Biomedical subjects

K B Storey

Publications and source records attributed to K B Storey.

At least 73 records · Page 4Linked to original sources

PubMed1997

Upregulation of a novel gene by freezing exposure in the freeze-tolerant wood frog (Rana sylvatica).

A novel gene responsive to freezing exposure was identified among five cDNA clones obtained through differential screening of a cDNA library constructed from liver of frozen wood frogs. The cDNA sequence of this gene, cloned in the recombinant plasmid, pBfFR14, showed no homology to any genes available in the Genbank database. The clone, designated as Fr10, carried a 457 bp cDNA sequence and contained a single open reading frame that could potentially encode a small protein of 90 amino acids with a molecular weight of about 10 kDa, named FR10. The putative protein contained a highly hydrophobic N-terminal region (21 residues) that carries a potential nuclear exporting signal (NES) sequence, LALVVLVIAISGL, similar to the NES found in PKI, an inhibitor of protein kinase A (PKA). A single mRNA transcript with a size of 550 nt was detected when the insert of the pBfFR14 was used as a probe against the Northern blot containing total RNA isolated from wood frog organs. RNA blotting analysis for gene expression in eight organs showed that transcription of the gene was highly induced by 24 h of freezing exposure at -2.5 degrees C in liver and gut, moderately elevated in heart, lung, brain and bladder but showed no change in skeletal muscle and decreased in kidney. A time-course analysis for freezing regulation of gene expression in liver showed that transcript levels were increased by 2-fold in 1 h of freezing exposure and the levels continued to increase up to 3.5-fold over the control after 24 h of freezing exposure, but had returned to control levels after 24 h thawing at 5 degrees C. Gene expression in liver was also up-regulated by whole animal dehydration at 5 degrees C but strongly down-regulated by anoxia exposure, indicating that the gene may respond to cell volume regulatory signals in vivo during natural freezing.

Adaptation, Physiological↗

PubMed1997

Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress.

The gene Aat coding for ADP/ATP translocase (AAT) was cloned from liver of the freeze-tolerant wood frog, Rana sylvatica, via differential screening of a cDNA library from liver of frozen frogs and using probes from control versus frozen frogs. Sequence analysis showed that clone pBfFR07 bearing the AAT cDNA contained a 1318-bp insert with one full-length open reading frame. The deduced amino acid sequence included 317 residues, with 81-86% identities to mammalian AAT. A 1750-nt transcript from the Aat gene was detected using pBfFR07 probe and a putative frog AAT of over 30 kDa was visualized by immunoblotting using a polyclonal antibody raised against chicken AAT. Analysis of liver samples from a time course of freezing showed a maximal 4.5-fold increase in mRNA after 8 h with AAT protein peaking in 24-h frozen frogs. Freezing also induced Aat expression in bladder and lung. In liver, mRNA expression also responded positively to anoxia stress but not to experimental dehydration of the animals. These results suggest that AAT induction during freezing may be stimulated by the ischemia that develops when plasma freezes; changes in AAT may contribute to stabilizing energetics in mitochondrial versus cytosolic pools over freeze/thaw cycles.

Amino Acid Sequence↗

PubMed1997

De novo protein biosynthesis responses to water stresses in wood frogs: freeze-thaw and dehydration-rehydration.

Protein biosynthesis responses occurring during the postthaw (after 12 h freezing at -1.4 degrees C), dehydration (to 27 or 40% of total body water lost), or rehydration (after the loss of 40% of body water) were monitored in tissues of spring-collected wood frogs (Rana sylvatica) after intraperitoneal injection of 35S-labeled methionine + cysteine. All six organs tested accumulated radiolabeled amino acids and organs of both thawing and rehydrating frogs held at 3-5 degrees C showed a linear increase in amino acid incorporation into the acid-precipitable protein fraction over time. By contrast, dehydrating animals showed little or no increment in protein bound radioactivity over the course of the stress, a result that may be indicative of metabolic suppression in organs when dehydration became severe. Isoelectrofocusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to characterize the proteins synthesized by liver under each experimental state. IEF revealed both new peaks of 35S-labeled proteins and enhanced labeling of others in extracts from experimental animals, compared with controls. In particular, new synthesis of proteins with isoelectric points of about 6.0 was prominent and labeled proteins in this IEF peak persisted at 5, 10, or 24 h postinjection, becoming proportionally more important over time. SDS-PAGE analysis of the p(I) 6.0 peaks from thawed, dehydrated, and rehydrated frogs revealed the presence of one major low molecular weight protein in each case with molecular masses of 15, 13, and 21 kDa, respectively. These data indicate that the biochemical adaptations supporting freeze tolerance and dehydration tolerance in anurans include the stress-induced biosynthesis of a suite of proteins including the novel synthesis of selected specific proteins. These proteins may represent stress-related (or shock) proteins or may have specific roles in metabolic adaptation in each state such as in water and ionic balance or cell volume regulation.

