Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Plasma growth hormone responses to repetitive administrations of growth hormone releasing factor in patients with pituitary dwarfism

Plasma growth hormone (GH) responses to the repetitive administrations of synthetic human pancreatic growth hormone releasing factor (hpGRF-44) were studied in 15 patients with GH deficiency (11 diagnosed as idiopathic and 4 diagnosed as secondary to hypothalamo-pituitary tumor). hpGRF-44 was administered by single iv bolus (2 micrograms/kg), repetitive im (100 micrograms, twice a day), and/or repetitive iv infusion (2.5 micrograms/min for 90 min, once a day) for three to six consecutive days. Three of the eleven idiopathic GH deficient patients had plasma GH responses to both single iv bolus injection and repetitive administrations by im, or iv infusion of hpGRF. In four of the remaining eight, who had not had peak plasma GH levels above 5 ng/ml to a single iv bolus of the peptide, repetitive administrations of hpGRF-44 by im injection and/or iv infusion induced GH responses to the peptide. In the four patients with secondary GH deficiency, three had plasma GH response to hpGRF administration but one patient, who had indications of pituitary disorder, did not show any plasma GH response to either single iv injection or repetitive administrations of hpGRF-44. These data show that repetitive administrations of hpGRF-44 can induce plasma GH responses in some GH deficient patients who do not respond to a single iv bolus of the peptide.     

Structure of the baboon endogenous virus genome: cloning of circular virus DNA in bacteriophage lambda.

Linear, small and large circular forms of unintegrated viral DNAs were detected in Hirt supernatant fraction of human cultured cells infected with baboon endogenous virus M7. The circular M7 DNAs were cloned in bacteriophage lambda, Charon 28. Seventeen independent clones were isolated and analyzed by restriction endonuclease mapping. Nine clones were carrying a viral sequence of 8.6 kilobase pairs (kb) with two tandem repeats of 0.6 kb, which correspond to the large circular form of the unintegrated M7 DNA. Eight other clones had the viral insert of 8.0 kb, i. e., the small circular form, and were deleted one of the repeated sequences. The repeated sequences correspond to the long terminal repeats of 0.6 kb, located at both ends of the linear M7 DNA of 8.6 kb. One of the recombinants of the large circular M7 DNA had an inversion of 2.5 kb. One end of the inverted sequence was near the terminus of the long terminal repeats and the other in the gag gene region. The inversion seems to be occurred by integration of a viral DNA within itself during early periods of infection. The mechanism of the processes leading to integration is discussed from the structure of these unintegrated M7 DNAs as the precursors.     

Intranuclear synthesized and native glycogen particles in human gastric cancer: Ultrastructure and histochemistry

Summary Ultrastructural and histochemical studies on human gastric cancer cells disclosed the presence of native and synthesized glycogen particles. The glycogen particles were investigated in the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system and non-incubated native glycogen in human gastric adenocarcinoma tubulare.It was observed that focal synthesis localized in the intracytoplasmic matrix and intranucleus. Intranuclear synthesized glycogen appeared as a rosette form ranging from 1100 to 1300 Å in diameter and free particles ranging from 325 to 900 Å in diameter. The synthesis of glycogen appeared in the nucleus as well as in the cytoplasm of the human gastric cancer cells, and the synthesized glycogen was observed as a group of particles. Newly formed glycogen particles appeared occasionally in the interchromatin area as a large macromolecular structure of rosette form.Native glycogen appeared as a free-particle (250–333 Å, medium=300 Å) and aggregated rosette from (694–1050 Å, medium=917 Å) in the autophagosome of gastric cancer cells. There was not, however, a native glycogen particle in the nuclei of gastric cancer cells.Under certain conditions the nuclei of gastric cancer cells can acquire the capacity to synthesize glycogen.     

Phosphorylation and subcellular distribution of calpastatin in human hematopoietic system cells

Calpastatin is an endogenous inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase). The phosphorylation of calpastatin was investigated in human hematopoietic system cell lines. Microheterogeneity of calpastatin was observed, in which 118- and 116-kDa forms were named calpastatin a and b, respectively. The phosphorylation of both calpastatins was identified in all cell lines examined and occurred mainly at serine residues with trace amounts of phosphothreonine in vivo. The incubation of cells with 12-O-tetradecanoylphorbol-13-acetate increased the incorporation of 32P-orthophosphate into calpastatin a. Two-dimensional maps of 32P-labeled phosphopeptide from both calpastatins were identical except for additional minor spots for calpastatin a. [35S]methionine-labeled calpastatins a and b were localized mainly in the cytosol, and only 6% of cellular calpastatins were detected in the membrane fraction. By contrast, more than 30% of the 32P-labeled calpastatins a and b were distributed in the membrane fraction. Thus, the phosphorylation of calpastatin may be involved in regulating the calpain-calpastatin protein kinase system by its subcellular distribution.     

Synthesis of lipopolysaccharide by Escherichia coli cells recovering from sublethal heat stress.

Escherichia coli cells exposed to a sublethal heat treatment at 55 degrees C for 15s synthesized lipopolysaccharide during their recovery period after heat stress. As chloramphenicol at least partly inhibited the synthesis of lipopolysaccharide, it is suggested that its synthesis might be in part due to the lipopolysaccharide-synthesizing enzymes produced de novo. The results obtained coincided with our previous finding that the permeability barrier function was repaired by heat-stressed cells.     

Possible involvement of protein kinase C and calcium in GSH efflux from Hep G2 cells.

We investigated the effects of protein kinase C modulations and calcium mobilization on GSH efflux in Hep G2 cells. GSH efflux from Hep G2 cells was increased by a phorbol ester. Staurosporine, an inhibitor of protein kinase C, diminished phorbol ester-stimulated GSH efflux from the cells. GSH efflux was negatively correlated with extracellular calcium concentrations. Verapamil enhanced GSH efflux, whereas ATP decreased GSH efflux. The latter effect was diminished in the absence of extracellular calcium. Protein kinase C and calcium mobilization may be crucial factors in GSH efflux from human hepatocytes.     

Demonstration of platelet activating factor receptor in guinea pig kidney.

Distribution of platelet activating factor (PAF) receptor was examined in the guinea pig kidney. Northern blot analysis showed a single band electrophoresed just below the 28S rRNA, and the mRNA was richest in the cortex with lesser amounts in the outer and then inner medulla. Scatchard analysis of membrane fraction using [3H]WEB 2086, a specific PAF receptor antagonist, revealed a single binding site with Bmax of 522, 228, 58 fmol/mg protein for the cortex, outer medulla and inner medulla, respectively. Kd values were in the same order of magnitude (10(-8) M). These results indicate the presence of a single class of PAF receptor in the guinea pig kidney which is most abundant in the cortex.