Detection of functional differences between different platelet membrane glycoprotein Ibalpha variable number tandem repeat and Kozak genotypes as shown by the PFA-100 system.
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Aspirin↗
Biomedical subjects
H Douglas
Publications and source records attributed to H Douglas.
Revision surgery for nonunion after early failure of fixation of fractures of the distal humerus.
Sixteen patients who underwent a revision operation for nonunion of fractures of the distal humerus following previous internal fixation were reviewed at a mean follow-up of 39 months (8 to 69). The Mayo elbow performance score was excellent in 11, good in two, fair in two and poor in one. In 15 patients union was achieved and in one with an infected nonunion a subsequent bone graft was necessary in order to obtain union.Age, gender, a history of smoking, mechanism of the injury and the AO classification of the initial fracture did not correlate with the development of nonunion. In 12 patients (75%), the initial fixation was assessed as being suboptimal. The primary surgery was regarded as adequate in only three patients. Our findings suggest that the most important determinant of nonunion of a distal humeral fracture after surgery is the adequacy of fixation.
Adult↗
Conserved glycines in the C terminus of MinC proteins are implicated in their functionality as cell division inhibitors.
Alignment of 36 MinC sequences revealed four completely conserved C-terminal glycines. As MinC inhibits cytokinesis in Neisseria gonorrhoeae and Escherichia coli, the functional importance of these glycines in N. gonorrhoeae MinC (MinC(Ng)) and E. coli MinC (MinC(Ec)) was investigated through amino acid substitution by using site-directed mutagenesis. Each mutant was evaluated for its ability to arrest cell division and to interact with itself and MinD. In contrast to overexpression of wild-type MinC, overexpression of mutant proteins in E. coli did not induce filamentation, indicating that they lost functionality. Yeast two-hybrid studies showed that MinC(Ec) interacts with itself and MinD(Ec); however, no interactions involving MinC(Ng) were detected. Therefore, a recombinant MinC protein, with the N terminus of MinC(Ec) and the C terminus of MinC(Ng), was designed to test for a MinC(Ng)-MinD(Ng) interaction. Each MinC mutant interacted with either MinC or MinD but not both, indicating the specificity of glycine residues for particular protein-protein interactions. Each glycine was mapped on the C-terminal surfaces (A, B, and C) of the solved Thermotoga maritima MinC structure. We found that MinC(Ec) G161, residing in close proximity to the A surface, is involved in homodimerization, which is essential for MinC function. Glycines corresponding to MinC(Ec) G135, G154, and G171, located within or adjacent to the B-C surface junction, are critical for MinC-MinD interactions. Circular dichroism revealed no gross structural perturbations of the mutant proteins, although the contribution of glycines to protein flexibility and stability cannot be discounted. Using molecular modeling, we propose that exposed conserved MinC glycines interact with exposed residues of the alpha-7 helix of MinD.
Amino Acid Sequence↗
Coronary angioplasty enhances platelet reactivity through von Willebrand factor release.
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Adult↗
Platelet membrane glycoprotein Ibalpha gene -5T/C Kozak sequence polymorphism as an independent risk factor for the occurrence of coronary thrombosis.
OBJECTIVE: To explore the potential of the GPIbalpha gene variable number tandem repeat (VNTR) and -5T/C Kozak polymorphisms to act as independent risk factors for myocardial infarction. METHODS: 256 patients aged 33-80 years (180 caucasian, 76 Indian Asian) were recruited at cardiac catheterisation for any diagnostic indication, and divided into two groups: group A, with confirmed previous myocardial infarction evident on ECG or ventriculogram (88 patients, 79 men, 9 women) and group B, with no evidence of myocardial infarction (168 patients, 101 men, 67 women). RESULTS: There was no significant difference in race, age, hypertension, smoking status, or family history between the infarct and non-infarct groups, though there was a significant difference in sex (89.8% male in group A, 60.1% male in group B, p < 0.001). Genotype analysis showed a strong association between the GPIbalpha Kozak homozygous TT genotype and the occurrence of myocardial infarction (group A: TT 85.2%, TC 12.5%, CC 2.3%; group B: TT 67.3%, TC 32.7%, p = 0.001). No significant association was found between myocardial infarction and the GPIbalpha VNTR, although analysis of the CC VNTR genotype against all other GPIbalpha VNTR genotypes showed a marginal association with myocardial infarction (p = 0.059). There was no association between the Kozak sequence polymorphism (p = 0.797) or GPIbalpha VNTR (p = 0.714) and the degree of vessel disease. CONCLUSIONS: The homozygous TT Kozak genotype may be a significant factor in the outcome of coronary artery disease completed by myocardial infarction. Conversely, the Kozak C allele in the heterozygous state TC may confer some protection against myocardial infarction.
