Hanane

Marcus Allan, M.Sc. Biology, 2010

Nuclear factor (NF)-kappaB regulation in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.

 

Abstract:

When environmental conditions become unfavorable, such as during winter, many small mammals are able to enter into a state of dormancy known as hibernation in order to conserve energy. Energy conservation is accomplished via a drastic decline in metabolic and physiological activity in association with a decrease in body temperature, which is periodically interspersed with brief bouts of arousal back to their euthermic values. These drastic changes in oxygen consumption and concentration, perfusion of tissues and energy consumption results in an elevated susceptibility to oxidative stress which can cause severe tissue damage. Hibernators are able to mitigate this damage using antioxidants and their associated pathways in a coordinated response. In the present study, the role of the redox sensitive transcription factor NF-κB was investigated to gain insight into its regulation during hibernation. NF-κB is an essential transcription factor which is known to regulate many targets including antioxidant, antiapoptotic/pro-survival and pro-inflammatory genes. The extent and duration of the NF-κBs response depends on its interactions with its multiple upstream effectors. During hibernation it was found that NF-κB and its signaling components have different expression patterns which are tissue dependant and change along the torpor–arousal cycle. Overall, NF-κB was found to be maximally activated during entrance into torpor, with its cytoprotective downstream genes being upregulated in time for next subsequent arousal in both liver and skeletal muscle tissue. Therefore, these results suggest that antioxidant defenses are upregulated throughout torpor-arousal and that NF-κB may help mediate such protective responses.

Shannon N. Tessier, M.Sc. Biology, 2010

Molecular adaptations of skeletal muscle and cardiac muscle in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.

 

Abstract:

Many small mammals face severe problems during the winter – little or no food supply and yet huge energy costs for keeping their bodies warm. To escape these problems, they hibernate, entering states of deep torpor where metabolic rate falls to just 2-4% of normal and body temperature falls to near 0°C. Remarkably, skeletal muscle sustains cell size and strength despite extended periods of disuse during torpor whereas cardiac muscle actually promotes cell growth (hypertrophy) to support the stronger cardiac contractions needed in the cold. Despite overall suppression of transcription and translation during hibernation, the present research identified and analyzed selected muscle genes and their products that were up-regulated during torpor in striated muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus). These changes in myocyte enhancer factor-2 (MEF2a, MEF2c) transcription factor levels as well as altered expression of selected downstream targets (e.g. glucose transporter 4, myogenic differentiation protein) aid skeletal and cardiac muscle in meeting metabolic challenges associated with hibernation. MEF2 transcription factors were significantly elevated at various points in the torpor-arousal cycle suggesting a significant role for MEF2-mediated gene transcription in the selective adjustment of striated muscle proteins. Muscle plasticity in the hibernator was also evidenced by torpor-responsive changes in the levels of important contractile (troponin I, α/β-tropomyosin), sarcomeric (myomesin) and cytoskeleton proteins (desmin, andvimentin). These data provides new insights into muscle remodeling during hibernation and the role of selected genes/proteins in balancing programs of atrophy, stasis andmyogenesis.

 

Helen Alyx Holden, M.Sc. Biology, 2011

Anuran adaptations to climatic stress: Immune responses and the SMAD family in the wood frog, Rana sylvatica, and the African clawed frog, Xenopus laevis

 

Abstract:

The wood frog, Rana sylvatica, survives freezing over winter. The African clawed frog,Xenopus laevis, withstands substantial dehydration seasonally. The effects of environment on these frogs‟ immunity were investigated with a focus on antimicrobial peptides. Expression of brevinin-1SY was analyzed during freezing, dehydration, anoxia, and development in R. sylvatica. Brevinin-1SY responded differently to each stress, suggesting environmentally regulated expression. Upregulation of hepcidin mRNA was demonstrated during dehydration in X. laevis liver, as were hepcidin agonists, STAT 3 andcMYC. Alternatively, hepcidin antagonizing TGF-β-mediated SMADs weredownregulated and the BMP-mediated SMADs, promoters of hepcidin expression, did not change. Molecular controls of X. laevis skeletal muscle growth were also explored during dehydration. Myostatin, a muscle growth antagonizer, was downregulated during dehydration, whereas cMYC, a muscle growth agonizer, and GLUT 4, a glucose transporter, were upregulated; differential control of SMADs was documented. The data suggest that, during estivation, muscle growth signals are promoted.

