Purification and characterization of a protein kinase from pine pollen

A kinase phosphorylating casein and phosvitin has been purified from pine pollen by a three-step procedure involving DEAE-cellulose chromatography, affinity chromatography on casein-Sepharose and Sephadex G-100. A purification of about 2000 fold was obtained by this procedure. The kinase is affected neither by cyclic nucleotides nor by Ca²⁺-calmodulin, whereas it is strongly inhibited by heparin. Using this purification procedure, we have isolated protein kinase exhibiting phosphorylating activity towards casein in the pollen of many other Pinaceae species.     

Antibiotic Susceptibility Profiles of Dairy Leuconostoc,Analysis of the Genetic Basis of Atypical Resistances and Transfer of Genes In Vitro and in a Food Matrix

In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ≈35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance.     

Disaccharides Protect Antigens from Drying-Induced Damage in Routinely Processed Tissue Sections

Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions.     

Changes in endogenous levels of three cytokinins during development of excised watermelon cotyledons

We have determined by an immunological method the endogenous levels of three cytokinins: dihydrozeatin riboside (DHZR), transzeatin riboside (tZR) and isope-ntenyladenosine (IPA) in watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either attached to the seedling or excised from the seed after imbibition and then grown on water. Both seedlings and cotyledons were grown either for 5 days in continuous light or for 3 days in the dark and 2 days in light. Our aim was to verify whether endogenous cytokinin levels are lower in excised than in attached cotyledons as could be expected since excised cotyledons are much more sensitive to exogenous cytokinin application. The levels of the three cytokinins were very low immediately after imbibition, but gradually increased during the following days. They were higher in excised cotyledons after 5 days of culture in the dark than in cotyledons of the same age that had developed on the seedling. Dihydrozeatin riboside was by far the most abundant of the three cytokinins in cotyledons as well as in the hypocotyl and the root.
Irradiation reduced the level of DHZR, negating the concept that light promotes cotyledon development by increasing endogenous cytokinins. Transzeatin riboside when supplied exogenously, stimulated cotyledon development at a lower concentration than the other two cytokinins. Exogenous supply of ben-zyladenine (BA) induced a strong increase in endogenous tZR already after 24 h.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.     

Effects of re‐oligotrophication and climate change on lake thermal structure

相似文献   

Blood cultures incorporating antibiotic absorbing resins as an index of effective chemotherapy

During a mean time of eight days, blood cultures made in duplicate with and without resins from nine patients with septicemia were evaluated. Data supporting the usefulness of blood cultures incorporating antibiotic absorbing resins as an index of chemotherapy are presented and discussed. The authors propose that this resin system be used not only to increase the sensitivity of blood cultures, but also as an index of effective chemotherapy.