Adult↗
A mixture of alfentanil and morphine for rapid postoperative loading with opioid: theoretical basis and initial clinical investigation.
Pharmacokinetic modelling of estimated central nervous system concentrations was used to devise the optimal mixture of morphine and alfentanil for the treatment of postoperative pain. Modelling revealed that an intravenous opioid pain protocol using an alfentanil-morphine mixture in the proportions 0.75 : 10 mg would provide a profile of analgesia of rapid onset, yet slow offset. The regimen was evaluated in 58 patients in the recovery ward who were randomly allocated to receive analgesia using pain protocols with either morphine or the mixture. Groups were well matched for age, weight and initial pain scores. The mean (SD) time to patient comfort was 27.6 (20.2) min for the mixture and 41.2 (18.6) min for morphine (p = 0.01). Multiple regression analysis revealed that that initial pain score (p = 0.009) and drug group (p = 0.02), but not age, weight or gender were independent predictors of the time to comfort. Drug group was not a significant predictor of adverse effects.
Adult↗
Mechanisms involved in the cardiovascular responses to opioid products of proenkephalin in the anaesthetised rat.
1. Cardiovascular effects of opioid peptide products of proenkephalin, [Met] enkephalin (ME), [Leu] enkephalin (LE), [Met] enkephalyl Arg6-Phe7 (MEAP) and [Met] enkephalyl Arg6-Gly7-Leu8 (MEAGL) have been studied in urethane-anaesthetised rats. 2. ME, LE, MEAP and MEAGL produced vasodepression and bradycardia mediated by mu-opioid receptors. 3. Atypical responses to MEAP were observed in a quarter of the animals studied showing tachycardia and pressor effects. This response was probably due to the release of the dipeptide Arg-Phe which exerted its effects at sympathetic ganglia. 4. Studies with the peptidase inhibitors captopril and bestatin showed a differential potentiation of the cardiovascular effects of the proenkephalin products by inhibition of angiotensin converting enzyme and aminopeptidase. 5. The effects of vagotomy, pithing and studies with atropine, and N-methyl levallorphan were used to demonstrate that, for all four proenkephalin peptides, cardiovascular effects were mediated by peripheral opioid receptors and transmission to the CNS via vagal afferents.
Anesthesia↗
Fibrin-enmeshed tobramycin liposomes: single application topical therapy of Pseudomonas keratitis.
Treatment of bacterial keratitis requires frequent application of topical antibiotics. We studied the efficacy of a single topical administration of tobramycin incorporated in large multivesicular liposomes and enmeshed in a fibrin sealant on rabbit corneas infected with Pseudomonas aeruginosa. One cornea each of 25 New Zealand albino rabbits was infected with P. aeruginosa. Twenty-four hours later, the animals were randomly divided into five groups of five. Group A received single hourly drops (50 microliters) of fortified tobramycin (14.5 mg/ml, total of 17.4 mg). Group B received a single topical application of 3.5 mg tobramycin, in 0.1 ml multivesicular liposomes, enmeshed in a fibrin sealant with an overlaying bandage contact lens. Group C was treated in the same manner as group B without the addition of fibrin sealant. Groups D and E served as nondrug-treated controls, with group D receiving topical fibrin-enmeshed liposomes devoid of tobramycin and group E receiving hourly topical balanced salt solution (BSS) drops. All animals were killed 24 h after initiation of therapy. Significantly fewer colonies of Pseudomonas were present in corneas of all three treated groups, as compared with the two nondrug-treated control groups (p less than 0.02). There were significantly fewer colonies of Pseudomonas in groups A and B as compared with group C (p less than 0.02). No significant difference was noted between a single administration of topical fibrinen-meshed tobramycin-encapsulated liposomes (group B) and 24 doses of hourly fortified topical tobramycin (group A, p greater than 0.05). Tobramycin-encapsulated megaliposomes may serve as a useful adjunct in treatment of Pseudomonas keratitis.
