The analysis of gene transcripts associated with conidiation in the insect pathogenic fungus, Metarhizium anisopliae /

Conidia of the insect pathogenic fungus, Metarhizium anisopliae play an important role in pathogenicity because they are the infective propagules that adhere to the surface of the insect, then germinate and give rise to hyphal penetration of the insect cuticle. Conidia are produced in the final stages of insect infection as the mycelia emerge from the insect cadaver. The genes associated with conidiation have not yet been studied in this fiingus. hi this study we used the PCR-based technique, suppression subtractive hybridization (SSH) to selectively amplify conidial-associated genes in M. anisopliae. We then identified the presence of these differentially expressed genes using the National Center for Biotechnology Information database. One of the transcripts encoded an extracellular subtilisin-like protease, Prl, which plays a fundamental role in cuticular protein degradation. Analysis of the patterns of gene expression of the transcripts using RT-PCR indicated that conidial-associated cDNAs are expressed during the development of the mature conidium. RT-PCR analysis was also performed to examine in vivo expression of Prl during infection of waxworm larvae {Galleria mellonelld). Results showed expression of Prl as mycelia emerge and produce conidia on the surface of the cadaver. It is well documented that Prl is produced during the initial stages of transcuticular penetration by M. anisopliae. We suggest that upregulation of Prl is part of the mechanism by which reverse (from inside to the outside of the host) transcuticular penetration of the insect cuticle allows subsequent conidiation on the cadaver.

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

TIME COURSE OF SIGNALING PROCESSES INVOLVED WITH EXTRACELLULAR OSMOTIC - INDUCED GLUCOSE UPTAKE IN MAMMALIAN SKELETAL MUSCLE

Extracellular hyper-osmotic (HYPER) stress increases glucose uptake to defend cell volume, when compared to iso-osmotic (ISO) conditions in skeletal muscle. The purpose of this study was to determine a time course for changes in common signaling proteins involved in glucose uptake during acute hyper-osmotic stress in isolated mammalian skeletal muscle. Rat extensor digitorum longus (EDL) muscles were excised and incubated in a media formulated to mimic ISO (290 ± 10 mmol/kg) or HYPER (400 ± 10 mmol/kg) extracellular condition (Sigma Media-199). Signaling mechanisms were investigated by determining the phosphorylation states of Akt, AMPK, AS160, cPKC and ERK after 30, 45 and 60 minutes of incubation. AS160 was found to be significantly more phosphorylated in HYPER conditions compared to ISO after 30 minutes (p<0.01). It is speculated that AS160 phosphorylation increases glucose transporter 4 (GLUT4) content at the cell surface thereby facilitating an increase in glucose uptake under hyper-osmotic stress.

Investigation of the Time Dependent Influence of Extracellular Osmotic Stress on Protein Turnover in Skeletal Muscle Cells

Acute alterations in cell volume can substantively modulate subsequent metabolism of substrates. However, how such alterations in skeletal muscle modulate protein metabolism is limited. The purpose of this study was to determine the time dependent influence of extracellular osmotic stress on protein turnover in skeletal muscle cells. L6 cells were incubated in hyperosmotic (HYPER; 425.3 ± 1.8mmol/kg), hypo-osmotic (HYPO; 235.4 ± 1.0mmol/kg) or control (CON; 333.5 ± 1.4mmol/kg) media for 4, 8, 12, or 24hrs. During the final 4hrs, incorporation of L-[ring-3,5-3H]-tyrosine was measured to estimate protein synthesis. Western blotting measured markers of protein synthesis and degradation. No differences were observed in any outcomes except p70S6K phosphorylation whereby HYPO was lower (p<0.05) than CON and HYPER; which remained similar except for a large increase at 8hrs for HYPER. These findings suggest that regardless of duration, extracellular osmotic stress does not significantly affect protein metabolism in L6 cells.

INFLUENCE OF INCREASED EXTRACELLULAR LEUCINE ON THE PROTEIN METABOLIC RESPONSES DURING OSMOTIC STRESS IN SKELETAL MUSCLE

The purpose of this study was to examine the effects of increased extracellular leucine concentration on protein metabolism in skeletal muscle cells when exposed to 3 different osmotic stresses. L6 skeletal muscle cells were incubated in either a normal or supplemental leucine (1.5mM) medium set to hypo-osmotic (230 ± 10 Osm), iso-osmotic (330 ± 10 Osm) or hyper-osmotic (440 ± 10 Osm) conditions. 3H-tyrosine was used to quantify protein synthesis. Western blotting analysis was performed to determine the activation of mTOR, p70S6k, ubiquitin, actin, and μ-calpain. Hypo-osmotic stress resulted in the greatest increase in protein synthesis rate under the normal-leucine condition while iso-osmotic stress has the greatest increase under the elevated-leucine condition. Elevated-leucine condition had a decreased rate in protein degradation over the normal condition within the ubiquitin proteasome pathway (p<0.05). Leucine and hypo-osmotic stress therefore creates a favourable environment for anabolic events to occur.

