Heat inactivation of catalase from Staphylococcus aureus MF-31.

The effects of heat on catalase from Staphylococcus aureus lysates were examined. Catalase activity increased with increasing concentrations of potassium phosphate buffer, when heated at temperatures between 50 and 65 degrees C for 10 min. Inactivation of catalase by NaCl during heating was demonstrated. Extended heating of S. aureus cells at 52 degrees C resulted in a slight decrease in catalase activity of the resultant lysates. This decrease was more pronounced in the presence of salt. Heating at 62 degrees C caused a decrease in catalase activity, but not complete inactivation. These results implicate the combined effects of heat, and NaCl in the inactivation of catalase from S. aureus. The findings are consistent with the hypothesis that H2O2 may accumulate as a result of decreased catalase activity and be responsible for the decreased colony-forming ability of stressed S. aureus.     

Usefulness of the stomacher in a microbiological regulatory laboratory.

The relative efficiency of the Waring blender, the Stomacher 400, and the Stomacher 3500 for preparing food samples for microbiological analysis was studied. Comparative aerobic plate count (APC) values were determined on 671 samples, representing 30 categories of foods. Of the 26 categories of nonfatty foods, the blender gave significantly higher geometric mean APC values than those given by the Stomacher 400 and the Stomacher 3500 in 65 and 69 percent of the categories, respectively. In a comparison of the two Stomacher models, the Stomacher 400 gave significantly higher geometric mean APC values than these given by the Stomacher 3500 in 73 percent of the food categories. Addition of Tween 80 to four categories of fatty foods at concentrations of 0.5, 1.0, and 2.0 percent did not raise the APC values given by either model of stomacher to the levels given by the Waring blender. Overall, the efficiency of both models of Stomacher, relative to the blender and to each other, was specific and depended upon the particular food being analyzed.     

The chemical nature of osmium tetroxide fixation and staining of membranes by x-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy was used to determine the oxidation states of osmium compounds present in erythrocyte ghost preparations and related systems treated with osmium tetroxide. Osmium tetroxide and cholesterol, codeposited at -100 degrees C, began to react at -70 degrees C, and Os(VI) was formed. Similarly, Os(VI) was detected for the known cholesterol-osmate ester prepared and purified chemically. However, osmium tetroxide applied in phosphate buffer (pH 7.2) gave rise to large proportions of Os(IV) and Os(III) species in addition to Os(VI) compounds. Egg phosphatidylcholine likewise produced a mixture of Os(VI), Os(IV), and Os(III), but dipalmitoyl phosphatidylcholine failed to give significant amounts of osmium containing products under identical conditions. Glutaraldehyde gave a mixture of compounds with the same osmium oxidation states when allowed to react with aqueous osmium tetroxide. Unfixed and glutaraldehyde-fixed erythrocyte ghosts also produced mixtures of Ss(VI), Os(IV) and Os(III) under conditions identical to those of normal tissue processing. Additionally, the mixture of adducts initially formed by treatment with osmium tetroxide was further reduced by dehydration of the tissue with ethanol, rpesulting in a final mixture which was 50-60% Os(III). The results support a scheme for the reaction os osmium tetroxide with tissues in which the initial reaction site is the double bonds of unsaturated lipids to form Os(VI) derivatives. Subsequent hydrolysis and further reduction yield complexes of Os(IV) and Os(III). A mixture of these three states is present in membrane specimens during microscopic observation. Os(VI) and Os(IV) could be present as osmate esters and osmium dioxide, respectively; Os(III) could be present as an oxo- or amino complex(es). The photoelectron spectrum of intact erythrocyte ghosts can be synthesized from the spectra of phospholipid and cholesterol only, suggesting the predominance of the reaction with lipids in the fixation process.     

An ultrastructural study of pyrenoids inLeptosiropsis torulosa

Summary The ultrastructure of the mature pyrenoid of the chaetophoracean algaLeptosiropsis torulosa is unlike any described for the green algae. The nucleus is surrounded by the pyrenoid, with only the endoplasmic reticulum interrupting the encircling pyrenoid. The pyrenoid possesses only the appressed plastid membranes that are in contact with the nucleus, with no traversing thylakoids present.     

Accurate measurement of microsatellite length by disrupting its tandem repeat structure

Tandem repeats of simple sequence motifs, also known as microsatellites, are abundant in the genome. Because their repeat structure makes replication error-prone, variant microsatellite lengths are often generated during germline and other somatic expansions. As such, microsatellite length variations can serve as markers for cancer. However, accurate error-free measurement of microsatellite lengths is difficult with current methods precisely because of this high error rate during amplification. We have solved this problem by using partial mutagenesis to disrupt enough of the repeat structure of initial templates so that their sequence lengths replicate faithfully. In this work, we use bisulfite mutagenesis to convert a C to a U, later read as T. Compared to untreated templates, we achieve three orders of magnitude reduction in the error rate per round of replication. By requiring agreement from two independent first copies of an initial template, we reach error rates below one in a million. We apply this method to a thousand microsatellite loci from the human genome, revealing microsatellite length distributions not observable without mutagenesis.     

