Proteins and Carbohydrates in Xylem Sap from Squash Root

The xylem sap from squash roots was collected from the cut surfaceof stems, and the proteins and carbohydrates in the sap wereanalyzed. The sap contained 18.6 µg ml^–1 proteinand the major polypeptides were as follows: 1) two polypeptides,of 75 and 40 kDa, with high-mannose glycans, the levels of whichincreased for about 24 h after cutting and then decreased; 2)a 32-kDa polypeptide, which appeared soon after cutting, disappearedand then reappeared again 48–64 h after cutting; and 3)a 19-kDa and a 14-kDa polypeptide, which were present constitutively.The carbohydrates contained in the xylem sap were fractionatedinto 80% ethanol-soluble and -insoluble material, and whichwere analyzed by high-performance liquid chromatography, gaschromatography and enzymatic mathods. The former fraction containedconsiderable amounts of myo-inositol and fructose as free sugarsand oligosaccharides composed mainly of galactose, arabinoseand glucose. The latter contained polysaccharides composed mainlyof uronic acids, galactose and arabinose. The possible significanceof these substances, which may mediate the interactions betweenthe root and the aerial organs, is discussed. (Received April 20, 1992; Accepted July 4, 1992)     

Lipase modification by various synthetic polymers for use in chloroform

Summary Lipase was modified with several hydrophilic and hydrophobic synthetic polymers. The modified lipase was solubilized into chloroform by. The catalytic esterification activity of modified lipase increased linearly with the increase of its solubility in chloroform.     

Deduced primary structure of a Xenopus proteasome subunit XC3 and expression of its mRNA during early development

Proteasome is a non-lysosomal proteinase complex ubiquitously distributed in eukaryotic cells. We isolated here the cDNA clone for one of the proteasome subunits (XC3) from Xenopus ovary cDNA libraries using rat RC3 cDNA as a prove. The cDNA is 885 bp long and encodes 234 amino acids. The deduced amino acid sequence is highly homologous (95.3%) to those of rat RC3 and human HC3 subunits. The mRNA for XC3 is one of the maternal mRNAs and detected at all the embryonic stages investigated, but its level changes in a characteristic way especially at the gastrula stage. We suggest that the highly conserved XC3 subunit plays an essential role in proteasome function and also that during Xenopus embryogenesis mRNA for XC3 subunit is replaced from maternal to newly-synthesized one probably around the gastrula stage.     

Expression of the pS2 gene in human gastric cancer cells derived from poorly differentiated adenocarcinoma

NH2-terminal amino acid sequence of the pS2 protein produced and secreted by human gastric cancer cells, MKN-45, was determined to be identical to that of MCF-7 cells. A clone encoding pS2 protein was isolated from the cDNA library constructed from MKN-45 cells. The nucleotide sequence was identical to that of pS2 cDNA previously isolated from human breast cancer cells, MCF-7, except for one nucleotide in the 3' untranslated region. Thus, in this cell line, the pS2 gene product is translated and secreted as in MCF-7 cells. RNA blot hybridization analysis revealed that pS2 gene was expressed well in two (MKN-45 and KATO-III; derived from poorly differentiated adenocarcinoma) but not in three cell lines (MKN-1, MKN-28 and MKN-74; from well differentiated adenocarcinoma), suggesting that expression of the pS2 gene depends on the state of cell differentiation. These results suggest that pS2 is expressed in human gastric cancer cells in an estrogen-independent manner and is possibly associated with the malignant state of cells.     

Effect of gastric distention on RNA synthesis in neonatal chick liver.

RNA synthesis in the nuclei of liver from newly hatched chicks was enhanced 1.25 fold at 10 min after intragastric administration of water. Differential inhibition of RNA synthesis by alpha-amanitin indicated that the enhancement mainly represented rRNA synthesis; the synthesis of mRNA and tRNA was scarcely affected. Enhanced RNA synthesis was accompanied by greater susceptibility of nuclei to digestion by micrococcal nuclease, indicating that the chromatin structure was modified. It was further shown that the "water effect" was mimicked by distention of the stomach by raising the pressure in the intragastric balloon. Since the prior administration of atropine abolished the "water effect", the enhancement of hepatic RNA synthesis may be mediated by hepatic nervous system.     

Maintenance of cell proliferation and steroidogenesis in cultured human fetal adrenal cells chronically exposed to adrenocorticotropic hormone: rationalization of in vitro and in vivo findings