Animals↗

PubMed1997

Second messenger and cAMP-dependent protein kinase responses to dehydration and anoxia stresses in frogs.

The effects of whole body dehydration (up to 40% of total body water lost) or anoxia exposure (up to 2 days under N2 gas) at 5 degrees C on tissue levels of adenosine 3'-5' cyclic monophosphate (cAMP) and the percentage of cAMP-dependent protein kinase present as the free catalytic subunit (PKAc), as well as the levels of the protein kinase C (PKC) second messenger, inositol 1,4,5-trisphosphate (IP3), were assessed in two anurans, the freeze-tolerant wood frog, Rana sylvatica, and the freeze-intolerant leopard frog, Rana pipiens. Dehydration of wood frogs resulted in a rapid elevation of liver cAMP and PKAc; cAMP was 3.4-fold greater than control values in animals that had lost 5% of total body water, whereas PKAc was elevated threefold in 20% dehydrated frogs. These results indicate protein kinase A mediation of the liver glycogenolysis and hyperglycemia that is induced by dehydration in this species. Skeletal muscle PKAc content also rose with dehydration but neither cAMP nor PKAc was affected by dehydration in leopard frog tissues. Anoxia exposure had different effects on signal transduction systems. PKAc was elevated after 1 h anoxia in R. sylvatica brain and was sustained over time but the enzyme was unaffected in other organs; by contrast, R. pipiens showed variable responses by PKAc to anoxia in three organs. Both species showed rapid (within 30 min) and large (3 to 7.8-fold) increases in IP3 in liver of anoxic frogs that decreased slowly with continued anoxia. IP3 also increased quickly in heart of anoxia-exposed wood frogs. This suggests that PKC may mediate various metabolic adjustments that promote hypoxia/anoxia resistance such as coordinating metabolic rate depression. A progressive rise in liver IP3 during dehydration in wood frogs (reaching fourfold higher than controls in 40% dehydrated animals) may also mediate similar hypoxia resistance adaptations under this stress since anurans experience progressive hypoxia due to increased blood viscosity when water loss reaches high values. The patterns of second messenger and PKAc changes in wood frog liver during dehydration closely parallel the changes seen in these same parameters during natural freezing suggesting that the freeze tolerance of selected terrestrially hibernating anurans may have evolved out of various anuran mechanisms of dehydration resistance.

Animals↗

PubMed1997

Protein kinase C from bat brain: the enzyme from a hibernating mammal.

Protein kinase C (PKC) from brain of euthermic and hibernating bats (Myotis lucifugus) showed only one form as determined by hydroxylapatite chromatography, compared with three forms found in rat brain. Cross-reaction with antibodies to rabbit alpha, beta, and gamma isozymes showed that bat brain contained only PKC(gamma). During hibernation the activity of PKC in bat brain decreased to 63% of the euthermic value but the percentage that was membrane-associated did not change. Bat and rat brain PKC(gamma) were purified to homogeneity. Both enzymes phosphorylated all three of the substrates tested (FKKSFKL-NH2 peptide substrate, histone H1, protamine), the bat enzyme having significantly higher K(m) values than rat PKC for both peptide and histone. Both enzymes required phospholipids and Ca2+ for activation with rat brain PKC depending almost exclusively on phosphatidylserine. Bat PKC, however, made use of other phospholipids and showed relative activities of 100:81:33:42 for euthermic PKC and 100:91:45:35 for hibernator PKC with phosphatidylserine, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine (each at 50 microM), respectively. Activation of bat PKC by phosphatidylserine was temperature sensitive, being 3.5-fold at 4 degrees C (hibernating body temperature) compared with 14-18-fold at 33 degrees C (near euthermic body temperature). Arrhenius plots for bat brain PKC showed a sharp break below 10 degrees C; activation energies below this temperature were 11.5- and 5.2-fold greater than at higher temperatures for the enzyme from hibernating versus euthermic animals. By contrast, plots for the rat enzyme were linear over the range 0-42 degrees C. The data suggest that a sharp suppression of PKC activity by several mechanisms (reduced total activity, low temperature effects on activity and sensitivity to phospholipids) may be important to overall metabolic rate suppression during hibernation.

