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.     

The Sabinas syndrome

We have defined a new autosomal recessive disorder in patients stemming from a small community in northern Mexico. Diagnosable at birth, its major symptoms include brittle hair, mental retardation, and nail dysplasia. Structural hair abnormalities are seen by both light and electron microscopy. Hair cystine content is reduced while the copper/zinc ratio in hair is increased.     

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.     

Occluding junctions and cytoskeletal components in a cultured transporting epithelium

To study the size and structure of the Na,K-pump molecule, the ultrastructure of phospholipid vesicles was examined after incorporation of purified Na,K-ATPase which catalyzes active coupled transport of Na+ and K+ in a ratio close to 3Na/2K. The vesicles were analyzed by thin sectioning and freeze-fracture electron microscopy after reconstitution with different ratios of Na,K-ATPase protein to lipid, and the ultrastructural observations were correlated to the cation transport capacity. The purified Na,K-ATPase reconstituted with phospholipids to form a very uniform population of vesicles. Thin sections of preparations fixed with glutaraldehyde and osmium tetroxide showed vesicles limited by a single membrane which in samples stained with tannic acid appeared triple-layered with a thickness of 70 A. Also, freeze-fracture electron microscopy demonstrated uniform vesicles with diameters in the range of 700-1,100 A and an average value close to 900 A. The vesicle diameter was independent of the amount of protein used for reconstitution. Intramembrane particles appeared only in the vesicle membrane after introduction of Na,K-ATPase and the frequency of intramembrane particles was proportional to the amount of Na,K-ATPase protein used in the reconstitution. The particles were evenly distributed on the inner and the outer leaflet of the vesicle membrane. The diameter of the particles was 90 A and similar to our previous values for the diameter of intramembrane particles in the purified Na,K-ATPase. The capacity for active cation transport in the reconstituted vesicles was proportional to the frequency of intramembrane particles over a range of 0.2-16 particles per vesicle. The data therefore show that active coupled Na,K transport can be carried out by units of Na,K-ATPase which appear as single intramembrane particles with diameters close fo 90 A in the freeze-fracture micrographs.     

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.