Administration, Topical↗
Non-opioid effects of N-methyl levallorphan in the anaesthetised rat.
1. The effects of the quaternary opioid antagonist N-methyl levallorphan upon cardiovascular responses to non-opioid agonists has been studied in the urethane-anaesthetised rat. 2. N-methyl levallorphan showed an initial nicotinic agonist effect followed by pronounced ganglion blocking activity after intravenous administration. These effects are observed at doses only 2-fold higher than those required to block cardiovascular responses to [Met] enkephalin. 3. The narrow selectivity of N-methyl levallorphan makes this compound inappropriate for opioid antagonist studies where nicotinic receptors may be involved.
Acetylcholine↗
Late referral for biliary atresia.
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Age Factors↗
Identification and localization of cyst-specific antigens of Giardia lamblia.
We induced Giardia lamblia trophozoites to encyst in vitro by exposure to conditions which are specific to the human small intestinal milieu. We now show that encystation entails the appearance of two new groups of antigens detected in Western blots by rabbit antiserum against cysts which had been purified from human feces. A heterodisperse group of lower-molecular-mass antigens (approximately 21 to 39 kilodaltons) was expressed relatively early (less than 19 h) in encystation. In contrast, discrete bands at approximately 66, 78, 92, and 103 kilodaltons only appeared after 24 h of incubation under conditions which lead to production of large numbers of water-resistant cysts. We also describe for the first time the appearance of prominent cytoplasmic vesicles, which were the earliest morphologic change in encysting trophozoites observable by light microscopy. Early in encystation, cyst wall antigens were concentrated in these vesicles, as shown by immunocytochemistry, suggesting that the vesicles function in export of cyst wall constituents.
Animals↗
Production of a heat stable enterotoxin by Plesiomonas shigelloides.
Eight strains of Plesiomonas shigelloides were assayed for enterotoxin production in the rabbit ileal loop assay. Seven strains required serial in vivo passage in the rabbit's intestine before enterotoxin activity was detected in the cells' filtrate. Enterotoxin production was readily lost with subculture of these toxinogenic cells. Heat treatment of the cells' filtrate from three strains that had never been passed in vivo led to detectable enterotoxin activity in three of six separate assays. Using LT, CT, STIa, STIb and STII as probes, no homology to the whole cell DNA of the eight strains was detected on Southern hybridization under low stringency conditions. The enterotoxin of P. shigelloides appears to be novel since production is induced by in vivo passage, activity is seen with heat treatment of cells' filtrate and there is no DNA homology to the cloned enterotoxin genes of Escherichia coli and Vibrio cholerae.
Animals↗
Effects of proenkephalin products on rat cardiac and vascular tissue in-vitro.
The cardiovascular effects of the four proenkephalin products [Met]-enkephalin (ME), [Leu]-enkephalin (LE), [Met]-enkephalyl-arg6phe7 (MEAP) and [Met]-enkephalyl-arg6-gly7-leu8 (MEAGL) have been studied on the isolated spontaneously beating rat atria and the perfused rat mesentery in-vitro. All four peptides (at concentrations up to 10(-6)M and in the presence of peptidase inhibitors) had no direct effect on atrial rate or contractility and did not alter responses to noradrenaline or field stimulation. In addition, the peptides had no effect on the perfusion pressure of the mesentery and did not alter vasoconstrictor responses to noradrenaline. The results show that proenkephalin products are without direct or modulating effects on atrial muscle or mesenteric vasculature of the rat, and suggest that there is no endogenous opioid control in these tissues.
Animals↗
Encystation and expression of cyst antigens by Giardia lamblia in vitro.
The cyst form of Giardia lamblia is responsible for transmission of giardiasis, a common waterborne intestinal disease. In these studies, encystation of Giardia lamblia in vitro was demonstrated by morphologic, immunologic, and biochemical criteria. In the suckling mouse model, the jejunum was shown to be a major site of encystation of the parasite. Small intestinal factors were therefore tested as stimuli of encystation. An antiserum that reacted with cysts, but not with cultured trophozoites was raised in rabbits and used as a sensitive probe for differentiation in vitro. Cultured trophozoites that were exposed to bile salts showed a more than 20-fold increase in the number of oval, refractile cells that reacted strongly with anticyst antibodies, and in the expression of major cyst antigens. Exposure to primary bile salts resulted in higher levels of encystation than exposure to secondary bile salts. These studies will aid in understanding the differentiation of an important protozoan pathogen.
