Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs

Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe(3+)-chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe(3+) complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe(3+) complexes, and toxicity of the chelators increased with oxygen tension. Fe(3+) complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)(2) did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)(2) in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe(3+) complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series.     

Prdm9 Intersubspecific Interactions in Hybrid Male Sterility of House Mouse

The classical definition posits hybrid sterility as a phenomenon when two parental taxa each of which is fertile produce a hybrid that is sterile. The first hybrid sterility gene in vertebrates, Prdm9, coding for a histone methyltransferase, was identified in crosses between two laboratory mouse strains derived from Mus mus musculus and M. m. domesticus subspecies. The unique function of PRDM9 protein in the initiation of meiotic recombination led to the discovery of the basic molecular mechanism of hybrid sterility in laboratory crosses. However, the role of this protein as a component of reproductive barrier outside the laboratory model remained unclear. Here, we show that the Prdm9 allelic incompatibilities represent the primary cause of reduced fertility in intersubspecific hybrids between M. m. musculus and M. m. domesticus including 16 musculus and domesticus wild-derived strains. Disruption of fertility phenotypes correlated with the rate of failure of synapsis between homologous chromosomes in meiosis I and with early meiotic arrest. All phenotypes were restored to normal when the domesticus Prdm9^dom2 allele was substituted with the Prdm9^dom2H humanized variant. To conclude, our data show for the first time the male infertility of wild-derived musculus and domesticus subspecies F1 hybrids controlled by Prdm9 as the major hybrid sterility gene. The impairment of fertility surrogates, testes weight and sperm count, correlated with increasing difficulties of meiotic synapsis of homologous chromosomes and with meiotic arrest, which we suppose reflect the increasing asymmetry of PRDM9-dependent DNA double-strand breaks.     

Phosphodiester Amidates of Unsaturated Nucleoside Analogues as Anti-HIV Agents

Abstract

Lipophilic phosphodiester L-alaninates of acyclic unsaturated nucleoside analogues 1d, 1e, 2d, 2e, 3d, 3e, 4d and 5d were prepared and their antiretroviral activity was examined in ATH8 cell culture infected with HIV-1. A possible mechanism of action of these analogues is discussed.     

Dispersal patterns of endemic alpine butterflies with contrasting population structures:<Emphasis Type="Italic"> Erebia epiphron</Emphasis> and<Emphasis Type="Italic"> E. sudetica</Emphasis>

We studied population sizes and mobility of Erebia epiphron and Erebia sudetica, two high mountain butterflies forming endemic subspecies in the Hrubý Jeseník Mountains, Czech Republic. E. epiphron formed two continuous populations containing 100,000 and 4,500 individuals on alpine grasslands. The butterflies moved freely within their habitats, but movements between the two populations were highly unlikely. E. sudetica formed a system of colonies at timberline sites on valley headwalls and in forest clearings. Two such colonies studied in detail contained 4,500 and 450 adults and were interconnected by limited dispersal. The negative exponential function and the sigmoid function (this assumes flat decrease of movements over short distances) were superior to the inverse power function in fitting mobility data for both species. For E. sudetica, the functions describing movements within a habitat differed significantly from total movements, suggesting different behaviours of dispersing individuals. The habitats of E. epiphron are uniform and highly isolated, favouring free within-habitat mobility but prohibiting leaving their boundaries. The habitats of E. sudetica are diverse and disturbance-dependent; leaving such habitats is less risky, and a source-sink model may explain the persistence of the species in the mountains.     

Changes in recreational catfish Silurus glanis harvest rates between years 1986–2017 in Central Europe

The European catfish Silurus glanis is an important fish species in both commercial and recreational fisheries. Catfish is a spreading species that was reported to potentially benefit from increasing temperatures. The goal of this study was to estimate long‐term changes in harvest rates of catfish in Central Europe. This study used individual mandatory angling logbooks collected by the Czech Fishing Union in the Czech Republic (Central Europe) over the course of years 1986–2017 (32 years) to assess harvest rates of catfish. This study discovered that harvest of catfish has been increasing over time. Moreover, rivers that previously showed zero harvested catfish are beginning to display higher harvest rates of catfish. Increasing average air temperature and angling effort in the study area have positively affected harvest rates of catfish. The increased harvest of catfish could not be reliably explained by intensive fish stocking. In conclusion, while other studies show that many fish species are negatively affected by human activities and therefore show decreased harvest rates, catfish angling seems to benefit from anthropogenic changes.     

Overaccumulation of γ-Glutamylcysteine in a Jasmonate-Hypersensitive Arabidopsis Mutant Causes Jasmonate-Dependent Growth Inhibition

