Hanane

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.

 

J. Dayre McNally, Ph.D. Chemistry 2002

Response to freeze exposure by the wood frog, Rana sylvatica: investigating the freeze induced changes to transcription and translation patterns in heart and liver

 

Abstract:

In recent years changes to gene and protein expression have been shown to underlie animal adaptation to environmental stresses. Similarly, recent studies have demonstrated significant changes to transcription and translation in wood frog tissues during freeze and thawing. The primary goal of this thesis was to further investigate these changes through the application of cDNA library screening and 2-D PAGE technologies. In total 7 additional genes and one protein were identified, significantly extending the list of known freeze up-regulated genes and proteins. From this expanded list the contractile protein, MLC2; two identifiable heart transcripts (hsp27 and anac); and the novel liver transcripts (li16 and ddx8) were selected for further investigation. The results implicate the heat shock and hypertrophic gene responses as part of the cellular response to freezing, and reveal that the wood frog heart responds to freezing with changes expected to alter AP-1 mediated gene transcription. Moreover, all of the identified cardiac responses, including specific increases in gene/protein levels and changes to protein phosphorylation states could be linked to the p38 MAPK pathway, confirming that it is the major signaling pathway activated in the wood frog heart following freezing exposure. The two novel liver clones were similarly investigated and studies were carried out to obtain full open reading frames, confirm the protein coding capacity and determine the factors regulating their expression. Although the cellular functions of the novel proteins remain a mystery the results obtained suggest distinctive cellular roles, and on the whole provide further evidence that novel gene development is an important component of acquired freeze tolerance. Secondary to the determination of the specific changes in gene and protein expression patterns following freeze exposure the impact of freezing on the overall rates of transcription and translation in wood frog heart tissue were evaluated. The data from the nuclear run-on assays and western blots analysis (phosphorylation status of eIF2a ), confirmed the presumed suppression of transcription and translation in the wood frog heart during freezing. This discovery highlights the importance of the aforementioned genes and proteins, as their up-regulation takes place against a background of suppressed macromolecular synthesis.

Sean F. Eddy, Ph.D. Chemistry 2003.

Role of differential gene and protein regulation in hibernating mammals

 

Abstract:

Winter survival for many mammals involves hibernation. By strongly suppressing metabolic rate, animals conserve energy and endure long months of subzero environmental temperatures and lack of food. The little brown bat, Myotis lucifugus, and thirteen-lined ground squirrel, Spermophilus tridecemlineatus, are two such hibernating mammals. Hibernation consists of long periods (1-2 weeks) of cold torpor with body temperature near ambient interspersed with brief periods of arousal to euthermia. Precise control over gene and protein expression is needed to prevent costly overuse of essential fuel reserves while ensuring that selected specific changes are made that aid survival in the cold, torpid state. The present studies evaluated signal transduction pathways and changes in gene expression in skeletal and cardiac muscle during hibernation. The p38 MAP kinase signal transduction pathway in skeletal muscle of both species appears triggered with increased amounts of active phosphorylated p38 found during hibernation along with activation of various downstream targets of p38 including ATF-2, CREB, HSP27 and IκB. The Akt-mediated insulin-signalling pathway was, by contrast, apparently suppressed in hibernator muscle; this may help restrict the use of carbohydrate fuels.cDNA arrays were used to compare gene expression in euthermic versus hibernating states; selected genes (0.5-2%) were up-regulated during hibernation but most were unaffected. A general suppression of protein synthesis is likely during hibernation and this was supported by elevated levels of phosphorylated initiation (eIF2α) and elongation (eEF2) ribosomal proteins. Despite the probable suppression of transcription and translation, selected genes and proteins were up-regulated during hibernation including two involved in fatty acid transport and metabolism: fatty acid-binding protein and carnitine palmitoyl transferase-1. Furthermore, two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry revealed the up-regulation of a thioredoxin peroxidase type enzyme in heart of M. lucifugus; both mRNA and protein levels rose during hibernation. This, along with elevated amounts of the active forms of oxidative stress markers, HSP27 and IκB, demonstrates that oxidative stress has a role in hibernating tissues. The data enhance our knowledge of the molecular mechanisms of hibernation with novel contributions to the understanding of the roles of fatty acid metabolism, oxidative stress, and muscle atrophy.

J.N. Amritha De Croos, Ph.D. Biology, 2003

Gene and protein regulation in liver of the freeze tolerant wood frog, Rana sylvatica.

