Spiders spinning electrically charged nano-fibres

Most spider threads are on the micrometre and sub-micrometre scale. Yet, there are some spiders that spin true nano-scale fibres such as the cribellate orb spider, Uloborus plumipes. Here, we analyse the highly specialized capture silk-spinning system of this spider and compare it with the silk extrusion systems of the more standard spider dragline threads. The cribellar silk extrusion system consists of tiny, morphologically basic glands each terminating through exceptionally long and narrow ducts in uniquely shaped silk outlets. Depending on spider size, hundreds to thousands of these outlet spigots cover the cribellum, a phylogenetically ancient spinning plate. We present details on the unique functional design of the cribellate gland–duct–spigot system and discuss design requirements for its specialist fibrils. The spinning of fibres on the nano-scale seems to have been facilitated by the evolution of a highly specialist way of direct spinning, which differs from the aqua-melt silk extrusion set-up more typical for other spiders.     

Homoeologous recombination within bread wheat to develop novel combinations of HMW-GS genes: transfer of the <Emphasis Type="Italic">Glu-A1</Emphasis> locus to chromosome 1D

In an attempt to improve the bread-making quality within hexaploid wheat by elaborating novel high-molecular weight glutenin subunits (HMW-GS) combinations useful in wheat-breeding programmes, a 1A chromosome fragment carrying the Glu-A1 locus encoding the subunit Ax2*, was translocated to the long arm of chromosome 1D. The partially isohomoeoallelic line, designated RR239, had a meiotic behaviour as regular as cv. Courtot. It was characterised using genomic in situ hybridization and microsatellite markers as well as biochemical and proteomic approaches. The translocated 1D chromosome had an interstitial 1AL segment representing in average 30% of the recombinant arm length that was confirmed by molecular analysis. The genetic length of the removed segment in chromosome 1DL was estimated to be at least 51 cM, and that of the interstitial 1AL translocation to be at least 33 cM. Proteome analysis performed on total endosperm proteins revealed variation in amounts, 8 spots and 1 spot being up- and downregulated, respectively. Quantitative variations in HMW-GS were observed for the Glu-A1 (Ax2*) and Glu-B1 (Bx7 + By8) loci in response to duplication of the Glu-A1 locus.     

Methods for homocysteine analysis and biological relevance of the results

It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B(2), B(6), B(9), B(12)) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.     

Characterization of aspartate transcarbamylase activity from gonads of the soft shell clam,Mya arenaria

Aspartate transcarbamylase (ATCase, EC 2.1.3.2) has been shown to be a good index of the reproductive cycle in marine molluscs. However, this enzyme has never been studied in the soft shell clam Mya arenaria. The characteristics of gonadal ATCase of the soft shell clam, Mya arenaria were therefore determined since we need powerful tools to assess the degree of effects of endocrine disruptors in this species at risk. Enzyme kinetic values observed at pH 8.3 were significantly lower than those measured at pH 9.4. The optimal conditions for the enzyme assays were reached in the presence of a 10 mM of substrate concentration and at pH 9.2 for 60 min at 37 °C. We have found that the enzyme was heat sensitive, markedly activated by DMSO and DMF, but no effect was observed with ethanol, ATP or CTP. However, clam ATCase activity was partly inhibited by the addition of CuSO₄ and PHMB to the medium, an inhibition that could be attributed to the presence of SH sites in cysteine residues localized in the catalytic site of this enzyme. All these results will be very useful in the near future to study the gametogenetic process of Mya arenaria, since little is known about the factors that control the physiological process of reproduction in this bivalve of ecological and economic importance. Studies of variations of the activity of aspartate transcarbamylase will also be useful as a potential biomarker to evaluate the disruption of gametogenesis in clams exposed to endocrine disruptors in situ.     

Engineering of phytase for improved activity at low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Effects of wounds on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

Summary The effect of wounds on the volatile terpene composition of the living bark of Pinus pinaster Ait. (maritime pine) twigs was investigated with respect to the processes of mono- and sesquiterpene hydrocarbon biosynthesis. The large increase in the amounts of - and -pinene is a characteristic feature after a mechanical injury, whereas the quantities of the other terpenes are only slightly increased. This is due to the reactivation of the resin duct secretory cells of primary origin located in cortical tissues. The effect of wounding is observed over a long period and the terpene profiles are very different at the end of the experiments as compared with the initial profiles of the same tissues. The traumatic essential oil (obtained after mechanical traumatism) resembles an oleoresin extracted from tissues of secondary origin. Statistical analysis underlines the effects of the between-tree variations and of the dates of application of the wound.     

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]-substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.     

Bicyclic heteroaryl inhibitors of stearoyl-CoA desaturase: from systemic to liver-targeting inhibitors

Optimization of a lead thiazole amide MF-152 led to the identification of potent bicyclic heteroaryl SCD1 inhibitors with good mouse pharmacokinetic profiles. In a view to target the liver for efficacy and to avoid SCD1 inhibition in the skin and eyes where adverse effects were previously observed in rodents, representative systemically-distributed SCD1 inhibitors were converted into liver-targeting SCD1 inhibitors.