Laboratory studies on salmonella-contaminated cheese involved in a major outbreak of gastroenteritis

Dried biomass of Streptomyces cyaneus NCIB 9616 harvested at 1, 3, 7, 10 and 14 d from a liquid culture was degraded by alkaline methanolysis and the fatty acid methyl esters released examined quantitatively by gas chromatography. Isoprenoid quinones were extracted with petroleum ether and methanolic saline, purified by thin-layer chromatography and quantified by high performance liquid chromatography. Data for each component were plotted against time. The results showed that at the beginning of logarithmic growth there was a small quantitative shift in the fatty acid composition but, the fatty acid profiles remained relatively constant during both the logarithmic and stationary phases of growth. In contrast, the isoprenoid quinone composition depended markedly on the stage of growth. These results indicate that valid comparison of isoprenoid quinone profiles requires that sampling take place at defined stages of the growth cycle.     

Steady state terrestrial ecosystems and the global carbon cycle

The assumption that landscapes dominated by mature vegetation are presently in carbon steady state with the atmosphere is challenged. Evidence suggests that the vegetation and soils of these landscapes are frequently disturbed and over short time periods (<300 yr) slowly sequester atmospheric carbon. The critical consideration in this argument is the time interval used to evaluate a steady state. Current models of carbon flux through the terrestrial biota limit their time considerations to 120 yr, a short and inadequate time interval for realistic assumptions about steady state in the carbon cycle of vegetation.Research performed under subcontract 19B-07762C with S. Brown and 19X-43326C with the Center for Energy and Environment Research of the University of Puerto Rico (A. E. Lugo) under Martin Marietta Energy Systems, Inc., contract DE-AC05-840R21400 with the U.S. Department of Energy.     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides

The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (ca. 500 000 daltons), highly phosphorylated protein containing multiple copies of filaggrin (26 000 daltons). The conversion of profilaggrin to filaggrin late in epidermal cell differentiation involves dephosphorylation and proteolysis to yield the unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight in the nature of these processes, we compared tryptic digests of profilaggrin with those of filaggrin by reverse-phase liquid chromatography. Approximately 80% of the profilaggrin mass consists of multiple copies of filaggrin. Twenty peptides purified in good yield from both profilaggrin and filaggrin accounted for most of the filaggrin sequence. A detailed analysis of the yield of several peptides provided an estimate of the size and frequency of the repeat unit within profilaggrin. These data indicate that the repeating substructure of profilaggrin contains about 265 amino acids and that about 50 residues are removed per filaggrin domain as the precursor is processed to filaggrin. Assuming a molecular weight of 500 000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin showed that 66% of the phosphate was located on peptides that are unphosphorylated in filaggrin. Analysis of peptide recoveries confirmed the repeat size and showed that every copy of filaggrin was phosphorylated in profilaggrin.     

Sequence of the OXA2 beta-lactamase: comparison with other penicillin-reactive enzymes

The nucleotide sequence of the unusual plasmid-mediated OXA2 beta-lactamase is presented, and compared with other beta-lactamases. The OXA2 enzyme has similar features at the presumed active site, but no other significant regions of homology with other penicillin-reactive enzymes. The active site homology may therefore represent convergent evolution of otherwise dissimilar genes.     

Population genetics of Biomphalaria straminea in Hong Kong. A neotropical schistosome-transmitting snail recently introduced into China

Since its introduction in the early 1970s Biomphalaria straminea (Gastropoda: Planorbidae) has spread rapidly and is locally the most abundant fresh water snail in Hong Kong. Studies of 19 electrophoretically detected loci in four populations show that the colonists retain high levels of variability (P = 0.26, H = 0.056 - 0.097), comparable with those found in autochthonous samples of related species. Genotype frequencies at the five polymorphic loci, and a comparison of maternal and progeny genotypes of individual field-collected snails, revealed no evidence for self-fertilization in these functional hermaphrodites. F statistics indicated minimal genetic structuring, presumably because of outcrossing and recency of origin of the populations. Geographic distribution of various alleles and their frequencies suggest that two southern populations were derived from the original colonists by dispersal but that a northern population represents a second introduction in about 1982. This interpretation (based on genetics) is consistent with the known history of the various populations. The Asian populations of this South American snail are interpreted as being in the "flush" phase of the colonization process. Finally, the probability of the secondary spread of this snail from Hong Kong, and the probability of its parasite, the human blood fluke Schistosoma mansoni, being introduced to Asia are discussed.     

Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.     

Tyrosine phosphorylation of a 94-kDa protein of human fibroblasts stimulated by streptococcal lipoteichoic acid

Lipoteichoic acid (LTA) is an amphipathic component of Gram-positive bacteria. Previous studies from this laboratory have shown that at low concentrations, ranging from 0.1 to 10.0 micrograms/ml, LTA binds to mammalian cells and stimulates mitogenic responses as demonstrated by increased DNA and RNA synthesis. Tyrosine kinase appears to be involved in the action of a number of mitogens including epidermal growth factor, platelet-derived growth factor, and insulin. In the present study, we report the novel finding that tyrosine protein kinase activity is increased in human fibroblasts treated with LTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the whole cell lysate of fibroblasts cultured with 32Pi showed increased phosphorylation of a 94-kDa polypeptide. Alkali treatment of the gel resulted in a decreased intensity of the 94-kDa phosphorylated protein in control cells, but not in LTA-treated cells, suggesting the addition of phosphate groups to threonine or tyrosine residues. High voltage electrophoresis of the acid hydrolysate of the excised and eluted 94-kDa protein revealed that LTA stimulated the phosphorylation of tyrosine but not threonine residues. These results suggest that LTA acts on mammalian cells by phosphorylating tyrosine residues of certain proteins and thereby may regulate diverse functions of these cells.     

Modification of the gene 5 DNA unwinding protein with ruthenium

A chemical modification of the gene 5 DNA binding protein (G5BP) from bacteriophage fd was investigated using X-ray diffraction and difference Fourier analysis. The crystalline protein was reacted with pentaammineruthenium (III) trichloride, Ru(NH₃)₅Cl₃, a reagent believed specific for histidine residues and useful in NMR and chemical modification studies of proteins. The major ruthenium site was found by difference Fourier analysis to be 4 Å from histidine 64, the only histidine residue in the molecule. A second bipartite site, believed to be a ruthenium-anion pair, appeared to be salt-bridged to glutamic acid 40 and arginine 16. Indications were present in the difference Fourier results to suggest that the loop containing tyrosine 41 had undergone a slight conformational rearrangement to accommodate this interaction.