Salivacin 140, a novel bacteriocin from Lactobacillus salivarius subsp. salicinius T140 active against pathogenic bacteria

K. ARIHARA, S. OGIHARA, T. MUKAI, M. ITOH AND Y. KONDO. 1996. Fifteen of 353 environmental isolates of lactic acid bacteria consistently showed activity against Listeria monocytogenes, Streptococcus mutans, Actinomyces viscosus , and/or Propionibacterium acnes . Strain T140, isolated from the surface of Japanese pampas grass leaves and identified as Lactobacillus salivarius subsp. salicinius , also had activity against several Lactobacillus species, Staphylococcus aureus and Yersinia enterocolitica . Since the antagonistic factor(s) produced by T140 was sensitive to a proteolytic enzyme, it was concluded that a bacteriocin (named salivacin 140) was involved in the inhibition activity. Strain T140 required a high initial pH (7.5–8.5) in agar plates for bacteriocin production.     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.     

Promotion of Zygote Formation by Protein Kinase Inhibitors during the Sexual Development of Dictyostelium mucoroides

To analyze the possible involvement of protein kinases in the sexual development (macrocyst formation) of the cellular slime mold Dictyostelium mucoroides-7 (Dm7), the effects of several protein kinase inhibitors were examined. K252a, a potent inhibitor of protein kinase activities, promoted the sexual cell fusion, through enhancement of gamete formation. In contrast, staurosporine (structurally and functionally similar to K252a) inhibited markedly the progress of development including cell aggregation, thus resulting in the failure of cells to form mature macrocysts. The effective period of K252a was 5–7 hr after starvation, during which Dm7 cells could acquire fusion competence, and the inhibitory effect of cAMP on zygote formation was nullified by the co-application of K252a. Although KT5720 (a specific inhibitor of cAMP-dependent protein kinase) and W7 (a calmodulin inhibitor) had no effects on zygote formation when applied separately, their combined application enhanced zygote formation like K252a did. Neither calphostin C (a specific inhibitor of Ca²⁺-dependent protein kinase) nor herbimycin A (a specific inhibitor of tyrosine kinase) exerted a stimulative influence upon macrocyst formation. These results strongly suggest that the two signal transduction pathways mediated by cAMP-dependent protein kinase (PKA) and calmodulin are closely related to zygote formation, their blockage being favorable to zygote formation.     

Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A₂ inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathioneS-transferase (leukotrienes LTA₄-LTC₄) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked withl-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB₄, LTC₄ and LTE₄). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.     

Suppressive Effect of Liposomes Containing DNA Coding for Diphtheria Toxin A-Chain on Cells Transformed with Bovine Leukemia Virus

A recombinant plasmid which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV-LTR) was constructed to test a novel application of liposomes as antiviral agents. The promoter activity of BLV-LTR was estimated by the chloramphenicol acetyltransferase (CAT) assay using a plasmid which contains the coding sequence of CAT under the control of BLV-LTR (pBLVCAT). When BLV-infected cells were transfected with pBLVCAT, CAT activity was detected. BLV-uninfected cell lines, however, showed no detectable CAT activity. The plasmid DNA entrapped in liposomes was added to BLV-infected cells in culture. Syncytium formation induced by BLV-infected cells was effectively suppressed by the liposomes containing the gene for DT-A under the control of BLV-LTR. Conversely, liposomes containing the gene for DT-A without a promoter showed no such effect. DT-A gene-containing liposomes with BLV-LTR did not affect formation of syncytium induced by bovine immunodeficiency virus. These observations indicate that BLV-infected cells were readily targeted on the level of gene expression. This strategy could be applied to the treatment of BLV-induced B-cell proliferation of cattle, and further to other viral/neoplastic diseases where specific gene expression is exerted.     