Intracellular trafficking of protease : Activated Receptor 2 (PAR2) by members of sorting nexins family

Le dogme voulant que les récepteurs couplés aux protéines G (GPCRs) activent des voies de signalisation seulement lorsqu’ils sont localisés à la membrane plasmatique, a récemment été remis en question. Des données récentes indiquent que certains GPCRs peuvent également induire une réponse intracellulaire à partir des compartiments intracellulaires dont le noyau. Les récepteurs activés par la protéase (PAR) sont des membres de la famille GPCR. Les PARs sont activés par le clivage de la partie N–terminale du récepteur ce qui permet au ligand attaché sur le récepteur de se lier à sa poche réceptrice. Quatre PARs ont été décrits : PAR1, PAR2, PAR3 et PAR4. PAR2 peut susciter des effets mitogéniques et participer aux processus comme l’angiogenèse et l'inflammation. Alors que beaucoup d'effets intracellulaires de PAR2 peuvent être expliqués lorsqu’il est localisé à la membrane plasmatique, une fonction intracrine de PAR2 a aussi été proposée. Pourtant les mécanismes par lesquels PAR2 peut provoquer l’expression de gènes ciblés sont toujours inconnus. Le but de notre étude était de vérifier l’existence d’une population nucléaire de PAR2. Nous avons également émis l’hypothèse que les voies activées par l’activation de PAR2 dépendent de sa localization cellulaire. En utilisant des techniques de microscopie confocale et de "Western Blot" nous avons démontré la présence d’une population nucléaire de PAR2. À la suite de la stimulation de PAR2, nous avons observé une augmentation de la translocation du récepteur de la membrane plasmatique au noyau. En utilisant la technique de "RT – PCR", nous avons observé des rôles différents de PAR2 à la surface de la cellule et du noyau dans l’initiation de l’expression des gènes. Afin d’identifier les mécanismes responsables de la translocation nucléaire de PAR2, nous avons évalué l’implication des membres de la famille de "Sorting Nexins (SNX)" dans la translocation nucléaire de PAR2. "Sorting Nexins" est un groupe de protéines avec des fonctions de transport bien établies. SNX1 et SNX2 ont été identifiés comme responsables du transfert de PAR1 vers les lysosomes. SNX11 n'a pas encore été étudié et nous avons émis l’hypothèse qu'il pourrait être un autre membre de la famille des SNX impliqué dans la signalisation de PAR2. Pour ce faire, nous avons développé des "knockdowns" stables pour SNX1, SNX2 et SNX11 dans les cellules HEK293. En utilisant les essais d’immunofluorescence, "Western Blot" et de cytométrie en flux, nous avons déterminé que tous les trois membres du groupe SNX sont des partenaires d'interaction de PAR2. Toutefois, seul SNX11 se co-localise avec son partenaire au noyau et est responsable de sa translocation nucléaire. Les expériences de "RT - PCR" sur les lignées de cellule de SNXs "knockdowns" ont démontré que la fonction de PAR2 nucléaire dépend surtout de SNX11; néanmoins SNX1 et SNX2 peuvent aussi l’influencer, suggérant qu'ils font aussi partie du réseau signalétique de PAR2. En conclusion, PAR2 est déplacé de la membrane plasmatique à la membrane nucléaire après sa stimulation avec un agoniste. La translocation nucléaire de PAR2 par un mécanisme impliquant SNX11, initie des effets intracellulaires différents de sa signalisation membranaire. Mots clés : récepteurs couplés à la protéine G, “Sorting Nexins”, récepteurs activés par la protéase, translocation nucléaire, membrane nucléaire, signal nucléaire.

Identification and characterization of a new adhesin involved in the binding of Streptococcus suis to the extracellular matrix proteins

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Increased extracellular brain glutamate in acute liver failure: decreased uptake or increased release?