Animals↗

PubMed1997

Organic solutes in freezing tolerance.

The accumulation of high levels of low-molecular-weight solutes (polyhydric alcohols, saccharides) provides cryoprotection to freeze-tolerant animals by minimizing, via colligative effects, the percentage of body water converted to extracellular ice and the extent of cell volume reduction. Many freeze-tolerant insects accumulate high levels of polyols during autumn cold hardening, whereas freeze-tolerant frogs respond to ice formation in peripheral tissues by synthesizing large amounts of glucose in the liver and rapidly distributing the sugar throughout the body. Seasonal patterns of enzymatic change occur in cold-hardy insects; activities associated with cryoprotectant synthesis rise in the fall, whereas enzymes associated with polyol degradation dominate in the spring. Enzyme profiles also revealed the route of glycerol degradation via polyol dehydrogenase and the novel enzyme, glyceraldehyde kinase. Proton magnetic resonance imaging of freezing and thawing in whole frogs showed a new adaptive effect of the very high glucose levels in core organs; during thawing, organs such as liver and heart melted first, allowing recovery of their vital functions to begin while the rest of the frog thawed. New studies have examined signal transduction in the stimulation of glucose production by wood frog liver, revealing the key role of beta-adrenergic receptors and cAMP-mediated activation of glycogenolysis for cryoprotectant synthesis. The seasonal elevation of plasma membrane glucose transporters was also shown to be key to cryoprotectant distribution during freezing. Other new work has shown that frog freeze tolerance probably grew out of preexisting mechanisms of amphibian dehydration tolerance and that both freeze-tolerant and -intolerant frogs show a hyperglycemic response to desiccation at 5 degrees C.

Adaptation, Physiological↗

PubMed1997

Glycolytic controls in estivation and anoxia: a comparison of metabolic arrest in land and marine molluscs.

Facultative metabolic rate depression is the common adaptive strategy underlying various animal mechanisms for surviving harsh environmental conditions. This strategy is common among molluscs, enabling animals to survive over days or even months in the absence of oxygen or under extremely dry conditions. The large reductions in metabolic rate during estivation and anoxia can translate into considerable energy savings when dormant animals are compared to active animals. A complex metabolic coordination is required during the transition into the dormant state to maintain cellular homeostasis and involves both energy-consuming and energy-producing pathways. With regard to energy-producing pathways, several different mechanisms have been identified that participate in controlling flux. One such mechanism, enzyme phosphorylation, can have a wide-ranging effect. For example, phosphorylated enzymes exhibit altered substrate, activator, and inhibitor affinities. This effect may be magnified by changes in the concentrations of allosteric effectors, such as fructose 2,6-bisphosphate, that occur during hypometabolic states. Changes in fructose 2,6-bisphosphate are related to changes in enzyme phosphorylation through changes in the relative activity of phosphofructokinase-2. Alterations in glycolytic enzyme binding can also be brought about through changes in enzyme phosphorylation. The present review focuses on identifying hypometabolism-related changes in enzyme phosphorylation as well as characterizing the mechanisms involved in mediating these phosphorylation events.

Animals↗

PubMed1997

Metabolic regulation in mammalian hibernation: enzyme and protein adaptations.

Mammalian hibernation requires specific regulatory controls on metabolism to coordinate entry, maintenance, and arousal stages, as well as adjustments to many metabolic functions to support long-term dormancy. Several mechanisms of metabolic regulation are involved in potentiating survival. One of these is the reversible phosphorylation of regulatory enzymes, including glycogen phosphorylase, phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. In particular, the sharp suppression of pyruvate dehydrogenase during hibernation shows the importance of control over mitochondrial oxidative metabolism for reducing metabolic rate. Fine control over specific enzymes also occurs via differential temperature effects on kinetic and allosteric properties. Analysis of temperature effects on the properties of pyruvate kinase, fructose-1,6-bisphosphatase, creatine kinase, and citrate synthase from ground squirrel or bat tissues shows a range of responses, some that would reduce enzyme activity in the hibernating state and some that would promote temperature-insensitive enzyme function. Reduced tissue phosphagen and adenylate levels, but not energy charge, may also contribute to overall metabolic suppression. New research is exploring the role of transcriptional and translational controls in hibernation via several approaches. For example, immunoblotting with antibodies to heat shock proteins (hsp 70 family) revealed the presence of constitutive hsc 70 in bat tissues but levels of the protein did not change between euthermic and hibernating states and neither the inducible hsp 70 nor the glucose-responsive protein grp 78 appeared during hibernation.

