Tyrosinase: polybrene noncovalent complexes in water-ethanol mixtures

Formation of noncovalent complexes between tyrosinase from mushrooms and a cationic polyelectrolyte, polybrene (PB, poly (1,5-dimethyl-1,5-diazaundecamethyelene) bromide), was shown to activate and stabilize tyrosinase in water-ethanol mixtures. In the reaction of catechol oxidation in aqueous solutions, catalytic activity (k(cat)) of tyrosinase-PB complex ([PB]/[tyrosinase] molar ratio 100:1, per mole of polymer) in a wide range of pH was higher than that of free tyrosinase. In aqueous solutions and in water-ethanol mixtures at moderate concentrations of ethanol (10-40% v/v), the value of k(cat) of tyrosinase-PB complex exceeded the activity of free enzyme, from 1.2-2-fold, accompanied by the essential (up to 10-fold) increase in the value of the specificity constant (k(cat)/K(m)). The results are of practical importance for the construction of biocatalysts working successfully in polar organic media.     

Metagenomic Exploration of Viruses throughout the Indian Ocean

The characterization of global marine microbial taxonomic and functional diversity is a primary goal of the Global Ocean Sampling Expedition. As part of this study, 19 water samples were collected aboard the Sorcerer II sailing vessel from the southern Indian Ocean in an effort to more thoroughly understand the lifestyle strategies of the microbial inhabitants of this ultra-oligotrophic region. No investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean or across multiple size fractions thus far. Therefore, the goals of this study were to examine the effect of size fractionation on viral consortia structure and function and understand the diversity and functional potential of the Indian Ocean virome. Five samples were selected for comprehensive metagenomic exploration; and sequencing was performed on the microbes captured on 3.0-, 0.8- and 0.1 µm membrane filters as well as the viral fraction (<0.1 µm). Phylogenetic approaches were also used to identify predicted proteins of viral origin in the larger fractions of data from all Indian Ocean samples, which were included in subsequent metagenomic analyses. Taxonomic profiling of viral sequences suggested that size fractionation of marine microbial communities enriches for specific groups of viruses within the different size classes and functional characterization further substantiated this observation. Functional analyses also revealed a relative enrichment for metabolic proteins of viral origin that potentially reflect the physiological condition of host cells in the Indian Ocean including those involved in nitrogen metabolism and oxidative phosphorylation. A novel classification method, MGTAXA, was used to assess virus-host relationships in the Indian Ocean by predicting the taxonomy of putative host genera, with Prochlorococcus, Acanthochlois and members of the SAR86 cluster comprising the most abundant predictions. This is the first study to holistically explore virioplankton dynamics across multiple size classes and provides unprecedented insight into virus diversity, metabolic potential and virus-host interactions.     

Phenological sensitivity to climate change is higher in resident than in migrant bird populations among European cavity breeders

Many organisms adjust their reproductive phenology in response to climate change, but phenological sensitivity to temperature may vary between species. For example, resident and migratory birds have vastly different annual cycles, which can cause differential temperature sensitivity at the breeding grounds, and may affect competitive dynamics. Currently, however, adjustment to climate change in resident and migratory birds have been studied separately or at relatively small geographical scales with varying time series durations and methodologies. Here, we studied differential effects of temperature on resident and migratory birds using the mean egg laying initiation dates from 10 European nest box schemes between 1991 and 2015 that had data on at least one resident tit species and at least one migratory flycatcher species. We found that both tits and flycatchers advanced laying in response to spring warming, but resident tit populations advanced more strongly in relation to temperature increases than migratory flycatchers. These different temperature responses have already led to a divergence in laying dates between tits and flycatchers of on average 0.94 days per decade over the current study period. Interestingly, this divergence was stronger at lower latitudes where the interval between tit and flycatcher phenology is smaller and winter conditions can be considered more favorable for resident birds. This could indicate that phenological adjustment to climate change by flycatchers is increasingly hampered by competition with resident species. Indeed, we found that tit laying date had an additional effect on flycatcher laying date after controlling for temperature, and this effect was strongest in areas with the shortest interval between both species groups. Combined, our results suggest that the differential effect of climate change on species groups with overlapping breeding ecology affects the phenological interval between them, potentially affecting interspecific interactions.     

