A role for nickel in osmotic adjustment in drought-stressed plants of the nickel hyperaccumulator <Emphasis Type="Italic">Stackhousia tryonii</Emphasis> Bailey

The hypothesis that hyperaccumulation of certain metals in plants may play a role in osmotic adjustment under water stress (drought) was tested in the context of nickel hyperaccumulator Stackhousia tryonii. Field-collected mature plants of S. tryonii, grown in native ultramafic soil, were pruned to soil level and the re-growth exposed to five levels of water stress (20, 40, 60, 80 and 100% field capacity; FC) for 20 weeks. Water stress had significant (P<0.05) influence on growth (biomass), water potential and shoot Ni concentrations, with progressively more impact as water stress was increased from 80 to 40% FC. Shoot Ni concentration increased significantly from 3,400 μg g⁻¹ dry weight (at 100% FC) to 9,400 μg g⁻¹ dry weight (at 20% FC). Assuming that Ni is uniformly distributed through the shoot tissue, the Ni concentration could account for 100% at the 80 and 60% FC conditions, and 50% at the 40 and 20% FC conditions of plant osmotic regulation. The results are consistent with a role of Ni in osmotic adjustment and protection of S. tryonii plants against drought.     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.     

Protective effect of 3,5,3′-triiodothyroacetic and 3,5,3′,5′-tetraiodothyroacetic acids on serum albumin fibrillation

Inhibition or reversion of protein self-aggregation has been suggested as a possible preventive mechanism against amyloid diseases, and many efforts are underway to found out molecules capable to restrain the protein aggregation process. In this paper, the inhibitory effects of thyroid hormone analogues on heat-induced fibrillation process of serum albumin are reported. Among the analogues tested, 3,5,3′,5′-tetraiodothyroacetic and 3,5,3′-triiodothyroacetic acid showed the most important inhibitory effects on amyloid formation. Thyroxine exhibits a lesser protective effect, while 3,5,3′-triiodothyronine showed no significant inhibition. The gaining of a negative charge together with a size reduction of the hormone molecule could play an essential role in the inhibition of fibrils formation. According to infrared spectroscopy results, the thyroid hormones analogues protective effects proceed via the stabilization of the protein native structure. The current work demonstrates the effectiveness of naturally occurring molecules in the inhibition of albumin fibril formation.     

Protective immunity of <Emphasis Type="Italic">E. coli</Emphasis>-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.     

Anti-HIV-1 efficacy of extracts from medicinal plants

The anti-HIV-1 activities of butanol, hexane, chloroform and water extracts from four widely used folk medicinal plants (Sophora flavescens, Tulipa edulis, Herba ephedra, and Pachyma hoelen Rumph) were evaluated in this study. The hexane extract of Pachyma hoelen Rumph, PH-4, showed effective inhibition against HIV-1. The 50% effective concentration (EC₅₀) of PH-4 was 37.3 μg/ml in the p24 antigen assay and 36.8% in the HIV-1 recombinant RT activity test (at 200 μg/ml). In addition, the PH-4 showed the protective effect on the infected MT-4 cells, with a 58.2% rate of protection. The 50% cytotoxic concentration (CC₅₀) of PH-4 was 100.6 μg/ml. These results suggest that PH-4 from Pachyma hoelen Rumph might be the candidate for the chemotherapy agent against HIV-1 infection with further study.     

Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.     

Do soil aggregates really protect encapsulated organic matter against microbial decomposition?

Soil aggregates can provide an effective protection of organic matter against microbial decomposition as reported by several macroaggregate disruption studies. However, research on the role of aggregation for carbon mineralization was mainly focused on arable soils. In the present study we aim to clarify the impact of aggregation on organic matter protection by measuring carbon mineralization in terms of microbial respiration rates of intact macroaggregates (2–4 and 4–8 mm) and corresponding crushed aggregates from seven topsoil horizons from both arable and forest sites. For two arable and one forest soil we found a significantly (P < 0.001) lower carbon mineralization from intact aggregates as compared to the corresponding crushed material. The portion of aggregate protected carbon reached up to 30% for a grassland soil. For the other arable and forest soils no significant effect of aggregation was found. Similarly, no clear trend could be found for the protective capacity of different size fractions. We conclude that protection by aggregation is effective primarily for soils with a large pool of labile organic matter regardless of their usage as arable land or forest.     

