Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.     

Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.     

Tumor necrosis factor antagonizes inhibitory effect of azidothymidine on human immunodeficiency virus (HIV) replication in vitro.

Tumor necrosis factor alpha (TNF-alpha) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-1) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-1, increased from 5.8 nM in the absence of TNF-alpha to greater than 125 microM in the presence of TNF-alpha (100 U/ml). TNF-alpha also antagonized the anti-HIV-1 activity of dideoxycytidine but did not markedly affect the anti-HIV-1 activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-alpha.     

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.     

Involvement of protein kinase C in prostaglandin E2-induced catecholamine release from cultured bovine adrenal chromaffin cells

We recently reported that prostaglandin (PG) E2 stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain induced a gradual secretion of catecholamines from the cells (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). Here we examined the involvement of two signal pathways, Ca2+ mobilization and protein kinase C activation resulting from phosphoinositide metabolism, in the PGE2-induced catecholamine release. Either the Ca2+ ionophore ionomycin or 12-O-tetradecanoylphorbol 13-acetate (TPA) could enhance the release in the presence of ouabain, and ionomycin-induced release was additive to PGE2-induced release, but TPA-induced release was not additive. PGE2 dose-dependently stimulated the formation of diacylglycerol and caused the translocation of 4% of the total protein kinase C activity to become membrane-bound within 5 min. These effects were specific for PGE2 and PGE1 among PGs tested (PGE2 = PGE1 greater than PGF2 alpha greater than PGD2). Furthermore, the phosphoinositide-specific phospholipase C inhibitor neomycin inhibited PGE2-induced accumulation of inositol phosphates, diacylglycerol formation, translocation of protein kinase C, and also stimulation of catecholamine release. Both PGE2- and TPA-induced release were inhibited by the depletion of protein kinase C caused by prolonged exposure to TPA, but ionomycin-induced release was not inhibited. We recently found that the amiloride-sensitive Na+, H+-antiport participates in PGE2-evoked catecholamine release (Tanaka, T., Yokohama, H., Negishi, M., Hayashi, H., Ito, S., and Hayaishi, O. (1990) J. Neurochem. 54, 86-95). In agreement with our recent report, PGE2 and TPA induced a sustained increase in intracellular pH that was abolished by the protein kinase C inhibitor staurosporine but not by the calmodulin inhibitor W-7. Ionomycin also induced a marked increase in intracellular pH, but this increase was abolished by W-7 but not by staurosporine. These results demonstrate that PGE2-induced activation of the Na+, H(+)-antiport and catecholamine release in the presence of ouabain are mediated by activation of protein kinase C, rather than by Ca2+ mobilization, resulting from phosphoinositide metabolism.     

Angeloylcumambrin-B,an antimicrobial sesquiterpene lactone from Chrysanthemum ornatum var. Spontaneum

Angeloylcumambrin-B, a new antimicrobial guaianolide sesquiterpene lactone, was isolated from Chrysanthemum ornatum and the structure was determined by a combination of chemical and physical methods.     

Seven sesquiterpene lactones from Inula britannica var. chinensis

Four known sesquiterpene lactones, tomentosin, ivalin, 4-epi-isoinuviscolide and gaillardin, together with three new lactones, inuchinenolides A, B and C, were identified in the whole plant of Inula britannica var. chinensis.     

Protein localization in E. coli: Is there a common step in the secretion of periplasmic and outer-membrane proteins?

An E. coli strain carrying a fusion of the malE and lacZ genes is induced for the synthesis of a hybrid protein, consisting of the N-terminal part of the maltose-binding protein and the enzymatically active C-terminal part of β-galactosidase, by addition of maltose to cells. The secretion of the protein is initiated by the signal peptide attached to the N terminus of the maltose-binding protein sequence, but is not completed, presumably because the β-galactosidase moiety of the hybrid protein interferes with the passage of the polypeptide through the cytoplasmic membrane. Thus the protein becomes stuck to the cytoplasmic membrane. Under such conditions, periplasmic proteins, including maltose-binding protein (encoded by the malE gene) and alkaline phosphatase, and the major outer-membrane proteins, including OmpF, OmpA and probably lipoprotein, are synthesized as precursor forms with unprocessed signal sequences. This effect is observed within 15 min after high levels of induction are achieved. The simplest explanation for these results and those of pulse-chase experiments is that specific sites in the cytoplasmic membrane become progressively occupied by the hybrid protein, resulting in an inhibition of normal localization and processing of periplasmic and outer-membrane proteins. These results suggest that most of the periplasmic and outer-membrane proteins share a common step in localization before the polypeptide becomes accessible to the processing enzyme. If this interpretation is correct, we can estimate that an E. coli cell has roughly 2 × 10⁴ such sites in the cytoplasmic membrane. A system is described for detecting the precursor of any exported protein.