Adaptation, Physiological↗

PubMed1997

Antioxidant systems and anoxia tolerance in a freshwater turtle Trachemys scripta elegans.

The effects of anoxic submergence (20 h at 5 degrees C) and subsequent 24 h aerobic recovery on the antioxidant systems of six organs were examined in freshwater turtles, Trachemys scripta elegans. Both xanthine oxidase and xanthine dehydrogenase were detected in turtle tissues with xanthine oxidase composing 36-75% of the total activity. Turtle organs displayed high constitutive activities of catalase (CAT), superoxide dismutase (SOD), and alkyl hydroperoxide reductase (AHR). Measurements of lipid peroxidation damage products (conjugated dienes, lipid hydroperoxides, thiobarbituric acid reactive substances) showed minimal changes during anoxia or recovery suggesting that natural anoxic-aerobic transitions occur without the free radical damage that is seen during ischemia-reperfusion in mammals. Anoxia exposure led to selected decreases in enzyme activities in organs, consistent with a reduced potential for oxidative damage during anoxia: SOD decreased in liver by 30%, CAT decreased in heart by 31%, CAT and total glutathione peroxidase (GPOX) decreased in kidney (by 68 and 41%), and CAT and SOD decreased in brain (by 80 and 15%). AHR, however, increased 2 and 3.5 fold during anoxia in heart and kidney respectively. Most anoxia-induced changes were reversed during aerobic recovery although brain enzyme activities remained suppressed. Some specific changes occurred during the recovery period: SOD increased from controls in heart by 45%, AHR increased to 200 and 168% of control values in red and white muscle respectively, and total GPOX decreased from controls in heart and white muscle by 75 and 77% respectively. The results show that biochemical adaptation for natural anoxia tolerance in turtles includes well-developed antioxidant defenses that minimize or prevent damage by reactive oxygen species during the reoxygenation of organs after anoxic submergence.

Acclimatization↗

PubMed1997

Glycolytic enzyme binding in Otala lactea hepatopancreas: effect of taxol, colchicine and cytochalasin B and D on the in vivo enzyme distribution.

The effect of cytoskeleton modulators on glycolytic enzyme binding was examined in the hepatopancreas of Otala lactea in an attempt to identify potential cellular binding sites. Binding was followed by measuring phosphofructokinase (PFK), aldolase (ALD), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) distribution between low speed pellets (12,000 xg), high speed pellets (100,000 xg) and high speed supernatants. Taxol (which stabilizes microtubules), colchicine (which destabilizes microtubules) and cytochalasin B and D (which destabilize F-actin filaments) were added to the homogenate prior to centrifugation. Addition of taxol increased the amount of PFK associated with the high speed pellet. Cytochalasin B and D reduced the binding of PFK and PK to the low speed pellet. ALD and GAPDH binding were unaffected by any treatment. Lowering the pH of the crude homogenate increased PFK binding to the low speed pellet by 33%. This effect could be reversed by addition of cytochalasin B and D suggesting that pH influences the PFK-F-actin interaction in vivo. The differential binding response of PFK, PK, ALD and GAPDH to added effectors suggests that, in the cell, PFK and PK are bound to different subcellular structural elements than are ALD and GAPDH.

Alkaloids↗

PubMed1997

Unusual AMP-deaminase solubilization from teleost fish white muscle.

A previous study described an unusual influence of neutral salts on the behavior of trout muscle AMP-deaminase (AMPD) in its interactions with subcellular particulate matter (Lushchak and Storey 1994, Fish Physiol. Biochem. 13: 356-368). The present study shows that this behavior is also shared by the muscle enzyme of two other fish species, sea scorpion (Scorpaena porcus) and corb (Sciena umbra), indicating that this describes a principle for AMPD interaction with cellular particulate material. AMPD binding to particulate matter increased with increasing KCl concentration through the physiological range (100-200 mM), but at higher salt concentrations the amount of bound enzyme was reduced. The pattern of binding was not influenced by hydrophobic interactions since addition of the nonionic detergents, Triton X-100 or Tween-80, did not alter the distribution of bound versus free enzyme although both detergents, at low concentrations, enhanced enzyme maximal activity. AMPD binding to particulate matter was also influenced by pH, the amount of free enzyme rising by nearly 3-fold as pH fell within the physiological range from 7.5 to 6.5. It is concluded that neither electrostatic nor hydrophobic forces alone can account for the unusual solubilization of AMPD from fish muscle and it is possible that the effect is also related to ion-induced conformational changes in the structure of AMPD and/or of the myosin to which the enzyme binds.

