Biomedical subjects
Publications and source records attributed to V K Brown.
In higher plants, fructose bisphosphate aldolase (EC 4.1.2.13) occurs in chloroplast, cytosol, and nucleus. Immunocytolocalization experiments with isozyme-directed antibodies indicate that both chloroplastic and cytosolic aldolase isoforms are present in the pea (Pisum sativum L.) leaf nucleus.
The arthropod species richness of pastures in three Azorean islands was used to examine the relationship between local and regional species richness over two years. Two groups of arthropods, spiders and sucking insects, representing two functionally different but common groups of pasture invertebrates were investigated. The local-regional species richness relationship was assessed over relatively fine scales: quadrats (= local scale) and within pastures (= regional scale). Mean plot species richness was used as a measure of local species richness (= alpha diversity) and regional species richness was estimated at the pasture level (= gamma diversity) with the 'first-order-Jackknife' estimator. Three related issues were addressed: (i). the role of estimated regional species richness and variables operating at the local scale (vegetation structure and diversity) in determining local species richness; (ii). quantification of the relative contributions of alpha and beta diversity to regional diversity using additive partitioning; and (iii). the occurrence of consistent patterns in different years by analysing independently between-year data. Species assemblages of spiders were saturated at the local scale (similar local species richness and increasing beta-diversity in richer regions) and were more dependent on vegetational structure than regional species richness. Sucking insect herbivores, by contrast, exhibited a linear relationship between local and regional species richness, consistent with the proportional sampling model. The patterns were consistent between years. These results imply that for spiders local processes are important, with assemblages in a particular patch being constrained by habitat structure. In contrast, for sucking insects, local processes may be insignificant in structuring communities.
Two different UK limestone grasslands were exposed to simulated climate change with the use of nonintrusive techniques to manipulate local climate over 5 years. Resistance to climate change, defined as the ability of a community to maintain its composition and biomass in response to environmental stress, could be explained by reference to the functional composition and successional status of the grasslands. The more fertile, early-successional grassland was much more responsive to climate change. Resistance could not be explained by the particular climates experienced by the two grasslands. Productive, disturbed landscapes created by modern human activity may prove more vulnerable to climate change than older, traditional landscapes.
BACKGROUND AND PURPOSE: Platelet glycoprotein IIb/IIa (GpIIb-IIIa), a membrane receptor for fibrinogen and von Willebrand factor, has been implicated in the pathogenesis of acute coronary syndromes but has not been previously investigated in relation to stroke in young adults. METHODS: We used a population-based case-control design to examine the association of the GpIIIa polymorphism P1A2 with stroke in young women. Subjects were 65 cerebral infarction cases (18 patients with and 47 without an identified probable etiology) 15 to 44 years of age from the Baltimore-Washington region and 122 controls frequency matched by age from the same geographic area. A face-to-face interview for vascular disease risk factors and a blood sample for the P1A2 allele and serum cholesterol were obtained from each participant. Logistic regression was used to estimate the odds ratio for one or more P1A2 alleles after adjustment for other risk factors. RESULTS: Among cases and controls, the prevalence rates of one or more P1A2 alleles were 21% and 22% among blacks and 36% and 28% among whites, respectively. This genotype was significantly associated with hypertension only in black control subjects but otherwise not with any of the established vascular risk factors. The adjusted odds ratio for cerebral infarction of one or more P1A2 alleles was 1.1 (confidence interval [CI], 0.6 to 2.3) overall, 0.5 (CI, 0.1 to 7.1) among blacks, and 1.4 (CI, 0.5 to 3.7) among whites. For the cases with an identified probable etiology, the corresponding odds ratios were 3.0 (CI, 0.9 to 10.4) overall, 0.7 (CI, 0.1 to 7.1) among blacks, and 12.8 (CI, 1.2 to 135.0) among whites. CONCLUSIONS: No association was found between the P1A2 polymorphism of GpIIIa and young women with stroke. However, subgroup analyses showed that the P1A2 polymorphism of GpIIIa appeared to be associated with stroke risk among white women, particularly those with a clinically identified probable etiology for their stroke. Further work with an emphasis on stroke subtypes and with multiracial populations is warranted.
Persons with a thermolabile form of the enzyme 5,10 methylenetetrahydrofolate reductase (MTHFR) have reduced enzyme activity and increased plasma homocysteine which can be lowered by supplemental folic acid. Thermolability of the enzyme has recently been shown to be caused by a common mutation (677C-->T) in the MTHFR gene. We studied 41 fibroblast cultures from NTD-affected fetuses and compared their genotypes with those of 109 blood specimens from individuals in the general population. 677C-->T homozygosity was associated with a 7.2 fold increased risk for NTDs (95% confidence interval: 1.8-30.3; p value: 0.001). These preliminary data suggest that the 677C-->T polymorphism of the MTHFR gene is a risk factor for spina bifida and anencephaly that may provide a partial biologic explanation for why folic acid prevents these types of NTD.