Glutamatergic dysfunction has been suggested to play an important role in the pathogenesis of hepatic encephalopathy (HE) in acute liver failure (ALF). Increased extracellular brain glutamate concentrations have consistently been described in different experimental animal models of ALF and in patients with increased intracranial pressure due to ALF. High brain ammonia levels remain the leading candidate in the pathogenesis of HE in ALF and studies have demonstrated a correlation between ammonia and increased concentrations of extracellular brain glutamate both clinically and in experimental animal models of ALE Inhibition of glutamate uptake or increased glutamate release from neurons and/or astrocytes could cause an increase in extracellular glutamate. This review analyses the effect of ammonia on glutamate release from (and uptake into) both neurons and astrocytes and how these pathophysiological mechanisms may be involved in the pathogenesis of HE in ALF.

Association of reduced extracellular brain ammonia, lactate, and intracranial pressure in pigs with acute liver failure.

We previously demonstrated in pigs with acute liver failure (ALF) that albumin dialysis using the molecular adsorbents recirculating system (MARS) attenuated a rise in intracranial pressure (ICP). This was independent of changes in arterial ammonia, cerebral blood flow and inflammation, allowing alternative hypotheses to be tested. The aims of the present study were to determine whether changes in cerebral extracellular ammonia, lactate, glutamine, glutamate, and energy metabolites were associated with the beneficial effects of MARS on ICP. Three randomized groups [sham, ALF (induced by portacaval anastomosis and hepatic artery ligation), and ALF+MARS] were studied over a 6-hour period with a 4-hour MARS treatment given beginning 2 hours after devascularization. Using cerebral microdialysis, the ALF-induced increase in extracellular brain ammonia, lactate, and glutamate was significantly attenuated in the ALF+MARS group as well as the increases in extracellular lactate/pyruvate and lactate/glucose ratios. The percent change in extracellular brain ammonia correlated with the percent change in ICP (r(2) = 0.511). Increases in brain lactate dehydrogenase activity and mitochondrial complex activity for complex IV were found in ALF compared with those in the sham, which was unaffected by MARS treatment. Brain oxygen consumption did not differ among the study groups. Conclusion: The observation that brain oxygen consumption and mitochondrial complex enzyme activity changed in parallel in both ALF- and MARS-treated animals indicates that the attenuation of increased extracellular brain ammonia (and extracellular brain glutamate) in the MARS-treated animals reduces energy demand and increases supply, resulting in attenuation of increased extracellular brain lactate. The mechanism of how MARS reduces extracellular brain ammonia requires further investigation.

L-ornithine phenylacetate attenuates increased arterial and extracellular brain ammonia and prevents intracranial hypertension in pigs with acute liver failure.

Hyperammonemia is a feature of acute liver failure (ALF), which is associated with increased intracranial pressure (ICP) and brain herniation. We hypothesized that a combination of L-ornithine and phenylacetate (OP) would synergistically reduce toxic levels of ammonia by (1) L-ornithine increasing glutamine production (ammonia removal) through muscle glutamine synthetase and (2) phenylacetate conjugating with the ornithine-derived glutamine to form phenylacetylglutamine, which is excreted into the urine. The aims of this study were to determine the effect of OP on arterial and extracellular brain ammonia concentrations as well as ICP in pigs with ALF (induced by liver devascularization). ALF pigs were treated with OP (L-ornithine 0.07 g/kg/hour intravenously; phenylbutyrate, prodrug for phenylacetate; 0.05 g/kg/hour intraduodenally) for 8 hours following ALF induction. ICP was monitored throughout, and arterial and extracellular brain ammonia were measured along with phenylacetylglutamine in the urine. Compared with ALF + saline pigs, treatment with OP significantly attenuated concentrations of arterial ammonia (589.6 +/- 56.7 versus 365.2 +/- 60.4 mumol/L [mean +/- SEM], P= 0.002) and extracellular brain ammonia (P= 0.01). The ALF-induced increase in ICP was prevented in ALF + OP-treated pigs (18.3 +/- 1.3 mmHg in ALF + saline versus 10.3 +/- 1.1 mmHg in ALF + OP-treated pigs;P= 0.001). The value of ICP significantly correlated with the concentration of extracellular brain ammonia (r(2) = 0.36,P< 0.001). Urine phenylacetylglutamine levels increased to 4.9 +/- 0.6 micromol/L in ALF + OP-treated pigs versus 0.5 +/- 0.04 micromol/L in ALF + saline-treated pigs (P< 0.001).Conclusion:L-Ornithine and phenylacetate act synergistically to successfully attenuate increases in arterial ammonia, which is accompanied by a significant decrease in extracellular brain ammonia and prevention of intracranial hypertension in pigs with ALF.

