M. Sc. Thesis Abstracts

Rabih Roufayel, M. Sc. Biology, 2009

Regulation of the Tb-E2F pathway in the freeze tolerant wood frog, Rana sylvatica

 

Abstract:   

The North American wood frog, Rana sylvatica, is a primary model animal used in the study of freeze tolerance in vertebrates. During Canadian winters, wood frogs endure the freezing of about 65-70% of their total body water. Anoxia, ischemia, oxidative stress and many other consequences are a result of freeze/thaw cycles. A variety of adaptations are known that protect the frogs against potential freezing injuries as well as regulate their intermediary metabolism and gene expression to support survival. Selected transcription factors have critical roles to play in freezing survival by regulating the expression of genes that control the adaptations needed to handle freezing stress. In my present study, the retinoblastoma (Rb) protein coupled with the E2F transcription factor family were demonstrated to have roles in controlling the cell cycle in wood frog liver and skeletal muscle during freezing and associated stresses (anoxia, dehydration). Western blotting was used to quantify total Rb, phosphorylation or acetylation at different sites on Rb, E2F members and selected downstream targets under E2F control. Other central regulators of the cell cycle were also quantified including Cyclins, Cyclin dependent kinases (Cdks), and checkpoint proteins. Nuclear distributions of Rb-E2F and Cdk:Cyclins were also assessed. RT-PCR was used to quantify mRNA transcript levels of Cyclin D1 which decreased significant during freezing as well as c-Myc, a downstream target of E2F. The data indicate that the cell cycle is under regulation during freezing through E2F upregulation and Rb phosphorylation via Cdk:Cyclin activity.

Allan Letourneau, M.Sc. Biology, 2010

Phosphorylation of glycolytic enzymes in hibernation.

 

Abstract:

Glycerol-3-phosphate dehydrogenase (G3PDH) and lactate dehydrogenase (LDH) were examined for differential phosphorylation, accompanying kinetics and stability inSpermophilus richardsonii liver and skeletal muscle. Hibernator G3PDH had a higher phosphate content and differential kinetics in both tissues which could be manipulated by kinase and phosphatase incubations in the G3P utilizing direction. Arrhenius plots and activation energies (Ea) showed that the hibernator form had a lower Ea in both tissues, especially in the liver.Euthermic LDH had a higher phosphate content and differential kinetics which could be manipulated by kinase and phosphatase incubations in both directions. Euthermic forms had lower Ea values in both tissues. G3PDH and LDH had differential urea I50 values with the hibernator more susceptible to urea denaturation in both tissues. Urea I50 values could be manipulated in LDH by kinase and phosphatase incubations in the liver but not in muscle.

Katrina J. Sullivan, M.Sc. Biology, 2011

Expression pattern of the novel freeze-responsive genes li16, fr10 and fr47 in the wood frog, Rana sylvatica

 

Abstract:

The ability of the wood frog (Rana sylvatica) to freeze up to 65% of its total body water allows it to endure subzero temperatures encountered during winter.  To survive the stresses associated with freezing, frogs have evolved multiple molecular adaptations including expression of three novel genes: li16, fr10 and fr47.  All three genes were found to be freeze responsive in a tissue-dependent manner.  They also all respond to anoxia and dehydration stresses, suggesting that the transcription of these genes is triggered by the low oxygen conditions common to all three stresses.  Protein levels were elevated after freezing for Li16; however FR10 and FR47 generally showed no change or a decrease in protein after freezing.  Transcripts of all three genes were also detected during tadpole development, indicating a possible additional role for metamorphosis.  Finally,bioinformatic analysis of their sequences provided insight into functional motifs and potential regulatory sites on the proteins.

Sandra Korycan, M.Sc. Biology 1984

Organ-specific metabolism during anoxia and recovery from anoxia in the cherrystone clam, Mercenaria mercenaria

 

Abstract:

The levels of intermediary metabolites and end products were quantified in the tissues of the cherrystone clam, Mercenaria mercenaria, over a time course of 96 h of anoxia followed by 48 h of aerobic recovery. Succinate and alanine accumulated as anaerobic products while glycogen and aspartate were utilized as substrates. Succinate accumulation ranged from 12-14 micromol/g wet weight in muscle (phasic and catch adductor, foot) to 25 micromol/g in gill and mantle with 32 micromol/mL released into the mantle cavity fluid. Lesser amounts of alanine were produced, the ratio succinate:alanine varying from 1.4:1 in phasic adductor to 3.2:1 in mantle at 96 h. Aspartate reserves apparently supply the carbon for succinate synthesis over the first 6-12 h of anoxia; subsequent succinate and alanine production probably results from glycogen fermentation. The imino acids alanopine and strombine were not produced in appreciable amounts (less than 1 micromol/g) during anoxia. When returned to aerated seawater, control levels of alanine and aspartate were reestablished within 24 h; accumulated succinate was catabolized within 48 h. Glycogen content of all tissues showed a sharp decline after 6 h of recovery, perhaps due to enhanced energy demands, but levels increased later in recovery. Tissue ATP levels, which were depressed during anoxia, were restored by 24 h.

