Expression,purification, phosphorylation and characterization of recombinant human statherin

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.     

Asexual reproduction in a population of a leafy hepatic species Lophozia silvicola Buch in central Norway

The seasonal pattern and degree of asexual reproduction of a boreal hepatic species, Lophozia silvicola Buch, were studied in a lowland population of the species in central Norway. Asexual reproduction was quantified as numbers of 1–2 -celled gemmae produced per individual shoot, with average production ranging from 1000 to 7000 gemmae per shoot. Despite a slightly increasing trend in early spring, average numbers of gemmae present on shoots were fairly constant during the growing season. Germinability of gemmae was high in early spring, but declined rapidly towards the end of the season, suggesting an increasing proportion of gemmae in the dormant stage in the diaspore bank. Despite repeated sampling, average shoot density remained fairly constant in L. silvicola colonies during the growth season. Gemma production did not correlate with shoot density. Local colonization efficiency was estimated as frequency of occupied substrate patches and proportion of substrate area occupied by L. silvicola in a 10×20 m plot. Colonizable substrates included decaying wood, bare soil and rock surfaces. Colonies of L. silvicola were small, and differences in colony sizes between the substrate types were not significant. Colonies were found in 38% of the substrate patches, but total cover was low, less than 0.1% of all colonizable substrate.     

Mendelian breeding units versus standard sampling strategies: Mitochondrial DNA variation in southwest Sardinia

We report a sampling strategy based on Mendelian Breeding Units (MBUs), representing an interbreeding group of individuals sharing a common gene pool. The identification of MBUs is crucial for case-control experimental design in association studies. The aim of this work was to evaluate the possible existence of bias in terms of genetic variability and haplogroup frequencies in the MBU sample, due to severe sample selection. In order to reach this goal, the MBU sampling strategy was compared to a standard selection of individuals according to their surname and place of birth. We analysed mitochondrial DNA variation (first hypervariable segment and coding region) in unrelated healthy subjects from two different areas of Sardinia: the area around the town of Cabras and the western Campidano area. No statistically significant differences were observed when the two sampling methods were compared, indicating that the stringent sample selection needed to establish a MBU does not alter original genetic variability and haplogroup distribution. Therefore, the MBU sampling strategy can be considered a useful tool in association studies of complex traits.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

Dynamic association of human insulin receptor with lipid rafts in cells lacking caveolae

Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.     

CD2AP contributes to cell migration and adhesion in cultured gastric epithelium

The potential association of CD2AP with the adherens junction protein E-cadherin, co-localization with the actin cytoskeleton, and involvement in cell migration was investigated in cultured rat gastric mucosal cells. In stationary cells, CD2AP was localized perinuclearly while E-cadherin was expressed along cell-cell contacts and F-actin formed a branched network and adhesion belts. In migrating cells, CD2AP appeared as thread-like accumulations in the leading edges, colocalizing with F-actin and occasionally with E-cadherin. Intracellular injection of anti-CD2AP significantly retarded the migration speed of the cells suggesting a crucial role for CD2AP in mucosal cell migration, possibly as a scaffolding protein between cell membrane proteins and actin cytoskeleton. Co-immunoprecipitation assays revealed that CD2AP and E-cadherin are in a complex in HGF stimulated cells. It is concluded that CD2AP interacts with E-cadherin and co-localizes with F-actin in the leading edge of migrating cells, and significantly contributes to cell migration in restituting gastric epithelium.     

Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH? group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.     

Microgeographic differentiation in historical Yemen inferred by morphometric distances

This study analyzed the variations in space of 8 body dimensions and 11 measures of the head of 1,244 adult Yemenite males, collected in 1933/34 by Coon in Yemen and in Hadhramawt. The aim was to evaluate the presence of geographic microdifferentiation of the populations settled in the different regions of Yemen at the time. Coon sub-divided the sample into six geographical areas according to birthplace and ethnicity of the individuals: Tihamah, the Western Mountains, the Central Plateau, the South Coast, the Eastern Mountains, and Hadhramawt. The results of ANCOVA (age as covariate) show that the observed differences of all variables among the six groups were highly statistically significant. Tukey's post-hoc test reveals higher statistically significant differences among four main groups: (1) Tihamah; (2) the Western Mountains and Central Plateau; (3) the Eastern Mountains; and (4) the Southern Coast and Hadhramawt. Multiple discriminant analysis carried out using only the data of the 11 measures of the head, the more "genetically" determined variables, confirmed these differences. Indeed, the first canonical variate well separates the groups with the Tihamah, Southern Coast and Hadhramawt on the one side and the Eastern Mountains, Western Mountains and Central Plateau on the other. The second canonical variate separates the Tihamah, Western Mountains and Central Plateau from the Eastern Mountains, Southern Coast and Hadhramawt. In conclusion, the Yemenite population seems to be composed of three morphologically distinct groups and an Eastern Mountains group which is positioned between the group formed by the Southern Coast and Hadhramawt and the Western Mountains and Central Plateau group. The Tihamah is the most distant from all the other groups. These differences are probably due to the presence/absence of geographical and cultural barriers that have favored/blocked the gene flow over the years. Indeed, the entire coastal bell, through the centuries, has constituted one of the principal commercial routes between the East, Africa, and the eastern Mediterranean, while the high inland mountains have isolated the remaining communities. This data is also confirmed by genetic studies. Finally, the average height (162.6 cm) of the global Yemenite population, compared to data from the other six middle-eastern Arab countries and Egypt, was found to be 3-6 cm less. This characteristic will be further studied, analyzing variations in average height according to the different age classes in order to evaluate any possible secular changes.