Hanane

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.

William G. Willmore, Ph.D. Biology 1997

Molecular adaptation to anoxia and recovery from anoxia in the freshwater turtle Trachemys scripta elegans

 

Abstract:

The responses to stresses associated with oxygen extremes, namely anoxia and oxidative stress associated with recovery from anoxia, were studied in an organism that must tolerate both extremes. The model organism used was the turtle Trachemys scripta elegans that can survive complete anoxia for up to 18 weeks at lower temperatures. Metabolic adaptations that support survival of oxygen extremes in this organism that were examined included: (a) tissue-specific changes in the maximal activities of enzymes of intermediary metabolism, (b) an assessment of the metabolite and enzymatic antioxidant defenses utilized to combat the oxidative stress encountered upon recovery from anoxia, (c) adaptations in specific enzymatic antioxidant defenses and (d) adaptations to anoxia and recovery from anoxia of a specific protease. Of the key enzymes of intermediary metabolism, 14 instances of reduced and only 3 instances of increased enzyme activities were observed in tissues during anoxia as compared to controls. Brain had changes related to both metabolic rate depression and neurotransmitter release. Maximal activities which increased during anoxia included G6PDH in heart, MDH in white muscle and CPT in kidney. Anoxia stress and aerobic recovery produced relatively few changes in organ antioxidant enzyme activities and levels of lipid peroxidation products. Changes included tissue specific decreases in antioxidant enzymatic activities during anoxia, particularly SOD and CAT. The levels of enzymatic antioxidant activities were high in turtles in comparison with other vertebrates and non-vertebrates. Turtles maintained high levels of total glutathione in tissues in comparison to other ectotherms. Tissue-specific changes in the maximal activities of the glutathione-related enzymes occurred, the most dramatic being a decrease in gamma-GTPase during anoxia to 2% of control values. Turtle liver contained one homodimeric alpha class and a unique heterodimeric alpha-like GST. Turtle liver GR revealed a high affinity for GSSG. No new isoforms of either GST or GR were formed during anoxia. MPC displayed an increase in peptidylglutamyl-peptide bond hydrolyzing activity during recovery from anoxia, possibly as a result of oxidative damage to proteins in this tissue. Overall, T. S. elegans maintains continually high defenses against the stresses of anoxia and oxidative stress upon recovery from anoxia.

Bradley J. Thatcher, Ph.D. Biology, 1997

Properties of enzymes from mammalian hibernators: Structure function relationships

 

Abstract:

The examination of four enzymes, glutathione S-transferase (GST), glutathione reductase (GR), aspartate aminotransferase (AspAT), and glutamate dehydrogenase (GDH), from two hibernators Cynomys leucurus andSpermophilus richardsonii were used to show three different responses to a hibernating life style. The production of a new (isozyme) of GST, alpha-class, in the liver and muscle tissues of Cynomys leucurus. N-terminal and amino acid compositions show strong homology alpha-class GSTs from a variety of species. The kinetic profile and structural studies suggest that this isozyme is similar to other alpha-class GSTs. PDM 18 showed a broader temperature optima and lower thermal stability compared to a comparable rabbit enzyme. A different form of GDH was found in the liver tissue of Spermophilus richardsonii in the hibernating animal. This new isozyme of GDH differed from the nonhibernating and bovine enzyme in both physical and kinetic parameters. There is a shift in the directional preference between the two ground squirrel forms of the enzyme in the presence of GTP, ATP, AMP. Further kinetic differences are also reflected in I50, Km, and Ka values between the two ground squirrel isozymes. The N-terminal sequence of the two isozymes is identical over the first 12 residues; there are differences in the molecular weight by size exclusion chromatography, and retention time on reverse phase HPLC suggesting small differences in the polypeptide chain. The final observed case was that there were no substantial kinetic and minor structural changes between the enzyme from the hibernator and from a nonhibernator. This was seen with two enzymes from the study GR (from prairie dog) and AspAT (from ground squirrel).

 

Kyra J. Cowan, Ph.D. Chemistry, 1998

Evidence for the reversible phosphorylation control of metabolism during prolonged environmental stress

 

Abstract:

I hypothesized that the environmental conditions endured during estivation in the spadefoot toad, Scaphiopus couchii, would dramatically affect metabolism and metabolic regulation in toad organs, and that reversible protein phosphorylation is important in the control of the necessary metabolic adaptations. Enzyme activities in tissues of control versus 2 month estivated toads revealed that glycolysis and ketone body metabolism dropped in liver, and that amino acid catabolism and NADPH production increased in brain and muscle. The influence of urea, which can build to 300 mM in estivating toads to aid desiccation resistance, was assessed on liver and muscle enzymes. Although known as a protein denaturant, urea had little effect on toad enzymes, being less disruptive of enzyme activities than 300 mM KCl. Estivation led to significant changes in the kinetic properties of both pyruvate kinase and phosphofructokinase that were mediated by reversible phosphorylation. To explore this mode of metabolic regulation, estivation effects on the activities of multiple protein kinases and protein phosphatases were studied. Protein kinase A (PKA) activities dropped in all tissues, and dormancy strongly affected type-1 and type-2 protein phosphatases. Estivation effects on the isozyme types, activities and distribution of protein tyrosine kinases (PTKs) and phosphatases (PTPs) revealed three major forms of PTKs and four PTPs in muscle and overall activities of PTKs decreased during estivation in the soluble fractions of liver, lung, and leg muscle, with the opposite pattern in heart. PTP activity in soluble fractions of gut, heart, and leg muscle also increased in estivation. Tissue specific changes in the enzyme activities and protein levels of mitogen-activated protein kinases (including Raf-1, ERK 1 & 2, MAPKAPK-1, JNK, p38) also occurred during estivation as did levels of various transcription factors (ATF-2, Elk-1, Egr-1, CREB). Involvement of these kinases and transcription factors implicates tissue-specific changes in gene expression during estivation and together with estivation-induced reversible phosphorylation of enzymes indicates that widespread, coordinated controls are used to readjust metabolism for the needs of the estivating state including metabolic rate depression, desiccation tolerance and long term starvation.

 

Justin A. MacDonald, Ph.D. Biology, 1998

Enzyme thermal adaptations and signal transduction involvement in ground squirrel hibernation

 

Abstract:

Regulatory enzymes are integral to the regulation of metabolism, and reversible protein phosphorylation by kinases and phosphatases controls most aspects of cell life. Brown adipose (BAT) PKA, liver PP-1 and muscle phosphofructokinase (PFK) from the ground squirrel were purified to homogeneity; physical and kinetic properties of these were assessed with respect to function at low temperature. An assessment of PKA catalytic subunit function via in vitro incubations of ground squirrel BAT extracts with 32P-ATP revealed differences in the patterns of phosphorylated proteins between euthermic and hibernating animals and between 37° and 5°C. The addition of 10% (w/v) polyethylene glycol reversed all negative effects of cold temperature, low pH and urea on PFK stability. A reduction in assay temperature from 37° to 5°C had numerous effects on ground squirrel enzymes including changes in pH optima, decreases in Km values for substrates, and reduced inhibition by salts. The percentage of membrane associated PKC increased during hibernation in liver, but the % active PKA was unaffected by hibernation in any tissue. Changes in PP-1 and -2 activities in tissues of euthermic and hibernating animals showed that phosphatases are regulated in hibernation. The subcellular localization of PP-1 in liver and muscle was affected by hibernation. The mitogen-activated protein kinase (MAPKs) MAPK-activated protein kinase (MAPKAPK-1 and -2) activities were affected by hibernation. p70S6Kkinase activity decreased in kidney during hibernation. c-jun kinase activity increased in 4 hibernating tissues but decreased slightly in brain. p38 was activated in hibernating muscle and heart. The responses of immediate-early gene products c-jun, egr-1, and c-myc were tissue specific with no apparent overall pattern. Maintenance of muscle energy status at the expense of the total adenylate pool was accompanied by a 60% decrease in the Na+K+-ATPase activity. Enzyme activity was reduced in vitro when incubated with protein kinase activators and was relieved by addition of alkaline phosphatase. A change in the ATP dependency of the enzyme also occurred in hibernation. Overall, protein phosphorylation is key to the regulation of hibernating metabolism in ground squirrels, and hibernator enzymes demonstrate functional adaptations to cold temperature.

 

Tolga Bilgen, Ph.D. Biology 1998

Differential gene expression in two cold hardy insects in response to low temperatures

 

Abstract:

The two cold hardy gall insects, Epiblema scudderiana andEurosta solidaginis, employ freeze avoidance and freeze tolerance, respectively, in coping with low seasonal temperatures. Biochemical and metabolic changes in these animals in response to low temperatures have been well documented, but the possibility that cold and subzero temperatures could upregulate genes in these animals has not been investigated. In this study, cDNA library screening and differential display PCR were used to address this question. Animals were sampled over a successive cold (2 weeks at 4°C) and subzero temperature (one week at -20°C) time course. Differential screening/PCR against RNA from control (15°C) animals and Northern analyses revealed a number of upregulated transcripts. All clones were sequenced, and all but two were unidentifiable through GenBank database comparisons, due to the novel and incomplete nature of their sequences. The two identifiable sequences had strong homologies to Drosophila genes mlp6OA andrpAl, encoding respectively, a factor crucial for myogenesis and an acidic ribosomal protein. Implicated in embryonic and adult development, the upregulation of these genes suggests a unique developmental regime in E. scudderiana and F. solidaginis,both of which overwinter as final instar larvae. These animals may be preparing in advance for impending morphogenic changes, allowing for a head-start with the onset of spring.

