Reversal of CD45R isoform switching in CD8+ T cells.

Both CD4+ and CD8+ T cells express either CD45RA or CD45R0 isoform of CD45R in an exclusive way. Recent reports have shown that CD45RA+ T cells lose CD45RA and gain CD45R0 upon activation. This switching has been suggested to be irreversible although more recently, examples of reversal of CD45R isotype switching in CD4+ T cells have been reported. We report here that freshly isolated unprimed CD8+ T cells, when activated with PHA, temporarily lose CD45RA but reexpress an intermediate level of CD45RA 2-3 weeks after activation with PHA. This reversal seems to take place much more slowly in unprimed CD4+ T cells: the majority of CD4+ T cells that had lost CD45RA and gained CD45R0 remained CD45RA-CD45R0+ in 3 weeks after the stimulation. Also, long-term CD8+ CD45RA+ T cell lines stimulated with PHA or OKT3 showed even more rapid recovery of CD45RA while PPD-specific CD4+ T cell clones retained the original CD45R0 phenotype 3 weeks after stimulation with PPD or PHA.     

EPR signals of cytochrome b558 purified from porcine neutrophils.

Cytochrome b558 of pig blood neutrophils was partially purified, and its EPR spectra were measured. The cytochrome b558 was solubilized from membranes with the detergent n-heptyl-beta-thioglucoside and purified by DEAE-Sepharose and heparin-Sepharose chromatographies. The small and large subunits of cytochrome b558 were detected on gel by immunoblotting. A solution of the purified, undenatured cytochrome b558 at 85-108 microM concentration was obtained. The concentrated cytochrome b558 showed an EPR signal at a g value of 3.26 with a bandwidth of 100 G at 10 K. Addition of 2 mM KCN had no effect on the low spin signal at g = 3.26 but caused disappearance of a minor high spin signal. The cyanide-insensitive signal at g = 3.26 disappeared completely on reduction with Na2S2O4. These results suggest that the g = 3.26 signal is characteristic of the low spin heme in cytochrome b558 of neutrophils.     

Tissue factor potentiates the factor VIIa-catalyzed hydrolysis of an ester substrate.

We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The Km value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.     

cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding

Suspension-cultured cells of carrot (Daucus carota L.) synthesize and secrete a glycoprotein that is normally found only in dermal tissues (epidermis, endodermis and periderm). This protein, previously called GP57, is now referred to as EDGP (E xtracellular D ermal G lyco P rotein). We purified sufficient quantities of EDGP to obtain amino-acid sequences on two internal tryptic peptides and screened a cDNA library of young carrot roots with antiserum to EDGP and with oligonucleotides corresponding to the peptides. Here we report the derived amino-acid sequence of EDGP. Sequence comparisons show that it has 40% amino-acid sequence identity with 7S basic globulin, a protein that is released when soybean seeds are soaked in hot water for a few hours. We suggest that these two proteins belong to a new family of dermal proteins. As far as we know, this is the first reported derived amino-acid sequence for protein that is specific to the epidermis and other dermal tissues. The level of EDGP mRNA is low in dry seeds, but increases rapidly in growing seedlings as they develop dermal tissues. The level of mRNA is low in storage roots, but increases rapidly in response to wounding. The presence of EDGP in dermal tissues and its up-regulation in response to wounding indicate a role in the response of plants to biotic and-or abiotic stresses. An unusual feature of the amino-acid sequence of EDGP is that it contains a short motif, which is present at the active site of aspartyl proteases such as pepsin and chymosin.Abbreviations cDNA copy DNA - 2,4-D 2,4-dichlorophen-oxyacetic acid - EDGP extracellular dermal glycoprotein - 7SBG 7S basic globulin Supported by a contract from the United States Department of Energy (Energy Biosciences) (to M.J.C.) and a Grant-in-Aid for Special Research on Priority Areas (01660002, Cellular and Molecular Basis for Reproductive Processes in Plants) from the Ministry of Education, Science and Culture, and by the Fund from Basic Research Core System of Science and Technology Agency, Japan (to S.S.).     

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)