A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Cardioprotective Effects of an Aminothiazole Compound on Isoproterenol-Induced Myocardial Injury in Mice

Lactate dehydrogenase (LDH), marker of anaerobic metabolism, is associated with highly invasive and metastatic breast cancer. Novel studies show that increased anaerobic metabolism (LDH), as well as activity of antioxidative enzymes (superoxide dismutase (SOD) and catalase (CAT)), is correlated with higher mammographic density, as known predictor of breast cancer risk. In this study, we measured LDH, MDH, and SOD activity in tumor and adjacent tissues of breast cancer patients by spectrophotometric assay. Mammograms were evaluated according to the American College of Radiology Breast Imaging Reporting and Data system. Mammographically dense breast tissue is associated with higher activity of LDH in tumor tissue of breast cancer patients. Moreover, patients with masses have significantly higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Patients with spiculated mass margin had higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Activity of LDH in patients significantly increases, while activity of CAT significantly decreases with the increase of BIRADS category. These results suggest that the association of activity of LDH and CAT in tumor tissue with mammographic characteristics could help in defining aggressive breast cancers.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Nicotinamide (vitamin B3) as an effective antioxidant against oxidative damage in rat brain mitochondria

Nicotinamide (vitamin B3) an endogenous metabolite, showed significant inhibition of oxidative damage induced by reactive oxygen species (ROS) generated by ascorbate-Fe2+ and photosensitization systems in rat brain mitochondria. It protected against both protein oxidation and lipid peroxidation, at millimolar concentrations. Inhibition was more pronounced against oxidation of proteins than peroxidation of lipids. Chemically related endogenous compounds, tryptophan and isonicotinic acid, showed comparable inhibitory properties. The protective effect observed, at biologically relevant concentrations, with nicotinamide was more than that of the endogenous antioxidants ascorbic acid and alpha-tocopherol. Hence our studies suggest that nicotinamide (vitamin B3) can be considered as a potent antioxidant capable of protecting the cellular membranes in brain, which is highly susceptible to prooxidants, against oxidative damage induced by ROS.     

DNA protective properties of vanillin against gamma-radiation under different conditions: possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of gamma-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to gamma-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of gamma-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, 'tail moment' and 'Olive tail moment'. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to gamma-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS*(+) radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against gamma-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

Enhanced one-carbon flux towards DNA methylation: Effect of dietary methyl supplements against γ-radiation-induced epigenetic modifications

Radiation exposure poses a major risk for workers in the nuclear power plants and other radiation related industry. In this context, we demonstrate that γ-radiation is an efficient DNA demethylating agent and its injurious effect can be minimized by dietary methyl supplements (folate, choline and vitamin B12). To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on normal control diet (NCD) and methyl-supplemented diet (MSD). After 2 weeks of NCD and MSD dietary regimen, we exposed the animals to γ-radiation (2, 4 and 6 Gy) and investigated the profile of downstream metabolites and activity levels of one-carbon (C₁) flux generating enzymes. In MSD fed and irradiated animals, hepatic folate levels increased (P < 0.01), while hepatic homocysteine levels decreased (P < 0.01) compared to NCD fed and irradiated animals. Although hepatic folate level increased significantly in MSD fed animals (P < 0.01), it showed a decrease in response to high doses of γ-irradiation. Under these conditions, a marked suppression of S-adenosylmethionine (SAM) levels occurred in NCD fed and irradiated animals, suggesting reduced conversion of homocysteine to SAM. Concomitant with decline in liver SAM Pool, activities of DNA methyltransferase (Dnmt, that methylates DNA) and methionine synthase (MSase, that regenerates methionine from homocysteine) were both decreased in NCD fed and irradiated mice. However, in MSD fed and irradiated mice, they were increased. These results strongly indicated that increased levels of dnmt and MSase may enhance C₁ flux towards DNA methylation reactions in MSD fed animals. These results were confirmed and further substantiated by measuring genomic DNA methylation levels, which were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals (P < 0.01). In conclusion, our results suggest that maintenance of genomic DNA methylation under γ-radiation stress might be a very dynamic, progressive diet dependent process that could involve increased one-carbon flux through various C₁ metabolites.     

