The epsilon-isoform of PKC mediates the hypertonic activation of cation channels in confluent monolayers of rat hepatocytes.

We were interested whether PKC alpha, delta, epsilon or zeta is the isoform actually employed in the activation of hypertonicity-induced cation channels (HICCs) in primary cultures of rat hepatocytes. Quantitative SDS-page and Western-blot experiments revealed that PKC alpha, delta and epsilon were stimulated by Indolactam V (as a DAG substitute for activation of c and nPKCs) but that only PKC delta and epsilon did respond to hypertonic stress. Furthermore, chelation of intracellular Ca(++) by BAPTA-AM did not alter HICC activation in cable-analysis experiments whereas Indolactam V as well as V8 (an Indolactam derivative specific for PKC delta and epsilon) activated HICC currents under isotonic conditions. Finally, by use of Rottlerin (as an inhibitor exhibiting a slight preference for PKC delta over epsilon) PKC epsilon could be identified as the most likely isoform responsible for the activation of the HICC.     

Heterogeneous connexin43 expression produces electrophysiological heterogeneities across ventricular wall

Recently we found that electrophysiological (EP) heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias. However, cell-to-cell coupling through gap junctions is expected to attenuate transmural heterogeneities between cell types spanning the ventricular wall. Because connexin43 (Cx43) is the principal ventricular gap junction protein, we hypothesized that transmural EP heterogeneities are in part produced by heterogeneous Cx43 expression across the ventricular wall. The left ventricles of eight dogs were sectioned to expose the transmural surface. To determine whether heterogeneous Cx43 expression influenced EP function, high-resolution transmural optical mapping of the arterially perfused canine wedge preparation was used to measure transmural conduction velocity (thetaTM), dV/dt(max), transmural space constant (lambdaTM), and transmural gradients of action potential duration (APD). Relative Cx43 expression, quantified by confocal immunofluorescence, was significantly lower (by 24 +/- 17%; P < 0.05) in subepicardial compared with deeper layers. Importantly, reduced subepicardial Cx43 was associated with transmural heterogeneities of EP function evidenced by selectively reduced subepicardial thetaTM (by 18 +/- 9%; P < 0.05) compared with deeper layers. In subepicardial regions, dV/dt(max) was fastest (by 19 +/- 15%) and lambdaTM was smallest (by 18.1 +/- 2%), which suggests that conduction slowing was attributable to localized uncoupling rather than reduced excitability. The maximum transmural APD gradients occurred in the same regions where Cx43 expression was lowest; this suggests that Cx43 expression patterns served to maintain APD gradients across the transmural wall. These data demonstrate that heterogeneous Cx43 expression is closely associated with functionally significant EP heterogeneities across the transmural wall. Therefore, Cx43 expression patterns can potentially contribute to arrhythmic substrates that are dependent on transmural electrophysiological heterogeneities.     

Pan-European regional-scale modelling of water and N efficiencies of rapeseed cultivation for biodiesel production

The energy produced from the investment in biofuel crops needs to account for the environmental impacts on soil, water, climate change and ecosystem services. A regionalized approach is needed to evaluate the environmental costs of large-scale biofuel production. We present a regional pan-European simulation of rapeseed ( Brassica napus ) cultivation. Rapeseed is the European Union's dominant biofuel crop with a share of about 80% of the feedstock. To improve the assessment of the environmental impact of this biodiesel production, we performed a pan-European simulation of rapeseed cultivation at a 10 × 10 km scale with Environmental Policy Integrated Climate (EPIC). The model runs with a daily time step and model input consists of spatialized meteorological measurements, and topographic, soil, land use, and farm management practices data and information. Default EPIC model parameters were calibrated based on literature. Modelled rapeseed yields were satisfactory compared with yields at regional level reported for 151 regions obtained for the period from 1995 to 2003 for 27 European Union member countries, along with consistent modelled and reported yield responses to precipitation, radiation and vapour pressure deficit at regional level. The model is currently set up so that plant nutrient stress is not occurring. Total fertilizer consumption at country level was compared with IFA/FAO data. This approach allows us to evaluate environmental pressures and efficiencies arising from and associated with rapeseed cultivation to further complete the environmental balance of biofuel production and consumption.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms

When outer-row dynein arms are extracted from Chlamydomonas flagellar axonemes, they dissociate into two ATPase complexes with sedimentation coefficients of 12S and 18S. We immunized mice with 18S dynein and generated a library of monoclonal antibodies against the polypeptides in this complex. Antibodies were selected which specifically recognize the 18S alpha- and beta-heavy chains and the 83,000-dalton and 70,000-dalton intermediate chains. These antibodies were isolated and characterized for their ability to recognize determinants on both denatured antigens and native 18S dynein; 18S dynein was dissociated in stepwise fashion into smaller aggregates with ionic and nonionic detergents and the resulting subcomplexes were isolated by precipitation with specific monoclonal antibodies. The smallest aggregates isolated were heterodimers between the alpha-chain and a 16,000-dalton light chain and between the two intermediate chains. Additional close associations of the beta-heavy chain with an 18,000-dalton light chain and 70,000-dalton intermediate chain, and a weaker interaction between the intermediate chain heterodimer and light chains of 21,000 daltons and 12,500 daltons, were also observed. We present a model of 18S dynein substructure based upon this information.     

Evolutionary distances for protein-coding sequences: modeling site- specific residue frequencies

Estimation of evolutionary distances from coding sequences must take into account protein-level selection to avoid relative underestimation of longer evolutionary distances. Current modeling of selection via site-to-site rate heterogeneity generally neglects another aspect of selection, namely position-specific amino acid frequencies. These frequencies determine the maximum dissimilarity expected for highly diverged but functionally and structurally conserved sequences, and hence are crucial for estimating long distances. We introduce a codon- level model of coding sequence evolution in which position-specific amino acid frequencies are free parameters. In our implementation, these are estimated from an alignment using methods described previously. We use simulations to demonstrate the importance and feasibility of modeling such behavior; our model produces linear distance estimates over a wide range of distances, while several alternative models underestimate long distances relative to short distances. Site-to-site differences in rates, as well as synonymous/nonsynonymous and first/second/third-codon-position differences, arise as a natural consequence of the site-to-site differences in amino acid frequencies.      

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.     

Relationships Between Adult Male and Maturing Mountain Gorillas (Gorilla beringei beringei) Persist Across Developmental Stages and Social Upheaval

Across the animal kingdom, long‐term social relationships outside the context of reproductive pair bonds are rare. However, they have been demonstrated in some mammals including primates, cetaceans, and social carnivores. The ontogeny of such relationships is likely to depend on the benefits individuals can gain by cultivating them. Previous studies demonstrated that young mountain gorillas (Gorilla beringei beringei) have strong relationships with adult males, but little is known about the longevity of these bonds. Here, we examine the temporal stability of proximity relationships between coresident adult male and maturing gorillas in the habituated population monitored by the Dian Fossey Gorilla Fund International's Karisoke Research Center in Volcanoes National Park, Rwanda. We used spatial proximity data to assess the strength of relationships between adult males and juveniles, and tracked these relationships as the juveniles matured into subadults (3–4 yr later; n = 229 dyads) and then young adults (7 yr later; n = 42 dyads). The proximity relationships of juveniles of both sexes predicted their proximity relationships with adult males in both subadulthood and young adulthood. However some young adult males who had lost their top adult male proximity partner from juvenilehood developed new relationships with older males that had risen in the dominance hierarchy. These data suggest that (1) kin selection may play a more important role in social relationships between potential fathers and adult female offspring than previously suspected, and (2) when maturing males' foremost adult male social partners remain available to them, their relationships can be stable past the age at which younger males become reproductive competitors.     

Climate impacts on transocean dispersal and habitat in gray whales from the Pleistocene to 2100

Arctic animals face dramatic habitat alteration due to ongoing climate change. Understanding how such species have responded to past glacial cycles can help us forecast their response to today's changing climate. Gray whales are among those marine species likely to be strongly affected by Arctic climate change, but a thorough analysis of past climate impacts on this species has been complicated by lack of information about an extinct population in the Atlantic. While little is known about the history of Atlantic gray whales or their relationship to the extant Pacific population, the extirpation of the Atlantic population during historical times has been attributed to whaling. We used a combination of ancient and modern DNA, radiocarbon dating and predictive habitat modelling to better understand the distribution of gray whales during the Pleistocene and Holocene. Our results reveal that dispersal between the Pacific and Atlantic was climate dependent and occurred both during the Pleistocene prior to the last glacial period and the early Holocene immediately following the opening of the Bering Strait. Genetic diversity in the Atlantic declined over an extended interval that predates the period of intensive commercial whaling, indicating this decline may have been precipitated by Holocene climate or other ecological causes. These first genetic data for Atlantic gray whales, particularly when combined with predictive habitat models for the year 2100, suggest that two recent sightings of gray whales in the Atlantic may represent the beginning of the expansion of this species' habitat beyond its currently realized range.