M. Sc. Thesis Abstracts

Differential gene expression in response to sub-zero temperatures in the freeze tolerant wood frog, Rana sylvatica

Abstract:

Exposure to subzero freezing temperatures causes a variety of biochemical changes in the freeze-tolerant wood frog, Rana sylvatica. Cellular responses to freezing commence a mere five minutes after the initial formation of epidermal ice. To obtain a general overview of differential protein expression in response to freezing, in vitro translation of mRNA isolated from tissues (liver, brain, heart, muscle, kidney and gut) was conducted and compared for control (5°C), frozen (24 hours at -2.5°C) and recovered (24 hour thaw at 5°C) wood frogs; ³⁵S-labeled proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and located by fluorography. Results showed changes in the translatable mRNA population of freezing exposed wood frogs. Differential protein expression was greatest in the comparison of liver from control-versus frozen frogs but other tissues each showed one or two differentially expressed proteins in either the frozen or thawed states. Subsequently, isolation of a specific cDNA clone, the R. sylvatica liver glucose transporter (glut2), was attempted via heterologous probing of the liver cDNA library from frozen frogs with a human glut2 cDNA fragment. Potential clones were chosen from a primary screening and further purification was achieved with secondary and tertiary screens. Analysis of the partial nucleic acid sequence of fifteen potential positive clones using the GenBank database did not reveal the presence of the wood frog glut2 cDNA. In another effort to find a wood frog glut2, TA cloning, a PCR based method, was attempted. A PCR product was amplified using a variety of degenerate primers and one product was cloned into a T-vector. The insert of the resulting recombinant plasmid was sequenced and comparison of this with the GenBank database did not reveal its identity as the liver glucose transporter.

Purification and characterization of the glycolytic enzymes hexokinase and pyruvate kinase from Eurosta solidaginis

Abstract:

Larvae of the insect Eurosta solidaginis spend the winter in galls on goldenrod plants and survive low subzero temperatures by allowing the formation of extracellular ice within their bodies. Different mechanisms are used to allow survival of these extreme conditions: some ensure that the physical integrity of cells is maintained whereas others suppress metabolism so that only basal metabolic functions subsist. Glycolysis, which is at an intersection of several important metabolic pathways, must be subjected to fine regulation to allow the synthesis of the two cryoprotectants of the larvae, glycerol and sorbitol, during the autumn and as well as to facilitate metabolic depression during the winter. In this study, hexokinase (EC 2.7.1.1) and pyruvate kinase (EC 2.7.1.40), two regulatory enzymes of glycolysis, were examined. Hexokinase was partially purified and kinetic studies were performed both at 6°C and 25°C and in the presence and the absence of the cryoprotectants of the insect, glycerol and sorbitol. The data indicate that besides a role in the regulation of glycolysis, hexokinase is probably involved in the control of the synthesis and catabolism of the two polyols. Pyruvate kinase (PK) was purified to near homogeneity and kinetic and structural studies were performed on the purified preparations. Kinetic studies showed that the activity of PK was reduced at cold temperatures and in the presence of the two polyols, with an increase in inhibition by citrate and the loss of activation by fructose-1,6-bisphosphate. Fluorescence and heat denaturation studies underlined the importance of the cryoprotectants, especially sorbitol, for the stabilization of the enzyme.

Gene expression supporting freeze tolerance in the wood frog, Rana sylvatica

Abstract :

