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Biomedical subjects

R E Mitchell

Publications and source records attributed to R E Mitchell.

At least 19 recordsLinked to original sources
PubMed1995

Stabilisation of purified human collagenase by site-directed mutagenesis.

During purification, human fibroblast collagenase breaks down into two major forms, an N-terminal 22000/25000-Mr fragment and a C-terminal 27000-Mr fragment; the most likely mechanism being autolysis. The cleavage site has been identified (Pro269- Ile270) and in an attempt to obtain full-length human collagenase (i.e., Mr 42570), this cleavage site and another potential cleavage site (Ala258- Ile259) have been mutated by PCR- directed mutagenesis: Ile270Ser and Ile259Leu. The mutated cDNA was then cloned into the expression vector, pGEX2T, and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). After cleavage with factor Xa, the mutated collagenase was purified on a peptide hydroxamic acid affinity column. The mutated recombinant collagenase is stable, remains full length and retains the ability to cleave collagen.

Alanine↗
PubMed1995

Recombinant porcine collagenase: purification and autolysis.

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage. The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.

Amino Acid Sequence↗
PubMed1995

Achalasia. A new modality for treatment.

This lesion of the esophagus, first described in 1682 by Thomas Willis, been subject to many forms of therapy. We feel botulinum toxin injection to be an acceptable alternative treatment modality for select patients with primary esophageal achalasia. Traditional methods of treating achalasia consist of medical therapy for short-term relief, balloon dilation and myotomy. Botulinum toxin injection is an alternative method of treatment, suggested by Pasricha et al and used successfully in our patient, which does not seem to cause the significant complications of perforation or gastroesophageal reflux and which may be more attractive to patients less able to undergo dilation or myotomy. This method of injecting botulinum toxin directly into the LES appears to be a relatively safe modality of treatment. Reports suggest symptoms of achalasia may recur (in up to a year's time) and repeated injections may be needed. Even so, this would seem to be acceptable in the overall management of achalasia. We agree that long-term follow-up of these patients is indicated. Data to date, plus our personal experiences, have been encouraging. We feel this represents an option for non-surgical patients and may even be considered prior to the endoscopic balloon surgery approach. It is certainly more cost effective. We are currently evaluating a second patient for botulinum toxin therapy.

Aged↗
PubMed1995

Orthopedic injuries experienced by U.S. prisoners of war during Operation Desert Storm: a descriptive analysis.

U.S. prisoners of war from Operation Desert Storm suffered significant orthopedic injuries. The repatriated prisoners of war (RPOWs) have been medically evaluated over a 3-year period with orthopedic follow-up. A significant proportion of the musculoskeletal injuries were located around the neck and spine, shoulder, and upper extremity. Aircraft ejection was the cause of the majority of these injuries. Lower extremity injuries after ejection, aside from the knee, were not reported. Flail injuries of the lower extremities were absent as well. These results were examined with reference to Vietnam RPOW data.

Adult↗
PubMed1992

Physical and functional characterization of the gene cluster encoding the polyketide phytotoxin coronatine in Pseudomonas syringae pv. glycinea.

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The coronatine synthesis genes in PG4180 were previously shown to reside on a 90-kb plasmid designated p4180A. In the present study, clones containing a 34-kb region of p4180A were saturated with Tn5, and 71 unique mutations were recombined into p4180A by marker exchange. The effect of each mutation on coronatine synthesis was determined by analyzing the organic acids produced by the mutants by reverse-phase high-performance liquid chromatography. The organic acids of selected mutants were derivatized to their methyl esters and analyzed by gas chromatography and gas chromatography-mass spectrometry. Mutations in a 20.5-kb region of p4180A completely blocked the synthesis of coronafacic acid and coronatine. Mutations within a 4.4-kb region of p4180A prevented the formation of coronatine but allowed for production of coronafacic acid, coronafacoylvaline, coronafacoylisoleucine, and coronafacoylalloisoleucine. The phenotypes of selected mutants were further confirmed in feeding experiments in which coronafacic acid or coronamic acid was added to the culture media. The results of this study allow us to speculate on the likely sequence of steps in the later stages of coronatine biosynthesis.

Amino Acids↗
PubMed1991

Implications of toxins in the ecology and evolution of plant pathogenic microorganisms: bacteria.

This review attempts to rationalise what is known about bacterial phytotoxins and associate it with the ecology and possible evolution of the producing organisms. Study of non-toxin producing variants gives insight into the ecological role of the toxin. Elucidation of chemical structures of phytotoxins has shown that many exist as families of analogous compounds. Studies on the variation of chemical structures and how they are distributed across species and genera can lead to development of hypotheses on evolutionary relationships. Knowledge on biosynthetic pathways to toxins allows recognition of specific enzymatic steps involved in developing the characteristic features of the structures. Phytotoxins often have a potent biochemical activity, and in some cases the producing organism has associated mechanisms to prevent action of the toxin upon itself; in such cases toxigenesis is clearly not a chance event. The various aspects of bacterial toxigenesis indicate that bacterial phytotoxins are special secondary metabolic products that play beneficial roles to the producing organisms in their various ecological niches.

Amino Acid Sequence↗
PubMed1991

Comparative evaluation of treated bovine pericardium as a xenograft for hernia repair.