William C. Plaxton, Ph.D. Biology, 1984

A study of the metabolic adaptations of marine gastropod molluscs to environmental anoxia stress

 

Abstract:

Catalytic and regulatory properties of alanopine dehydrogenase (ADH) and pyruvate kinase (PK) from tissues of anoxia tolerant marine gastropods were studied. Particular attention was given to those properties of the enzymes which could help explain their potential role(s) in anaerobic energy metabolism. The physical and kinetic properties of the terminal glycolytic dehydrogenase, ADH, purified to homogeneity from foot muscle of the common periwinkle, Littorina littorea, were examined. The kinetic properties of ADH favor enzyme function in cytoplasmic redox balance during the recovery period following long-term environmental anoxia. Tissue specific isozymes of ADH were found in another marine gastropod, the channelled whelk,Busycotypus canaliculatum. Three isozymic forms, specific for muscle, gill/kidney and hepatopancreas were identified. The three tissue specific isozymes of ADH were purified to homogeneity from foot muscle, gill and hepatopancreas and their kinetic and physical properties were studied. Muscle ADH showed properties which appear to gear this isozyme for alanopine synthesis as an end product of glycolysis. The hepatopancreas isozyme appears suited for a role in alanopine oxidation in vivo. The properties of gill ADH are intermediate between those of the other two forms. Tissue specific forms of PK were also found in B. canaliculatum. Three isozymic forms, specific for red muscle, white muscle and soft tissues, were identified. Furthermore, each PK isozyme was modified in animals subjected to 21 h of anoxic stress such that several physical and kinetic characteristics were altered. Aerobic and anoxic forms of red muscle PK (RPK-AER and RPK-ANX) were purified to homogeneity from radular retractor tissue of B canaliculatum and the physical and kinetic properties of the enzyme were extensively studied. The differences in kinetic properties between RPK-AER and RPK-ANX indicates that red muscle PK activity is probably greatly depressed in vivo during long-term anoxic stress. The anoxia-dependent, in vivo, covalent incorporation of injected (’32)P orthophosphate into RPK-ANX demonstrated that the enzyme is a phosphoprotein. Evidence for the reversibility of this phosphorylation was provided by the kinetic similarities between purified RPK-AER and homogenous alkaline phosphatase treated RPK-ANX.

Thomas A. Churchill, Ph.D. Biology 1992

Metabolic biochemistry of freeze tolerance in vertebrates

 

Abstract:

A unique group of vertebrate animals has developed complex metabolic adaptations that enable them to survive freezing. This thesis investigates: i) cryoprotectant synthesis in freeze tolerant frogs, ii) freeze tolerance in a newly identified freeze tolerant vertebrate, the garter snake, and iii) metabolic responses elicited by other stresses (anoxia and dehydration) in the garter snake and two frog species. Investigation of cryoprotectant synthesis in spring frogs,Pseudacris crucifer, revealed large amounts of glucose produced during freezing; approximately 0.1 molar. Changes in the levels of glycolytic intermediates indicated that an activation of glycogen phosphorylase and phosphofructokinase (PFK) directed glycolytic flux to cryoprotectant synthesis. Tissue glucose distribution was much lower than in fall animals. These results suggested seasonal variation in glucose transport mechanisms. A similar investigation of cryoprotectant synthesis in spring Hyla versicolorshowed a maintenance of regulatory enzyme controls at glycogen phosphorylase and PFK directing glycogen carbon to cryoprotectant. Only glucose was synthesized as cryoprotectant; quantities of glycerol (the major cryoprotectant of winter H. versicolor) showed no increase. The amount of cryoprotectant produced was directed correlated to glycogen content in the liver. Investigation of freeze tolerance in garter snakes revealed that these snakes were only partially freeze tolerant. Survival of brief freezing exposures (5-10 h at -2.5°C; 30-50% ice) was possible. Two amino acids, glutamate and taurine, were implicated as possible cryoprotective agents. Comparison of the metabolic responses (adenylate levels, anaerobic end products, glycolytic flux) to freezing in garter snakes were similar to those elicited by anoxia. Dehydration timecourses were investigated in two freeze tolerant frog species, Rana sylvatica andPseudacris crucifer. Even though whole body water contents dropped by 50-60 %, individual tissues exhibited little or no change in water content. There were many similarities between metabolic responses to dehydration and those to freezing. The most remarkable similarity between freezing and dehydration was the accumulation of glucose, presumably acting as a cellular protectant; quantities in liver rose to 127 and 220 micromole per gram in R. sylvatica and P. crucifer, respectively.