Biocontrol Ability of <Emphasis Type="Italic">Lysobacter antibioticus</Emphasis> HS124 Against <Emphasis Type="Italic">Phytophthora</Emphasis> Blight Is Mediated by the Production of 4-Hydroxyphenylacetic Acid and Several Lytic Enzymes

Several rhizobacteria play a vital role in plant protection, plant growth promotion and the improvement of soil health. In this study, we have isolated a strain of Lysobacter antibioticus HS124 from rhizosphere and demonstrate its antifungal activity against various pathogens including Phytophthora capsici, a destructive pathogen of pepper plants. L. antibioticus HS124 produced lytic enzymes such as chitinase, β-1,3-glucanase, lipase, protease, and an antibiotic compound. This antibiotic compound was purified by diaion HP-20, silica gel, sephadex LH-20 column chromatography and high performance liquid chromatography. The purified compound was identified as 4-hydroxyphenylacetic acid by gas chromatography-electron ionization (GC-EI) and gas chromatography-chemical ionization (GC-CI) mass spectrometry. This antibiotic exhibited destructive activity toward P. capsici hyphae. In vivo experiments utilizing green house grown pepper plants demonstrated the protective effect of L. antibioticus HS124 against P. capsici. The growth of pepper plants treated with L. antibioticus culture was enhanced, resulting in greater protection from fungal disease. Optimum growth and protection was found when cultures were grown in presence of Fe(III). Additionally, the activities of pathogenesis-related proteins such as chitinase and β-1,3-glucanase decreased in roots, but increased in leaves with time after treatment compared to controls. Our results demonstrate L. antibioticus HS124 as a promising candidate for biocontrol of P. capsici in pepper plants.     

Commercial baker's yeast stability as affected by intracellular content of trehalose, dehydration procedure and the physical properties of external matrices

The effects of vacuum-drying and freeze- drying on the cell viability of a commercial baker's yeast, Saccharomyces cerevisiae, strain with different endogenous contents of trehalose were analyzed. An osmotolerant Zygosaccharomyces rouxii strain was used for comparative purposes. Higher viability values were observed in cells after vacuum-drying than after freeze-drying. Internal concentrations of trehalose in the range 10–20% protected cells in both dehydration processes. Endogenous trehalose concentrations did not affect the water sorption isotherm nor the T _g values. The effect of external matrices of trehalose and maltodextrin was also studied. The addition of external trehalose improved the survival of S. cerevisiae cells containing 5% internal trehalose during dehydration. Maltodextrin (1.8 kDa) failed to protect vacuum-dried samples at 40 °C. The major reduction in the viability during the freeze-drying process of the sensitive yeast cells studied was attributed to the freezing step. The suggested protective mechanisms for each particular system are vitrification and the specific interactions of trehalose with membranes and/or proteins. The failure of maltodextrins to protect cells was attributed to the fact that none of the suggested mechanisms of protection could operate in these systems. Received: 6 December 1999 / Received revision: 8 May 2000 / Accepted: 19 May 2000     

Clovamide-rich extract from <Emphasis Type="Italic">Trifolium pallidum</Emphasis> reduces oxidative stress-induced damage to blood platelets and plasma