Ajoy Chakrabarti, M.Sc. Biology 1988

Immobilization of Cellulase Using Polyurethane Foam

 

Abstract: 

Cellulase was covalently immobilized using a hydrophilic polyurethane foam (Hypol FHP 2002). Compared to the free enzyme immobilized cellulase showed a dramatic decrease (7.5-fold) in the Michaelis constant for carboxymethylcellulose. The immobilized enzyme also had a broader and more basic pH optimum (pH 5.5-6.0) a greater stability under heat-denaturing or liquid nitrogen-freezing conditions and was relatively more efficient in utilizing insoluble cellulose substrates. High molecular weight compounds (Blue Dextran) could move throughout the foam matrix indicating permeability to insoluble celluloses; activity could be further improved 2.4-fold after powdering foams under liquid nitrogen. The improved kinetic and stability features of the immobilized cellulase combined with advantageous properties of the polyurethane foam (resistance to enzymatic degradation plasticity of shape and size) suggest that this mechanism of cellulase immobilization has high potential for application in the industrial degradation of celluloses.

John Duncan, M.Sc. Biology 1988

Role of enzyme binding in muscle metabolism of the goldfish

 

Abstract:

Regulation of glycolytic metabolism in muscle by the reversible association of enzymes with the particulate fraction of the cell was assessed in heart and white skeletal muscle of the goldfish Carassius auratus. Three physiological states were compared: rested aerobic controls, 21 h of anoxia exposure in water bubbled with N2 and CO, and exhaustive swimming. Heart muscle responded to the anoxia exposure with an increase in the percentage bound for phosphofructokinase (PFK), aldolase, and pyruvate kinase, the greatest increase being for PFK (from 35 to 48%). When fish swam to exhaustion, however, no changes in the percentage bound occurred for the eight enzymes assayed in heart. In white muscle neither anoxia nor exhaustive swimming altered the binding of the eight enzymes to the particulate fraction of the cell, except for a significant decrease in the percentage of alcohol dehydrogenase bound in anoxic muscle. PFK binding was particularly high in white muscle (63-72% bound, assessed by two methods). PFK binding was sensitive to pH in both organs, with binding increasing as pH decreased. This suggests that binding of PFK could be enhanced when metabolism is highly dependent on glycolytic ATP production due to the fall in cellular pH as a result of metabolic acidosis.

Doris Schafhauser, M.Sc. Chemistry 1990

Immobilization of Amyloglucosidase onto Granular Chicken Bone

 

Abstract:

Amyloglucosidase was immobilized onto granular chicken bone (BIOBONE TM) by noncovalent interactions. The amount of activity bound relative to an equal amount of free enzyme was 13.6+0.4%. The estimated specific activity for amyloglucosidase decreased from 75.3+0.8 to 43.5+9.6 U/mg protein upon immobilization. The Km value of the bone-immobilized enzyme using glycogen as substrate increased from 3.04+0.38 mg/mL (free) to 9.04+l.51 mg/mL (immobilized), but Km showed no change upon immobilization when starches were used as substrates. A decrease in Vmax values occurred upon enzyme immobilization for all substrates, but this largely reflected the percentage of enzyme initially bound to the bone. Immobilization also improved enzyme stability in the presence of various additives (e.g., detergent, KCl, and ethanol) or under low or high pH reaction conditions. Bound amyloglucosidase maintained high activity (greater than 90%) following five cycles of continuous use at moderate (23°C) and high (55°C) temperatures. Data derived from Lineweaver-Burk and Arrhenius plots indicated that substrate and product diffusion limitation were minimal.

Elizabeth Russell, M.Sc. Chemistry 1993

Regulation of enzymes of carbohydrate metabolism during anoxia in the salt marsh bivalve Geukensia demissus

 

Abstract:

The effects of anoxia exposure (2 or 12 h at 5°C) on the tissue-specific responses by enzymes of carbohydrate metabolism were analyzed in mantle, gill, hepatopancreas and adductor muscle of the intertidal bivalve Geukensia demissus demissus. Reversible enzyme phosphorylation has been identified in other species of marine molluscs as a mechanism for coordinating the suppression of metabolic rate and the redirection of carbon into fermentative pathways under anoxic conditions. The present study shows patterns of response to anoxia by five enzymes of carbohydrate metabolism including glycogen synthetase (GS) and pyruvate dehydrogenase (PDH) whose responses to anoxia have not before been analyzed in marine molluscs. Anoxia-induced changes in properties, consistent with reversible phosphorylation modification of the enzymes, were found for pyruvate kinase (PK) and pyruvate dehydrogenase (PDH) in all tissues and in selected tissues for glycogen phosphorylase (GP) and GS. However, phosphofructokinase did not appear to be modified in any tissue during anoxia. Within 2 h of anoxia exposure, PK showed a sharp drop in the activity ratio (at subsaturating vs. saturating PEP concentrations) that indicated a stable modification of enzyme Km for PEP; for example, in gill the ratio (determined at 0.75 & 7.5 mM PEP) fell from 0.39 + 0.08 for aerobic controls to 0.08 + 0.01 after 2 h anoxia. The percentage of PDH in the active a form also dropped significantly in anoxia from 80-84 % a in controls to 65-75% a in anoxic tissues. Changes in both of these enzymes are consistent with anoxia-induced metabolic rate suppression. By contrast, anoxia exposure increased GP activity in gill and adductor muscle indicating a need for increased glycogenolysis during anoxia in these tissues. Total phosphorylase (a + b) activity increased in both tissues as also did the %a in adductor; for example, in adductor, active GPa content rose from 0.09 + 0.02 U/g wet weight in controls to 0.24 + 0.01 U/gww after 2 h and 0.16 + 0.02 U/gww after 12 h anoxia exposure. GS behaved oppositely in hepatopancreas showing a significant decrease in total activity in 12 h anoxic tissue but GS was unaffected by anoxia in gill or adductor muscle. In mantle, oppositely directed changes in total GS activity and the % active resulted in no change in the activity of the active I form during anoxia.

Donna Douglas, M.Sc. Chemistry 1993

Anoxia induces changes in translatable mRNA populations in turtle organs: a possible adaptave strategy for anaerobiosis

 

Abstract:

The effects of anoxic submergence (16 h at 15°C) on cellular mRNA contents were assessed in five organs of anoxia tolerant turtles Trachemys scripta elegans. Poly(A)+ RNA was extracted from liver, red and white skeletal muscle, kidney and heart of control and anoxic turtles, as well as from heart and kidney of turtles allowed 24 h aerobic recovery (at 15°C) after anoxia exposure. Poly(A)+ RNA content increased by 30 % in white muscle from anoxic turtles relative to control animals but was unchanged by metabolic state in other organs. Extracted mRNA was translated in vitro in a wheat germ lysate system and the 35S-labelled polypeptides that were produced were separated by SDS-polyacrylamide gel electrophoresis. Overall translational activity of the mRNA pool [cpm 35S-methionine incorporated per microgram poly(A)+ RNA] was altered by anoxia exposure in 3 organs, increasing by 38 and 18 % in liver and kidney and decreasing by 42 % in red muscle. Anoxia exposure also led to qualitative changes in the protein products that resulted from in vitro translation. SDS-PAGE revealed the presence of a novel 19.5 kDa polypeptide in liver of anoxia-exposed animals as well as increased amounts of two other proteins at 28.6 and 79.9 kDa. In heart, a new translation product of 26.8 kDa appeared in anoxia, and in kidney a 32.8 kDa polypeptide was produced during the aerobic recovery period after anoxia exposure. Anoxia stimulated the appearance of a 37.5 kDa protein in red skeletal muscle but anoxic red muscle also lost proteins of 40, 32, and 28.2 kDa that were present in aerobic controls. Anoxia exposure did not change the proteins produced by in vitro translation in white muscle. The results suggest that anoxia exposure triggers rapid cellular responses in T. s. elegans that modify translatable mRNA populations in organs, leading to new protein transcripts. This response may be one of the important molecular adaptations that support the natural anoxia tolerance of this species.

Marc A. de la Roche, M. Sc. Chemistry, 1995

Metabolic adjustment and biochemical adaptation to torpor in mammalian hibernators.

 

Abstract:

Hibernating mammals have evolved the ability to lower their metabolic rate in response to temperate conditions and/or lack of food and water. At the level of enzyme control, this is accomplished by the coordinated suppression of all metabolic processes and the rearrangement of metabolic flux to maximize fuel stores. The quantification of 32 enzyme activities in 5 tissues of hibernating versus euthermic golden-mantled ground squirrels (Spermophilus lateralis) demonstrated a decreased potential in glycolysis, biosynthetic processes and carbon entry from glycolysis into the TCA cycle relative to an increased potential in fatty acid oxidation during hibernation. This was further supported by studies on ketone body metabolism, a product of fatty acid oxidation. Kinetic characterization of beta-hydroxybutyrate dehydrogenase (betaDH) and glycerol-3-phosphate dehydrogenase (G3PDH) established unique functional adaptations of these enzymes in S. lateralis and Cynomys ludovicianus. Using temperature and chemical denaturants as probes, purified G3PDH from a hibernator was found to have a greater chemical and thermal stability, and maintained both structural and functional integrity at lower temperatures relative to the enzyme from rabbits. These results purport a unique metabolic state during hibernation and demonstrate both structural and functional adaptations of hibernator enzymes to cold stress.