Natural freezing survival by the wood frog, Rana sylvatica, is supported by multiple metabolic and gene expression adaptations. The present study explored the responses by protein chaperones, both heat shock proteins (Hsps) and glucose-regulated proteins (Grps), as well as the hypoxia-inducible transcription factor (HIF-1α), to freezing and two component stresses of freezing (anoxia, dehydration) in seven organs of wood frogs. Sequence analysis of Grp78, Grp94 and HIF-1α found high identity between the frog proteins and other species but HIF-1α showed unique amino acid substitutions that might aid low temperature function. RT-PCR and Western blotting were used to monitor mRNA transcript levels and protein levels, respectively. All three stresses stimulated up-regulation of HIF-1α at transcriptional and translational levels in liver. Other organs showed stress-specific responses and time course studies showed maximum hif-1α mRNA levels by 2 hours of freezing which suggests that hypoxia-induced proteins may contribute tocryoprotection. Levels of all seven shock proteins (Grp78, Grp94, Hsp110, Hsc70, Hsp60, Hsp40, Hsp10) rose during freezing in liver; six responded in skeletal muscle and kidney, and four in heart. The distribution of shock proteins and HIF-1α between liver cytosolic and nuclear fractions also changed during freezing. Most shock proteins responded to anoxia stress in liver and to dehydration stress in skin but other organs showed mixed responses to these stresses. The mitochondrial chaperones Hsp60 and Hsp10 were consistently elevated during freezing and/or anoxia in most organs. Up-regulation of chaperones during freezing suggests that they play a cryoprotective role in stabilizing proteins against the multiple stresses on cells imposed by extracellular freezing.

Glucose regulated protein and heat shock protein expression in hibernating mammals

Abstract:

During mammalian hibernation, most physiological activities are dramatically suppressed, but selected genes are up-regulated to provide protein products that protect cells and organs for long term survival in the hypometabolic, hypothermic state. In this study, the roles of chaperone proteins including glucose regulated proteins and heat shock proteins were assessed in two species: thirteen-lined ground squirrels, Spermophilus tridecemlineatus, and little brown bats, Myotis lucifugus. RT-PCR and Western blot techniques were used to examine gene and protein expression. Compared with euthermic control squirrels and bats, glucose regulated protein 75 (Grp75), Grp94 and Grp170 were elevated in some tissues at the mRNA and/or protein levels with organ-specific patterns of response by grp transcripts and GRP protein. The up-regulation of grp mRNA may be important for rapidly elevating the protein content of these chaperones during hibernation and arousal; elevated GRP protein would then aid in the folding of other proteins that are newly synthesized during hibernation and/or in the renaturation of proteins that become misfolded at low body temperatures. Heat shock proteins (Hsps) including Hsp40, Hsp72, Hsp73 and Hsp90 were elevated in some tissues of hibernating ground squirrels and bats. Analysis of partial amino acid sequences of Grps and Hsps showed very high identities (88-100%) compared with human or mouse sequences which indicates similar structures and functions of Grps and Hsps among mammalian species. The data support the idea that Grps and Hsps are up-regulated during hibernation to function as chaperones to bind non-native polypeptides and suppress protein aggregations caused by low temperature during hibernation.

Anti-apoptotic and antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica.

Abstract:

Multiple biochemical adaptations support natural freeze tolerance by wood frogs, Rana sylvatica. The present research explored the role of anti-apoptotic and antioxidant defenses in organ survival of freeze/thaw stresses using PCR and Western blotting to analyze the expression of selected genes and proteins. The STAT family of transcription factors mediate both pro- and anti-apoptotic gene responses. Elevated amounts of phosphorylated (active) Stat5 (Tyr694) and/or phospho-Stat3 (Ser727) in selected frog organs during freeze/thaw suggest activation of anti-apoptotic defenses to help organs recover from metabolic insults caused by freezing. However, levels of phospho-Stat1 (Tyr701), a pro-apoptotic signal, also rose in kidney and muscle during thawing. Increased amounts of anti-apoptotic proteins including Bcl-2 and phospho- Bcl-2 (Ser70) in liver and skeletal muscle and Bcl-xL (and phospho-Bcl-2) in kidney could help counteract freeze-induced apoptotic signals that were evidenced by higher levels of Bad protein (liver, muscle, kidney) and phospho-Bad (Ser112) (kidney) and enhanced DNA laddering. Antioxidant defense via glutathione S-transferase (GST) was evaluated by analyzing the expression of GST isozymes. GST Pi protein rose in four organs during freeze/thaw and GST Pi mRNA was freeze up-regulated in liver. GST M1/2, M5, A3 and A5 were freeze- or thaw- responsive in selected organs. Freeze-induced changes in the transcription factors, Nrf2 and MafG, and elevated MafG in the nucleus suggest that these regulate the freeze up-regulation of antioxidant enzymes by targeting the antioxidant response element of genes. Both anti-apoptotic and antioxidant defenses are important aspects of natural freezing survival.