Endothelin 1 induces beta 1Pix translocation and Cdc42 activation via protein kinase A-dependent pathway

p21-activated kinase (Pak)-interacting exchange factor (Pix), a Rho family guanine nucleotide exchange factor (GEF), has been shown to co-localize with Pak and form activated Cdc42- and Rac1-driven focal complexes. In this study we have presented evidence that treatment of human mesangial cells (HMC) with endothelin 1 (ET-1) and stimulation of adenylate cyclase with either forskolin or with the cAMP analog 8-Br-cAMP activated the GTP loading of Cdc42. Transient expression of constitutively active G alpha(s) also stimulated Cdc42. In addition, overexpression of beta(1)Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of beta(1)Pix SH3m(W43K), which lacks the ability to bind Pak, and beta(1)PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Furthermore, ET-1 stimulation induced beta(1)Pix translocation to focal complexes. Interestingly, pretreatment of HMC with protein kinase A (PKA) inhibitors blocked both Cdc42 activation and beta(1)Pix translocation induced by ET-1, indicating the involvement of the PKA pathway. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assay, we have shown that beta(1)Pix is phosphorylated by PKA. Using purified recombinant beta(1)Pix(wt) and beta(1)Pix mutants, we have identified Ser-516 and Thr-526 as the major phosphorylation sites by PKA. beta(1)Pix(S516A/T526A), in which both phosphorylation sites are replaced by alanine, blocks beta(1)Pix translocation and Cdc42 activation. Our results have provided evidence that stimulation of PKA pathway by ET-1 or cAMP analog results in beta(1)Pix phosphorylation, which in turn controls beta(1)Pix translocation to focal complexes and Cdc42 activation.     

Detecting and accounting for multiple sources of positional variance in peak list registration analysis and spin system grouping

Peak lists derived from nuclear magnetic resonance (NMR) spectra are commonly used as input data for a variety of computer assisted and automated analyses. These include automated protein resonance assignment and protein structure calculation software tools. Prior to these analyses, peak lists must be aligned to each other and sets of related peaks must be grouped based on common chemical shift dimensions. Even when programs can perform peak grouping, they require the user to provide uniform match tolerances or use default values. However, peak grouping is further complicated by multiple sources of variance in peak position limiting the effectiveness of grouping methods that utilize uniform match tolerances. In addition, no method currently exists for deriving peak positional variances from single peak lists for grouping peaks into spin systems, i.e. spin system grouping within a single peak list. Therefore, we developed a complementary pair of peak list registration analysis and spin system grouping algorithms designed to overcome these limitations. We have implemented these algorithms into an approach that can identify multiple dimension-specific positional variances that exist in a single peak list and group peaks from a single peak list into spin systems. The resulting software tools generate a variety of useful statistics on both a single peak list and pairwise peak list alignment, especially for quality assessment of peak list datasets. We used a range of low and high quality experimental solution NMR and solid-state NMR peak lists to assess performance of our registration analysis and grouping algorithms. Analyses show that an algorithm using a single iteration and uniform match tolerances approach is only able to recover from 50 to 80% of the spin systems due to the presence of multiple sources of variance. Our algorithm recovers additional spin systems by reevaluating match tolerances in multiple iterations. To facilitate evaluation of the algorithms, we developed a peak list simulator within our nmrstarlib package that generates user-defined assigned peak lists from a given BMRB entry or database of entries. In addition, over 100,000 simulated peak lists with one or two sources of variance were generated to evaluate the performance and robustness of these new registration analysis and peak grouping algorithms.     

Synthesis and structure-activity relationship studies of 4,11-diaminonaphtho[2,3-f]indole-5,10-diones

We describe the synthesis of derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione and their cytotoxicity for human tumor cells that express major determinants of altered anticancer drug response, the efflux pump P-glycoprotein, and non-functional p53. Nucleophilic substitution of methoxy groups in 4,11-dimethoxynaphtho[2,3-f]indole-5,10-dione with various ethylenediamines yielded the derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione, the indole containing analogues of the antitumor agent ametantrone. The cytotoxicity of novel compounds for multidrug resistant, P-glycoprotein-expressing tumor cells is highly dependent on the N-substituent at the terminal amino group of the ethylenediamine moiety. Whereas p53 null colon carcinoma cells were less sensitive to the reference drug doxorubicin than their counterparts with wild type p53, the majority of novel naphthoindole derivatives were equally potent for both cell lines, regardless of the p53 status.