Chemically-induced affinity star restriction specificity: a novel TspGWI/sinefungin endonuclease with theoretical 3-bp cleavage frequency

The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes and cleaves at least 12 degenerate variants of the original recognition sequence that vary by single base pair changes from the original 5-bp restriction site with only a single degeneracy per variant appearing to be allowed. In addition, sinefungin was found to have a stimulatory effect on cleavage at these nondegenerate TspGWI recognition sites, irrespective of their number or the DNA topology. Interestingly, no fixed "core" could be identified among the new recognition sequences. Theoretically, TspGWI cleaves DNA every 1024 bp, while sinefungin-induced activity cleaves every 78.8 bp, corresponding to a putative 3-bp long recognition site. Thus, the combination of sinefungin and TspGWI represents a novel frequent cutter, next only to CviJI/CviJI*, that should prove useful in DNA cloning methodologies.     

Kinetic model of oxidation catalyzed by xanthine oxidase-the final enzyme in degradation of purine nucleosides and nucleotides

A new kinetic model is presented for analysis of experimental data of oxidation process catalyzed by milk xanthine oxidase. The kinetics for two substrates, xanthine and its analog 2-chloroadenine, in a broad pH range (5.8-9.0) are best described by an equation which is a rational function of degree 2:3 and 2:2, respectively.     

Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors

Caveolae are present in almost all cells and concentrate a wide variety of signaling molecules, receptors, transporters, and ion pumps. We have investigated the distribution of the ryanodine receptor, the Na(+)/Ca(2+) exchanger, the predominant Na(+) channel isoform rH1, and the L-type calcium channel, Ca(v)1.2, relative to the muscle-specific caveolin isoform, caveolin-3, in adult rat ventricular myocytes. Three-dimensional immunofluorescence images were deconvolved and analyzed. Caveolin-3 colocalizes with all of these molecules at the surface of the cell, but there is no significant colocalization between caveolin-3 and either the Na(+)/Ca(2+) exchanger or the Na(+) channel in the cell interior. The distribution of the surface colocalization indicates that the caveolae that colocalize with each molecule form distinct populations. This organization indicates that there are multiple populations of caveolae separable by location and occupants. In the interior of the cell, caveolin-3 shows a marked colocalization with a population of ryanodine receptors that are separate from those within the dyad. Because of their location, the signaling molecules contained within these caveolae may have preferred access to the neighboring nondyadic ryanodine receptors.     

Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities

Gilthead sea bream (Sparus aurata) is a euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-adaptive processes and their hormonal control. Immature sea bream acclimated to different salinities, i.e. SW (38 per thousand), LSW (5 per thousand) and HSW (55 per thousand), were kept at 18 degrees C under natural photoperiod. Arginine vasotocin (AVT) and isotocin (IT) in plasma and pituitary were determined by HPLC. Plasma melatonin (Mel) was assayed by RIA. Plasma osmolality, ion concentrations (Na(+), K(+), Ca(2+), Cl(-)) and Na(+),K(+)-ATPase activity in gill were measured. A steady increase in plasma AVT, along with increasing water salinity was observed. Pituitary IT concentration in HSW-acclimated fish was significantly higher than that in LSW group. AVT/IT secretory system of sea bream does appear to be involved in the mechanism of long-term acclimation to different salinities. The distinct roles and control mechanisms of both nonapeptides are suggested. Plasma Mel was significantly higher in LSW compared with both HSW and SW groups. Data indicate that the changes in Mel level are linked to osmoregulation. Further studies are required to elucidate a complex role of AVT, IT and Mel in sea bream osmoregulation.     

Glycoprotein hypersecretion alters the cell wall in Trichoderma reesei strains expressing the Saccharomyces cerevisiae dolichylphosphate mannose synthase gene

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.     

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.     

Lateral root organogenesis - from cell to organ

Unlike locomotive organisms capable of actively approaching essential resources, sessile plants must efficiently exploit their habitat for water and nutrients. This involves root-mediated underground interactions allowing plants to adapt to soils of diverse qualities. The root system of plants is a dynamic structure that modulates primary root growth and root branching by continuous integration of environmental inputs, such as nutrition availability, soil aeration, humidity, or salinity. Root branching is an extremely flexible means to rapidly adjust the overall surface of the root system and plants have evolved efficient control mechanisms, including, firstly initiation, when and where to start lateral root formation; secondly lateral root primordia organogenesis, during which the development of primordia can be arrested for a certain time; and thirdly lateral root emergence. Our review will focus on the most recent advances in understanding the molecular mechanisms involved in the regulation of lateral root initiation and organogenesis with the main focus on root system of the model plant Arabidopsis thaliana.     

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site

The aim of this study was to improve a useful molecular tool—TaqII restriction endonuclease-methyltransferase—by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue—TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.