Incidence of toxigenic vibrios in foods available in Taiwan.

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-O1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.     

IL-4 regulation of a protein kinase C independent pathway for the generation of alpha CD3-induced activated killer cells.

alpha CD3 induced the generation of activated killer cells from resting T cells. Pretreatment of the splenic responders with PMA, a phorbol ester, depleted protein kinase C and induced unresponsiveness to the generation of alpha CD3-induced activated killer (CD3-AK) cells. Addition of exogenous IL-4 (1 U/ml) restored the cytotoxic response, with the maximal effect achieved with 30 to 100 U/ml. The phenotypes of CD3-AK cells maintained in IL-2 or in IL-4, with or without PMA, were the same: Thy1+ and CD8+. These results were reproduced with purified T cells and purified CD8+ cells, indicating that both the effectors and precursors were CD8+ cells and IL-4 had a selective effect to upregulate the CD8+ cells. Similar results were obtained by using SSP (staurosporine), another PKC inhibitor. At 2 days prior to testing, switching the lymphokine added to 2-week PMA- and IL-2-maintained CD3-AK cells reversed their cytolytic activity: switching from IL-2 to IL-4 restored cytolytic activity, and switching from IL-4 to IL-2 reduced cytolytic activity. The cytolytic activity of these CD3-AK cells correlated with their ability to produce BLT-esterase. In the absence of PMA, CD3-AK cells cultured in either IL-2 or IL-4 were cytolytic and contained high levels of BLT-esterase. In contrast, in the presence of PMA, only the IL-4-maintained CD3-AK cells were cytolytic and produced significant amounts of BLT-esterase. The effect of IL-4 was abrogated by the alpha IL-4 antibody 11B11, which reduced the cytolytic activity of CD3-AK and the ability to produce BLT-esterase. The requirement of IL-2 was less stringent and its major role appeared to be maintaining the cell growth. These findings indicate that IL-4 may participate in the regulation of a PKC-independent pathway for the generation of CD3-AK cells by regulating the production of cytolytic granules.     

Usage of primary cells to delineate IFN-gamma-responsive DNA elements in the HLA-DRA promoter and to identify a novel IFN-gamma-enhanced nuclear factor.

The IFN-gamma regulation of the HLA-DRA gene was examined in a primary cell type, the astrocyte. Site-specific mutagenesis of the DRA promoter reveals that three known sequences, S, X, and Y, are required for an optimal IFN-gamma response. Specifically for the X sequence, the X1, but not the X2, site is involved in IFN-gamma regulation of HLA-DRA in the astrocyte. Most interesting, a novel IFN-gamma-enhanced protein (IFNEX) with specificity for the X element has consistently been observed in nuclear extracts made from primary astrocytes. The correlation of the functional importance of X1 in IFN-gamma-regulated DRA expression and the enhancement of IFNEX by IFN-gamma strongly suggests that IFNEX may play a crucial role in IFN-gamma-regulated class II MHC gene expression.     

Solvent effect on binding thermodynamics of biopolymers

The indirect solvent-induced effect on the free energy of binding of biopolymers is examined within the framework of classical statistical mechanics. We focus specifically on the role of the solute-solvent hydrogen bonding. In particular, we have estimated the first order solvent effect on the indirect interaction between two biopolymers. We find that the solvent-induced interactions between two hydrophilic groups through water-bridged hydrogen bonds could significantly enhance the binding free energy. Some preliminary estimates indicate that this effect is significant and perhaps could be crucial in molecular recognition processes. Furthermore, we have calculated, from crystal structure data, the distance distribution between all the oxygens and nitrogens on the surface of some proteins that do not belong to the binding domain. In most cases we found an enhanced peak in the range of 4-5 A, which is where we expect to find strong solvent-induced interactions.     

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [³⁵S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m -guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [³⁵S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of ³⁵S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more ³⁵S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m -urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.     

2,4,6-Trinitrobenzenesulfonate labels an essential amino group near the bound phosphate at the catalytic site of mitochondrial F1-ATPase

The amino reagent 2,4,6-trinitrobenzenesulfonate (TNBS) was found to inactivate mitochondrial F₁-ATPase through covalent labeling, which was not reversed by dithiothreitol. The observed rate of inactivation was retarded by inorganic phosphate, but enhanced by prior labeling of F₁ with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1). These observations are consistent with the presence of an essential amino group near the bound inorganic phosphate at the catalytic site of F₁. A comparison of the observed protection of F₁ from NBD-C1 and 5′-p-fluorosulfonyl-benzoyladenosine (FSBA) respectively by inorganic phosphate and by 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP) suggests that NBD-C1 labels an essential Tyr residue in the positively charged locus for binding the polyphosphate end of ATP, and that FSBA labels an essential Tyr residue in the more hydrophobic locus for binding the adenosine moiety of ATP at the catalytic site of F₁.     

Studies of the mechanisms for the induction of in vivo tumor immunity. V. A comparison of the generation of the primary cell-mediated cytotoxic response using in vitro mixed-lymphocyte--tumor cell culture and an in vivo technique

In vivo immunization of C57BL/6 mice with FBL-3, a syngeneic Friend-virus-induced leukemia, can induce a specific, T-cell-mediated cytotoxic response, as measured by the ¹²⁵IUdR release assay. In vitro it was difficult to generate an analogous, primary cytotoxic response, using a normal spleen responder population. After modification of the splenic responders by adding normal peritoneal cells, this modified population then had the capacity to mount a primary cytotoxic response in the mixed-lymphocyte-tumor cell culture reaction to FBL-3. We have characterized the effector population as well as the helper (peritoneal) cells which were responsible for elevating the cytolytic response to FBL-3. The results indicate that there are at least two populations of cells which are essential for inducing a primary cell-mediated cytotoxic response. First, the effectors which are directly responsible for mediating the cytotoxic reactions and are derived from radiosensitive T cells and second, a helper cell population which is radioresistant and has the characteristics of macrophages.     

Microbody Malate Dehydrogenase Isozyme in Cotyledons of Cucumis sativus L. during Development

The properties of the microbody malate dehydrogenase (EC 1.1.1.37) (MDH) isozyme from cotyledons of Cucumus sativus L. were compared during development. It is concluded that the isozyme remains unaltered, despite the transition from glyoxysomal to peroxisomal function that occurs during greening of the cotyledons. This conclusion is based on electrophoretic behavior, chromatographic elution from DEAE-cellulose, molecular weight, kinetic behavior, and immunological identity. In most cases, the distinct properties of the other MDH isozymes in the tissue during development provide additional support for an unchanging microbody isozyme. A method for assaying specifically the microbody isozyme was developed; a diluted preparation was assayed spectrophotometrically before and after complete immunological precipitation. The turnover of the microbody MDH isozyme was investigated by a radioactive labeling study. There is incorporation into both glyoxysomal and peroxisomal MDH. Degradation rates do not correspond with either decline of glyoxysomal activity or the continuation of peroxisomal activity. Apparently, the microbody MDH isozyme is continually turned over throughout cotyledon development.