Myosin Vb Mediated Plasma Membrane Homeostasis Regulates Peridermal Cell Size and Maintains Tissue Homeostasis in the Zebrafish Epidermis

The epidermis is a stratified epithelium, which forms a barrier to maintain the internal milieu in metazoans. Being the outermost tissue, growth of the epidermis has to be strictly coordinated with the growth of the embryo. The key parameters that determine tissue growth are cell number and cell size. So far, it has remained unclear how the size of epidermal cells is maintained and whether it contributes towards epidermal homeostasis. We have used genetic analysis in combination with cellular imaging to show that zebrafish goosepimples/myosin Vb regulates plasma membrane homeostasis and is involved in maintenance of cell size in the periderm, the outermost epidermal layer. The decrease in peridermal cell size in Myosin Vb deficient embryos is compensated by an increase in cell number whereas decrease in cell number results in the expansion of peridermal cells, which requires myosin Vb (myoVb) function. Inhibition of cell proliferation as well as cell size expansion results in increased lethality in larval stages suggesting that this two-way compensatory mechanism is essential for growing larvae. Our analyses unravel the importance of Myosin Vb dependent cell size regulation in epidermal homeostasis and demonstrate that the epidermis has the ability to maintain a dynamic balance between cell size and cell number.     

Organization of the Human Mitochondrial Hydrogen Sulfide Oxidation Pathway

Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.     

An unusual cyanobacterium from saline thermal waters with relatives from unexpected habitats

Cyanobacteria that grow above seawater salinity at temperatures above 45°C have rarely been studied. Cyanobacteria of this type of thermo-halophilic extremophile were isolated from siliceous crusts at 40–45°C in a geothermal seawater lagoon in southwest Iceland. Iceland Clone 2e, a Leptolyngbya morphotype, was selected for further study. This culture grew only at 45–50°C, in medium ranging from 28 to 94 g L⁻¹ TDS, It showed 3 doublings 24 h⁻¹ under continuous illumination. This rate at 54°C was somewhat reduced, and death occurred at 58°C. A comparison of the 16S rDNA sequence with all others in the NCBI database revealed 2 related Leptolyngbya isolates from a Greenland hot spring (13–16 g L⁻¹ TDS). Three other similar sequences were from Leptolyngbya isolates from dry, endolithic habitats in Yellowstone National Park. All 6 formed a phylogenetic clade, suggesting common ancestry. These strains shared many similarities to Iceland Clone 2e with respect to temperature and salinity ranges and optima. Two endolithic Leptolyngbya isolates, grown previously at 23°C in freshwater medium, grew well at 50°C but only in saline medium. This study shows that limited genotypic similarity may reveal some salient phenotypic similarities, even when the related cyanobacteria are from vastly different and remote habitats.     

Cloning and expression of mannosylphospho dolichol synthase from bovine adrenal medullary capillary endothelial cells

Mannosylphospho dolichol synthase (DPMS) is a critical enzyme in the biosynthesis of lipid-linked oligosaccharide (LLO; Glc₃Man₉GlcNAc₂-PP-Dol), a pre-requisite for asparagine-linked (N-linked) protein glycosylation. We have shown earlier that DPMS is important for angiogenesis, i.e., endothelial cell proliferation. This is true when cAMP is used for intracellular signaling. During cAMP signaling, DPMS is activated and ER stress is reduced. To understand the activation of DPMS at the molecular level we have isolated a cDNA clone for the DPMS gene (bDPMS) from the capillary endothelial cells of bovine adrenal medulla. DNA sequencing and the deduced amino acid sequence have established that bDPMS has a motif to be phosphorylated by cAMP-dependent protein kinase (PKA). Based on the sequence information Serine 165 has been found to be the phosphorylation target in bDPMS. Hydropathy Index when plotted against amino acid number indicates the presence of a hydrophobic region around the amino acid residues 120–160, supporting that bDPMS has one membrane spanning region. The recombinant bDPMS has now been purified as His-tag protein with an apparent molecular weight of M _r 33 kDa. Additionally, we show here that overexpression of DPMS is indeed angiogenic. The capillary endothelial cells proliferate at a higher rate carrying the DPMS overexpression plasmid over the parental cells or the vector.     

