Characterization of thrombin binding to alpha 2-macroglobulin

The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.3:1 (thrombin:alpha 2M). Measurements of the ability of alpha 2M to bind trypsin after prior reaction with thrombin indicated that thrombin binds rapidly at one site on alpha 2M, but occupies the second site with some difficulty. Intrinsic fluorescence studies of trypsin binding to alpha 2M at pH 5.0, 6.5, and 8.0 not only revealed striking differences in trypsin's behavior over this pH range, but also some similarities between the behavior of thrombin and trypsin not heretofore recognized. Structural studies, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure alpha 2M-125I-thrombin covalent complex formation, indicated that covalency reached a maximum at a mole ratio of approximately 1.5:1. At this ratio, only 1 mol of thrombin is bound covalently per mol of alpha 2M. These gel studies and those of proteolytic digests of denatured alpha 2M-125I-trypsin and alpha 2M-125I-thrombin complexes suggest that proteinases form covalent bonds with uncleaved alpha 2M subunits. The sum of our results is consistent with a mechanism of proteinase binding to alpha 2M in which the affinity of the proteinase for alpha 2M during an initial reversible interaction determines its binding ratio to the inhibitor.     

Numerical taxonomy of some non-saccharolytic and saccharolytic Bacteroides species

Various Bacteroides spp. were examined by physiological tests, presence of specific enzymes, antibiotic sensitivity, menaquinone composition and a few miscellaneous tests. The data matrix containing 58 strains and 55 unit characters was examined using Gower's similarity coefficients (SG) and included matching negative character states and multistate characters. The highly saccharolytic strains were separated from the less saccharolytic and non-fermentative strains at the 55% similarity level; while at the slightly higher level of 63% strains of Capnocytophaga (formerly Bact. ochraceus) were recovered as a compact phenon distinct from other saccharolytic species. The phenogram was divided into 6 clusters at 72% similarity level. Most of the 'Bact. fragilis group' of species clustered in one phenon while Bact. melaninogenicus ssp. melaninogenicus, Bact. bivius and a new species, Bact. denticola, formed another group. Another phenon comprised the saccharolytic non-pigmented species closely related to Bact. oralis such as Bact. buccalis and Bact. pentosaceus. The less saccharolytic strains of Bact. melaninogenicus ssp. intemedius and Bact. disiens were recovered in a distinct phenon. The low affinity (less than 55% similarity) between the two subspecies of Bact. melaninogenicus emphasised the need for reclassifying these taxa into separate species. The non-fermentative and very weakly saccharolytic strains formed good taxospecies. The separation of this cluster into three subclusters is in excellent agreement with chemotaxonomic data now available.     

The mutagenic potential of high flash aromatic naphtha

Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 aromatics, primarily isomers of ethyltoluene and trimethylbenzene), which is removed and used as a solvent — high-flash aromatic naphtha. A program was initiated to assess the toxicological properties of high-flash aromatic naphtha since there may be human exposure through inhalation or external body contact. The current study was conducted partly to assess the potential for mutagenic activity and also to assist in an assessment of carcinogenic potential. The specific tests utilized included the Salmonella/mammalian microsome mutagenicity assay, the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward mutation assay in CHO cells, in vitro chromosome aberration and sister chromatid exchange (SCE) assays in CHO cells, and an in vivo chromosome aberration assay in rat bone marrow.There was no evidence that high-flash aromatic naphtha was either a gene or chromosomal mutagen. Thus it is unlikely to be a genotoxic carcinogen.Abbreviations Brdu 5-Bromo-2-deoxyuridine - C9 Aromatic species with 9 carbons (i.e., ethyl toluene and trimethyl benzene isomers) - CE Cloning efficiency - CHO Chinese hamster embryo - CP Cyclophosphamide - DMSO Dimethyl sulfoxide - HGPRT Hypoxanthine-guanine phosphoribosyl transferase - HVAC Heating, Ventilation, Air Conditioning - 3MC 3 Methylcholanthrene - MMC Mitomycin C - MMS Methyl methanesulfonate - S9 S9 Mammalian microsomal enzyme activation mixture - SCE Sister chromatid exchange     

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Summary Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition,³⁵S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues usingin situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in ‘cement lines’. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results ofin situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites ofde novo bone formation. More moderate expression of BSP was observed in ‘older’ osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.     