Regulation of enzymes of energy metabolism – AMP deaminase and creatine kinase – in an anoxia tolerant turtle.

Abstract:

The red-eared slider turtle (Trachemys scripta elegans) is one of the few vertebrate species that can survive long term oxygen deprivation. Maintenance of viable cellular energetics is key to anoxia survival and, in response to low oxygen, most anoxia tolerant animals show a drop in total adenylate levels while energy charge remains stable. To better understand how turtles regulate their energy metabolism when deprived of oxygen, the present studies focused on the control of two important enzymes in muscle energy metabolism: AMP deaminase (AMPD) and creatine kinase (CK). AMPD activity increased under anoxia in turtle skeletal muscle and the effects of ATP∙Mg and ions indicated that allosteric controls are part of the mechanism of AMPD regulation. In vitro incubations to stimulate the actions of endogenous protein kinases and phosphatases showed that AMPD is a phosphoenzyme and suggested that reversible phosphorylation has a central role in AMPD regulation under aerobic versus anoxic conditions. CK from turtle heart is also a phosphoprotein and anoxia-induced metabolic rate depression was accompanied by a strong increase in the fraction of dephosphorylated CK that showed increased affinity for creatine. Incubation studies implicated selected protein kinases (PKA, PKG, and AMPK) and phosphatases (PP1) as responsible for heart CK regulation. However, anoxia-responsive changes in kinetic properties of skeletal muscle CK did not appear to be caused by a change in phosphorylation state. Regulation of muscle CK under anoxia may be linked with changes in the binding of CK with myofibrils and the effects of binding on enzyme properties.

Antioxidant and anti-apoptotic defenses in the anoxia-tolerant turtle, Trachemys scripta elegans.  

Abstract:

The freshwater turtle, Trachemys scripta elegans, utilizes various biochemical adaptations to survive anoxia-reoxygenation cycles without apparent tissue damage. This thesis focused on changes in the expression and regulation of selected enzymes and proteins involved in antioxidant and anti-apoptotic defense in turtle tissues in response to anoxic submergence and reoxygenation recovery. Western blotting showed that levels of the antioxidant enzyme, manganese superoxide dismutase (Mn SOD), were significantly higher (P<0.05) in heart and skeletal muscle during anoxia. Among four isozymes of glutathione S-transferase (GST), GST K1 expression level was enhanced in kidney, liver and muscle during anoxia, but remained stable in heart. GSTT1, GSTP1 and GSTM3 were elevated in a tissue-specific manner. Anoxia-induced upregulation of the transcription factor, Nrf2, coupled with translocation of Nrf2 into the nucleus in anoxia, indicated that Nrf2 is probably involved in activating downstream antioxidant genes such as GST. Analysis of antiapoptotic proteins (Bcl-XL, Bcl-2 and Mcl-1) also showed enhanced expression in selected tissues during anoxia exposure whereas the pro-apoptotic protein, Bad, was suppressed via phosphorylation during anoxia in muscle. Levels of bcl-xl mRNA were also quantified to assess the relationship between bcl-xl gene and Bcl-XL protein expression under anoxia. The results indicate that enhancement of antioxidant and anti-apoptotic defenses is an important adaptive mechanism for effectively dealing with low oxygen and oxidative stresses over cycles of anoxia/reoxygenation in turtles.

Freeze tolerant frogs: expression and regulation of transcription factors of the unfolded protein response and the ER-associated degradation.  