Optimization of extraction and purification of glucoamylase produced by Aspergillus awamori in solid-state fermentation

Various parameters such as solvent selection, concentration, soaking time, and temperature were tested in a single bioreactor in order to determine optimum extraction conditions of glucoamylase, when produced simultaneously with protease by Aspergillus awamari nakazawa MTCC 6652. Optimum conditions were achieved in a 10% glycerol solution soaked for 2 h at 40°C, followed by concentration of extracted glucoamylase (9,157 U/gds) by acetone precipitation (1:2, v/v), which yielded 51.9% recovery. Ion exchange chromatography and gel filtration showed specific activities of 270.5 and 337.5 U/mg, respectively, while SDS-PAGE and zymogram analysis of glucoamylase indicated the presence of three starch-hydrolyzing isoforms with molecular weights of approximately 109.6, 87.1, and 59.4 kDa, respectively     

Linear polyethyleneimine grafted to a hyperbranched poly(ethylene glycol)-like core: a copolymer for gene delivery

A block copolymer of a hyperbranched poly(ethylene glycol)-like core and linear polyethylenimine (HBP) was synthesized by a facile synthetic route that included (1) a single-step cationic copolymerization of diepoxy and polyhydroxyl monomers, (2) derivatization of hydroxyl groups of the core HBPEG copolymer with either tosyl or chloromethylbenzoyl chlorides resulting in a corresponding macroinitiator, and (3) synthesis of HBPEG-block-poly(alkyl oxazolines). HBPEG-block-linear polyethyleneimine (HBP) was obtained by hydrolysis of HBPEG-block-poly(alkyl oxazolines). Linear PEI-bearing hyperbranched polycations (HBP) had lower inherent toxicity in cell culture than PEG-grafted linear polyethyleneimines (PEGLPEI). PEGLPEI formed a complex with DNA with an average diameter of 250 nm. The complexes were loosely condensed and formed aggregates and precipitates during storage. By contrast, hyperbranched polycations (HBP) formed approximately 50 nm nanocomplexes with DNA that were stable for several weeks and showed resistance to DNAse I-mediated degradation. The 'inverted' block copolymers showed several orders of magnitude higher transfection efficiency than PEGLPEI in vitro. Because of the biocompatibility and higher transfection efficiency, the 'inverted' block copolymer merits further investigation as a gene carrier.     

Defining the molecular role of gp91phox in the immune manifestation of acute allergic asthma using a preclinical murine model

ABSTRACT: OBJECTIVE: The phenomena manifested during inflammation require interplay between circulating effector cells, local resident cells, soluble mediators and genetic host factors to establish, develop and maintain itself. Of the molecues involed in the initiation and perpetuation of acute allergic inflammation in asthma, the involvement of effector cells in redox reactions for producing O2- (superoxide anion) through the mediation of NADPH oxidase is a critical step. Prior data suggest that reactive oxygen species (ROS) produced by NADPH oxidase homologues in non-phagocytic cells play an important role in the regulation of signal transduction, while macrophages use a membrane-associated NADPH oxidase to generate an array of oxidizing intermediates which inactivate MMPs on or near them. MATERIALS AND METHODS AND TREATMENT: To clarify the role of gp91phox subunit of NADPH oxidase in the development and progression of an acute allergic asthma phenotype, we induced allergen dependent inflammation in a gp91phox-/- single knockout and a gp91phox-/-MMP-12-/- double knockout mouse models. RESULTS: In the knockout mice, both inflammation and airway hyperreactivity were more extensive than in wildtype mice post-OVA. Although OVA-specific IgE in plasma were comparable in wildtype and knockout mice, enhanced inflammatory cell recruitment from circulation and cytokine release in lung and BALf, accompanied by higher airway resistance as well as Penh in response to methacholine, indicate a regulatory role for NADPH oxidase in development of allergic asthma. While T cell mediated functions like Th2 cytokine secretion, and proliferation to OVA were upregulated synchronous with the overall robustness of the asthma phenotype, macrophage upregulation in functions such as proliferation, and mixed lymphocyte reaction indicate a regulatory role for gp91phox and an overall non-involvement or synergistic involvement of MMP12 in the response pathway (comparing data from gp91phox-/- and gp91phox-/-MMP-12-/- mice).     

