19F-n.m.r. study of trifluoperazine-S100 protein interaction: effects of Ca2+ and Zn2+

19F-n.m.r. spectra were measured to investigate the effects of Ca2+ and Zn2+ on the interaction of trifluoperazine (TFP) with three S100 proteins. It was found that TFP binds to S100a and S100ao proteins irrespective of the presence of Ca2+ and Zn2+, while in the presence of Ca2+ the apparent affinity of TFP to the proteins was greater than that in its absence or in the presence of Zn2+. In contrast, the binding affinity of TRP to S100b protein in the presence and absence of metal ions was lower than to S100a and S100ao proteins. These results suggested that TFP binds to each S100 protein in two ways: one is Ca2(+)- or Zn2(+)-dependent specific manner and another is Ca2(+)- or Zn2(+)-independent non-specific manner.     

Free Radical Formation by Ultrasound in Aqueous Solutions. A Spin Trapping Study

Our recent spin trapping studies of free radical generation by ultrasound in aqueous solutions are reviewed. The very high temperatures and pressures induced by acoustic cavitation in collapsing gas bubbles in aqueous solutions exposed to ultrasound lead to the thermal dissociation of water vapor into H atoms and OH radicals. Their formation has been confirmed by spin trapping. Sonochemical reactions occur in the gas phase (pyrolysis reactions), in the gas-liquid interfacial region, and in the bulk of the solution (radiation-chemistry reactions). The high temperature gradients in the interfacial regions lead to pyrolysis products from non-volatile solutes present at sufficiently high concentrations. The sonochemically generated radicals from carboxylic acids, amino acids, dipeptides. sugars, pyrimidine bases. nucleosides and nucleo-tides were identified by spin trapping with the non-volatile spin trap 3.5-dibromo-2.6-dideuterio-4-nitrosobenzenesulfonate. At low concentrations of the non-volatile solutes. the spin-trapped radicals produced by sonolysis are due to H atom and OH radical reactions. At higher concentrations of these non-volatile solutes, sonolysis leads to the formation of additional radicals due to pyrolysis processes (typically methyl radicals). A preferred localization of non-volatile surfactants (compared to analogous non-surfactant solutes) was demonstrated by the detection of pyrolysis radicals at 500-fold lower concentrations. Pyrolysis radicals were also found in the sonolysis of aqueous solutions containing only certain nitrone spin traps. The more hydrophobic the spin trap, the lower the concentration at which the pyrolysis radicals can be observed. The effect of varying the temperature of collapsing transient cavities in aqueous solutions of different rare gases and of N₂O on radical yields and on cell lysis of mammalian cells was investigated.     

Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 μMCdCl₂did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

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.     

The Role in Ozone Phytotoxicity of the Evolution of Ethylene upon Induction of 1-Aminocydopropane-1-carboxylic Acid Synthase by Ozone Fumigation in Tomato Plants

The rate of evolution of ethylene by tomato plants was rapidlyincreased by O₃ fumigation. The time course of the increasein 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activitywas the same as that in the rate of evolution of ethylene, suggestingthat ACC synthase activity might be a rate-limiting step inthe evolution of ethylene that is caused by O₃ fumigation. Therate of the O₃-induced evolution of ethylene was increased bythe application of ACC to tomato plants, suggesting the involvementof ACC oxidase in the O₃-induced evolution of ethylene. Treatmentof plants with tiron inhibited the evolution of ethane, butnot of ethylene. These results indicated that evolution of ethylenein O₃-treated tomato plants might result from enzymatic reactionscatalyzed by both ACC synthase and ACC oxidase, but not fromstimulation by O₃ of the peroxidation of lipids mediated byfree radicals. Pretreatment of leaves with aminoethoxyvinylglycine (AVG), aninhibitor of ACC synthase, significantly inhibited the evolutionof ethylene that was induced by O₃ and concomitantly reducedthe extent of O₃-induced visible damage to leaves. Treatmentwith 2,5-norbonadiene, an inhibitor of the action of ethylene,strongly reduced the extent of visible damage caused by O₃,even though it did not suppress the evloution of ethylene. Theseresults indicate that ethylene acts on certain metabolic processesto cause visible damage. (Received September 7, 1995; Accepted December 18, 1995)     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG immunoglobulin G - IPTG isopropylthio--d-galactoside - PAL phenylalanine ammonia-lyase The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008).