Purification and characterization of an acetylcholinesterase from the infective juveniles of Heterorhabditis bacteriophora

Acetylcholinesterases (AChEs) have been estimated in the infective juveniles (IJs) of eight different strains of heterorhabditid nematodes. The enzyme content ranged from 45.6 to 421.3 units/10(5) IJs with specific activity 34.0 to 82.6 units/mg protein. The isoenzyme patterns revealed the existence of two-slow-moving isoforms. Heterorhabditis bacteriophora AChE1A has been purified from the IJs of the heterorhabditid nematode strain of the highest enzymatic activity to homogeneity by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and DEAE-Sepharose. The specific activity of the purified enzyme was 1378.1 units/mg protein with purification fold 17.5 over crude extract. The enzyme has a pH optimum at 7.5. The optimum temperature for enzyme activity and stability was 35 degrees C. The activation energy was calculated to be 9.0 kcal/mol. The enzyme hydrolyzes acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), S-butyrylthiocholine (BuSCh) and benzoylthiocholine (BzSCh) iodides with relative rate 100, 74.6, 41.7 and 22.2%, respectively. It displayed an apparent Michaelis-Menten behavior in the concentration range from 0.1 to 2 mM for the three former substrates with Km values 0.27, 0.42 and 0.59 mM, respectively. H. bacteriophora ChE1A is an AChE since it hydrolyzed AcSChI at higher rate than the other substrates and displayed excess substrate inhibition with AcSChI at concentrations over 2 mM. It was inhibited by eserine and BW284C51, but not by iso-OMPA. Its biochemical properties were compared with those reported for different species of insects as target hosts for heterorhabditid nematodes and animal parasitic nematodes.     

Myrionema strangulans (Chordariales, Phaeophyceae) epiphyte on Ulva spp. (Ulvophyceae) from Patagonian Atlantic coasts

Fronds of Ulva spp. from Patagonian Atlantic coasts exhibited brown spots produced by the presence of Myrionema strangulans (Chordariales, Phaeophyceae). The occurrence of M. strangulans on Ulva spp. is widely reported from several regions of the world, but there were no detailed studies about the subject. In the present study, we describe the morphology and interactions of M. strangulans with Ulva spp. as observed under light and electron microscopes, and we reconstruct all stages of its life cycle based upon in vitro experiments. The prevalence of infection by M. strangulans was 100%. In case of the strongest epiphytism, the host cuticle exhibited perforations, massive depigmentation, cellular disorganization, and cuticle rupture. It was possible to demonstrate a purely epiphytic life strategy of the organism by transmission electron microscopy. M. strangulans formed discoid thalli constituted by vegetative filaments and radiating from a central zone to a peripheral zone. Transversally, the discs were formed by two strata: a basal monostromatic and a filamentous erect stratum. From the monostromatic stratum, hyaline hairs and reproductive structures were produced. Both plurilocular and unilocular sporangia were present. Zoids from both plurilocular and unilocular sporangia were able to germinate in culture. M. strangulans exhibited a haploid–diploid, heteromorphic life cycle with thalli with three different morphologies. The haploid chromosome number was 12?±?2 chromosomes.     

Muscle cell depolarization induces a gain in surface GLUT4 via reduced endocytosis independently of AMPK

Contracting skeletal muscle increases glucose uptake to sustain energy demand. This is achieved through a gain in GLUT4 at the membrane, but the traffic mechanisms and regulatory signals involved are unknown. Muscle contraction is elicited by membrane depolarization followed by a rise in cytosolic Ca2+ and actomyosin activation, drawing on ATP stores. It is unknown whether one or more of these events triggers the rise in surface GLUT4. Here, we investigate the effect of membrane depolarization on GLUT4 cycling using GLUT4myc-expressing L6 myotubes devoid of sarcomeres and thus unable to contract. K+-induced membrane depolarization elevated surface GLUT4myc, and this effect was additive to that of insulin, was not prevented by inhibiting phosphatidylinositol 3-kinase (PI3K) or actin polymerization, and did not involve Akt activation. Instead, depolarization elevated cytosolic Ca2+, and the surface GLUT4myc elevation was prevented by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum) and by extracellular Ca2+ chelation. Ca2+-calmodulin-dependent protein kinase-II (CaMKII) was not phosphorylated after 10 min of K+ depolarization, and the CaMK inhibitor KN62 did not prevent the gain in surface GLUT4myc. Interestingly, although 5'-AMP-activated protein kinase (AMPK) was phosphorylated upon depolarization, lowering AMPKalpha via siRNA did not alter the surface GLUT4myc gain. Conversely, the latter response was abolished by the PKC inhibitors bisindolylmaleimide I and calphostin C. Unlike insulin, K+ depolarization caused only a small increase in GLUT4myc exocytosis and a major reduction in its endocytosis. We propose that K+ depolarization reduces GLUT4 internalization through signals and mechanisms distinct from those engaged by insulin. Such a pathway(s) is largely independent of PI3K, Akt, AMPK, and CaMKII but may involve PKC.     

