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.     

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ca2+-sequestering smooth endoplasmic reticulum in an invertebrate photoreceptor. I. Intracellular topography as revealed by OsFeCN staining and in situ Ca accumulation

Two ultrastructural approaches were used in photoreceptor cells of the leech, Hirudo medicinalis, to (a) investigate the intracellular topography of the smooth endoplasmic reticulum (SER) and (b) identify among the various subregions of the SER those which might function as Ca-sequestering sites. When the cells are prefixed with CaCl2-containing glutaraldehyde and postfixed with osmium tetroxide-ferricyanide (OsFeCN), only a part of the total SER is specifically stained. The stained SER cisternae include the submicrovillar cisternae (SMC), subsurface cisternae (SSC), the nuclear envelope, Golgi-associated SER, paracrystalline SER, and SER associated with glycogen areas. An extensive tubular SER cisternal system always remains unstained. When the cells are permeabilized by saponin and subsequently incubated with Ca2+, MgATP, and oxalate, the SMC (Walz, 1979, Eur. J. Cell Biol. 20:83-91), the SSC and the nuclear envelope contain electron-opaque Ca-oxalate precipitates indicating their ability to function as an effective Ca2+ sink. The results show that the very elaborate SER in this photoreceptor cell includes many functionally heterogeneous subregions. Of special physiological significance are those components (SMC and SSC) which are effective in Ca2+-buffering in the immediate vicinity of the plasma membrane.     

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.