Steroidal dihydrocarbothioic acid amido pyrazoles: synthesis,characterization, cytotoxicity and genotoxicity studies

A new series of steroidal dihydrocarbothioic acid amido pyrazole analogues were synthesized, and after characterization, evaluation for cytotoxicity, comet assay and western blotting was carried out. The synthesis of these analogues is convenient and involves two steps, i.e. aldol condensation as first step followed by nucleophilic addition of thiosemicarbazide across α, β-unsaturated carbonyl as a later step. Quantitative yields of more than 80 % are obtained in both the steps. After characterization by IR, ¹H NMR, ¹³C NMR, MS and analytical data, all the compounds of both series were tested for cytotoxic activity against a panel of different human cancer cell lines by MTT assay, during which compound 3e, 3f, 4e, 4f and 4h are very potent especially against HepG2 and MCF-7 cancer cell lines. Cell cycle analysis depicted the cell death in S-phase while as annexin V-FITC/PI analysis showed that compounds effectively induce apoptosis. Apoptotic degradation of DNA of MCF-7 cells in the presence of different steroidal derivatives was analysed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay). In western blotting analysis, the relative expressions of relevant apoptotic markers depicted an apoptosis by steroidal dihydropyrazole in MCF-7 cancer cells.     

Analysis of Site Frequency Spectra from Arabidopsis with Context-Dependent Corrections for Ancestral Misinference

Previous studies have shown that the pattern of single nucleotide polymorphism (SNP) in Arabidopsis (Arabidopsis thaliana) deviates from the distribution expected under a neutral model. Here, we test whether or not ancestral misinference could explain this deviation. We start by showing that there are significant and complex influences of context on mutation dynamics as inferred from SNP frequency, in Arabidopsis, and compare the results to observations about context dependency that have been made on a previous analysis of a maize (Zea mays) SNP dataset. The data concerning heterogeneity across sites are then used to make corrections for ancestral misinference in a context-dependent manner. Using Arabidopsis lyrata to infer the ancestral state for SNPs, we show that the resulting unfolded site frequency spectrum (SFS) in Arabidopsis is skewed toward sites with high frequency derived nucleotides. Sites are also partitioned into two general functional classes, second codon position and 4-fold degenerate sites. These two classes show different SFS; although both show an overrepresentation of high frequency derived sites, low frequency derived sites are vastly overrepresented at the second codon position, but significantly underrepresented at 4-fold degenerate sites. We find that these results are robust to corrections for ancestral misinference, even when context-dependent variation in mutation properties is taken into consideration. The data suggest that, in addition to purifying selection, complex demographic events and/or linked positive selection need to be invoked to explain the SFS, and they highlight the importance of sequence context in analyses of genome-wide variation.Analyses of site frequency spectra (SFS) from single nucleotide polymorphism (SNP) datasets provide a powerful method for making inferences about selection (Akashi, 1999; Bustamante et al., 2001; Hernandez et al., 2007). The allele frequency distribution expected under a neutral model (Tajima, 1989) can be applied to datasets for which an outgroup is available by unfolding the distribution using the assumption of parsimony. Deviation of this distribution from the neutral model provides insights about the role of selection or demographics; an overabundance of high frequency derived sites is frequently attributed to either recurrent positive selection (Bustamante et al., 2001; Caicedo et al., 2007), a population bottleneck (Caicedo et al., 2007), or hidden population substructure (Wakeley and Aliacar, 2001; Hernandez et al., 2007), whereas an excess of low frequency derived sites is commonly explained as a result of constraining selection or a recent population expansion (Slatkin and Hudson, 1991; Hernandez et al., 2007).Arabidopsis (Arabidopsis thaliana) represents one of the most intensively studied model organisms for molecular population genetics, and several genome-scale patterns of nucleotide variation have been generated (Nordborg et al., 2005; Schmid et al., 2005; Borevitz et al., 2007; Clark et al., 2007). These studies have shown evidence for genome-wide departures from a standard neutral population genetic model assuming constant population size. One recurring pattern is that minor allele frequencies tend to be skewed such that there is an excess of rare variants across the genome (Nordborg et al., 2005; Schmid et al., 2005). This pattern has typically been interpreted as evidence for population expansion, although other aspects of the genome-wide data, including a high variance in diversity across loci (Nordborg et al., 2005), appear inconsistent with a simple model of population growth. Furthermore, amino acid substitutions typically show a larger excess of rare variants (Foxe et al., 2008), suggestive of weak purifying selection across the genome.One limitation with these analyses is that outgroup data have rarely been available, restricting the ability to infer the derived frequency spectrum and thus distinguish new low frequency mutations from high frequency derived variants. Instead, these analyses implicitly rely on the theoretical prediction that the probability that an allele is ancestral is equal to its frequency (Watterson and Guess, 1977). In principle, the polarized frequency spectrum should provide considerably more information on the genome-wide patterns of variation and more power to infer the direction and strength of selection (Sawyer and Hartl, 1992; Akashi, 1999). However, a potential difficulty with the use of an outgroup to infer the ancestral and derived states at a given site is that the outgroup state is typically taken as ancestral under a parsimony assumption. This means that parallel changes could result in a misinference of the ancestral state, and this would generally lead to a skew toward sites with a high frequency of the derived state and, therefore, a potential for generating a spurious signature of positive selection or demographic effect (Baudry and Depaulis, 2003; Hernandez et al., 2007). Furthermore, given differences in effective mutation rates across different classes of sites, there may be biased rates of ancestral misinference, which can also lead to problems when inferring the strength of selection on different types of substitution. Given this potentially confounding effect of ancestral misinference, methods have been proposed to correct the SFS (e.g. Baudry and Depaulis, 2003; Hernandez et al., 2007).Any correction for ancestral misinference must be based on an adequate substitution model. In the case of plant genomes, including the maize (Zea mays) nuclear genome, it is well established that relative mutation rates vary significantly across sites as a function of context or the composition of surrounding nucleotides (Morton, 1995, 2003; Morton et al., 2006; Moore and Stevens, 2008) and similar context dependency has been observed in other genomes (Blake et al., 1992; Hess et al., 1994; Krawczak et al., 1998; Zhao and Boerwinkle, 2002). One prominent feature of context dependency is the CpG effect, or an increased rate of transitions at CG dinucleotides as a result of the relatively rapid deamination of methylated cytosines at many such sites (Bulmer, 1986; Zhao and Boerwinkle, 2002; Morton et al., 2006). More complex patterns of context dependency have also been observed in nuclear DNA of maize, where it has been shown that transition and transversion rates are significantly influenced by local and regional composition, but in different manners, and that the rate of mutation of GC and AT base pairs are affected differently by context (Morton et al., 2006).When complex context dependency exists, correcting for ancestral misinference would require that site context be taken into consideration (Hernandez et al., 2007). Therefore, we begin by analyzing heterogeneity across sites in Arabidopsis as a function of context. We find that mutation dynamics are influenced in a complex manner by both composition of flanking nucleotides and regional A+T content. These findings are compared to the context effects that have been observed in maize (Morton et al., 2006). We then analyze the unfolded SFS, with Arabidopsis lyrata as the outgroup, using the method of Baudry and Depaulis (2003) to account for ancestral misinference. To account for the influence of context on mutation dynamics, sites are partitioned by the number of flanking A/T base pairs because this was found to be a major contributing factor to context effects. Sites were also partitioned by codon position and degeneracy to account, approximately, for functional effects. An SFS was then generated for sites within each of the separate partitions and each spectrum was corrected using mutation parameters for that partition.We find that the excess of high frequency sites cannot be explained by ancestral misinference. In addition, second codon position sites show an excess of low frequency sites and 4-fold degenerate sites show a significant deficit of low frequency sites; both of these features remain after the correction. We suggest that complex demographic history and/or the action of positive selection have had a major effect on genome-wide patterns of variation, and we confirm the predominance of slightly deleterious amino acid polymorphisms in the Arabidopsis genome.     