Yanjing Su, Ph.D. Chemistry, 1992

Phosphofructokinase from white skeletal muscle and liver of rainbow trout (Oncorhynchus mykiss): isolation, characterization and study of enzymatic regulation

 

Abstract:

Phosphofructokinase (PFK) isozymes from white skeletal muscle and liver of rainbow trout Oncorhynchus mykiss were purified to electrophoretic homogeneity. Muscle PFK was purified 175-fold using phosphocellulose, hydroxylapatite, and ATP-agarose affinity chromatography whereas liver PFK was purified 13,400-fold using acetone precipitation, heat treatment, ammonium sulfate fractionation, and ATP-agarose chromatography. Muscle PFK was a homohexamer having a native molecular mass of 478,000. The enzyme was regulated by the levels of fructose-6-phosphate (F6P), ATP, pH, and allosteric effectors including activators (NH4+, inorganic phosphate, AMP, ADP, and fructose-2,6-bisphosphate [F2,6P2]) and inhibitors (citrate, phosphoenolpyruvate [PEP], and ATP). Activators increased the enzyme affinity for F6P and released the inhibition by ATP or citrate. Citrate inhibited the enzyme synergistically with ATP. Arrhenius plots of the enzyme activity showed discontinuity at 15 to 16°C, presumably due to conformational alterations in the enzyme. The kinetic behavior of muscle PFK was significantly altered by protein kinase – mediated phosphorylation. The high-phosphate form of the enzyme showed higher activity with increased affinity for F6P and less inhibition by ATP. Protein concentration affected enzyme activity as assessed by two different methods. PFK showed a higher Vmax, lower S0.5 F6P and higher I50 values for ATP as enzyme concentration increased. The association of PFK with myofibrils of trout muscle was affected by pH, ionic strength, protein concentration, and the levels of metabolites or the effectors of the enzyme with binding favored by lower pH values and increased protein concentration. During exercise, muscle PFK is probably activated by increases in the levels of enzyme activators and enzyme phosphorylation state, and enhanced PFK association with myofibrils. Trout liver PFK was also regulated by the levels of F6P, ATP, NH4+, inorganic phosphate, AMP, and F2,6P2. However, the liver enzyme was not sensitive to citrate inhibition. Contrary to its muscle counterpart, liver PFK was inhibited by protein phosphorylation catalyzed by the catalytic subunit of cAMP-dependent protein kinase and activated by the removal of phosphate through acid phosphatase. The high-phosphate form of liver PFK exhibited a lower Vmax, an increased S0.5 F6P, and higher I50 values for ATP.

 

Hossein Mehrani, Ph.D. Chemistry, 1994

Regulation of glycogen metabolism by protein phosphorylation during environmental stress

 

Abstract:

