-1-substituted- ethyl]-acetamides: the use of conformational analysis in the development of a novel series of potent opioid kappa agonists.

This paper describes the synthesis of a series of N-[2-(1-pyrrolidinyl)ethyl]acetamides (1), methylated at C1 and/or C2 of the ethyl linking group, and their biological evaluation as opioid kappa agonists. Conformational analysis of corresponding desaryl analogues 2 suggested that only those compounds capable of occupying an energy minimum close to that of the known kappa agonist N-[2-(1-pyrrolidinyl)cyclohexyl] acetamide U-50488 might possess kappa agonist properties. Starting from chiral amino acids, other alkyl and aryl substituents were introduced at C1 of the ethyl-linking moiety, giving compounds capable of adopting the same conformation as U-50488. The most potent of these, 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl] acetamide (8), was 146-fold more active than U-50488 in vitro in the mouse vas deferens model and exhibited potent naloxone-reversible analgesic effects (ED50 = 0.004 mg/kg sc) in an abdominal constriction model.

Acetamides↗
PubMed1991

Structure/activity studies related to 2-(3,4-dichlorophenyl)-N-methyl-N-[2-(1-pyrrolidinyl)-1-substituted- ethyl]acetamides: a novel series of potent and selective kappa-opioid agonists.

This paper describes the synthesis of a series of N-[2-(1-pyrrolidinyl)ethyl]acetamides 1, variously substituted at the carbon adjacent to the amide nitrogen (C1), and related analogues, together with their biological evaluation as opioid kappa agonists. In the first part of the study, the variants in N-acyl, N-alkyl, and amino functions were explored when the substituent at C1 was 1-methylethyl and the optimum was found to be exemplified by 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(1-methylethyl)-2- (1-pyrrolidinyl)ethyl]acetamide (13). Subsequently, racemic or chiral amino acids were used to introduce other alkyl and aryl substituents at C1 of the ethyl linking moiety. A series of potent compounds, bearing substituted-aryl groups at C1, were discovered, typified by 2-(3,4-dichloro-phenyl)-N-methyl-N-[(1R,S)-1-(3-aminophenyl)-2-(1- pyrrolidinyl)ethyl]acetamide (48), which was 5-fold more active as the racemate than 13 in vitro and exhibited potent naloxone-reversible analgesic effects (ED50 = 0.04 mg/kg sc) in a mouse abdominal constriction model.

Acetamides↗
PubMed1991

Cloning and characterization of the ColE7 plasmid.

The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.

Amino Acid Sequence↗
PubMed1991

Identification of a gene, closely linked to dnaK, which is required for high-temperature growth of Escherichia coli.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2.

Amino Acid Sequence↗
PubMed1991

Investigation of the specificity of the interaction between colicin E9 and its immunity protein by site-directed mutagenesis.

Comparison of the amino acid sequences of the C-terminal domain of three DNAase type E colicins has identified six candidate specificity determinants for the interaction of these E colicins with their homologous immunity proteins. We have changed these candidate specificity determinants of colicin E9, using site-directed mutagenesis, to the corresponding amino-acids of colicin E8. A 'mutant' colicin E9, in which four of the six candidate specificity determinants have been changed, demonstrated colicin activity against Escherichia coli indicator strains which carried either the E8imm or the E9imm genes, indicative of a 'novel' E. colicin. After changing all six of the candidate specificity determinants, the resulting colicin E9 'mutant' exhibited a phenotype very similar to that of colicin E8.

Amino Acid Sequence↗