Two forms of bovine pericardium (BPC) were assessed as hernia repair materials: non-cross-linked (lyophilized) and cross-linked through treatment with glutaraldehyde (GA). These were compared with polypropylene mesh (Marlex) in a rabbit model. Over 52 wk implantation, the GA BPC grafts developed a strong, stable, fibrous tissue replacement with good incorporation into the abdominal muscle wall. The lyophilized BPC grafts were substantially resorbed within 12 wk of implantation, however the thin, fibrous replacement tissue was inadequate for abdominal wall support. Marlex grafts provided sufficient abdominal support, however these grafts were associated with extensive adhesion formation and, in this model, fat deposition around the perimeter of the graft. Control (ungrafted) rabbit abdominal muscle in the transverse orientation had an ultimate tensile load (UTL) of 11.4 +/- 5.1 N (x +/- s.d.) and a strain at UTL of 35 +/- 12% (n = 169). At 52 weeks the UTL of the repair sites was 7.3 +/- 4.5 N (n = 6), 5.1 +/- 3.5 N (n = 6) and 5.6 +/- 2.7 N (n = 6) for GA BPC, lypophilized BPC and Marlex grafts, respectively.

Animals↗
PubMed1989

Plasmid-mediated production of the phytotoxin coronatine in Pseudomonas syringae pv. tomato.

Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were previously generated by using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants either were missing pPT23A (29 mutants) or contained deletions in this plasmid (4 mutants). These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce coronatine. A bioassay for coronatine suggested that PS61(pPT23A) transconjugants were able to make this phytotoxin. In a chemical analysis, organic acids were isolated from PT23.2, PS61, and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The derivatized organic acids extracted from PT23.2 and PS61(pPT23A) contained peaks that corresponded to coronafacic acid, coronafacoylvaline, and coronatine, but these were absent in the extracts from the wild-type strain PS61. The identification of these components was confirmed by combined gas chromatography-mass spectrophotometry. Therefore, the acquisition of pPT23A by PS61 resulted in biosynthesis of coronafacic acid, coronafacoylvaline, and coronatine, clearly demonstrating the involvement of pPT23A in coronatine production in P. syringae pv. tomato.

Amino Acids↗
PubMed1986

Cardiovascular epidemiology, exercise, and health: 40-year followup of the U.S. Navy's "1000 aviators".

The interrelationship of aging, performance, and stress modification has been the subject of investigations in the U.S. Navy. Beginning in 1940, a study of 1,056 student and instructor pilots lowered previously high attrition rates in training by emphasizing both physical and psychological screening. After World War II, when 208 pilots in the group died, followup studies of the survivors were conducted in 1951, 1957, 1963, 1969-71, 1977, and 1980-81. In February 1981, 715 questionnaires were mailed to known survivors, with 500 replies subsequently analyzed. Additionally, 114 of the respondents who had previously been examined during 1969, were again examined in 1980-81; those individuals were markedly different in their lifestyle, particularly in exercising regularly, abstaining from cigarette smoking, and drinking alcoholic beverages moderately, as contrasted to 28 aviators also examined in 1969 who died in the interim. Healthy lifestyle may alter cardiovascular risk, preventing premature death.

Aerospace Medicine↗
PubMed1985

The inactivation of ornithine transcarbamoylase by N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine.

Phaseolotoxin, a tripeptide inhibitor of ornithine transcarbamoylase, is a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, the causal agent of halo-blight in beans. In vivo the toxin is cleaved to release N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine, the major toxic chemical species present in diseased leaf tissue. This paper reports on the interaction between N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine and ornithine transcarbamoylase. N delta-(N'-Sulpho-diaminophosphinyl)-L-ornithine was found to be a potent inactivator of the enzyme, in contrast with phaseolotoxin, which previously has been reported to inhibit the enzyme reversibly. Inactivation by N delta-(N'-[35S]sulpho-diaminophosphinyl)-L-ornithine resulted in the incorporation of 35S into ethanol-precipitated protein. The stoicheiometry of 35S incorporation was approximately 1 mol/mol of active sites. Inactivation was second-order and a rate constant of 10(6) M-1 X s-1 at 0 degree C in 50 mM-Tris/HCl, pH 9.0, was obtained. Carbamoyl phosphate, a substrate of ornithine transcarbamoylase, protected the enzyme from inactivation. A dissociation constant of 3 microM for the enzyme-carbamoyl phosphate complex was calculated. L-Ornithine, the second substrate for ornithine transcarbamoylase, protected the enzyme only at high concentrations. The results are consistent with N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine being a potent affinity label that binds via the carbamoyl phosphate-binding site of ornithine transcarbamoylase. Cleavage of phaseolotoxin to N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine in vivo appears to be an important function in the physiology of the disease.

Binding Sites↗
PubMed1984

Prediction of subsequent ischemic heart disease using serial resting electrocardiograms.

To test the hypothesis that subtle changes in the resting electrocardiogram can have predictive value for subsequent cardiovascular disease, the authors evaluated serial electrocardiograms from a cohort of initially healthy men and related these to later development of ischemic heart disease. The cohort of 1056 men originally physically qualified for naval aviation were followed from 24-61 years of age between 1940 and 1977. Resting electrocardiograms were obtained at 24, 36, 42, and 54 years of age. Particular emphasis was directed to measurement of electrocardiographic intervals, amplitudes, and vectorial orientation. The potential effects of weight, blood pressure, and other major risk factors on electrocardiographic changes were controlled in analysis. At younger ages, the amplitude of the T wave in lead 2 and the change in P-R interval were predictive of cardiovascular disease. With aging of the cohort, more leftward orientation of the frontal plane QRS and T forces were predictive, as were the serial changes in the angle between QRS and T. When major risk factors (smoking, blood pressures, and serum cholesterol) were included in a predictive model, addition of electrocardiographic changes significantly improved prediction of ischemic heart disease. These subtle serial changes may be helpful to epidemiologists and clinical investigators in assessing risk of subsequent cardiovascular disease.

Adult↗
PubMed1984

Herbal medicine.

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Europe↗