A Nitrate-Inducible Plasma Membrane Protein of a Marine Alga, Heterosigma akashiwo

The rate of nitrate uptake by Heterosigma akashiwo cells thathad been cultured in medium with nitrate or ammonium ions asthe source of nitrogen was measured using¹⁵NO_3– The ratioof ¹⁵N/¹⁴N increased dramatically in nitrate-grown cells. Inammonium-grown cells, the ratio of ¹⁵N/¹⁴N did not increasefor 3 h but then it began to increase. Even when nitrate reductaseactivity was inhibited by tungstate, nitrate-grown cells couldtake up nitrate. Plasma membranes from nitrate-grown and ammonium-grown cellswere purified by the silica-microbead method, and polypeptidesassociated with the membranes were analyzed by SDS-PAGE andimmunostaining. A major polypeptide with a molecular mass of26 kDa appeared 3 h after the transfer of ammonium-grown cellsto nitrate-containing medium, and it disappeared 2 d after thetransfer of nitrate-grown cells to ammonium-containing medium.The 26 kDa polypeptide also appeared when cell growth shiftedfrom the logarithmic phase to the stationary phase and the ammoniumcontent of the medium decreased, even when the cells were culturedin ammonium-containing medium. (Received April 10, 1992; Accepted July 30, 1992)     

Identification of 19-nor-deoxycorticosterone in normal rat serum by enzyme immunoassay and gas chromatography-mass spectrometry

Recent reports on the isolation and identification of 19-nor-deoxycorticosterone from the urine of rats with adrenal regeneration hypertension give rise to the possibility of a role of this steroid in the pathogenesis of low renin essential hypertension. The present study was undertaken to investigate the presence of 19-nor-deoxycorticosterone in normal rat serum both by a sensitive enzyme immunoassay and by gas chromatography-mass spectrometry (GC/MS). 19-Nor-deoxycorticosterone in rat serum was separated from other steroids prior to enzyme immunoassay by using high performance liquid chromatography (HPLC). The average concentration of 19-nor-deoxycorticosterone in normal rat serum was 137 +/- 62 ng/dl (mean +/- SD, n = 32). Pooled normal rat serum (50 ml) was purified by HPLC and the purified sample was acetylated with acetic anhydride for GC/MS measurement. The retention time and m/z ions (358, 285, and 257) on the resulting mass fragmentogram coincided in position with those of authentic 21-acetoxy-19-nor-deoxycorticosterone and acetylated normal rat serum extract. The combined characteristics of HPLC elution, antigen-antibody reaction, GC retention and selected ion responses provided strongly evidence supporting the presence of 19-nor-deoxycorticosterone in normal rat serum.     

Molecular Analysis of the Hot Spot Region Related to Length Mutations in Wheat Chloroplast Dnas. I. Nucleotide Divergence of Genes and Intergenic Spacer Regions Located in the Hot Spot Region

The nucleotide divergence of chloroplast DNAs around the hot spot region related to length mutation in Triticum (wheat) and Aegilops was analyzed. DNA sequences (ca. 4.5 kbp) of three chloroplast genome types of wheat complex were compared with one another and with the corresponding region of other grasses. The sequences region contained rbcL and psaI, two open reading frames, and a pseudogene, rpl23' (pseudogene for ribosomal protein L23) disrupted by AT-rich intergic spacer regions. The evolution of these genes in the closely related wheat complex is characterized by nonbiased nucleotide substitutions in terms of being synonymous/nonsynonymous, having A-T pressure transitions over transversions, and frequent changes at the third codon position, in contrast with the gene evolution among more distant plant groups where biased nucleotide substitutions have frequently occurred. The sequences of these genes had diverged almost in proportion to taxonomic distance. The sequence of the pseudogene rpl23' changed approximately two times faster than that of the coding region. Sequence comparison between the pseudogene and its protein-coding counterpart revealed different degrees of nucleotide homology in wheat, rice and maize, suggesting that the transposition timing of the pseudogene differed and/or that different rates of gene conversion operated on the pseudogene in the cpDNA of the three plant groups in Gramineae. The intergenic spacer regions diverged approximately ten times faster than the genes. The divergence of wheat from barley, and that from rice are estimated based on the nucleotide similarity to be 1.5, 10 and 40 million years, respectively.