Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.     

Turbo-mixing in microplates

How to effectively mix small volumes of liquids within microplate wells is a still underestimated and often neglected challenge. The method the authors introduce here relies on violent turbulent motion within a liquid caused by spotting an organic solvent drop onto its surface. The amount needed, less than 1 to 3 microL, is generally small enough not to alter bioactive molecules. Moreover, a solvent may be selected for its compatibility with assay components. The method was tested with layers of aqueous liquids that differ in pH and concentration of a pH-dependent dye, allowing mixing to be monitored optically. Rapid mixing was caused by spotting drops of alcohols, acetone, acetonitrile, and aqueous solutions of these, as long as the difference of surface tension between the drop and the uppermost layer of the bulk liquid surpassed 30 dynes/cm. Along with this difference, position and velocity of spotting, as well as viscosity and geometry of the bulk liquid volume, may influence the turbulence evoked. No significant difference was found for the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase when measured after mixing by shaking and after mixing by spotting 1 microL of methanol onto assays within 96-well microplates.     

Generation of mice deficient for Lbx2, a gene expressed in the urogenital system, nervous system, and Pax3 dependent tissues

Lbx2 is a member of the ladybird family of homeobox genes. The first murine ortholog identified, Lbx1, is required for hypaxial musculature and dorsal spinal cord neuron development. The second murine ortholog, Lbx2, is expressed in the developing urogenital and nervous systems. To elucidate the function of Lbx2, we generated a gene-targeted allele of Lbx2 in mice. Lbx2 deficiency did not impair mouse development, and Lbx2 null mice appeared healthy and fertile. Replacement of Lbx2 by the lacZ gene provides a valuable histological marker for Lbx2-expressing cells. Given the important role of Pax3 in neural crest, we intercrossed our Lbx2 deficient mice with Splotch Pax3 mutant mice to determine if Pax3 affects Lbx2 expression. There was reduced Lbx2 expression in dorsal root ganglia and cranial nerve ganglia with Pax3 deficiency, but not in the genital tubercle. This suggested that Pax3 is required for Lbx2 expression in affected neural crest-derived tissues.     

Grazing conserves threatened carabid beetles in semi-natural calcareous grasslands better than mowing,especially at low intensities

Biodiversity and Conservation - Semi-natural grasslands are biodiverse ecosystems that support many threatened species, but they require management interventions to maintain their habitat...     

Mating success at high temperature in highland‐ and lowland‐derived populations as well as in heat knock‐down selected Drosophila buzzatii

Thermal‐stress selection can affect multiple fitness components including mating success. Reproductive success is one of the most inclusive measures of overall fitness, and mating success is a major component of reproduction. However, almost no attention has been spent to test how mating success can be affected by thermal‐stress selection. In this study, we examine the mating success in the cactophilic Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae) derived from two natural populations that nearly represent the ends of an altitudinal cline for heat knock‐down resistance. Furthermore, we extended the analysis using laboratory lines artificially selected for high and low heat knock‐down resistance. Mating success at high temperature was found to be higher in the lowland than the highland population after a heat pre‐treatment. Moreover, individuals selected for heat knock‐down resistance showed higher mating success at high temperature than did individuals selected for low knock‐down resistance. These results indicate that adaptation to thermal stress can confer an advantage on fitness‐related traits including mating success and highlight the benefits of earlier heat exposure as an adaptive plastic response affecting mating success under stress of higher temperature.     

Reconstructing a puzzle: existence of cyanophages containing both photosystem‐I and photosystem‐II gene suites inferred from oceanic metagenomic datasets

Cyanobacteria play a key role in marine photosynthesis, which contributes to the global carbon cycle and to the world oxygen supply. Genes encoding the photosystem‐II (PSII) reaction centre are found in many cyanophage genomes, and it was suggested that the horizontal transfer of these genes might be involved in increasing phage fitness. Recently, evidence for the existence of phages carrying Photosystem‐I (PSI) genes was also reported. Here, using a combination of different marine metagenomic datasets and a unique crossing of the datasets, we now describe the finding of phages that, as in plants and cyanobacteria, contain both PSII and PSI genes. In addition, these phages also contain NADH dehydrogenase genes. The presence of modified PSII and PSI genes in the same viral entities in combination with electron transfer proteins like NAD(P)H dehydrogenase (NDH‐1) strongly points to a role in perturbation of the cyanobacterial host photosynthetic electron flow. We therefore suggest that, depending on the physiological condition of the infected cyanobacterial host, the viruses may use different options to maximize survival. The modified PSI may alternate between functioning with PSII in linear electron transfer and contributing to the production of both NADPH and ATP or functioning independently of PSII in cyclic mode via the NDH‐1 complex and thus producing only ATP.     

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.     

BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.