Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Empirical choice of sampling procedures for optimal research design in the longitudinal study of primate behavior

A method is suggested to evaluate on an empirical basis sampling plans for the longitudinal study of primate behavior in those very common situations in which the mathematical structure of behavior is unknown. The method is based on a randomization procedure applied to a pilot sample of the behavior organized so that cost of implementation of a sampling plan can be evaluated vis á vis the sampling error intrinsic to the plan.     

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

Summary Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.Abbreviations BSA bovine serum albumin - EM electron microscope - GOGAT glutamate synthase - GS glutamine synthetase - GUS -glucuronidase - IgG immunoglobulin - PBS phosphate buffer saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

ImmobilizedAspergillus terreus in itaconic acid production from glucose

Summary The production of itaconic acid by immobilizedAspergillus terreus TKK 200-5-1 was studied both in shake flask cultures, and in continuous column bioreactors. The effect of glucose and ammonium nitrate concentrations, and of pH were examined using a statistical experimental plan. The highest itaconic acid product concentration could be reached at the highest investigated glucose concentration of 150 g/l and the highest initial pH of 3.75, in the absence of ammonium nitrate. In a continuous packed bed column system operated fro 4.5 months itaconic acid was obtained at a productivity of 328 mg/d per gram of polyurethane foam carrier.     

Plasmodium falciparum: Comparative analysis of erythrocyte stage-dependent protein antigens

Proteins of erythrocytic stages of Plasmodium falciparum were biosynthetically labeled at different times during the first cycle of in vitro synchronous cultivation after collection from patients in the Madang region of Papua New Guinea. Proteins were immunoprecipitated with a pool of hyperimmune serum collected in the region then analyzed by sodium dodecyl sulfate-gel electrophoresis. Antigens were recognized in all life cycle stages but the majority of antigens, particularly those of high molecular weight, were present in the mature forms of the parasite.     

Rescue of embryonic cells homozygous for a lethal haplotype of the T/t complex: tw12

A series of recessive mutations which arrest embryonic development are located within the T/t region of chromosome 17 in the mouse. To assess whether these mutations cause death in specific differentiating cells or in all cells of the embryo, we removed the embryonic cells from normal developmental constraints and attempted to grow them ectopically in vivo and in vitro. We have succeeded in producing teratomas and teratocarcinomas by transplantation of inner cell masses from blastocysts of $\text{t}{}^{\mathrm{w12}}\text{+}$ and $\text{t}{}^{\mathrm{w12}}\text{t}{}^{\mathrm{w12}}$ genotypes. The ability of embryonic cells to grow as tumors was not affected by their genotype; 7 of the 17 tumors were homozygous for t^w12, 7 were heterozygous, and 3 could not be analyzed. Virtually all the tumors of both genotypes contained derivatives of all three germ layers. Neuroepithelial and mature nervous tissue was present in all homozygous tumors and all except one heterozygous tumor. However, no cartilage or bone was found in 5 of 5 t^w12 homozygous tumors, while both tissues were present in 3 of 4 t^w12 heterozygous tumors. This observation is compatible with the abnormalities characteristic of $\text{t}{}^{\mathrm{w12}}\text{t}{}^{\mathrm{w12}}$ embryos, which show very localized effects in nervous tissue and more general effects on bone and cartilage formation. Cells derived from homozygous tumors were capable of at least limited growth in culture and a cell line has been derived from one of them. The p63/6.9a marker protein was used to determine the presence of the t^w12 haplotype in the tumor and cultured cells. We conclude that the lethality associated with the t^w12 haplotype is due to lethality of specific cells, and not all cell types.     

Hormonal control of uteroglobin secretion in rabbit uterus. Inhibition of uteroglobin synthesis and messenger ribonucleic acid accumulation by oestrogen and anti-oestrogen administration

Investigations were conducted to quantify activity of uteroglobin mRNA and secretion of uteroglobin in rabbit uterus after administration of progesterone and 5alpha-dihydrotestosterone, either alone or concomitantly with oestradiol-17beta and tamoxifen, a non-steroidal anti-oestrogen. Poly(A)-containing mRNA was isolated from the uterine tissue by extraction with phenol/chloroform, precipitation with ethanol and chromatography on oligo(dT)-cellulose. Cell-free translation in vitro of the poly(A)-containing mRNA was carried out in a wheat-germ lysate, and the product isolated by specific immuno-precipitation with anti-uteroglobin antiserum purified by affinity chromatography. Radioimmunoassay was utilized to determine uteroglobin content in the uterine flushings and tissue preparations. When given for 5 days, both progesterone (1mg/kg per day) and 5alpha-dihydrotestosterone (25mg/kg per day) elicited a marked induction of uteroglobin secretion, which was accompanied with accumulation of uteroglobin mRNA in the tissue. Concomitant administration of oestradiol-17beta (50mug/kg per day) or tamoxifen (12.5mg/kg per day) significantly decreased both progesterone- and 5alpha-dihydrotestosterone-induced uteroglobin secretion, with a parallel decrease in the uteroglobin-mRNA activity. The decline in the uteroglobin content of the uterine flushes brought about by oestradiol-17beta or tamoxifen administration was not due to inhibition of secretion of this protein by the endometrial cells, since a simultaneous decrease occurred in the tissue uteroglobin content. After a 5-day pretreatment with progesterone (1mg/kg per day), administration of oestradiol-17beta (50mug/kg per day) during the ensuing 4 days greatly accelerated the decay of the uteroglobin content in the uterine fluid.     

Induction of mutants in Saccharomyces cerevisiae by guanidine hydrochloride II. Conditions that prevent induction

The induction of ⁻ “petite” mutants by guanidine hydrochloride (GuHCl) is inhibited in several conditions. Anaerobiosis inhibited the induction either with or without cell multiplication. Both nalidixic acid (NA) and cycloheximide (CH) inhibited the induction of mutants. On the other hand, chloramphenicol (CAP) produced a dual effect: at low concentration it stimulated, at high concentration it inhibited, the induction. The effect of these different inhibitors on the transformation of ⁺ mother cells into ⁻ by GuHCl is discussed.