Glycolipid core structure switching from globo- to lacto- and ganglio-series during retinoic acid-induced differentiation of TERA-2-derived human embryonal carcinoma cells

We have analyzed the glycolipid markers of a recently cloned human embryonal carcinoma (EC) cell line, NTERA-2, which differentiates extensively into a variety of somatic cell types when exposed to retinoic acid. These tumor cells provide a model system that can be used to study the ontogeny of glycolipid diversity during human embryonic development. Glycolipid antigens were identified by cell surface immunofluorescence and thin-layer chromatography immunostaining using a comprehensive set of anticarbohydrate monoclonal antibodies. Undifferentiated NTERA-2 cells were found to express predominantly globo-series glycolipids, including Gb3, Gb5 (IV3GalGb4), globo-ganglioside (IV3NeuAc alpha 2----3GalGb4), globo-H (IV3Fuc alpha 1----2GalGb4), and globo-A (IV3GalNAc alpha 1----3[Fuc alpha 1----2]GalGb4). When NTERA-2 cells were induced to differentiate by culturing in the presence of 10(-5) M retinoic acid, a remarkable shift of cellular glycolipids from globo-series to lacto- and ganglio-series was observed: Globo-series structures declined, particularly during the period 7-20 days after first exposure to retinoic acid, while lacto-series structures, including fucosyl alpha 1----3 type 2 chain (Lex) and sialosyl type 2 chain, and ganglio-series structures, including GM3, GD3, 9-O-acetyl-GD3, GM2, GD2, and GT3, increased. The presence of globo-A and globo-H as the major ABH blood group antigens in undifferentiated NTERA-2 cells suggests that globo-series blood group antigens are embryonic antigens, synthesis of which switches to lacto-series during human development. Two-color immunofluorescence analysis indicated preferential expression of several ganglio- and lacto-series antigens on different subsets of differentiated cells and permitted the relationship of these subsets to the development of neurons in NTERA-2 cultures to be determined. The results suggest that glycosyltransferase, particularly those involved in controlling glycoconjugate core structure assembly, are key enzymes regulated during the differentiation of human EC cells and, by implication, during human embryogenesis.     

Design and testing for a nontagged F1-V fusion protein as vaccine antigen against bubonic and pneumonic plague

A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.     

Evidence that Early Carboniferous ostracods colonised coastal flood plain brackish water environments

A study of the stable isotope composition (δ¹⁸O, δ¹³C) of biogenic (ostracod, mollusc) and authigenic carbonates in the Ballagan Formation, Lower Carboniferous of Scotland, coupled with evidence from sedimentology and associated fossil fauna and flora, supports the argument that this formation was deposited in a coastal flood plain setting, in brackish (0.5 < 30‰ NaCl) and hypersaline (> 40‰ NaCl) waters, but in the absence of persistent normal marine conditions. The oxygen isotope data from the Ballagan Formation divide into three clusters: a diagenetic field defined by low δ¹⁸O (< − 11‰ VPDB); an intermediary field (δ¹⁸O − 11‰ to − 9‰) composed of a mixture of known primary and secondary (diagenetic) carbonates; and samples within the range of − 9‰ to − 4‰ which, as far as we can ascertain, are largely unaltered. No samples give typical Early Carboniferous δ¹⁸O marine values. Average marine carbonates from Europe have δ¹⁸O between − 4‰ to − 3‰. The Ballagan Formation carbonates were probably deposited in evaporated freshwater and/or brackish water. This conclusion is supported by the presence of evaporites (gypsum, anhydrite, halite pseudomorphs) and common desiccation-cracked mudstone surfaces throughout the Ballagan Formation, suggesting conditions of fluctuating salinity in ephemeral bodies of water. The stable isotope data support the notion that the ostracod assemblages of the Ballagan Formation were colonising brackish water and hypersaline ecologies on a coastal flood plain during the Early Carboniferous, a stage of development that may have encouraged their colonisation of fully non-marine (limnetic) environments during the later Carboniferous. The ostracods include cytherellacean and kloedenellacean species known from marginal marine sites elsewhere, but probably tolerant of brackish water, podocopid species such as ‘Bythocypris’ aequalis that may have been adapted for brackish water settings on coastal flood plains (ephemeral lakes and lagoons), and paraparchitacean-dominated assemblages that may signal harsh (hypersaline or desiccating) environments.