Hanane

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.

Tamara E. English, Ph.D. Biology 2000

Differential gene expression in response to freezing and anoxia in the intertidal marine gastropod, Littorina littorea.

 

Abstract:

The intertidal zone is a highly variable environment where temperature, salinity and oxygen availability all fluctuate on a daily and seasonal basis. As a result, animals that inhabit this zone possess a high degree of metabolic plasticity. Littorina littorea, an intertidal marine gastropod, is tolerant of both freezing and anoxia. This study searched for changes in gene expression that may underlie the animal’s ability to endure these stresses. Screening of cDNA libraries was employed to identify freezing- and anoxia-induced genes in foot muscle of L.littorea; two libraries were synthesized from mRNA isolated from foot muscle of animals exposed to either 1, 12 and 24 hours of freezing or 1, 12 and 24 hours of anoxia. Differential screening of the frozen or anoxic libraries with mRNA isolated from stressed versus control (5°C)snails, followed by northern blot analysis, resulted in six transcripts that were confirmed as stress-upregulated. DNA sequence analysis identified 3 clones as myosin heavy chain, beta actin and cytochrome oxidase subunit 2. These clones were isolated from the freezing library but showed greatest transcript accumulation during recovery after stress. The translated amino acid sequence of a fourth clone, LLMET, elicited a putative identification of metallothionein, a heavy metal binding protein with a possible antioxidant role. The remaining two clones, LLGRP and LLAFW, were novel, showing little or no homology to DNA or amino acid sequences in various databases. Analysis of their translated amino acid sequences indicated that both proteins possessed a secretory signal at the N terminus, suggesting that they had either a membrane location or are excreted from the cell. Structural predictions based on previously analyzed proteins, suggested that LLGRP is a membrane channel protein of the porin class whereas LLAFW fits the criteria of an anti-parallel bundle (apb) protein of the alpha-class. None of the six genes/proteins found by this study has a cellular function that integrates well with any of the previously-identified biochemical adaptations that support anoxia or freezing tolerance but each suggests that there is still much more to learn about the types of molecular adjustments that are needed to support natural stress tolerance.

Dustin S. Hittel, Ph.D. Biology, 2001

The physiological role of differentially expressed genes and their protein products in the hibernating thirteen-lined ground squirrel Spermophilus tridecemlineatus

 

Abstract:

The role of differential gene expression in supporting the survival of the hibernation phenotype was investigated using a variety of “gene discovery” techniques. A cDNA library constructed from kidney of the thirteen-lined squirrel,Spermophilus tridecemlineatus, was differentially screened for genes that were up-regulated during hibernation. A clone encoding cytochrome c oxidase subunit 1 (Cox1) was found and confirmed as up-regulated by Northern and Western blotting. This revealed the differential expression of Cox1 mRNA in multiple organs during hibernation. It is hypothesized that hibernating mammals increase the expression of the mitochondrial genome in general and Cox1specifically during torpor, to prevent or minimize the damage caused by the cold and ischemia experienced during a hibernation bout. The up-regulation of heart and adipose type fatty acid binding proteins (FABPs) was detected during hibernation in brown adipose tissue (BAT) using a commercial rat cDNA array. Full length cDNAs encoding heart and adipose-type FABPs were subsequently retrieved from a BAT cDNA library. H-FABP mRNA transcripts increased in BAT, skeletal muscle and heart of hibernating animals whereas A-FABP transcripts, which are normally expressed exclusively in adipose tissue, increased in both BAT and heart during torpor. The translation status of differentially expressed mRNAs during hibernation was also investigated in kidney and brown adipose tissue. Polysome profile analysis revealed a significant de-aggregation of polyribosomes during hibernation and a shift of housekeeping gene mRNAs and the up-regulated organic cation transporter 2 (OCT2) mRNA to the translationally silent monosome and mRNP fractions of kidney cytoplasmic extracts. In vitrotranslation rate and immunoreactive OCT2 protein were also significantly decreased in hibernating kidney. By contrast, the increased translation status of H-FABP mRNA, increase in immunoreactive H-FABP protein and unchanging in vitrotranslation rate reflect the important role of active brown adipose tissue during hibernation. The decrease in protein synthesis and de-aggregation of polysomes in kidney but not in brown adipose tissue, is linked to the phosphorylation of eIF2“ . Additionally, redistribution of Cox4 but not H-FABP mRNA to the monosome fraction in hibernating BAT may indicate a mechanism for the preferential translation of a subset of genes physiologically relevant to the survival of hibernation.

 

Kevin F. Larade, Ph.D. Biology 2002

Response to anoxia exposure by the marine snail, Littorina littorea: transcription and translation patterns of differentially expressed genes and proteins

 

Abstract:

Situated in the intertidal zone, the marine periwinkle Littorina littoreahas modified a number of biochemical mechanisms in order to endure extended periods of oxygen deprivation that accompany aerial exposure at low tide. To cope with decreased ATP production during anaerobiosis, organisms often suppress energy-consuming processes such as those involved in macromolecular synthesis. Analysis of hepatopancreas samples from snails exposed to 24-96 h anoxia showed a gradual disaggregation of polysomes into monosomes, signifying a decrease in protein synthesis, with a subsequent re-aggregation of polysomes observed after 3 h of aerobic recovery. Upon examination of protein synthesis, the rate of [3H] leucine incorporation into newly translated protein of hepatopancreas isolated from 48 h anoxic snails was determined to be 49% relative to normoxic controls. Western blots examining the phosphorylation state of eIF-2alpha, a factor involved with initiation of protein translation, also supported the proposal that metabolic suppression during anoxia in L. littorea involves a decrease in protein translation. The rate of overall mRNA synthesis in anoxia was also examined; [32P] UTP incorporation into RNA transcripts of nuclei obtained from 48 h anoxic snails was determined to be 31.7% relative to normoxic controls. Although these data show that transcription and translation are suppressed overall during anoxic exposure, specific RNA transcripts were up-regulated during anoxia as identified via differential screening of a hepatopancreas cDNA library. Several anoxia-induced clones were identified as homologues of known genes (e.g. ribosomal protein L26, ferritin heavy chain) and others were deemed novel (e.g.kvn). Northern blots showed gene-specific patterns of transcript elevation over a time course of anoxia. Nuclear run-off assays confirmed transcriptional up-regulation during anoxia, whereas organ culture experiments implicated selected second messengers and protein kinases in signal transduction pathways regulating gene expression. Examination of the protein products of anoxia-induced genes was accomplished with western blotting when antibodies to specific proteins were available. These results suggest that metabolic suppression in L. littoreainvolves a general decrease in macromolecular synthesis, whereas specific transcripts that may help cope with oxygen lack are up-regulated during anoxia exposure.