 

Abstract:

The wood frog Rana sylvaticahas the ability to tolerate whole body freezing in nature. Biochemical mechanisms supporting freezing survival have been widely studied in this species, but much remains to be explored. The present research demonstrates the involvement of specific gene and protein level changes in the response to freezing by liver. Screening of a cDNA library prepared from liver of R. sylvatica identified a freeze-responsive clone containing a 1370 nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over time during freezing, reaching >70-fold up-regulation after 24 hours frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase inpic transcript levels during dehydration (9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers of the same family were not affected. Southern blot analysis showed that the increase in PiC was not due to an increase in mitochondrial numbers. Analysis of polysome profiles revealed disaggregation of polysomes into translationally less active monosomes upon freezing. PiC transcripts segregated into monosomes in control liver, but would aggregate into actively transcribing polysomes during freezing. Western blot analysis showed that the activity of specific transcription factors in frogs is temporally controlled. The transcription factors CREB and NFk B were significantly activated after two hours of freezing exposure whereas protein kinase B (Akt) was activated after 24 hours of freezing. Screening of a cDNA microarray identified an anti-apoptotic factor that is up-regulated during freezing in liver. Western blot analysis showed time dependent changes in anti-apoptotic activity mitigated by significant changes in AP-2a , BAX-inhibitor and p53 protein levels. These results suggest that freeze tolerance in R. sylvatica involves control of gene and protein levels through the up-regulation of selected transcripts and the activation of kinases and transcription factors. This gene and protein control, or regulation, combined with specific anti-apoptotic actions, allows hepatocyte survival of the multiple stresses imposed by freezing.

David C. McMullen, Ph.D. Chemistry 2004.

Molecular and biochemical adaptations in two cold-hardy gall insects.

 

Abstract:

Terrestrial insects have evolved molecular and biochemical adaptations that have allowed them to exploit virtually all climates on earth.  Insects living in temperate and polar climates are forced to deal with freezing temperatures head-on.  Two strategies of cold-tolerance in insects have emerged, freeze-tolerance and freeze-avoidance.  Freeze-tolerantallow extracellular ice formation, whereas freeze-avoiding species depress the supercooling point of their body fluids below temperatures that they would normally encounter as part of their life-cycle.  This research used a model species from each category of cold-tolerant insects.  The model species chosen wereEpiblema scudderiana, a freeze-avoiding gall moth, and Eurosta solidaginis, a freeze-tolerant gall fly.  Previous studies on these systems were based on measuring physiological parameters such as substrate utilization of product build-up.  Results presented here are from molecular and biochemical studies completed to further our understanding of cold-tolerance and hypometabolism. Studies also included the effects of hypoxia on the two model species because this stress is often associated with freezing.  The first step in each type of experiment was to examine how these species responded in nature and then see if we could reproduce that response under laboratory conditions to identify the trigger for such a response.  The mechanism of ion pump regulation was examined and possible mediators of control were proposed.  Three different gene expression analysis techniques were employed in an effort to identify key gene product that contribute to cold and hypoxic tolerance.  Gene expression studies and enzyme assays have indicated that the role of the mitochondria in cold and hypoxic tolerance is more dynamic that previously understood.  In addition, the MAP kinase superfamily of signal transduction has clearly been implicated in the survival success of these insects. The p38 family of MAPK is of particular interest because there were clear differences in this pathway between the freeze-tolerant and freeze-avoiding species.  Reversible phosphorylation in undoubtedly a key mechanism of control in insect hypometabolism and stress tolerance.   Finally, the role of NF-κB and the inflammatory response appears to be significant in the over winter survival of both species.

Pier Morin, Ph.D. Chemistry, 2006

Transcriptional regulation in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.

 

Abstract:  

Many small mammals enter hibernation to survive the winter. Metabolic rate during torpor can drop to just 1-5 % of the euthermic rate providing energy savings of ~90% compared to the costs of remaining euthermic. Hibernation consists cycles of torpor bouts interspersed by short arousal periods. Regulation of torpor-arousal requires tight overall control of energy-consuming metabolic processes as well as selective expression of genes to accomplish these transitions and readjust metabolism for long term viability in a hypometabolic, hypothermic state. Hence, despite overall metabolic rate depression in hibernation, certain processes must be activated to ensure survival. The present studies examined transcriptional control in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus. Regulation of gene expression by the hypoxia-inducible transcription factor (HIF-1) pathway was shown to be important in hibernation; HIF-1α subunit levels rose by 60-70 % in skeletal muscle and BAT during hibernation and HIF-1 DNA-binding activity increased 6-fold in hibernating BAT.  By contrast, the overall transcriptional state in muscle was strongly suppressed during torpor.  Both activity and protein levels of histonedeacetylases, enzymes involved in transcriptional repression, were elevated during torpor whereas the activity of RNA Polymerase II, a key enzyme of gene transcription, was strongly reduced by 43 %.  To evaluate the involvement of oxidative stress and antioxidant defenses in hibernation, the responses of the Nrf2 transcription factor, involved in the oxidative stress response pathway, were evaluated as well as antioxidant genes/proteins under Nrf2 control. Nrf2 protein levels were elevated (by 1.4-fold) in all heterothermic parts of the hibernation cycle whereas the protein contents of three downstream gene targets of Nrf2 were only elevated during entrance into hibernation. This suggests aNrf2-mediated anticipatory enhancement of antioxidant defenses to deal with oxidative stress during torpor and/or arousal. Other antioxidant enzymes, the 2-Cys peroxiredoxins, also showed enhanced protein levels in torpor and increased enzyme activity (1.5-fold in heart and 3.4-fold in BAT) indicating a potential role in ROS detoxification during hibernation.  The data reported in this thesis provides new insights on the roles of selected transcription factors and on the importance of their tight regulation during all stages of hibernation.