Glutathione (GSH) is essential for many aspects of plant biology and is associated with jasmonate signaling in stress responses. We characterized an Arabidopsis (Arabidopsis thaliana) jasmonate-hypersensitive mutant (jah2) with seedling root growth 100-fold more sensitive to inhibition by the hormone jasmonyl-isoleucine than the wild type. Genetic mapping and genome sequencing determined that the mutation is in intron 6 of GLUTATHIONE SYNTHETASE2, encoding the enzyme that converts γ-glutamylcysteine (γ-EC) to GSH. The level of GSH in jah2 was 71% of the wild type, while the phytoalexin-deficient2-1 (pad2-1) mutant, defective in GSH1 and having only 27% of wild-type GSH level, was not jasmonate hypersensitive. Growth defects for jah2, but not pad2, were also seen in plants grown to maturity. Surprisingly, all phenotypes in the jah2 pad2-1 double mutant were weaker than in jah2. Quantification of γ-EC indicated these defects result from hyperaccumulation of this GSH precursor by 294- and 65-fold in jah2 and the double mutant, respectively. γ-EC reportedly partially substitutes for loss of GSH, but growth inhibition seen here was likely not due to an excess of total glutathione plus γ-EC because their sum in jah2 pad2-1 was only 16% greater than in the wild type. Further, the jah2 phenotypes were lost in a jasmonic acid biosynthesis mutant background, indicating the effect of γ-EC is mediated through jasmonate signaling and not as a direct result of perturbed redox status.Glutathione (GSH) is an essential thiol of most higher organisms, including plants. Primarily found in the reduced form, its roles in maintaining a reduced intracellular state are numerous and well characterized (Foyer and Noctor, 2011; Noctor et al., 2011). Additionally, GSH is involved in detoxifying reactive oxygen species, heavy metal detoxification through phytochelatins, elimination of xenobiotics, and signaling of plant development and stress responses (Rouhier et al., 2008).GSH is synthesized in two steps. The first links Cys to the γ-carboxyl group of Glu through an amide bond catalyzed by γ-glutamylcysteine (γ-EC) synthetase, encoded by the single gene GSH1 in Arabidopsis (Arabidopsis thaliana). Gly is then added by GSH synthetase (GSH-S), also encoded by a single gene (GSH2). GSH is typically present at millimolar levels in plants, and although γ-EC is normally present at only a few percent of this amount, there is evidence that γ-EC has redox activities in Arabidopsis (Pasternak et al., 2008).Insertional knockouts of GSH1 are embryo lethal, and rootmeristemless1, with only 5% of wild-type GSH level, lacks a root apical meristem due to cell cycle arrest (Vernoux et al., 2000; Cairns et al., 2006). Other mutants producing 25% to 50% of wild-type GSH levels grow normally but exhibit defects under various stress conditions. For example, phytoalexin-deficient2-1 (pad2-1) and cadmium sensitive2 mutants are susceptible to pathogens and hypersensitive to Cd, respectively, while regulator of axillary meristems1 causes elevated expression of ASCORBATE PEROXIDASE2 under non-photooxidative-stress conditions (Glazebrook and Ausubel, 1994; Cobbett et al., 1998; Ball et al., 2004).GSH2 null alleles (gsh2-1 and gsh2-2) are also lethal, although plants survive to the early seedling stage (Pasternak et al., 2008). Survival past the embryo stage was attributed to partial complementation of GSH activity by γ-EC, which accumulates to excessive levels in gsh2-1, and the mutant is partially rescued by GSH supplementation. Missense and nonsense GSH2 alleles of membrane trafficking mutants (gsh2-3–gsh2-5) disrupt endoplasmic reticulum (ER) organization and also arrest growth in early seedling development, while a weaker allele (gsh2-6) reached maturity but was smaller than the wild type (Au et al., 2012). A screen for reduced response to Cd also yielded a viable missense mutant of GSH2 (nonresponse or reduced response to Cd2) with approximately 75% of the wild-type GSH level (Jobe et al., 2012).Plant oxidative stress responses involve both redox signaling through GSH and jasmonate hormonal signaling, and gene expression studies have clearly linked these two signaling systems. GSH biosynthesis and metabolism genes are induced by jasmonate, while manipulating GSH level or redox status in various mutants alters expression of genes for jasmonate biosynthesis and signaling (Xiang and Oliver, 1998; Mhamdi et al., 2010; Han et al., 2013). GSH and jasmonate are also associated with protective glucosinolate production in response to insect feeding (Noctor et al., 2011). For example, pad2-1 is deficient in glucosinolates and more susceptible to insects, while several studies have shown jasmonate induces glucosinolates (Brader et al., 2001; Mikkelsen et al., 2003; Sasaki-Sekimoto et al., 2005; Schlaeppi et al., 2008). Liu et al. (2010) isolated jasmonic acid hypersensitive1 (jah1), an Arabidopsis mutant with greater inhibition of root growth than the wild type in the presence of jasmonic acid (JA). The affected gene encodes a cytochrome P450 (CYP82C3) involved in indole glucosinolate production, and this mutant was more susceptible to Botrytis cinerea.The basic mechanism of jasmonate signal transduction and some of the downstream responses emanating from it are now well understood (Browse, 2009; Wasternack and Hause, 2013). However, the mechanisms by which jasmonate and GSH coordinate their activities to mediate oxidative stress and other responses are not known. This study characterized, to our knowledge, a new jasmonate-hypersensitive mutant that accumulates excess γ-EC due to a defect in GSH2, but GSH is only modestly reduced. Results show that elevated γ-EC is deleterious to plant growth through a jasmonate-dependent mechanism.     

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants

There is great interest in increasing proteins’ stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt’s reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔT _m = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.     

Alpha-tocopheryl succinate epitomizes a compound with a shift in biological activity due to pro-vitamin-to-vitamin conversion

With the advent of the third millennium, a number of pathologies have been eradicated or taken under control. However, the incidences, of cancer and atherosclerosis, the two most common causes of death in developed countries, have increased or, in some instances, only stagnated. Therefore there has been an intensive search for agents effective against such life-threatening conditions. Accordingly, the potential anti-atherogenic activity of vitamin E analogs has been studied extensively. Interestingly, recent reports strongly suggest that certain vitamin E analogs, represented in particular by alpha-tocopheryl succinate (alpha-TOS), also possess anti-neoplastic activity. In this communication, we review our current understanding of the molecular basis for these double effects of alpha-TOS and propose a testable hypothesis, according to which this semi-synthetic analog exerts both anti-atherogenic and anti-neoplastic activities. We propose that the prevalence of each activity depends on the actual form of the vitamin E analog. That is, the conversion of the pro-vitamin E form, alpha-TOS, to the corresponding vitamin form, alpha-tocopherol, makes this anti-neoplastic agent active against inflammatory diseases like atherosclerosis.