Some novel approaches for radioprotection and the beneficial effect of natural products

Due to the increased use of ionizing radiation in various aspects of human life especially in areas pertaining to radiotherapy of cancer, food preservation, agriculture, industry and power generation, there is a need to develop an effective and non-toxic radioprotector. The currently available ones have many drawbacks including high cost, side effects and toxicity. Several novel approaches are on to locate a potent radioprotector. These include mimics of antioxidant enzymes, nitroxides, melatonin, growth factors, gene therapy, hyperthermia apart from natural products. The latter has several advantages since they are non-toxic with proven therapeutic benefits. These can be classified as natural compounds and plant extracts; polyherbal formulations; besides natural and semi-natural compounds of plant origin. A review of the above agents, their efficacy in radioprotection and possible mechanisms responsible has been carried out. As India and many Eastern countries have an enormous heritage of vast natural dietary and time tested medicinal resources it is worth exploring the possibility of developing efficient, economically viable and clinically acceptable radioprotectors for human application from these resources.     

Study on lipid peroxidation potential in different tissues induced by ascorbate-Fe2+: Possible factors involved in their differential susceptibility

Susceptibility of four major rat tissues to oxidative damage in terms of lipid peroxidation induced by in vitro by ascorbate-Fe²⁺ in homogenates and mitochondria has been examined. Lipid peroxidation, as assessed by thiobarbituric acid reactive substances (TBARS) and conjugated dienes was maximum in brain followed by liver, kidney and heart. However, the time course of lipid peroxidation showed different patterns in tissues examined. The higher susceptibilities of brain and liver can be explained by substrate availability and to a lesser extent the level of antioxidants. The differences observed in the tissues studied may reflect their susceptibility to degenerative diseases and xenobiotic toxicity which are considered as a result of oxidative damage to membranes.     

Free radical reactions of a naturally occurring flavone baicalein and possible mechanisms towards its membrane protective properties

Baicalein (5, 6, 7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavone present in some of the medicinal plants is known for its potential therapeutic effects, such as cardioprotective, anticancer and anti-inflammatory properties. However, detailed role and mechanisms behind its protective properties against different generators for oxidative stress have not been examined. In the present study, we investigated the possible protective ability of baicalein against the membrane damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the mechanisms involved using pulse radiolysis technique. Baicalein offered efficient protection even at a concentration of 10 microM towards membrane damage caused by lipid peroxidation induced by the gamma-radiation, peroxyl radicals, ascorbate-Fe2+ and peroxynitrite in rat liver mitochondria and heart homogenate. To elucidate its reaction mechanisms with biologically relevant radicals, transient absorption spectroscopy employing pulse radiolysis technique was used. Baicalein showed fairly high rate constants (3.7 x 10(9), 1.3 x 10(9) and 8.0 x 10(8) dm3 mol(-1) s(-1) for hydroxyl, azidyl and alkylchloroperoxyl radicals, respectively), suggesting that baicalein can act as an effective scavenger of these radicals. In each case, the phenoxyl radical of baicalein was generated. Thus, it was evident that the phenolic moiety of baicalein was responsible for the free radical scavenging process. Baicalein also reacts with linoleic acid peroxyl radical (LOO*), indicating its ability to act as a chain breaking antioxidant. Peroxynitrite-mediated radicals were shown to be reactive towards baicalein and the bimolecular rate constants were 2.5 x 10(7) and 3 x 10(8) dm3 mol(-1) s(-1) for *NO2 and CO3*(-) radicals, respectively. In conclusion, our results revealed the potential of baicalein in protecting mitochondrial membrane against oxidative damage induced by the four different agents. We propose that the protective effect is mediated via scavenging of primary and secondary radicals generated during oxidative stress.     

Oxidative damage induced by a novel porphyrin on rat brain mitochondria and its possible implications in therapy

Summary

Free radical-induced oxidative damage is involved in several pathological disorders. On the other hand, selective induction of peroxidation in diseased tissue is a promising approach to the treatment of cancer by photodynamic therapy. In this study we have used rat brain mitochondria as a model to evaluate the ability of a new water soluble porphyrin, 5,10,15,20-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (T4CPP), to induce peroxidative damage during photosensitization. Peroxidation in mitochondria, one of the crucial targets of the photodynamic effect, was assessed from the formation of thiobarbituric acid reactive substances and lipid hydroperoxides. The effect on mitochondrial function was estimated from the loss of a mitochondrial marker enzyme, succinate dehydrogenase (SDH). The photodamage was observed to be time- and concentration-dependent of T4CPP. Inhibition studies suggested involvement of singlet oxygen (¹O₂) and, to a lesser extent, of hydroxyl (OH), peroxyl (ROO^?) and superoxide radicals (O₂^?) in the photodamage. The addition of γ-linolenic acid (a promoter of lipid peroxidation) to the system led to an enhancement of the T4CPP-induced peroxidative damage. Thus, our study indicated that the combination of γ-linolenic acid and T4CPP could enhance the photodynamic effect and has potential applications in photodynamic therapy.