Christopher J. Ramnanan, Ph.D. Biology, 2006

Regulation of metabolism in estivating land snails: role of reversible protein phosphorylation.

 

Abstract:

Estivation is a state of aerobic dormancy used by animals such as the desert land snail, Otala lactea, to endure harsh environmental conditions. Metabolic rate depression is key to survival during estivation and requires coordinated suppression of ATP generating and ATP-consuming cellular functions by stable regulatory mechanisms. Current studies examined the role of reversible protein phosphorylation in metabolic arrest in snail organs focusing on a major ATP-consuming process, the Na+/K+-ATPase; a key enzyme that produces NADPH for antioxidant defense, glucose-6-phosphate dehydrogenase (G6PDH); and selected protein kinases and protein phosphatases that could regulate the process. Studies documented a decrease in foot muscle and hepatopancreas Na+/K+-ATPase activity, and an increase in hepatopancreas G6PDH activity during estivation, as indicated by changes in kinetic parameters (e.g. maximal velocity, substrate affinity, Arrhenius activation energy). Furthermore, in vitro incubations stimulating specific endogenous kinases and phosphatases implicated roles for PKG and PP1 in estivation-dependent changes in Na+/K+-ATPase and G6PDH. Ion exchange chromatography of G6PDH revealed two enzyme forms – a high phosphate, high activity form and a low phosphate, low activity form – whose proportions changed in dormant snails. The peak profiles of G6PDH from active and estivating snails were also interconverted after incubations promoting PKG or PP1 activities. Profiles of protein phosphatases in O. lacteatissues revealed a general suppression of activity during estivation. For PP1 and PP2A the differential activity in estivation was linked to altered enzyme elution profiles from gel filtration chromatography, indicating that differential association into phosphatase holoenzymes is partly responsible for reduced phosphatases activities in estivation. Type-1 and type-2 phosphatases were purified and analyzed; the data generally indicated that the mammalian phosphatase classification system was applicable to O. lacteaphosphatases. Examination of several protein kinases, utilizing a relatively novel assay method with P81 paper array/phosphor imaging, revealed increased activities of AMPK, PKB, and PKG in estivating O. lactea. Increased activities of AMPK and PKB were related to changes in their phosphorylation state and confirmed by changes in activities and/or phosphorylation status of downstream targets. Overall, these studies confirm the integral role of reversible protein phosphorylation in the suppression and reorganization of metabolism during estivation.

Hapsatou Mamady, Ph.D. Biology, 2006

Mammalian hibernation: gene expression and transcription factor regulation of the unfolded protein response, apoptosis and atrophy in ground squirrels.

 

Abstract:

Various mammalian species hibernate as a way to survive extended winter periods of food scarcity and cold environmental conditions. Hibernation is an energy-conserving strategy,characterized by periods of torpor with extreme decreases in core body temperature and strong metabolic rate depression, interrupted by brief periods of arousal to euthermia. To endure the conditions of cold torpor, as well as the wide fluctuations over cycles of torpor-arousal, differential expression of genes and their tight regulation is required. The present studies examined changes in the expression and regulation of selected genes involved in the unfolded protein response, muscle atrophy and anti-apoptotic defense during hibernation in thirteen-lined ground squirrels,Spermophilus tridecemlineatus. Despite overall suppression of transcription and translation during torpor, selected genes and their products were up-regulated. Themolecular chaperone GRP78 increased in BAT and brain of torpid animals, indicating endoplasmic reticulum stress and a role for GRP78 in alleviating stresses that cause protein misfoldingduring hibernation. Regulation of thegrp78 gene by the activating transcription factor ATF4 via the PERK/eIF2a/ATF4 pathway was shown to be important in hibernation; ATF4 protein expression increased in BAT, brain and skeletal muscle of hibernating squirrels and ATF4 DNA-binding activity increased in hibernating brain. Subcellular localization of ATF4 showed that this transcription factor and its cofactor, pCREB-1, weretranslocated into the nucleus during hibernation where they could activate downstream genes. Another transcription factor, FoxO1a, and the downstream genes that it controls via the PI3K/AKT/FOXO pathway can induce muscle atrophy. Hibernators appear to counteract this by phosphorylating and inactivating FoxO1a in heart and skeletal muscle and strongly suppressing FoxO1a DNA binding activity by 76% in muscle during torpor. Finally, the anti-apoptotic proteins, Bcl-XL and Bcl-2, showed enhanced expression in tissues of ground squirrels whereas the pro-apoptotic protein, BAD, was suppressed viaphosphorylation during torpor. These results show that anti-apoptotic defense is also important to cell survival in hibernation. The data in this thesis enhance our knowledge of the molecular mechanisms that govern hibernation and the role played by selected transcription factors in regulating subsets of genes that are physiologically relevant to the hibernation phenotype.