Shaobo Wu, Ph.D. Biology 1999

Differential gene expression under environmental stress in the freeze tolerant wood frog, Rana sylvatica

 

Abstract:

Freeze survival of wood frog, Rana sylvatica, involves adaptations including control over extracellular ice formation, production of glucose cryoprotectant, and resistance to freezing-caused intracellular dehydration and ischemia. Gene expression associated with stress survival was investigated in this freeze tolerant species. Freeze-inducible genes were found by differential screening of frog brain and liver cDNA libraries; these included mitochondrial genes [encoding ATPase subunit 6 & 8, 16S rRNA and NADH-ubiquinone oxidoreductase subunit 4 (ND4)], the phosphoglycerate kinase 1 (PGK1) gene, and genes whose products are involved in translational processes [acidic ribosomal phosphoprotein (P0), elongation factor 1 gamma subunit (EF-1 gamma)]. Another ribosome-associated gene, encoding ribosomal protein L7 (RPL7), was identified in skin via differential display of polymerase chain reaction (DD-PCR). This gene was up-regulated in skin of cold-acclimated frogs and brain of freeze-exposed frogs. Freezing stimulated the upregulation of the above genes in selected frog organs. Tissue-specific gene expression also occurred in frog brain and liver in response to anoxia or dehydration. Anoxia stimulated P0, PGK1, RPL7, 16S rRNA and ATPase 6 & 8 gene expression and modulated ND4and EF-l gamma expression, whereas dehydration enhanced the expression of genes such as PGK1.Upregulation of genes whose products are directly involved in energy generation (PGK1, ATPase subunit 6 & 8, ND4) and whose products are related to protein biosynthesis suggested that maintenance of minimal ATP levels and functional translation machinery may be critical for freezing survival. Freezing-induced ischemia may be a primary signal that triggers the upregulation of most of the isolated genes. However, low temperature seemed to play a role in the expression of ribosome associated protein genes, whereas freezing-related cell water stress may also regulate other selected genes (e.g. PGK1).Immunoblotting confirmed that elevated PGK1 transcripts resulted in increased enzyme protein and showed the potential physiological significance of up-regulated genes in response to stress. Immunoblotting also showed the elevation of Ca2+/ca1modulin-dependent protein kinase and phosphatase under freezing, anoxia and/or dehydration stresses which suggests that a Ca2~signaling pathway plays a role in stress-mediated gene expression. These cellular responses may play an important role in survival of environmental stress.

Andreas Fahlman, Ph.D. Biology 2000

On the physiology of hydrogen diving and its implication for hydrogen biochemical decompression

 

Abstract:

Biochemical decompression, a novel approach for decreasing decompression sickness (DCS) risk by increasing the tissue washout rate of the inert gas, was tested in pigs during simulated H2dives. Since there is only limited physiological data on the use of H2as a diving gas, direct calorimetry and respirometry were used to determine whether physiological responses to hyperbaric H2 and He are different in guinea pigs. The data suggested that responses in hyperbaric heliox and hydrox cannot be explained solely by the thermal properties of the two gas mixtures. To increase the washout rate of H2, a H2-metabolizing microbe (Methanobrevibacter smithii) was tested that converts H2 to H2O and CH4. Using pigs (Sus scrofa) comparisons were made between untreated controls, saline-injected controls, and animals injected with M. smithiiinto the large intestine. To simulate a H2 dive, pigs were placed in a dry hyperbaric chamber and compressed to different pressures (22.3-25.7 atm) for times of 30-1440 min. Subsequently, pigs were decompressed to 11 atm at varying rates (0.45-1.80 atm · min-1), and observed for severe symptoms of DCS for 1 h. Chamber gases (O2, N2, He, H2, CH4) were monitored using gas chromatography throughout the dive. Release of CH4 in untreated pigs indicated that H2 was being metabolized by native intestinal microbes andresults indicated that native H2-metabolizing microbes may provide some protection against DCS following hyperbaric H2exposure. M. smithii injection further enhanced CH4 output and lowered DCS incidence. A probabilistic model estimated the effect of H2-metabolism on the probability of DCS, P(DCS), after hyperbaric H2 exposure. The data set included varying compression and decompression sequences for controls and animals with intestinal injections of H2metabolizing microbes. The model showed that increasing total activity of M. smithii injected into the animals reduced P(DCS). Reducing the tissue concentration of the inert gas significantly reduced the risk of DCS in pigs, further supporting the hypothesis that DCS is primarily caused by elevated tissue inert gas tension. The data provide promising support for the development of biochemical decompression as an aid to human diving.