Calreticulin-melatonin. An unexpected relationship.

Increasing evidence suggests that melatonin can exert some effect at nuclear level. Previous experiments using binding techniques clearly showed the existence of specific melatonin binding sites in cell nucleus of rat liver. To further identify these sites, nuclear extracts from rat hepatocytes were treated with different percentages of ammonium sulfate and purified by affinity chromatography. Subsequent ligand blot analysis shows the presence of two polypeptides of approximately 60 and approximately 74 kDa that bind specifically to melatonin. N-Terminal sequence analysis showed that the 60 kDa protein shares a high homology with rat calreticulin, whereas the 74 kDa protein shows no homology with any known protein. The binding of melatonin to calreticulin was further characterized incubating 2-[125I]melatonin with recombinant calreticulin. Binding kinetics show a Kd = 1.08 +/- 0.2 nm and Bmax = 290 +/- 34 fmol.mg protein-1, compatible with other binding sites of melatonin in the cell. The presence of calreticulin was further identified by Western blot analysis, and the lack of endoplasmic reticulum contamination in our material was assessed by Western blot and immunostaining with anti-calnexin Ig. The results suggest that calreticulin may represent a new class of high-affinity melatonin binding sites involved in some functions of the indoleamine including genomic regulation.     

Proteomic approaches to identifying carbonylated proteins in brain tissue

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.     

Removal of chromium on Polyalthia longifolia leaves biomass

Adsorption is an environmental friendly process for removal and/or recovery of heavy metals from wastewater. In recent years, it has been substantiated as a popular technique to treat industrial waste effluents, with significant advantages. In this work, batchwise removal of chromium (III) ions from water by Polyalthia longifolia leaves was studied as a function of adsorbent dose, pH, contact time, and agitation speed. Surface characteristics of the leaves were evaluated by recording IR spectra. The Langmuir, Freundlich, and Temkin adsorption isotherms were employed to explain the sorption process. It was found that one gram of leaves can remove 1.87 mg of trivalent chromium when working at pH 3.0. It has been concluded that Polyalthia longifolia leaves can be used as cost-effective and benign adsorbents for removal of Cr(III) ions from wastewater.     

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.     

Possible role of adenosine in the CNS effects of ethanol

The ability of adenosine to modify the CNS effects of acute and chronic ethanol was studied by using theophylline, an adenosine antagonist, and dipyridamole, a blocker of adenosine reuptake. We also studied the binding characteristics of adenosine using crude membranes of whole brain. Theophylline pretreatment prior to acute ethanol administration markedly reduced the duration of ethanol-induced sleep and similarly decreased the intensity and duration of motor incoordination. In chronic ethanol treated mice the effect of theophylline on ethanol-induced hypnosis and motor incoordination was similar to the acute experiment. Dipyridamole markedly prolonged the duration of ethanol-induced hypnosis and potentiated the motor incoordination produced by acute ethanol. However, in chronic ethanol treated mice dipyridamole was not able to potentiate the motor incoordinating effect of ethanol although it was able to prolong ethanol hypnosis similar to the results obtained in the acute ethanol study. Neither drug had any effect on ethanol-induced hypothermia, in either the acute or chronic studies.After 10 days of ethanol ingestion the adenosine dissociation constant was unchanged whereas the number of brain adenosine receptors was increased 28% although the increase did not reach statistical significance. The number of the adenosine receoptors was reduced 40% at 24 and 48 h after withdrawal and returned to prewithdrawal levels at 72 h. The dissociation constant was reduced at 24 and 48 h but by 72 h had returned to prewithdrawal levels. The marked changes in adenosine binding characteristics as well as the modification of some CNS effect of ethanol by drugs which influence either adenosine binding to its receptor or the availability of adenosine suggest that adenosine may be involved in the expression of some of the CNS effects of ethanol.     

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.