Accessory cell competence of ovine oligodendrocytes in mitogenic activation of human peripheral T cells

The multiple sclerosis (MS) plaque is characterized by mononuclear inflammatory cell infiltration, demyelination, loss of oligodendrocytes (OGC), and proliferation of astrocytes. Although antigen-specific, Ia-dependent cellular immune mechanisms have been sought in plaque pathogenesis, Ia-independent T cell activation has not been actively investigated. We examined a potential role of OGC in accessory cell-dependent T cell mitogenesis with the anti-T3 monoclonal antibody OKT3. OGC isolated from ovine white matter on sucrose density gradients were uniformly negative for esterase activity, unlike ovine monocytes. Purified human T cells did not exhibit significant proliferation in 3-day cultures with OKT3, autologous peripheral blood adherent cells (PBAC), or ovine OGC. When T cells were cultured with either PABC or OGC in the presence of OKT3, brisk mitogenesis was observed. Thus, OGC have the capacity to function as accessory cells in the mitogen-induced proliferation of T cells.     

Application of lipase to concentrate the docosahexaenoic acid (DHA) fraction of fish oil

A commercial lipase preparation from Rhizopus niveus was used to concentrate the omega-3 fatty acid, docosahexaenoic acid (DHA) component in fish oil. The DHA content of cod-liver oil was 9.64% (w/w) of total fatty acids. Enzymatic digestion conditions were established which produced a DHA content in the monoglycerides fraction of 29.17% (w/w) of total fatty acid, triglyceride, and diglyceride components were 5.72, 9.95, and 15316%, respectively.     

Steroid bioconversion in a microemulsion system

The Delta(1,2)-dehydrogenation of high concentrations of the steroid -methyl-Reichstein's compound S-21-acetate (16MRSA) in a microemulsion system was studied using heat-dried and thawed Arthrobacter simplex cells as biocatalyst. The microemulsion system consists of an organic phase [75-95% (v/v)] with steroid (1-60 g/L(tot)), an aqueous phase [5-25% (v/v)] containing the cells (5-30 g/L(tot)), and a neutral surfactant (5-20 g/L organic solvent). Benzene derivatives, which solubilize 16MRSA up to 94 g/L, and phospholipids were used as organic solvents and surfactants, respectively, and menadione was added as an external electron acceptor. Factors affecting the dehydrogenation rate in the microemulsion system were studied. The influences of the 16MRSA and the menadione concentration on the dehydrogenation rate were described by Michaelis-Menten kinetics, apparent V'(max) and K'(m) values of 2.06 g/g dry weight h and 18.9 g/L for 16MRSA and 4.97 g/g dry weight h and 1.91 g/L for menadione being obtained. Optimal menadione concentration was dependent on the steroid concentration was dependent on the steroid concentration used. The reaction was strongly inhibited by high product concentrations. Much higher activities were obtained with the thawed cells than with the dried cells, conversions of 98% being reached within 14-16 h. for 16MRSA and cell dry weight concentrations of 40 and 10 g/L, respectively. Activity retention in a batch stirred tank reactor remained constant during the first 16-24 h of operation and then decreased, depending on the stirring rate; 22 to 65% of the initial reaction rate was obtained after 48 h at stirring rates of 650 and 2000 rpm, respectively.     

Use of Horseradish Peroxidase-Labeled Antibody for Light and Electron Microscope Localization of Reovirus Antigen

Horseradish peroxidase-labeled antibody was used for light and electron microscopic localization of reovirus antigen in tissue culture. Reaction product in infected cells was easily detected in the cytoplasm, and the procedure was as sensitive as the fluorescent-antibody technique. At the electron microscopic level, infected and enzyme-labeled antibody-treated cells showed accumulations of reaction product at the sites of viral replication and around the viral particles. Reaction product was not detected in unstained infected cells, in stained uninfected cells, or in cells infected with an unrelated virus.     

A general method for characterizing naturally occurring folate compounds, illustrated by characterizing Torula yeast (Candida utilis) folates

A method previously found suitable for the chromatographic separation and identification of simpler folates has been extended and found suitable for separating and characterizing all the complex polyglutamyl folyl derivatives occurring naturally. Folates were characterized employing the combined use of analytical DEAE-cellulose chromatography, folylpoly-γ-glutamyl carboxypeptidase digestion, and differential microbiological growth response studies. An observed relation between the log phosphate concentration of the eluting buffer and the number of γ-glutamyl residues in successive pteroylpoly-γ-glutamyl derivatives provides a simple tool for a rapid and accurate identification of folate compounds from their elution profile. Twelve folate compunds present in Torula yeast (Candida utilis) were characterized employing this method; 80% of the total folates appeared to be pteroylpolyglutamates. The method characterizes not only the number of γ-glutamyl residues but also the state of reduction of the pteridine ring and the nature of the 1-C substituents attached.     

In vivo proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the host plant Brassica oleracea

The genus Xanthomonas is composed of several species that cause severe crop losses around the world. In Latin America, one of the most relevant species is Xanthomonas campestris pv. campestris, which is responsible for black rot in cruciferous plants. This pathogen causes yield losses in several cultures, including cabbage, cauliflower and broccoli. Although the complete structural genome of X. campestris pv. campestris has been elucidated, little is known about the protein expression of this pathogen in close interaction with the host plant. Recently, a method for in vivo analysis of Xanthomonas axonopodis pv. citri was developed. In the present study, this technique was employed for the characterization of the protein expression of X. campestris pv. campestris in close interaction with the host plant Brassica oleracea. The bacterium was infiltrated into leaves of the susceptible cultivar and later recovered for proteome analysis. Recovered cells were used for protein extraction and separated by two-dimensional electrophoresis. Proteins were analysed by peptide mass fingerprinting or de novo sequencing and identified by searches in public databases. The approach used in this study may be extremely useful in further analyses in order to develop novel strategies to control this important plant pathogen.     

Synthesis and characterization of DNA duplexes containing an N3T-ethyl-N3T interstrand crosslink in opposite orientations

DNA duplexes containing an ethyl interstrand crosslink that bridges the N3 atoms of thymidines on the opposite strands have been synthesized using an approach that combines conventional solid phase oligonucleotide synthesis and the selective removal of protecting groups of a crosslinked thymidine dimer. This approach allows for the assembly of a crosslinked duplex directly on the solid support. Duplexes that contain a N3T-ethyl-N3T interstrand crosslink in a staggered orientation at either a -TA- or -AT-step in a duplex have been prepared. When placed in an -AT- step of a duplex the effect was stabilizing relative to the non-crosslinked control duplex (deltaTm= +24 degrees C) and this crosslinked duplex was found to efficiently form multimers in the presence of T4 ligase. In the case of the -TA- crosslinked duplex the stabilizing effect was less pronounced (deltaT.= +6 degrees C) and likewise did not undergo self ligation under identical conditions. Molecular modeling studies suggested that the -AT- containing lesion had little deviation in structure relative to the non-crosslinked duplex DNA control, whereas the -TA- crosslinked duplex exhibited significant buckling of the base pairs flanking the lesion.