The enzymes involved in the phosphorylation controlled glycogen catabolic pathway were studied in two different model systems involving anoxia: functional anoxia in exercised fish and environmental anoxia in turtle. Glycogen phosphorylase b from rainbow trout,Oncorhynchus mykiss, white skeletal muscle was purified to near homogeneity. Glucose and ATP inhibited the enzyme; glucose inhibition decreased at lower pH values. Michaelis constants for glycogen, phosphate, and AMP were 128 micromolar, 31 millimolar, and 142 micromolar respectively, at pH 7.2; maximum enzyme activity was obtained at pH 7.5 and 25°C Exhaustive swimming exercise altered tissue glycogen phosphorylase kinase (GPK) and protein kinase A (PKA), GPK activity increasing by 60% in liver and 40% in white muscle of exercised fish. The amount of active PKA rose from 12% to 21% in liver and from 32% to 57% in white muscle after exhaustive swimming coupled with 50% and 70% increases in cellular cyclic AMP levels, respectively. Three forms of alpha-glucosidase were identified in trout liver. Two forms showed acid pH optima, hydrolyzed glycogen, maltose and 4-methylumbelliferyl alpha-glucoside (MUalphaG), and were associated with lysosomes whereas the third was microsomal, had a neutral pH optimum and did not hydrolyze glycogen. Properties of acid alpha-glucosidase type I changed significantly during exercise; maximal activity increased by 80% and Km values for glycogen and maltose dropped by 50% in exercised, versus control, fish. Exposure of turtles, Trachemys scripta elegans, to submergence anoxia at 7°C, elevated activities of phosphorolytic and glucosidic enzymes in some organs. Phosphorylase a in liver and heart increased significantly after 5 h of anoxia. PKA activity increased 2.3-fold in liver within 1 h of anoxia accompanied by a 60% increase in cAMP levels; however, with longer anoxia active PKA was suppressed to 2.1-3.7% of the total. Protein phosphatase-1 (PP-1) activity in liver decreased to 63% of controls within 1 h and remained suppressed over the subsequent 20 h of anoxia. PP-1 activity also fell in anoxic red muscle and decreased transiently in brain. Within one hour of anoxia, 40% of protein kinase C beta isomer (PKC-beta) and over 80% of PKC-alpha were translocated from cytosol to the membrane fraction. Activity of acid alpha-glucosidase also increased in liver of anoxic turtles. PKA, PP-1, PKC-alpha, and PKC-beta from control turtle liver were purified to homogeneity; physical and kinetic properties of these are presented.

 

Clark P. Holden, Ph.D. Biology, 1995

Signal transduction and the function of second messengers and protein kinases in the control of glycogenolysis and cryoprotectant production in freeze tolerant vertebrates

 

Abstract:

A unique group of lower vertebrates, termed freeze tolerant, have developed complex biochemical mechanisms that allow them to survive whole body freezing. Perhaps the most important of these mechanisms is the production of cryoprotectant, usually in the form of glucose, via an activation of glycogenolysis as a response to freezing. This thesis investigates: (1) whether protein kinase enzymes are responsible for the early activation of glycolysis that stimulates cryoprotectant production in freeze tolerant vertebrates and/or the subsequent inhibition of the pathway in the later stages of a freezing episode, (2) the comparative aspects of the responses of protein kinases elicited by the environmental stresses of dehydration and anoxia, to those of freezing, and (3) the role of the free catalytic subunit of cyclic AMP-dependent protein kinase (PKAc) and protein kinase C (PKC) in cryoprotectant production in Rana sylvatica liver. Short freezing episodes caused a dramatic increase in the percentage of free catalytic PKA (PKAc). This response diminished with increased time frozen in tissues of freeze tolerant vertebrates. The measurement of second messengers over a freezing time course revealed significant increases in adenosine 3′,5′- cyclic monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) in liver and muscle of 4 freeze tolerant animals. An activation of PKA and PKC by these molecules, in response to freezing, is likely. Dehydration stress. not anoxia stress, elicited a remarkably similar protein kinase response to that seen during freezing. Levels of PKAc and its second messenger cAMP, as well as the second messenger for PKC, IP3, rose in dehydrated liver tissue whereas anoxia did not activate PKAc and only increased liver IP3. Purified R. sylvatica liver PKAc was not inhibited by high concentrations of salt or glucose and showed an increased affinity for substrates at low temperatures. Additional evidence showed that a freezing stress did not initiate a translocation of PKC to the plasma membrane in R. sylvatica liver and brain tissues. Freeze tolerant vertebrates possess an adaptive strategy to combat freezing which links second messengers and protein kinases to the post-translational modification of glycolytic enzymes conducting the biosynthesis of cryoprotectant.