Signal transduction via the B cell antigen receptor complex is regulated by changes in tyrosine phosphorylation of several proteins. The equilibrium between tyrosine phosphorylation and dephosphorylation is regulated by the combined action of protein tyrosine kinase and protein tyrosine phosphatase enzymes. In particular, the protein tyrosine phosphatase, CD45, has been shown to play an essential role in signal transduction via the B cell antigen receptor. Therefore, experiments were performed to examine the intermolecular associations between CD45 and phosphotyrosine-containing proteins in the B cell to identify potential substrates for CD45. Based on coprecipitation experiments, CD45 was found to be physically associated with multiple components of the B cell antigen receptor complex including the MB-1/B29 heterodimer. Additionally, CD45 was selectively associated with the src family protein tyrosine kinase, lyn. Neither blk nor fyn were observed to interact with CD45 even though they have been implicated in antigen receptor signal transduction. This finding suggests that CD45 may preferentially regulate the phosphorylation of lyn and thus, its activity. In summary, these studies provide evidence to support the hypothesis that CD45 regulates antigen receptor-mediated signal transduction by controlling the tyrosine phosphorylation of multiple components of the antigen receptor complex.
Recent studies have demonstrated that the protein tyrosine phosphatase CD45 plays an integral role in regulation of B-cell function. Most notably, expression of this phosphatase is required for activation of B lymphocytes and entry into the cell cycle. Here, Louis Justement and colleagues review current information concerning the function of CD45 in the B cell. The discussion focuses on two questions that are of central importance: what are the physiological substrates for CD45 and how does reversible tyrosine phosphorylation affect their function?
Signal transduction via the B cell AgR complex has recently been shown to be dependent on the activation of one or more protein tyrosine kinases. Similarly, it has been found that signal transduction requires the expression of the protein tyrosine phosphatase CD45. Thus, transduction of a signal after AgR cross-linking must involve the coordinate interaction of these two enzymatic activities. It is therefore logical to hypothesize that the competence of the B cell to respond to ligands that bind the AgR may be dependent on the maintenance of an equilibrium between the tyrosine phosphorylation and dephosphorylation of specific signal transduction components. We have demonstrated in the present study that in resting B cells, the basal level of AgR complex tyrosine phosphorylation is regulated by cellular protein tyrosine phosphatases. Treatment of cells with the protein tyrosine phosphatase inhibitor, Na3VO4, resulted in rapid hyperphosphorylation of the receptor complex. Based on this observation, experiments were designed to examine the role of CD45 in regulation of AgR complex phosphorylation. Treatment of B cells with anti-CD45 mAb alone was found to have no effect on cytoskeletal association of CD45 or on its distribution within the membrane. Addition of a secondary cross-linking reagent, however, induced the association of CD45 with the cytoskeleton and caused capping. Subsequent studies demonstrated that increased tyrosine phosphorylation of the mIg-associated proteins MB-1 and B29 could be induced after incubating cells with anti-CD45 mAb and a secondary cross-linker, but not after the addition of anti-CD45 mAb alone. Changes in tyrosine phosphorylation of MB-1 and B29 were found to correlate with the cytoskeletal association of CD45. Interestingly, although cross-linking CD45 induced alterations in its association with the cytoskeleton and in its distribution within the membrane, no significant change in the level of protein tyrosine phosphatase activity could be detected under these conditions. These findings support the possibility that ligand binding to CD45 can induce biochemical and/or physical alterations in the molecule that presumably inhibit its ability to interact with specific substrates in the cell, thereby shifting the established equilibrium between tyrosine-specific phosphorylation and dephosphorylation.
Toxigenic strains of Clostridium difficile are causative agents of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis. The toxigenicity is routinely assayed by using highly sensitive cell cultures. We used a simple and rapid polymerase chain reaction (PCR) assay to differentiate toxigenic and nontoxigenic strains of C. difficile. Two sets of oligonucleotide primer pairs derived from nonrepeating sequences of the toxin A gene were used to amplify 546- and 252-bp DNA fragments. A primer pair derived from repeating sequences of the toxin A gene was used to amplify a 1,266-bp DNA product. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 35 cytotoxic strains of C. difficile tested generated the expected amplified DNA. In contrast, none of the 26 noncytotoxic strains tested gave positive results. Although the toxins of C. difficile have been demonstrated to cross-react serologically with the toxins of Clostridium sordellii, we did not detect any amplified DNA in two cytotoxic strains or seven noncytotoxic strains of C. sordellii. PCR was negative in all 30 strains of 20 other Clostridium species. Southern hybridization of HindIII-digested genomic DNA by use of subgenomic probes showed a single hybridization band in toxigenic strains but not in nontoxigenic strains. PCR appears to be a sensitive and specific assay for the rapid identification of toxigenic C. difficile. Nontoxigenic C. difficile appeared to lack the C. difficile toxin A gene.