Slipped-strand DNAs formed by long (CAG)*(CTG) repeats: slipped-out repeats and slip-out junctions

The disease-associated expansion of (CTG)·(CAG) repeats is likely to involve slipped-strand DNAs. There are two types of slipped DNAs (S-DNAs): slipped homoduplex S-DNAs are formed between two strands having the same number of repeats; and heteroduplex slipped intermediates (SI-DNAs) are formed between two strands having different numbers of repeats. We present the first characterization of S-DNAs formed by disease-relevant lengths of (CTG)·(CAG) repeats which contained all predicted components including slipped-out repeats and slip-out junctions, where two arms of the three-way junction were composed of complementary paired repeats. In S-DNAs multiple short slip-outs of CTG or CAG repeats occurred throughout the repeat tract. Strikingly, in SI-DNAs most of the excess repeats slipped-out at preferred locations along the fully base-paired Watson–Crick duplex, forming defined three-way slip-out junctions. Unexpectedly, slipped-out CAG and slipped-out CTG repeats were predominantly in the random-coil and hairpin conformations, respectively. Both the junctions and the slip-outs could be recognized by DNA metabolizing proteins: only the strand with the excess repeats was hypersensitive to cleavage by the junction-specific T7 endonuclease I, while slipped-out CAG was preferentially bound by single-strand binding protein. An excellent correlation was observed for the size of the slip-outs in S-DNAs and SI-DNAs with the size of the tract length changes observed in quiescent and proliferating tissues of affected patients—suggesting that S-DNAs and SI-DNAs are mutagenic intermediates in those tissues, occurring during error-prone DNA metabolism and replication fork errors.     