 

 

Denis R. Joanisse, Ph.D. Biology, 1995

Metabolism during overwintering in two species of cold-hardy goldenrod gall insects

 

Abstract:

Metabolism was examined in the larvae of two cold-hardy goldenrod gall insects, the freeze tolerant fly Eurosta solidaginis (Fitch) (Diptera, Tephritidae) and the freeze avoiding moth Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae) during overwintering. Acclimatization resulted in specific changes in enzyme activities and cellular metabolites. Enzyme activity data suggested metabolic shifts concurrent with the requirements of both species. Larvae shifted metabolism towards cryoprotectant synthesis in the fall, as witnessed by increased activities of glycerol and sorbitol producing enzymes, and towards the removal of these polyols in the spring with increased activities of catabolic enzymes. Measured increases in lipid unsaturation in the fall helped to maintain membrane fluidity during cold-exposure. Decreased activities of enzymes of the tricarboxylic acid cycle paralleled the reduced demands on oxidative metabolism over the winter. In Eurosta solidaginis, decreased oxidative metabolism and the anoxic state of the animal when frozen further resulted in the lack of lipid reserve depletion over the winter, whereas freeze avoiding Epiblema scudderiana larvae appeared to use lipid reserves to support basal metabolism over the winter. Decreased activities of antioxidant enzymes in Eurosta solidaginisover the winter suggested that these larvae do not experience increased reactive oxygen species formation during freezing and thawing. Increasing antioxidant enzyme activities in Epiblema scudderianaover the winter suggested that these larvae may be subject to oxidative stress, perhaps due to the maintenance of aerobic metabolism during cold exposure.

 

Jean E. Grundy, Ph.D. Chemistry, 1996

Antioxidant defenses during estivation in the spadefoot toad, Scaphiopus couchii

 

Abstract:

Antioxidant enzymes and lipid peroxidation damage were analyzed in an estivating species, to determine whether estivation (a dormancy induced by hot, dry environmental conditions) represented an oxidative stress and to determine whether changes in organ antioxidant systems were made in response to estivation. The model animal used was Scaphiopus couchii, the spadefoot toad, that estivates for about 10 months per year in the Arizona desert during which it loses about 50% of its body water and builds up high levels of electrolytes and osmolytes in body fluids. Activities of six antioxidant enzymes (AOE) and xanthine oxidase/xanthine dehydrogenase as well as the small molecular weight free radical scavenger glutathione were measured in liver, lung, heart, kidney, gut and leg muscle of control and dormant toads. Damage to membranes was assessed by measurement of extent of lipid peroxidation by three different methods. The glutathione ratio, (GSSG) /(GSH), rose significantly in several tissues during estivation. In addition, activities of key AOE were reduced in several tissues, perhaps indicating that the metabolic cost of keeping antioxidant defenses high during estivation was greater than the cost of maintaining AOE at their previous level. Three antioxidant enzymes were further examined: Catalase (CAT) and glutathione reductase (GR) were purified and characterized from liver and glutathione S-transferase (GST) from liver and muscle of awake and estivated toads. CAT activity was lower in liver during estivation but the kinetic properties of catalase were the same in estivated and awake toads. Catalase from toad liver was an active dimer, which has not been demonstrated previously in a eukaryote, to our knowledge. GR and GST of toad liver had altered kinetic properties during estivation, indicating pre- or post-translational modification of the enzyme in the dormant animal. However, GST in muscle was the same for awake and estivated toads. GST is a multiclass family of enzymes which generally render lipid-soluble material soluble in the cytosol, via conjugation to glutathione. Three class of GST, Mu Pi and Alpha, have different substrate specificities; these parameters were used to classify frog and toad GSTs. GST, GR and CAT were relatively insensitive to physiological urea concentrations but were strongly modulated by KCl, suggesting that urea buildup during estivation may serve to modulate passive KCl accumulation as a result of dehydration.