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Iodination of highly purified hepatitis A (HAV) virus results in the selective labeling of two viral polypeptides, which are identified as the the VP 1 and VP 2 capsid polypeptides. Based upon the kinetics of labeling, the exposed region of VP 1 appears to be more accessible to iodination, although the ultimate proportion of label present within VP 1 and VP 2 is approximately equal. By utilizing iodinated whole virions, isolated VP 1, VP 2, and the tryptic digest derived from VP 1 and VP 2, binding by heterologous anti-160 S antibody indicated that a significant portion of the antibodies was directed against an epitope on VP 2 that was not affected by denaturation. Identification of the regions exposed for iodination on these two polypeptides was accomplished by tryptic digestion of the isolated polypeptides followed by characterization of the iodinated tryptic peptide by gel filtration and reverse-phase chromatography. The results indicate that tyrosine 100 on VP 2 and a large tryptic peptide composed of amino acids 222 through 260 on VP 1 which contains four tyrosine residues are two regions that are surface-exposed on these molecules.
The published sequence of hepatitis A virus (HAV), strain HAS-15, after 20-30 cell culture passages contains an 18 nucleotide deletion (Ovchinnikov et al., 1985) within the VP1 genome region. This results in a significant amino acid difference of the VP1 protein when this strain of HAV is compared with other published HAV sequences. Comparison of the polyacrylamide gel electrophoretic migration of HAS-15 HAV and two other strains of HAV revealed that the HAS-15 VP1 molecule migrated faster than the VP1 molecule of the other two strains. Enzymatic amplification of viral RNA derived from the original stool suspension and cell culture adapted HAS-15 using the polymerase chain reaction followed by hybridization analyses with selected synthetic oligonucleotide probes revealed that the original wild type virus did not contain the deletion. These results confirm that cell culture adapted HAS-15 contains an eighteen nucleotide deletion which apparently was selected during cell culture adaptation.
Approaches to cell culture propagation of hepatitis A virus (HAV) have used either acute infection by passage of infected cell lysates or supernatants into uninfected cells or the passage of persistently infected cells. The findings presented here demonstrate that the growth and recovery of purified virus from foetal rhesus monkey kidney (FRhK4) cells persistently infected with HAV isolate HAS-15 decreased over a 2 to 3 month period. In contrast, high multiplicity acute infection of FRhK4 cells with purified HAS-15 HAV resulted in degeneration of the cell monolayer 2 to 3 weeks later. Large scale propagation of acutely infected cells followed by traditional picornavirus purification procedures reproducibly yielded milligram amounts of purified virus.
The nucleotide sequence of the VP1 region of marmoset-attenuated hepatitis A virus (HAV), MS-1, was determined by incorporative dideoxynucleotide sequencing of the RNA obtained from purified, liver-derived virus. Comparison of this nucleotide sequence to those of four previously published isolates revealed that one of the isolates, HM-175, which was obtained from Australia and passed three times in marmosets, had a 8.5-11% nucleotide variability compared to the remaining four isolates which were isolated from North American sources. This nucleotide variability does not result in amino acid differences with the exception of two of the North American isolates, which were derived from tissue culture passage. These isolates have been shown to contain regions of variability generated by nucleotide insertions and/or deletions, while the remaining three isolates, including the Australian isolate, demonstrate limited amino acid differences within the VP1 molecule.
Twelve mineral oils, originating from naphthenic and paraffinic stocks and variously refined, were evaluated for their potential to induce cutaneous neoplasia in female CF1 mice. The oils were applied to the shorn dorsal skin for up to 78 weeks, using several different treatment regimes. The sole acid/earth refined naphthenic spindle oil was a moderately potent cutaneous carcinogen. By comparison, the 11 oils, processed by other refining routes, were less carcinogenic or non-carcinogenic to murine skin. Two of the 11 oils were weak cutaneous carcinogens viz, a naphthenic spindle oil refined only by mild hydrotreatment and a paraffinic spindle oil refined by mild solvent extraction and 'Ferrofining'. All 9 remaining oils had been solvent-extracted as part of the secondary refining process; none induced malignant tumours, although solitary benign tumours of the treated site were recorded after exposure to 3 oils. The cutaneous carcinogenic potential of the test oils did not correlate well with their potential to induce epidermal hyperplasia at the treated site. Consequently, hyperplasia caused after short term exposure is of little value for distinguishing between carcinogenic and non-carcinogenic oils.
By definition pesticides are biocidal products and this implies a probability that pesticides may be acutely toxic to species other than the designated target species. The ways in which pesticides are manufactured, formulated, packaged, distributed and used necessitates a potential for the exposure of non-target species although the technology exists to minimize adventitious exposure. The occurrence of deliberate exposure of non-target species due to the misuse of pesticides is known to happen. The array of predictive acute toxicity tests carried out on pesticides and involving the use of laboratory animals can be justified as providing data on which hazard assessment can be based. This paper addresses the justification and rationale of this statement.
Spraying or swabbing with a mixture of polyethylene glycol 300/industrial methylated spirits (PEG-300/IMS) (2:1 by volume) has been shown to substantially reduce mortality, systemic effects, and skin burns resulting from skin contamination by phenol, cumene hydroperoxide, or phenol/acetone cleavage product. The skin-damaging potentials of sodium hydroxide and sulfuric acid have also been investigated. PEG-300/IMS(2:1 by volume) mixture was found, in rats, to be slightly less effective than water as means of decontamination. The PEG-300/IMS mixture has been shown not to cause eye irritation, and so should not present a hazard where this mixture is used as a decontaminant spray.
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