Increased vascular smooth muscle contractility in TRPC6-/- mice

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6(-)(/)(-) smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6(-/-) smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.     

Toward an accurate statistics of gapped alignments

Sequence alignment has been an invaluable tool for finding homologous sequences. The significance of the homology found is often quantified statistically by p-values. Theory for computing p-values exists for gapless alignments [Karlin, S., Altschul, S.F., 1990. Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc. Natl. Acad. Sci. USA 87, 2264–2268; Karlin, S., Dembo A., 1992. Limit distributions of maximal segmental score among Markov-dependent partial sums. Adv. Appl. Probab. 24, 13–140], but a full generalization to alignments with gaps is not yet complete. We present a unified statistical analysis of two common sequence comparison algorithms: maximum-score (Smith-Waterman) alignments and their generalized probabilistic counterparts, including maximum-likelihood alignments and hidden Markov models. The most important statistical characteristic of these algorithms is the distribution function of the maximum score S _max, resp. the maximum free energy F _max, for mutually uncorrelated random sequences. This distribution is known empirically to be of the Gumbel form with an exponential tail P(S _max > x) ∼ exp(−λx) for maximum-score alignment and P(F _max > x) ∼ exp(−λx) for some classes of probabilistic alignment. We derive an exact expression for λ for particular probabilistic alignments. This result is then used to obtain accurate λ values for generic probabilistic and maximum-score alignments. Although the result demonstrated uses a simple match-mismatch scoring system, it is expected to be a good starting point for more general scoring functions.     

cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity

Signaling pathways targeting mitochondria are poorly understood. We here examine phosphorylation by the cAMP-dependent pathway of subunits of cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain. Using anti-phospho antibodies, we show that cow liver COX subunit I is tyrosinephosphorylated in the presence of theophylline, a phosphodiesterase inhibitor that creates high cAMP levels, but not in its absence. The site of phosphorylation, identified by mass spectrometry, is tyrosine 304 of COX catalytic subunit I. Subunit I phosphorylation leads to a decrease of V(max) and an increase of K(m) for cytochrome c and shifts the reaction kinetics from hyperbolic to sigmoidal such that COX is fully or strongly inhibited up to 10 mum cytochrome c substrate concentrations, even in the presence of allosteric activator ADP. To assess our findings with the isolated enzyme in a physiological context, we tested the starvation signal glucagon on human HepG2 cells and cow liver tissue. Glucagon leads to COX inactivation, an effect also observed after incubation with adenylyl cyclase activator forskolin. Thus, the glucagon receptor/G-protein/cAMP pathway regulates COX activity. At therapeutic concentrations used for asthma relief, theophylline causes lung COX inhibition and decreases cellular ATP levels, suggesting a mechanism for its clinical action.     

Target specificity analysis of the Abl kinase using peptide microarray data

Protein kinases play an important role in cellular signalling. The reliable prediction of their substrates is of high importance for the deciphering of signalling pathways. A recently developed peptide microarray technology for the charcterisation of protein kinases delivers data on the individual phosphorylation status of each single member of a large peptide library. This data can be used to approximate the substrate specificity of the investigated kinase. We present an approach to process the collected information using a combination of a weight matrix approach and a nearest neighbor approach. Experiments with the protein-tyrosine kinase Abl are conducted to validate the results. Randomly selected peptides (1433) are used to estimate the substrate preferences of the kinase. The obtained prediction results are compared with standard methods. The new approach is tested further on bona fide Abl phosphorylation sites.     

Common patterns in type II restriction enzyme binding sites

Restriction enzymes are among the best studied examples of DNA binding proteins. In order to find general patterns in DNA recognition sites, which may reflect important properties of protein–DNA interaction, we analyse the binding sites of all known type II restriction endonucleases. We find a significantly enhanced GC content and discuss three explanations for this phenomenon. Moreover, we study patterns of nucleotide order in recognition sites. Our analysis reveals a striking accumulation of adjacent purines (R) or pyrimidines (Y). We discuss three possible reasons: RR/YY dinucleotides are characterized by (i) stronger H-bond donor and acceptor clusters, (ii) specific geometrical properties and (iii) a low stacking energy. These features make RR/YY steps particularly accessible for specific protein–DNA interactions. Finally, we show that the recognition sites of type II restriction enzymes are underrepresented in host genomes and in phage genomes.     

