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.     

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.     

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)     

In Vitro Bleaching of Hardwood Kraft Pulp by Extracellular Enzymes Excreted from White Rot Fungi in a Cultivation System Using a Membrane Filter

To clarify the role of excreted extracellular enzymes during long-term incubation in a pulp biobleaching system with white rot fungi, we developed a cultivation system in which a membrane filter is used; this membrane filter can prevent direct contact between hyphae and kraft pulp, but allows extracellular enzymes to attack the kraft pulp. Phanerochaete sordida YK-624 brightened the pulp 21.4 points to 54.0% brightness after a 5-day in vitro treatment; this value was significantly higher than the values obtained with Phanerochaete chrysosporium and Coriolus versicolor after a 7-day treatment. Our results indicate that cell-free, membrane-filtered components from the in vitro bleaching system are capable of delignifying unbleached kraft pulp. Obvious candidates for filterable reagents capable of delignifying and bleaching kraft pulp are peroxidase and phenoloxidase proteins. The level of secreted manganese peroxidase activity in the filterable components was substantial during strain YK-624 in vitro bleaching. A positive correlation between the level of manganese peroxidase and brightening of the pulp was observed.     

Antioxidant Properties of Bromocriptine, a Dopamine Agonist

Abstract: It has been suggested that free radicals may adversely influence the pathogenesis of Parkinson's disease. We conducted this study to determine whether bro-mocriptine, an agent widely used for treating parkinsonism, possesses antioxidant effects. Bromocriptine scavenged superoxide produced from a superoxide generating system (hypoxanthine-xanthine oxidase) by the spin-trapping method using electron spin resonance. Bromocriptine had a strong scavenging effect on the 5,5-dimethyl-1-pyrroline- N -oxide hydroxide signal produced from Fenton's reaction. Bromocriptine also attenuated the stable free radical diphenyl- p -picrylhydrazyl signal. This drug inhibited the autooxidation of rat brain homogenates in a dose-dependent manner in vitro. Autooxidation of brain homogenates collected from rats treated with bromocriptine (2.5 mg/kg, i.p., daily for 3 days) was significantly reduced as compared with values in untreated rat homogenates. These observations suggest that bromocriptine is a free radical scavenger and a potent antioxidant.     

Microheterogeneity of PSI-E Subunit of Photosystem I in Nicotiana sylvestris

Microheterogeneity of a photosystem I (PSI) subunit encodedby a nuclear gene psaE was examined in Nicotiana sylvestris,with the aid of cDNA cloning, peptide mapping analysis and proteinsequencing. The psaE product of this plant has four isoformswhose mobilities in PAGE are slightly different from each other.We isolated two types of psaE cDNAs from a N. sylvestris cDNAlibrary, and designated the corresponding genes as psaEa andpsaEb, respectively. The psaEa and psaEb genes are 77% homologousat DNA level, and their translation products share 80.4% homologyfor the precursor proteins and 89.1% for the mature forms. Comparativeanalysis of the four isoproteins and the putative products ofthe two psaE genes revealed that two isoproteins out of fourare derived from psaEa gene, and the difference between thesetwo isoproteins lies in the respective presence or absence ofN-terminal alanine. Likewise, the other two proteins are derivedfrom psaEb with similar N-terminal heterogeneity. These resultsindicate that multi-gene organization and heterogeneous N-terminalformation at post-translational level are two possible causesfor PSI subunit polymorphism in isogenic plant lines. (Received October 8, 1993; Accepted November 30, 1993)     

Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana

Two cDNA clones, AATCDPK1 and cATCDPK2, encoding Ca²⁺-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51 % and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca²⁺-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca²⁺ for activation.