Fungal growth promotor endophytes: a pragmatic approach towards sustainable food and agriculture

Agricultural productivity suffers a heavy loss due to plant pathogens, insect pests and various abiotic stresses. Agriculture being the world’s largest economic sector, it is the need of time to find and establish the ideal strategy for sustainable agriculture and improvement in crop growth. Endophytes are microorganisms that asymptomatically grow within the plant tissues without causing any disease to the host. Endophytic fungi live in symbiotic association with plants and play an important role in plant growth promotion, higher seed yield and plants resistant to various biotic, abiotic stresses and diseases. Many are able to produce antimicrobial compounds, plant growth hormones and various agrochemical bioactive metabolites. These mycoendophytes hold enormous potential for the development of eco-friendly and economically viable agricultural products. In this review we focused on the endophytic fungi recovered from different medicinal plants, their active principles involved in plant growth enhancement and the applications of fungal endophytes in agriculture. Moreover, we also discussed about endophytic fungi and their pragmatic approach towards sustainable food and agriculture.     

Characterisation of Pseudosuccinea columella and Radix natalensis (Gastropoda: Lymnaeidae) in Egypt using shell and molecular data

ABSTRACT

Pseudosuccinea columella and Radix natalensis live in the same habitat in Egypt and are important intermediate hosts of Fasciola hepatica and F. gigantica. Our study aimed to characterise both snail species using molecular analysis and shell measurements. The ranges of morphometric parameters overlapped in the two lymnaeids, indicating that they do not clearly differentiate the two species. PCR-sequence analysis of the nuclear ribosomal small subunit rRNA and the polymorphic mitochondrial cytochrome oxidase subunit 1 (CO1) genes were used to determine the genetic identity and the potential diversity of the snails. Little intrasequence variations were detected in the sequences of both gene loci, indicating the potential homogeneity of lymnaeid populations in Egypt. Generated sequences of the mitochondrial CO1 gene locus for R. natalensis showed obvious heterogeneity compared to other sequences in GenBank. Molecular characterisation of these lymnaeids might help to understand the snails’ biodiversity in a bid to control these populations and their related diseases.     

Chromosome aberration and environmental physical activity: Down syndrome and solar and cosmic ray activity, Israel, 1990–2000

The possibility that environmental effects are associated with chromosome aberrations and various congenital pathologies has been discussed previously. Recent advances in the collection and computerization of data make studying these potential associations more feasible. The aim of this study was to investigate a possible link between the number of Down syndrome (DS) cases detected prenatally or at birth yearly in Israel over a 10-year period compared with the levels of solar and cosmic ray activity 1 year before the detection or birth of each affected child. Information about 1,108,449 births was collected for the years 1990–2000, excluding 1991, when data were unavailable. A total of 1,310 cases of DS were detected prenatally or at birth—138 in the non-Jewish community and 1,172 in the Jewish population. Solar activity indices—sunspot number and solar radio flux 2,800 MHz at 10.7 cm wavelength for 1989–1999—were compared with the number of DS cases detected. Pearson correlation coefficients (r) and their probabilities (P) were established for the percentage of DS cases in the whole population. There was a significant inverse correlation between the indices of solar activity and the number of cases of DS detected—r=–0.78, P=0.008 for sunspot number and r=–0.76, P=0.01 for solar flux. The possibility that cosmophysical factors inversely related to solar activity play a role in the pathogenesis of chromosome aberrations should be considered. We have confirmed a strong trend towards an association between the cosmic ray activity level and the incidence of DS.     

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H₂O₂) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H₂O₂ preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H₂O₂ stimulated divergent pathways, with caspases being activated only by H₂O₂. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H₂O₂, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H₂O₂. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.     

Effects of potyvirus effective population size in inoculated leaves on viral accumulation and the onset of symptoms

Effective population size (N(e)) is a key parameter for understanding evolutionary processes, but it is generally not considered in epidemiological studies or in studying infections of individual hosts. Whether N(e) has an effect on the onset of symptoms and viral accumulation in Tobacco etch virus (TEV) infection of Nicotiana tabacum plants is considered here. Using mixtures of TEV variants carrying fluorescent markers, the dose dependence of N(e) was confirmed, and the inoculation procedure was found to be the main source of variation in these experiments. Whereas the onset of symptoms was independent of N(e), there was less and more variable accumulation at 6 days postinoculation for small N(e) values (N(e) < 5). The observed variation in accumulation was not heritable, however, suggesting that this variation was not due to the fixation of deleterious mutations in the small founder populations. On the other hand, virus-induced fluorescence and accumulation in the inoculated leaf were strongly N(e) dependent. Systemic accumulation was independent of N(e), although removal of the inoculated leaf led to a small reduction in systemic accumulation for small N(e) values. For whole plants, N(e)-dependent effects on accumulation were no longer observed at 9 days postinoculation. Therefore, the effects of N(e) on accumulation are due mainly to limited expansion in the inoculated leaf and are transient. In this system, N(e)-dependent effects will be strongest at low doses and early in infection. We conclude that N(e) can have implications for epidemiology and infection at the individual host level, beyond determining the rate of mixed-genotype infection.     

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system.

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.