Abstract:

Wood frogs (Rana sylvatica) are the primary model used in the study of freeze tolerance in vertebrates and much is known about the adaptations of physiology, biochemistry and gene expression that support winter freezing survival, particularly their natural cryoprotective processes. Freezing and/or its components (anoxia and dehydration) places multiple stresses on cells; one of these is endoplasmic reticulum (ER) stress, a condition caused by accumulation of unfolded or misfolded proteins in the ER. The regulated expression of selected transcription factors, such as ATF4, that trigger genes that protect against ER stress is important for cell survival of freezing. During ER stress, the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway are triggered, which can potentially lead to apoptosis.  Western blots were used to evaluate the responses by key protein components of the UPR and the ERAD under freezing, anoxia and dehydration stresses in two major organs of wood frogs (skeletal muscle and liver).  The proteins analyzed included the activating transcription factors (ATF3, ATF4, ATF6), the growth arrest and DNA damage proteins (GADD34, GADD153), and the EDEM and XBP1 proteins.  Stress-induced redistribution of transcription factors between cytoplasmic and nuclear fractions was also evaluated. All three stresses triggered the UPR in both tissues but only freezing of skeletal muscle seemed to trigger the ERAD.  Only anoxic treated skeletal muscle showed metabolic signs of potential apoptosis. It was concluded that wood frog organs activate the UPR as a means of stabilizing cellular proteins and shutting down global protein synthesis in order to survive freezing exposures without irreparable injury.

Regulation of glutamate dehydrogenase in hypometabolic states

Abstract:

Glutamate dehydrogenase (GDH) is a key enzyme in nitrogen metabolism and has significant roles in amino acid catabolism and urea biosynthesis. The role of this enzyme in important metabolic processes suggests that it may be regulated in animals that survive in unforgiving environments. Such animals typically respond to harsh environmental conditions with strong overall metabolic rate suppression as well as species- and tissue specific metabolic alterations. To better understand the role of amino acid metabolism in hypometabolism, GDH was investigated in hibernating Richardson’s ground squirrels (Spermophilus richardsonii), anoxia-tolerant freshwater turtles (Trachemys scripta elegans), and estivating land snails (Otala lactea). Studies analyzed GDH substrate affinities, effects of metabolite activators and inhibitors, pH and/or temperature effects, and the actions of protein kinases and protein phosphatases in modifying enzyme properties. Liver GDH from ground squirrels was regulated by reversible protein phosphorylation in a manner that could activate the oxidation of glutamate to assist in energy production and contribute to gluconeogenesis during hibernation. Similarly, foot muscle GDH from land snails was regulated by reversible phosphorylation with the subsequent activation of the glutamate-oxidizing reaction to aid in energy production and urea biosynthesis in the estivating state. Conversely, under conditions of oxygen deprivation, the freshwater turtle utilizes reversible phosphorylation to inactivate both the glutamate-oxidizing and glutamate-synthesizing reactions of GDH. Thus, it appears that GDH and its effects on amino acid metabolism play an important role in animals during hypometabolism.

Regulation of tail muscle energetics during anoxia in the freshwater crayfish, Orconectes virilis

Abstract:

Metabolic rate depression is vital to the survival of many organisms in the face of low oxygen levels. This is achieved by a coordinated suppression of both ATP-consuming and ATP-producing metabolic pathways. The role of reversible protein phosphorylation in metabolic rate depression during anoxia was explored in the tail muscle of the anoxia-tolerant freshwater crayfish, Orconectes virilis. This study investigated glutamate dehydrogenase (GDH), the enzymatic bridge between amino acid and carbohydrate metabolism, arginine kinase (AK), an important enzyme involved in regulation of phosphagen reserves, and hexokinase (HK), the enzyme at the forefront of carbohydrate metabolism. The data obtained showed that GDH and AK are regulated by reversible phosphorylation during anoxia, resulting in less phosphorylated, less active forms of these enzymes. Experiments were performed under normoxic and anoxic conditions, and protein expression levels, susceptibility to urea denaturation, structural stability, response to specific protein kinase and phosphatase incubations as well as elution profiles from an ion-exchange column were explored. The data from GDH suggests that amino acid metabolism is left largely separate from carbohydrate metabolism by the reduction of this vital bridge point. AK results suggest that precious ATP is not involved in the regeneration of phosphagen reserves during anoxia. HK was also explored using similar experiments, and it seems that HK protein levels increase during anoxia, and reversible phosphorylation seems to increase protein stability and affect cellular localization. Overall, these studies suggest that reversible phosphorylation plays a key role in the regulation of muscle energetics in the freshwater crayfish, O. virilis.