Eye lens proteomics: from global approach to detailed information about phakinin and gamma E and F crystallin genes

Exploration of the lenticular proteome poses a challenging and worthwhile undertaking as cataracts, the products of a disease phenotype elicited by this proteome, remains the leading cause of vision impairment worldwide. The complete ten day old lens proteome of Mus musculus C57BL/6J was resolved into 900 distinct spots by large gel carrier ampholyte based 2-DE. The predicted amino acid sequences of all 16 crystallins ubiquitous in mammals were corroborated by mass spectrometry (MS). In detailed individual spot analyses, the primary structure of the full murine C57BL/6J beaded filament component phakinin CP49 was sequenced by liquid chromatography/electrospray ionization-tandem MS and amended at two positions. This definitive polypeptide sequence was aligned to the mouse genome, thus identifying the entire C57BL/6J genomic coding region. Also, two murine C57/6J polypeptides, both previously classified as gamma F crystallin, were clearly distinguished by MS and electrophoretic mobility. Both were assigned to their respective genes, one of the polypeptides was reclassified as C57BL/6J gamma E crystallin. Building on these data and previous investigations an updated crystallin reference map was put forth and several non crystallin lenticular components were examined. These results represent the first part of a comprehensive investigation of the mouse lens proteome (http://www.mpiib-berlin.mpg.de/2D-PAGE) with emphasis on understanding genetic effects on proteins and disease development.     

Life Cycle Assessment of Novel Aircraft Interior Panels Made from Renewable or Recyclable Polymers with Natural Fiber Reinforcements and Non‐Halogenated Flame Retardants

A comprehensive life cycle assessment of panels for aircraft interiors was conducted, including both a conventional glass fiber‐reinforced panel and different novel sustainable panels. The conventional panel is made of a glass fiber‐reinforced thermoset composite with halogenated flame retardant, whereas the sustainable panels are made of renewable or recyclable polymers, natural fiber reinforcements, and nonhalogenated flame retardants. Four different sustainable panels were investigated: a geopolymer‐based panel; a linseed‐oil–based biopolymer panel; and two thermoplastic panels, one with polypropylene (PP) and another with polylactic acid (PLA). All of the sustainable panels were developed to fulfil fire resistance requirements and to be lighter than the conventional panels in order to reduce fuel consumption and air pollutant emissions from the aircraft. The environmental impacts associated with energy consumption and air emissions were assessed, as well as other environmental impacts resulting from the extraction and processing of materials, transportation of materials and waste, panel manufacturing, use, maintenance, and end of life (EoL). All the sustainable panels showed better environmental performance than the conventional panel. The overall impacts of the sustainable panels were offset by the environmental benefits in the use stage attributed to weight reduction. One square meter of the novel panels could save to 6,000 kilograms of carbon dioxide equivalents. The break‐even point (in months) at which the use of sustainable panels would yield an environmental benefit relative to the impacts arising in production and EoL was as follows: 1.2 for the geopolymer panel; 1.7 for the biopolymer panel; 10.4 for the PLA panel; and 54.5 for the PP panel.     

The effect of environmental gradients on the bed site selection of roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

A wildlife species’ selection of bedding sites is often characterised by strong trade-offs, as habitat quality, predator avoidance and foraging needs should be achieved simultaneously. Human activities often represent major threats in addition. In areas of intensive agriculture, e.g. mowing is one of the main causes of mortality of roe deer (Capreolus capreolus) fawns due their hiding strategy. For a species’ offspring, the selection of bedding sites is particularly crucial and thus, identifying how and when animals use such habitats is important for management. We used a long-term dataset of marked roe deer fawns in Switzerland (1971–2015) to reveal the characteristics of optimal bedding sites within the first two weeks of a fawn’s life in three contrasting landscapes and the potential trade-offs that may occur. We hypothesised that roe deer adjust the selection of bedding sites to current environmental conditions and available habitat to achieve sufficient levels of predator avoidance and thermoregulation necessary for the fawn’s survival, as well as the availability of sufficient food resources for the mother doe. We found that, in general, grassland habitats with medium vegetation height (20–50 cm) and habitats in close proximity to the edge of the forest were favoured to achieve those basic requirements. However, the use of bed site habitats differed between the three contrasting landscapes in dependence of elevation and hence vegetation phenology. Our results provide essential information to reduce mortality rates caused by mowing and improve the reproductive success of this species.