Animals↗
A new method for purification of Giardia lamblia cysts.
One of the major problems in studying Giardia cysts is separating this form of the parasite from faecal debris and bacteria. We approached this problem by passing filtered stool suspensions over a column of Sephadex G-50 followed by low speed centrifugations. The Sephadex retained most of the faecal debris and the centrifugation reduced the bacterial flora more than 10(5)-fold to less than 3 X 10(4)/ml. Cyst recovery was 61% (+/- 28%). This new method of separating G. lamblia from faecal material is simple, rapid, and effective.
Centrifugation↗
In vitro susceptibility of fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and opportunism.
Pathogenic fungi, according to their propensity to cause infection of apparently normal individuals, can be grouped into either primary pathogens (e.g., Coccidioides, Histoplasma, Paracoccidioides, Blastomyces, and Sporothrix) or opportunists (e.g., Candida, Mucoraceae, Aspergillus spp., Petriellidium, and Trichosporon). There is, however, no unifying concept explaining the difference between the virulence of the two fungal categories. Previously we have speculated that neutrophils are the common denominator of the high natural resistance to opportunistic fungi. Accordingly, we then compared the susceptibility to killing by neutrophil granulocytes of Histoplasma, Blastomyces, Paracoccidioides, and Sporothrix with that of 14 opportunistic fungi. We found the four virulent dimorphic yeasts, in contrast to opportunistic fungi, to be resistant to killing by neutrophils. Virulent dimorphic yeasts were ingested by neutrophils, and triggered a respiratory burst comparably to opportunists but were less susceptible to hydrogen peroxide, suggesting that differences in the susceptibility to microbicidal products of leukocytes may explain the difference in virulence.
Blastomycosis↗
Growth and antigenic variation of Trypanosoma brucei, T. rhodesiense and T. gambiense in subcutaneous millipore chambers.
The inability to cultivate infective bloodstream forms of the African trypanosomes in cell-free media has complicated studies of the biology of trypanosomes and the pathogenesis of trypanosomiasis. We attempted to overcome this problem by subcutaneous implantation in mice of Millipore chambers that isolate trypanosomes from cells but permit diffusion of soluble substances across their membranes. Chambers were inoculated with 5 X 10(4) to 5 X 10(5) per ml Trypanosoma brucei, T. rhodesiense or T. gambiense; the trypanosomes multiplied rapidly, persisted for as long as five weeks, and remained infective, even when the original inocula were freed of donor cells by ion-exchange. The presence of anti-trypanosomal IgG and IgM in the sera and chambers of recipient mice proved that trypanosomal and mammalian products crossed the membranes. Chamber trypanosomes also expressed two important aspects of normal in vivo biological behaviour: (i) differentiation from long slender to short stumpy bloodstream forms and (ii) antigenic variation. Death of trypanosomes was associated with the presence of IgM antibody in the chambers. This model provides a system for study of an entire population of trypanosomes in an extravascular, cell-free environment.
Animals↗
Protection against gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies.
Hybridomas producing human monoclonal IgM antibodies (mAbs) against bacterial lipopolysaccharide (LPS) were generated by fusion of B lymphocytes from sensitized human spleen with heteromyeloma cells. The splenocytes were from patients undergoing splenectomy during staging for Hodgkin disease after vaccination with the J5 mutant of Escherichia coli, which is deficient in O antigenic side chains. This deficiency exposes the core oligosaccharide, common to LPS of all Gram-negative bacteria. The mAbs cross-reacted strongly with endotoxins from a wide range of unrelated species of Gram-negative bacteria. The mAbs also gave strong protection against LPS in the dermal Shwartzman reaction and against lethal Gram-negative bacteremia in mice. These findings indicate that monoclonal IgM against LPS endotoxin can neutralize its toxicity in vivo and might be valuable for treatment of patients with Gram-negative bacteremia. Analysis of one of the hybridoma clones, A6(H4C5), showed that the IgM mAb is directed against the covalently bound lipid A, which represents the most conservative and least variable structural element of LPS.
Animals↗