Alkaline protease production by marine fungus engyodontium BTMFS 10

The thesis entitled “Alkaline Protease Production by Marine Fungus Engyodontium BTMFS 10”.Proteases are the single class of enzymes, which occupy a pivotal position with respect to their application in both physiological and commercial filed. Protease in the industrial market is expected to increase further in the coming year. The current trend is to use microbial enzymes since they provide a greater diversity of catalytic activities and can be produced more economically. Main objective of theses studies are the optimization of various physicochemical factors in the solid state fermentation for the production of alkaline protease enzyme, characterization of the enzyme, evaluation of the enzyme for various industrial application. The result obtained the during the course of theses study indicate the scope for the utilization of this study Marine Fungus E. Album for extra cellular protease production employing solid state fermentation

Isolation, purification and characterization of Protease Inhibitor from Moringa oleifera Lam.

Protease inhibitors are one of the most important tools of nature for regulating the proteolytic activity of their target proteases. They are synthesized in biological systems and they play a critical role in controlling a number of diverse physiological functions. The current investigation focused on the isolation, purification and characterization of a novel protease inhibitor from Moringa oleifera. The results obtained during the course of study opens new perspectives for the utilization of protease inhibitor from Moringa oleifera for various pharmaceutical, agricultural and food industries. The biological and physicochemical properties exhibited by the novel protease inhibitor from Moringa oleifera clearly testify its suitability for the development as a drug for application in pharmaceutical industries such as anticoagulant agent or biocontrol agent in agriculture and even as a food preservant. There is a scope for further research on the structure elucidation and protein engineering towards a wide range of further applications. Detailed structure/function analysis of these proteins is important to facilitate their use in genetic engineering for various applications.

Production of Alkaline Protease by Free and Immobilised Cells of VIBRIO sp. Under Different Fermentation Systems and Its Application on Deproteinisation of Prawn Shell Waste for Chitin Recovery

This thesis presents a detailed account of a cost - effective approach towards enhanced production of alkaline protease at profitable levels using different fermentation designs employing cheap agro-industrial residues. It involves the optimisation of process parameters for the production of a thermostable alkaline protease by Vibrio sp. V26 under solid state, submerged and biphasic fermentations, production of the enzyme using cell immobilisation technology and the application of the crude enzyme on the deproteinisation of crustacean waste.The present investigation suggests an economic move towards Improved production of alkaline protease at gainful altitudes employing different fermentation designs utilising inexpensive agro-industrial residues. Moreover, the use of agro-industrial and other solid waste substrates for fermentation helps to provide a substitute in conserving the already dwindling global energy resources. Another alternative for accomplishing economically feasible production is by the use of immobilisation technique. This method avoids the wasteful expense of continually growing microorganisms. The high protease producing potential of the organism under study ascertains their exploitation in the utilisation and management of wastes. However, strain improvement studies for the production of high yielding variants using mutagens or by gene transfer are required before recommending them to Industries.Industries, all over the world, have made several attempts to exploit the microbial diversity of this planet. For sustainable development, it is essential to discover, develop and defend this natural prosperity. The Industrial development of any country is critically dependent on the intellectual and financial investment in this area. The need of the hour is to harness the beneficial uses of microbes for maximum utilisation of natural resources and technological yields. Owing to the multitude of applications in a variety of industrial sectors, there has always been an increasing demand for novel producers and resources of alkaline proteases as well as for innovative methods of production at a commercial altitude. This investigation forms a humble endeavour towards this perspective and bequeaths hope and inspiration for inventions to follow.

Alkaline Protease from a Non-toxigenic Vibrio sp.(V26) and its Applications

The thesis presents a detailed account of the alkaline protease produced by Vibrio sp.(V26) a mangrove isolate,and the application of this enzyme in different fields.The protease producer strain was identified on the basis of biochemical characteristice,putative virulence traits and 16S rRNA gene sequencing.The purification and characterization of the protease has been carried out. Along with this, an attempt has been made to identifiy the protease gene. The physical parameters as well as the media components influencing protease production were optimized using Response Surfce Methodology(RSM).The scale up of the application of the protease from Vibrio sp.(V26) in the dissociation of cells in animal cell culture,in the recovery of silver from used X-ray films as well as an ingredient in commercial detergents were investigated.