Effect of beta-mercaptoethanol or epidermal growth factor supplementation on in vitro maturation of canine oocytes collected from dogs with different stages of the estrus cycle

Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.

Physico-chemical properties of thymidylate synthase from Lactobacillus leichmannii: thiol groups, amino acid composition and the terminal end-groups.

Thymidylate synthase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyltransferase, EC 2.1.1.45) from Lactobacillus leichmannii was completely inactivated after 5 min of heat treatment at 55 degrees C. A remarkable synergistic effect with no loss in activity was noted when 10(-3) M dUMP was added to the enzyme before subjecting to heat treatment. The enzyme got activated in the presence of 2-mercaptoethanol (75 mM) and inhibited by pCMB (I50 = 5 microM). It had 2 free sulfhydryl groups and a single disulfide bond. The two identical subunits of the 74 kDa dimer were possibly bonded by a single disulfide linkage. It had a total of 652 amino acids with methionine as the amino-terminal and alanine as the carboxy-terminal amino acid residues. The carboxy-terminal end-group alanine was preceded by valine, lysine and proline sequentially in that order.

An axenic culture system for the transformation of bloodstream forms to procyclic forms of Trypanosoma brucei brucei in vitro.

An axenic culture system for continuous cultivation of bloodstream forms of Trypanosoma brucei GUT at 3.1 and subsequent transformation of bloodstream forms to procyclic forms is described. Bloodstream forms were continuously grown at 37 degrees C in Iscove's modification of Dulbecco's medium (M-DMEM, with bovine serum albumin, transferrin and soybean lecithin supplemented with 100 microM hypoxanthine, 30 microns thymidine, 40 microM adenosine, 1mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% (v/v) heat-inactivated fetal bovine serum. In this system, 2-mercaptoethanol (2-ME) was essential and in the absence of 2-ME, 100 microM L-cysteine and 10 microM bathocuproine sulfonate could not be substituted for 2-ME. This culture system was useful for long-term culture of bloodstream forms of this clone. Axenic cultivation of bloodstream forms at 27 degrees C resulted in transformation to procyclic forms within 5 days in the same medium supplemented with 5 mM L-proline, 8 micrograms/ml hemin and 4 micrograms/ml hematin, respectively and, instead of FBS, 20% (v/v) hemoglobin-poor fraction of fetal bovine serum.

Western blot analysis of antigens specifically recognized by natural immune responses of patients with Japanese encephalitis infections.

Specific recognition of antigenic proteins of Japanese encephalitis virus (JEV) by JE patients was investigated by using non-reducing and reducing Western immunoblot analysis. Under non-reducing conditions, the profile of JEV proteins recognized comprised E (52 kDa), NS1 (45 and 41 kDa), NS3 (66.2 kDa) and NS5 (103 and 97.4 kDa). When recognition patterns of sera from JE and dengue patients were compared, only slight differences between JE and dengue sera were found (under non-reducing conditions), involving only the 66.2 kDa protein: to this protein, JE sera exhibited greater reactivity, but not in greater frequency, than did dengue sera. In contrast, cerebrospinal fluid (CSF) from JE patients showed more differences from JE sera: CSF antibody lacked recognition of the 41 kDa protein and had lower frequencies, as well as less reactivities to several other proteins. These results suggested that restricted populations of lymphocytes were localized in the central nervous system of JE patients. The effect of reducing agent (2 beta-mercaptoethanol) on the recognition patterns of those groups of sera was also analysed: the reducing agent affected all the proteins mentioned above, however, the effects were not uniform. It is proposed that JE and dengue sera may recognize different epitopes on some or all of these proteins. Such differences cannot be detected by Western immunoblot analysis, but it would be feasible to test this hypothesis using epitope mapping with synthetic peptides in a multi-pin ELISA. Analysis in this fine detail is essential for designing improved JE vaccines.

Keratinase of a streptomycete.

Keratinase produced from Streptomyces Sp.A11 decomposed human hair, chicken feather, wool, silk and pure keratin extracted from human epidermis. Purification of the enzyme by DEAE-cellulose column chromatography resulted in 7.5-fold increase in activity relative to the activity of the culture filtrate. The enzyme was inducible, extracellular, homogeneous with a molecular weight of 49,000. The enzyme activity was inhibited by reduced glutathione, phenylmethyl sulphonyl fluoride and 2-mercaptoethanol.

Hemolisinas de Listeria monocytogenes demostrables por activación con 2- mercaptoctanol / Demonstrable Listeria monocytogenes hemolysins by 2- mercaptocthanol activation

Jones y Seeliger, con el apoyo del Comité Internacional sobre Bacteriología Sistemática, Subcomité sobre Taxonomía de Listeria, han propuesto la necesidad de reemplazar la cepa de L. monocytogenes ATCC 15313 como prototipo en virtud de su incapacida de inducir hemólisis en medios gelificados que contienen 5 o 10% de hematíes de origen ovino o equino (método clásico). En este trabajo se demuestra que, si bien, la cepa ATCC 15313 no induce hemólisis en las condiciones anteriores es capaz de hemolizar hematíes de carnero cuando los sobrenadantes de cultivos en infusión cerebro corazón adicionado de 0,5% de glucosa son previamente activados con 2-mercaptoetanol, condición que resulta general para todas las cepas de L. monocytogenes ensayadas. Igualmente la cepa ATCC 15313 induce lisis en medio gelificado de agar base sangre con hematíes de carnero cuando es cultivada en microaerofilia. Se estudiarion 13 cepas caracterizadas originalmente com L. monocytogenes. Se demuestra que la presencia de hemolisinas por el método directo y/o detección por activación con 2-mercaptoetanol es compatible con la identificación como L. monocytogenes, por lo cual se recomienda la inclusión de estaspruebas en todo estudio identificatorio. Además el estudio de capacidad hemolítica no puede obviar la prueba en microaerofilia. La ausencia de capacidad hemolítica en todas estas condiciones de ensayo es un factor de importancia para cuestionar la identificación de una cepa como L. monocytogenes

Humoral immune responses in hamsters infected with Opisthorchis viverrini.

The kinetics and nature of humoral immune responses to somatic and excretory-secretory (ES) antigens were investigated in hamsters experimentally infected with different numbers of Opisthorchis viverrini. ES antigens were obtained from the in vitro culture of adult flukes and somatic antigens were aqueous extracts of adult flukes. Antibodies in the serum and bile of infected animals were determined by the microhaemagglutination technique, using glutaraldehyde fixed sheep red blood cells sensitized with these parasite antigens. Antibody responses to both somatic and ES antigens were detected in the serum from the second week of infection onward. The peak response was noted at the end of the second month and declined slowly thereafter. Antibody levels in animals with heavy infections (100 metacercariae) appeared earlier but declined more rapidly than in animals with light infections (25 metacercariae). The serum antibodies were highly sensitive to mercaptoethanol throughout the course of infection (23 weeks). Antibodies also appeared in the bile obtained at the time of sacrifice but their titres were rather low compared with those in the serum. Like serum antibodies, biliary antibodies were reactive with both somatic and ES antigens. Biliary antibodies were of the secondary IgA type. These findings are discussed in relation to pathogenesis of the disease process and to the possible usefulness in immunodiagnosis.