SDS-soluble and peptidoglycan-bound proteins in the outer membrane-peptidoglycan complex of Brucella abortus

Outer membrane-peptidoglycan complex from Brucella abortus was separated from cytoplasmic membrane and cytosol by either sucrose density gradient fractionation or differential (rate) centrifugation of surface labeled cells disrupted by sonication without the use of detergents. The outer membrane-peptidoglycan complex had a buoyant density of 1.22 gm/ml and contained 67 labeled SDS-soluble proteins when examined by SDS-PAGE. Included were four major bands exhibiting molecular masses of 88k, 40k, 35.7k and 26k daltons corresponding to previously described group 1, 2 and 3 outer membrane proteins. Lysozyme treatment of outer membrane-peptidoglycan complex increased its buoyant density to 1.25 gm/ml and released eight additional peptidoglycan-linked proteins.     

Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus

The outer membrane-peptidoglycan complex (OM-PG) from rough strains of Brucella abortus was tested for its ability to induce lymphocyte responsiveness in cattle. Six groups of heifers were immunized with varying doses and administration schedules of rough OM-PG and assayed for responsiveness of their lymphocytes in proliferation assays in vitro. All OM-PG preparations were emulsified in a commercial adjuvant for administration. Two other groups of heifers were immunized with strain 19 vaccine or adjuvant alone. Three groups of heifers received two inoculations of OM-PG antigens from a naturally-occurring rough strain at a 57-day interval. The doses of OM-PG given in these three groups were 400 micrograms, 1200 micrograms, and 4000 micrograms at each inoculation. The frequency of cows that responded in lymphocyte proliferation assays increased with the dose of OM-PG given. Two groups received single inoculations of OM-PG, either 2400 micrograms or 8000 micrograms. Although there were responsive cows in these immunization groups, their frequency was lower than in the groups receiving the same total dose in two inoculations. A sixth group of cows was inoculated with OM-PG from a rough transposon mutant of B. abortus, and the frequency of responsive cows in this immunization group was comparable to that of responsive cows immunized with the same dose of OM-PG from the spontaneous rough mutant. In comparisons of cows inoculated with strain 19 to those inoculated with OM-PG preparations, differences were observed in the relative responsiveness of their lymphocytes to whole cells and OM-PG in the in vitro lymphocyte proliferation assays. These differences suggested that lymphocytes stimulated by strain 19 vaccination have different specificities than those stimulated by immunization with OM-PG of rough mutant strains of B. abortus.     

Physicochemical Parameters and Antioxidant Activity of Bee Honey Enriched With Herbs

Three groups of products enriched with herbs were studied: (1) commercial herb honeys (n?=?5) produced by bees fed a syrup with an herbal extract, (2) natural herbal honey (n?=?3) produced by bees from the nectar of herbs, and (3) creamed multifloral honey with added dried herbs (n?=?5). As a control, multifloral honey (n?=?5) was used. The physicochemical parameters (i.e., sugar extract, water content, specific rotation, conductivity, hydroxymethylfurfural content, pH and acidity), sugar profiles (HPLC analysis), antioxidant activity and total phenolic compounds content of the studied samples were compared. Although great diversity in the basic properties of the studied products was observed, they were comparable to multifloral honey and complied with honey regulations. Significant differences in sugar composition were observed, and adversely positive rotation (excluding nettle herb honey) was detected in group 1, likely resulting from the change in bee feeding. The best antioxidant activity for creamed honeys with dried herbs (group 2) was investigated, whereas herb honeys (group 1) exhibited similar antioxidant properties as multifloral honey. The use of controlled feeding of bees appears to be an effective method of enriching honey with desirable plant bioactive components to create innovative bee products.     

Smenospongine,a Sesquiterpene Aminoquinone from a Marine Sponge,Induces G1 Arrest or Apoptosis in Different Leukemia Cells

Smenospongine, a sesquiterpene aminoquinone isolated from the marine sponge Dactylospongia elegans, was previously reported by us to induce erythroid differentiation and G1 phase arrest of K562 chronic myelogenous leukemia cells. In this study, we investigated the effect of smenospongine on the cell cycles of other leukemia cells, including HL60 human acute promyelocytic leukemia cells and U937 human histiocytic lymphoma cells by flow cytometric analysis. Smenospongine induced apoptosis dose-dependently in HL60 and U937 cells. The smenospongine treatment increased expression of p21 and inhibited phosphorylation of Rb in K562 cells, suggesting the p21-Rb pathway play an important role in G1 arrest in K562 cells. However, the p21 promoter was not activated by the smenospongine treatment based on a luciferase assay using the transfected K562 cells. Smenospongine might induce p21 expression via another mechanism than transactivation of p21 promoter.     

Immunogenicity of subcellular fractions of Brucella abortus: measurement by in vitro lymphocyte proliferative responses

Five groups of heifers were immunized with various subcellular fractions of Brucella abortus and tested for their responsiveness in lymphocyte proliferative responses in vitro. The five subcellular fractions used as immunogens were: (1) a mixture of recombinant outer membrane proteins fused to Escherichia coli beta-galactosidase, (2) a mixture of outer membrane proteins BaomI, BaomIIB1, and BaomIII1, (3) a mixture of outer membrane proteins 7.5 kDa and 8.8 kDa, (4) a complex of smooth lipopolysaccharide and proteins, and (5) a complex of outer membranes and peptidoglycan (OM-PG complex) from a rough strain. All immunogens were emulsified in adjuvant and administered twice at a 61-day interval. Two other groups of cows were included; one immunized with strain 19 and the other with adjuvant only. Strain 19 and the rough OM-PG complex induced responsiveness in lymphocyte proliferation assays in a high percentage of immunized cows. The smooth lipopolysaccharide-protein complex induced responsiveness in fewer cows. The lowest frequencies of responding cows were found in groups that received either recombinant proteins or purified protein mixtures. Based on these results, we concluded: (1) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced with subcellular fractions of B. abortus and (2) the more complex the immunogen, the greater the frequency of responding cows.     

Cancer proliferation gene discovery through functional genomics

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.     

First detailed report on Puccinia graminis f. sp. avenae virulence structure and Pg resistance genes effective in Poland

European Journal of Plant Pathology - Occurrence of stem rust, caused by Puccinia graminis f. sp. avenae, on oat fields in Europe may lead to significant yield losses. The last P. graminis...     

Preliminary investigations on the effects of a Strongylus vulgaris larval extract,mononuclear factors and platelet factors on equine smooth muscle cells in vitro

Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smooth muscle cells exposed to other factors. Equine mononuclear leukocytes exposed to LAE developed prominent morphological changes, including enlargement, clumping and increased numbers of mitotic figures.     

Immunoglobulin G1 Fc in colostral whey.

Two IgG1 fragments were isolated from bovine colostral whey. Based on gel diffusion analysis, both fragments originated from the Fc portion of the molecule but were not identical to IgG1 Fc prepared by papain cleavage. The molecular weights were determined to be 66,000 and 14,000 daltons and it was hypothesized that the larger fragment could be a polymer of the smaller. No IgG1 Fab or IgG2 fragments could be demonstrated.