Numerous plants (including clovers) have been widely used in folk medicine for the treatment of different disorders. This in vitro study was designed to examine the antioxidative effects of the clovamide-rich fraction, obtained from aerial parts of Trifolium pallidum, in the protection of blood platelets and plasma against the nitrative and oxidative damage, caused by peroxynitrite (ONOO⁻). Carbonyl groups and 3-nitrotyrosine in blood platelet and plasma proteins were determined by ELISA tests. Thiol groups level was estimated by using 5,5′-dithio-bis(2-nitro-benzoic acid, DTNB). Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric acid reactive substances. The results from our work indicate that clovamide-rich T. pallidum extract may reveal the protective properties in the prevention against oxidative stress. The presence of clovamide-rich T. pallidum extract (12.5–100 μg/ml) partly inhibited ONOO⁻-mediated protein carbonylation and nitration. All the used concentrations of T. pallidum extract reduced lipid peroxidation in plasma. The antioxidative action of the tested extract in the protection of blood platelet lipids was less effective; the extract at the lowest final concentration (12.5 μg/ml) had no protective effect against lipid peroxidation. The present results indicate that the extract from T. pallidum is likely to be a source of compounds with the antioxidative properties, useful in the prevention against the oxidative stress-related diseases.     

Interaction of a functionalized complex of the flavonoid hesperetin with the AhR pathway and CYP1A1 expression: involvement in its protective effects against benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene-induced oxidative stress in human skin

Skin cancer pathogenesis is partially associated to the oxidative stress conditions induced by environmentally carcinogens such as benzo[a]pyrene (BaP). The protective effects against BaP-induced oxidative stress of the flavonoid hesperetin as a complex with the 2-hydroxypropyl-β-cyclodextrin (HE/HP-β-CyD) have been evaluated using an ex vivo human skin model. Human healthy skin has been pre-treated with the functionalized complex HE/HP-β-CyD (0.5–50 μM) before BaP (5 μM) application simulating occupational and environmental exposure. Oxidative stress was evaluated in terms of 3-(4, 5-dimethylthiazol−2-yl)-2, 5-dipheyltetrazolium bromide reduction, protein peroxidation and reactive oxygen species (ROS) formation. Additionally, it has been investigated whether the potential protective effects of HE/HP-β-CyD may be correlated to the interaction with aryl hydrocarbon receptor (AhR) pathway. A significant protection by HE/HP-β-CyD against the BaP-induced increase in ROS and carbonyl compound production, as well as reduction in tissue viability, has been observed (p < 0.001). Results obtained showed that HE/HP-β-CyD was also able to reduce BaP-induced AhR and CYP1A1 protein expression (p < 0.001). Experimental evidences provided from this study suggest significant preventive properties of HE/HP-β-CyD in the toxicity caused by environmental carcinogens such as PAHs.     

The influence of melatonin and N-acetylcysteine in δ-aminolevulinic acid and lead induced genotoxicity in lymphocytes in vitro

As is well known from earlier studies, the genotoxic effect of lead exposure was partly attributed to the formation of the highly reactive oxygen metabolites (ROMs) in the blood. However, lead ions have no ability to generate ROMs. Therefore, the recently published studies paid more attention to the role of δ-aminolevulinic acid (ALA) accumulation in lead-induced DNA damage. If the above-mentioned assumptions were taken into consideration, it seemed a reasonable approach to study the possible protective effects of antioxidants against genotoxic effects of lead. According to our results, N-acetylcysteine (NAC) and melatonin (MEL) were able to reduce significantly (p < 0.05) the lead- and ALA-induced sister chromatid exchange frequencies in human lymphocytes in vitro. In spite of a relative reduction in the lead- and ALA-induced micronucleus formation in human lymphocytes, the reduction was not statistically significant (p > 0.05). These results could be evaluated as supportive evidence for the hypothesis that increased antioxidant capacity of cells might fortify the efficiency of protective pathways against cytogenetic damage in lead exposure.     