Food restriction induced thyroid changes and their reversal after refeeding in female rats and their pups

In the present study, two groups of pregnant female rats were submitted to food restriction (24 h fast versus 24 h diet intake) from the 14th day of pregnancy until either the 14th day (group B) or the 4th day after parturition (group C). All pups and their mothers were sacrificed on day 14 after delivery. The body weight of the 14-day-old pups (group B) was 46% less than the controls (group A). Free thyroxine and free triiodothyronine levels in the plasma were reduced by 44 and 16% in pups and by 20 and 36% in their mothers, respectively. These reductions were correlated with a decrease in thyroid iodine content of the pups (-50%) and their mothers (-24%). Radioiodine uptake (131I) by the thyroid gland of pups was significantly increased by 27%. Plasma TSH levels were decreased by 38% in pups and by 44% in dams. Morphological changes in thyroid glands were observed in energy restricted dams and in their pups. Some of follicles in pups were empty. Moroever in dams, we noted the presence of peripheral resorbed vacuoles, sign of thyroid hyperactivity. After a refeeding (group C) period of ten days, total recovery occurred in plasma thyroid hormone levels (FT4 and FT3) and in thyroid iodine contents of pups in spite of a partial recovery of body weights and plasma TSH levels. In dams, a partial recovery occurred in plasma thyroid hormone levels in spite of total recovery in thyroid iodine contents, while plasma TSH levels exceeded control values. A significant amelioration in thyroid histological aspects was observed in pups and their dams.     

Loss of CHSY1, a secreted FRINGE enzyme, causes syndromic brachydactyly in humans via increased NOTCH signaling

We delineated a syndromic recessive preaxial brachydactyly with partial duplication of proximal phalanges to 16.8 Mb over 4 chromosomes. High-throughput sequencing of all 177 candidate genes detected a truncating frameshift mutation in the gene CHSY1 encoding a chondroitin synthase with a Fringe domain. CHSY1 was secreted from patients' fibroblasts and was required for synthesis of chondroitin sulfate moieties. Noticeably, its absence triggered massive production of JAG1 and subsequent NOTCH activation, which could only be reversed with a wild-type but not a Fringe catalytically dead CHSY1 construct. In vitro, depletion of CHSY1 by RNAi knockdown resulted in enhanced osteogenesis in fetal osteoblasts and remarkable upregulation of JAG2 in glioblastoma cells. In vivo, chsy1 knockdown in zebrafish embryos partially phenocopied the human disorder; it increased NOTCH output and impaired skeletal, pectoral-fin, and retinal development. We conclude that CHSY1 is a secreted FRINGE enzyme required for adjustment of NOTCH signaling throughout human and fish embryogenesis and particularly during limb patterning.     

Synthesis of carboxymethyl cellulose (CMC) and starch-based hybrids and their applications in flocculation and sizing

Starch and hydrolyzed starches along with carboxymethyl cellulose were independently subjected to cationization by reacting them with N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (known commercially as Quat 188) in presence of alkaline catalyst. The cationization reaction was carried out under different conditions Optimal reaction conditions could be achieved at Quat 188 (2 mmol), NaOH (1 mmol) at temperature (70 °C) 60 min for CMC or 120 min for starch. The cationic products were characterized by FTIR spectroscopy, and nitrogen percentage. Application of the cationized products as well as their parent materials in textile sizing was also undertaken. Moreover, hybrids of starch-Quat 188 and CMC-Quat 188 were used as flocculants; flocculation was carried at pH 6 using different flocculant dose. Results obtained indicate that the cationic-based hybrids – under investigation display characteristics, which qualify them to function as an excellent sizing and flocculating agents.     

Individual Based Model for Grouper Populations

Dusky groupers (Epinephelus marginatus) are characterized by a complex sex allocation strategies and overexploitation of bigger individuals. We developed an individual based model to investigate the long-term effects of density dependence on grouper population dynamics and to analyze the variabilities of extinction probabilities as a result of interacting mortalities at different life stages. We conduct several simulations with different forms of sex allocation functions and different combinations of mortality rates. The model was parametrized using data on dusky grouper populations from the literature. The most important insights produced by this simulation study are that density dependence of sex allocation is an evolutionarily stable strategy, increases the population biomass, mitigates the effect of the removal of large male and indicates a need for protection of females and flexible stages.     

Inhibition of myostatin in adult mice increases skeletal muscle mass and strength

A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.