Previous studies indicated that acute exposure of adrenal cells to adrenocorticotropic hormone (ACTH) markedly stimulates steroidogenic capacity in vitro but also inhibits cell proliferation. However, in vivo, ACTH is known to stimulate adrenal cell growth. To address this discrepancy, we determined the effect of long-term (9-11 days) continuous or intermittent exposure to ACTH on human fetal adrenal cell proliferation and steroidogenesis. Adrenal glands from fetuses 18-22 wk gestation were studied. Fetal zone cells were plated either on plastic or on an extracellular matrix (ECM) in the presence and absence of basic fibroblast growth factor (bFGF) (0.5 ng/ml) and 1 or 10 nM ACTH. As determined by cell counting, bFGF stimulated cell proliferation during 9 days in culture. In the presence of bFGF, the average doubling time decreased from 44 to 30 h on plastic and from 37 to 26 h on ECM. Under these conditions, ACTH did not inhibit cell proliferation. Proliferation of fetal adrenal corticosteroid-producing cells in the ACTH-treated cultures also was assessed by histochemical staining for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). The number of positive cells increased more than 4-fold between Days 5 and 9 in culture. Continuous treatment with 1 nM ACTH increased dehydroepiandrosterone sulfate (DHAS) production 5- to 10-fold during the first 5 days in culture. Thereafter, the stimulated hormone production decreased over time, although there was still a difference of almost 100-fold between the control and ACTH-treated cultures at the end of 9 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

9 alpha,11 beta-prostaglandin F2 formation in various bovine tissues. Different isozymes of prostaglandin D2 11-ketoreductase, contribution of prostaglandin F synthetase and its cellular localization

9 alpha,11 beta-prostaglandin F2 was formed from prostaglandin D2 by its 11-ketoreductases in 100,000 x g supernatants of various bovine tissues in the presence of an NADPH-generating system. The reductase activities were high in liver (51.09 nmol/h/mg of protein), lung (24.99), and spleen (14.20); moderate in heart and pancreas (3.09-3.61); weak in stomach, intestine, colon, kidney, uterus, adrenal gland, and thymus (0.11-2.63); and undetectable in brain, retina, carotid artery, and blood (less than 0.10). No formation of prostaglandin F2 alpha from prostaglandin D2 was detected in all tissues. In immunotitration analyses with a polyclonal antibody specific for prostaglandin F synthetase, the reductase activities in lung and spleen showed identical titration curves to that of the purified synthetase and decreased to less than 15% of the initial activity under the condition of antibody excess. Prostaglandin F synthetase-immunoreactive protein in these two tissues showed peptide fingerprints identical to that of the purified enzyme after partial digestion with Staphylococcus aureus V8 protease. The antibody was partially cross-reactive to the reductase in liver (about 20% of that to the synthetase) but not to the reductase(s) in other tissues. The Km value for prostaglandin D2 of the reductase activity was the same in lung and spleen as that of the purified prostaglandin F synthetase (120 microM) but differed in liver (6 microM), heart, and pancreas (15 microM). The predominant distribution of prostaglandin F synthetase in lung and spleen was confirmed by radioimmunoassay (2.8 and 1.0 micrograms/mg protein, respectively) and Northern blot analyses. In immunoperoxidase staining, this enzyme was localized in alveolar interstitial cells and nonciliated epithelial cells in lung, histiocytes and/or dendritic cells in spleen, and a few interstitial cells in kidney and adrenal cortex.     

The human ryanodine receptor gene: Its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy

The recent cloning of cDNA encoding the Ca++ release channel (ryanodine receptor) of human sarcoplasmic reticulum has enabled us to use somatic cell hybrids to localize the ryanodine receptor gene (RYR) to the proximal long arm of human chromosome 19. Studies with additional hybrids containing deletions or translocations in chromosome 19 enabled us to localize RYR to 19q13.1 in a region distal to GPI/MAG and proximal to D19S18/DNF11. On the basis that the myotonic dystrophy (DM) locus maps near this region and that myotonia could result from a defect in the ryanodine receptor, we examined the linkage between the DM locus and RYR. Our results, showing several DM-RYR recombinants, rule out an RYR defect as the cause of DM. However, localization of RYR to a region of human chromosome 19 which is syntenic to an area of pig chromosome 6 containing the HAL gene responsible for porcine malignant hyperthermia supports the candidacy of RYR for this disorder.     

Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity.

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

Effects of neuromedin B and GRP-10 on gastrin and insulin release from cultured tumor cells of a malignant gastrinoma

A study relating to gastrin release from gastrinoma cells by neuromedin B and C-terminal decapeptide of gastrin releasing peptide (GRP-10) has not yet been reported. Therefore, we studied the effects of neuromedin B and GRP-10 on gastrin release from cultured dispersed cells prepared from both the primary tumor in the pancreas and the metastatic tumor in the liver from a case of malignant Zollinger-Ellison syndrome. Both the primary and metastatic tumors obtained by a curative operation contained similar concentrations of gastrin and glucagon, whereas the primary tumor contained 10 times more insulin than the metastatic tumor. Gastrin release from cultured cells of both tumors was suppressed by 0.1 and 10 nM neuromedin B and tended to be suppressed by 0.1-10 nM GRP-10. However, insulin release from cultured cells of the pancreatic tumor was stimulated by GRP-10, but not by neuromedin B. These results might suggest that receptor function for the bombesin family peptides is abnormal in gastrinoma cells in both primary and metastatic tumors, and that a major source of insulin secretary cells is the contaminated normal islet cells in the primary tumor.