Gamma radiation induced mutagenesis in <Emphasis Type="Italic">Aspergillus niger</Emphasis> to enhance its microbial fermentation activity for industrial enzyme production

α- and β-Galactosidases find application in food processing, health and nutrition. Aspergillus niger is one of the potent producer of these enzymes and was genotypically improved using gamma-ray induced mutagenesis. The mutant-derivative produced two-fold higher α- and β-galactosidases. For testing genetic variability and its relationship with phenotypic properties of the two organisms, DNA samples of the mutant and parental strains of A. niger were amplified with 28 deca-nucleotide synthetic primers. RAPD analysis showed significantly different pattern between parental and mutant cultures. The mutant derivative yielded homogeneous while parental strain formed heterogeneous amplification patterns. Seven primers identified 42.9% polymorphism in the amplification products, indicating that these primers determined some genetic variability between the two strains. Thus RAPD was found to be an efficient technique to determine genetic variability in the mutant and wild organisms. Both wild and mutant strains were analyzed for their potential to produce galactosidases. Comparison of different carbon sources on enzyme yield revealed that wheat bran is significant (P < 0.01) effective producer and economical source followed by rice bran, rice polishing and lactose. The mutant was significantly better enzyme producer and could be considered for its prospective application in food, nutrition and health and that RAPD can be effectively used to differentiate mutant strain from the parental strain based on the RAPD patterns.     

Anticonvulsant and Antioxidant Effects of Cyano-carvone and Its Action on Acetylcholinesterase Activity in Mice Hippocampus

The anticonvulsant effect of cyano-carvone, a monoterpene monocyclic, was investigated in epilepsy model induced by pilocarpine. Cyano-carvone at doses of 25, 50 or 75 mg/kg promoted a reduction of 16.7, 33 and 66.7%, respectively, against pilocarpine-induced seizures, and it was efficacious in increasing both the latency to first seizures and the survival percentage, resulting in 33.3, 67 and 91.7% of protection against death induced by seizures, respectively (P < 0.05). The reference drug atropine (25 mg/kg) also produced a significant protection (100%). Its monoterpene, at 25, 50 and 75 mg/kg, was also capable to increase the latency for installation of status epilepticus induced by pilocarpine, and presented a significant protection against lipid peroxidation and nitrite formation in mice hippocampus (P < 0.05). In addition, it was observed that the cyano-carvone pretreatment increased the acetylcholinesterase activity in mice hippocampus after pilocarpine-induced seizures. The present results clearly indicate the anticonvulsant ability of cyano-carvone, which can be, at least in part, explained by the increased activity of the acetylcholinesterase enzyme. Our data suggest that the action mechanism can also be due to a direct activation of the antioxidant enzymes that could be associated with a reduction observed in oxidative stress in mice hippocampus, probably involving an inhibition of free radical production.     

Intranasally administered Lactobacillus gasseri TMC0356 protects mice from H1N1 influenza virus infection by stimulating respiratory immune responses

We conducted a study to evaluate the possibility that intranasal administration of a new probiotic strain Lactobacillus gasseri TMC0356 (TMC0356) may protect host animals from influenza virus (IFV) infection, which was indicated by enhanced respiratory immune responses in a mouse model. After 3 days of exposure to TMC0356, BALB/c mice were intranasally infected with IFVA/PR/8/34 (H1N1). Lung cells were isolated from the tested mice and evaluated for cytotoxicity against YAC-1 cells. After intranasal treatment with TMC0356, mice showed a lower morbidity and higher survival rate compared to control mice (P < 0.05). The cytotoxicity of lung cells isolated from mice after intranasal treatment against YAC-1 cells was statistically higher than that of lung cells isolated from control mice (P < 0.05). Intranasal administration of TMC0356 significantly increased mRNA expression of interleukin (IL)-1β, tumor necrosis factor, IL-10, and monocyte chemotactic protein-1 (P < 0.01). These results suggest that intranasal administration of TMC0356 may protect the host animal from IFV infection. They also indicate that TMC0356 can enhance respiratory cell-mediated immune responses of host animals characteristically with up-regulated activation of lung natural killer cells. Further studies will evaluate the possible role of the immune stimulatory effects of TMC0356 within the protective effects of this bacterium against IFV, as observed in the present study.     

Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

Background  

DNA polymerase ε (Pol ε) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect.     

Antimutagenic activity of wheat β-purothionin Tk-AMP-BP

Antimutagenic activity on human RD cells was studied for β-purothionin Tk-AMP-BP isolated from seeds of wheat Triticum kiharae, which has high stress resistance. Cadmium chloride at 5 × 10⁻⁶ M was used as a mutagen. The numbers of DNA breaks in mutagen-treated and intact cells were inferred from the single-stranded to double-stranded DNA ratio and expressed as protection coefficients. The protective effect was simultaneously assayed for aqueous plant extracts known to possess antioxidant properties. Wheat thionin was the most active among all of the antimutagens examined; its protection coefficient reached 85–88% at micromolar peptide concentrations (8–32 μg/ml). Thus, wheat β-purothionin was for the first time demonstrated to be highly efficient in protecting human cell DNA from the damaging effect of cadmium chloride.     

Immunization with murine breast cancer cells treated with antisense oligodeoxynucleotides to type I insulin-like growth factor receptor induced an antitumoral effect mediated by a CD8+ response involving Fas/Fas ligand cytotoxic pathway

We have demonstrated that in vivo administration of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to type I insulin-like growth factor receptor (IGF-IR) mRNA resulted in inhibition of C4HD breast cancer growth in BALB/c mice. The present study focused on whether in vivo administration of C4HD tumor cells pretreated with IGF-IR AS[S]ODN and irradiated could provide protection against C4HD wild-type tumor challenge and also on elucidating the mechanism mediating this effect. Our results showed that mice immunized with IGF-IR AS[S]ODN-treated C4HD cells experienced a growth inhibition of 53.4%, 61.6%, and 60.2% when compared with PBS-treated mice, wild-type C4HD cell-injected mice, or phosphorothioate sense oligodeoxynucleotide-treated C4HD cell-injected mice, respectively. The protective effect was C4HD-specific, because no cross-protection was observed against other syngeneic mammary tumor lines. The lack of protection against tumor formation in nude mice indicated that T cells were involved in the antitumoral response. Furthermore, cytotoxicity and splenocyte proliferation assays demonstrated that a cellular CD8(+)-dependent immune response, acting through the Fas/Fas ligand death pathway, could be mediating the antitumor effect induced by immunization with AS[S]ODN-treated cells. Immunization also induced splenocytes to produce Ag-dependent IFN-gamma, indicating the presence of a type 1 response. We demonstrated for the first time that IGF-IR AS[S]ODN treatment of breast cancer cells induced expression of CD86 and heat shock protein 70 molecules, both involved in the induction of the immunogenic phenotype. Immunization with these tumor immunogens imparted protection against parental tumor growth through activation of a specific immune response.     

Microcystin-induced 8-hydroxydeoxyguanosine in DNA and its reduction by melatonin, vitamin C, and vitamin E in mice

Microcystin LR (MC-LR), a liver-specific toxin synthesized by Microcystis aeruginosa, was investigated. MC-LR initiated reactive oxygen species formation followed by damaging DNA and some other cellular components. We investigated the ability of MC-LR to induce oxidative DNA damage by examining the formation of 8-hydroxydeoxyguanosine (8-OH-dG) using HPLC with electrochemical detection. Melatonin, vitamin C (ascorbate), and vitamin E (as Trolox), all of which are free radical scavengers, markedly inhibited the formation of 8-OH-dG in a concentration-dependent manner. The concentration that reduced DNA damage by 50% (IC₅₀) was 0.55, 31.4, and 36.8 μM for melatonin, ascorbate, and Trolox, respectively. The results show that melatonin is 60-and 70-fold more effective than vitamin C or vitamin E, respectively, in reducing oxidative DNA damage. These findings are consistent with the conclusion that melatonin’s highly protective effect against microcystin toxicity relates, at least in part, to its direct hydroxyl radical scavenging ability. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 10, pp. 1377–1382.