Vaccination to prevent persistent viral infection.

Persistent virus infections are increasingly being recognized as a significant cause of human morbidity and mortality. To establish persistence, a virus must establish infection and evade eradication by the host immune response, in particular by cytotoxic T lymphocytes (CTL). We have studied a virus that establishes persistence in part by suppressing the CTL response of the infected host. The virus persists in many cell types, including lymphocytes and macrophages. We show that prior immunization with a vaccine designed to induce CTL (in the absence of antiviral antibody) confers complete protection against subsequent establishment of persistence in all tissues analyzed. The vaccine can be designed to express as few as 10 amino acids of a viral protein that comprise the CTL epitope. Further, two CTL epitopes for two discrete MHC haplotypes can be successfully used in a single vaccine that protects both strains of mice. Hence, a "string of CTL epitopes" (beads) concept for vaccination is feasible. Finally, the CTL vaccine provided protection against the establishment of persistence by an immunosuppressive virus.     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Seedling recruitment patterns in a Belizean mangrove forest: effects of establishment ability and physico-chemical factors

A field study was conducted to evaluate the relative importance of factors affecting seedling establishment and survival on a mangrove-dominated island in Belize. An examination of spatial patterns of seedling relative densities in relation to reproductive adults and physico-chemical conditions provided correlative information on factors affecting mangrove regeneration patterns. Distance from reproductive adults explained 89–94% of the variation in relative density of Rhizophora mangle seedlings, whereas availability of resources (light and NH₄) explained 73–80% of variation in Avicennia germinans seedling relative density. Just after dispersal (December), 89% of the variation in Laguncularia racemosa seedling relative density was attributable to distance from reproductive adults, but 7 months later (July) 74% of the variation was explained by intensity of flooding- and salinity-related stresses. Survivorship (after 2.5 years) of propagules and seedlings of R. mangle and A. germinans transplanted to zones of contrasting physico-chemical conditions demonstrated that: (1) mortality was highest during the establishment phase and major causes were failure to strand before viability was lost, consumption by predators and desiccation; and (2) after establishment, differences in sensitivity to physicochemical stress factors such as flooding (A. germinans) and initial orientation of the seedling axis (R. mangle) exerted a further influence on seedling survival. The results indicate that seedling recruitment in these neotropical forests is strongly influenced by dispersal patterns, differential establishment abilities and effects of physico-chemical factors that vary with elevation and distance from the shoreline.     

Faunal assemblage seriation of southern African Pliocene and Pleistocene fossil deposits

Fossil assemblages from the Pliocene and Pleistocene of southern Africa were seriated in order to give a better idea of their relative chronology. Time-sensitive mammals were selected for calculation of the Faunal Resemblance Index among 17 site units. On the basis of a logistical seriation and subsequent site analysis, the following sequence of sites was deemed most probable: Makapansgat Member 3, Makapansgat Member 4, Taung Dart deposits, Sterkfontein Member 4 and Taung Hrdli?ka deposits, Sterkfontein Member 5 (in part) and Kromdraai B, Kromdraai A and Swartkrans Member 1, Swartkrans Member 2, Swartkrans Member 3, Plovers Lake, Cornelia, Elandsfontein Main Site, Cave of Hearths Acheulian levels, Florisbad and Equus Cave and Klasies River Mouth. © 1995 Wiley-Liss, Inc.     

Modelling coccidial infection in chickens: emphasis on vaccination by in-feed delivery of oocysts.

Trickle immunization of poultry by incorporating wildtype strains of coccidia in the feed has been shown to be an effective and practical means of controlling coccidiosis. Here we develop a mathematical model of the life cycle of Eimeria tenella and estimate the model parameters from extant experimental studies. Numerical solutions of the model compare well with experiment. The model provides quantitative estimates of the required trickle immunization hitherto only available after extensive time-consuming trials.