Effect of heart rate on hemodynamic endpoints under concomitant microvascular disease in a porcine model

Diagnosis of the ischemic power of epicardial stenosis with concomitant microvascular disease (MVD) is challenging during coronary interventions, especially under variable hemodynamic factors like heart rate (HR). The goal of this study is to assess the influence of variable HR and percent area stenosis (%AS) in the presence of MVD on pressure drop coefficient (CDP; ratio of transstenotic pressure drop to the distal dynamic pressure) and lesion flow coefficient (LFC; ratio of %AS to the CDP at the throat region). We hypothesize that CDP and LFC are independent of HR. %AS and MVD were created using angioplasty balloons and 90-μm microspheres, respectively. Simultaneous measurements of pressure drop (DP) and velocity were done in 11 Yorkshire pigs. Fractional flow reserve (FFR), CDP, and LFC were calculated for the groups HR < 120 and HR > 120 beats/min, %AS < 50 and %AS > 50, and additionally for DP < 14 and DP > 14 mmHg, and analyzed using regression and ANOVA analysis. Regression analysis showed independence between HR and the FFR, CDP, and LFC while it showed dependence between %AS and the FFR, CDP, and LFC. In the ANOVA analysis, for the HR < 120 beats/min and HR > 120 beats/min groups, the values of FFR (0.82 ± 0.02 and 0.82 ± 0.02), CDP (83.15 ± 26.19 and 98.62 ± 26.04), and LFC (0.16 ± 0.03 and 0.15 ± 0.03) were not significantly different (P > 0.05). However, for %AS < 50 and %AS > 50, the FFR (0.89 ± 0.02 and 0.75 ± 0.02), CDP (35.97 ± 25.79.10 and 143.80 ± 25.41), and LFC (0.09 ± 0.03 and 0.22 ± 0.03) were significantly different (P < 0.05). A similar trend was observed between the DP groups. Under MVD conditions, FFR, CDP, and LFC were not significantly influenced by changes in HR, while they can significantly distinguish %AS and DP groups.     

Ovotoxic effects of galactose involve attenuation of follicle-stimulating hormone bioactivity and up-regulation of granulosa cell p53 expression

Clinical evidence suggests an association between galactosaemia and premature ovarian insufficiency (POI); however, the mechanism still remains unresolved. Experimental galactose toxicity in rats produces an array of ovarian dysfunction including ovarian development with deficient follicular reserve and follicular resistance to gonadotrophins that characterize the basic tenets of human POI. The present investigation explores if galactose toxicity in rats attenuates the bioactivity of gonadotrophins or interferes with their receptor competency, and accelerates the rate of follicular atresia. Pregnant rats were fed isocaloric food-pellets supplemented with or without 35% D-galactose from day-3 of gestation and continuing through weaning of the litters. The 35-day old female litters were autopsied. Serum galactose-binding capacity, galactosyltransferase (GalTase) activity, and bioactivity of FSH and LH together with their receptor competency were assessed. Ovarian follicular atresia was evaluated in situ by TUNEL. The in vitro effects of galactose were studied in isolated whole follicles in respect of generation of reactive oxygen species (ROS) and expression of caspase 3, and in isolated granulosa cells in respect of mitochondrial membrane potential, expression of p53, and apoptosis. The rats prenatally exposed to galactose exhibited significantly decreased serum GalTase activity and greater degree of galactose-incorporation capacity of sera proteins. LH biopotency and LH-FSH receptor competency were comparable between the control and study population, but the latter group showed significantly attenuated FSH bioactivity and increased rate of follicular atresia. In culture, galactose increased follicular generation of ROS and expression of caspase 3. In isolated granulosa cells, galactose disrupted mitochondrial membrane potential, stimulated p53 expression, and induced apoptosis in vitro; however co-treatment with either FSH or estradiol significantly prevented galactose-induced granulosa cell p53 expression. We conclude that the ovotoxic effects of galactose involves attenuation of FSH bioactivity that renders the ovary resistant to gonadotrophins leading to increased granulosa cell expression of p53 and follicular atresia.     

Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex

Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.