AMP Deaminase↗

PubMed1997

A novel RNA species from the turtle mitochondrial genome: induction and regulation of transcription and processing under anoxic and freezing stresses.

The present study identifies a previously cloned cDNA, pBTaR914, as homologous to the mitochondrial WANCY (tryptophan, alanine, asparagine, cysteine, and tyrosine) tRNA gene cluster. This cDNA clone has a 304-bp sequence and its homologue, pBTaR09, has a 158-bp sequence with a long poly(A)+ tail (more than 60 adenosines). RNA blotting analysis using pBTaR914 probe against the total RNA from the tissues of adult and hatchling turtles revealed five bands: 540, 1800, 2200, 3200, and 3900 nucleotides (nt). The 540-nt transcript is considered to be an intact mtRNA unit from a novel mtDNA gene designated WANCYHP that overlaps the WANCY tRNA gene cluster region. This transcript was highly induced by both anoxic and freezing stresses in turtle heart. The other transcripts are considered to be the processed intermediates of mtRNA transcripts with WANCYHP sequence. All these transcripts were differentially regulated by anoxia and freezing in different organs. The data suggest that mtRNA processing is sensitive to regulation by external stresses, oxygen deprivation, and freezing. Furthermore, the fact that the WANCYHP transcript is highly induced during anoxic exposure suggests that it may play an important role in the regulation of mitochondrial activities to coordinate the physiological adaptation to anoxia.

Adaptation, Physiological↗

PubMed1997

Freezing-induced genes in wood frog (Rana sylvatica): fibrinogen upregulation by freezing and dehydration.

Differential screening of a cDNA library produced from liver of the freeze-tolerant wood frog, Rana sylvatica, was used to search for freezing-induced genes. Five freezing-responsive cDNA clones representing different genes were isolated when approximately 80,000 plaques of a cDNA library, prepared from liver of frozen frogs (24 h at -2.5 degrees C), were screened with 32P-labeled total cDNA probes from control (5 degrees C) versus freezing-exposed frogs. Two clones, pBfFR45 and pBfFR04, are reported here in detail and were found to be homologous with the genes for the alpha- and gamma-subunits of fibrinogen, respectively. The clone pBfFR45 carried a 2,305-hp cDNA sequence that was of bipartite structure, containing two open reading frames (ORFs). The first ORF potentially encoded a 332-residue polypeptide, covering a partial sequence of the NH2-terminal region of the alpha-chain. The second ORF encoded a 247-amino acid sequence, covering the whole COOH-terminal region of the alpha-chain; this was highly homologous to the FASORF (fibrinogen-alpha second ORF) of chicken alpha-fibrinogen and the extended alpha-chain of the human protein. Under control (5 degrees C) conditions, moderate levels of fibrinogen alpha- and gamma-transcripts were exclusively found in liver. When frogs were given survivable freezing exposures, levels of these transcripts in liver were highly induced. Transcription of these genes was also elevated in gut and lung during freezing, but mRNA levels in these tissues were lower than in liver. A time course assay confirmed that the transcript levels of both alpha- and gamma-subunit genes were dramatically elevated within the early hours of freezing and reached a maximum threefold increase over control levels after 8 h of freezing exposure. Two other physiological stresses, whole body dehydration and anoxia exposure, mimic individual elements of freezing stress in wood frogs. Northern blot hybridization analysis showed that the expression of both the alpha- and gamma-genes was also upregulated in response to dehydration in vivo (20% of total body water lost), but both were completely inhibited by anoxia exposure.

Amino Acid Sequence↗

PubMed1997

Glutathione systems and anoxia tolerance in turtles.

Effects of anoxic submergence (20 h at 5 degrees C) and subsequent 24 h aerobic recovery on glutathione levels and the activities of glutathione-related enzymes were examined in six tissues of Trachemys scripta elegans. Anoxia exposure resulted in tissue-specific changes in enzyme maximal activities, the most dramatic being suppression of gamma-glutamyl transpeptidase (gamma-GTPase) activity in anoxic kidney to only 2% of control. Anoxia exposure also caused significant decreases in activities of liver and heart glutathione-S-transferase (GST) (by 25 and 42%), heart glutathione reductase (GR) (by 67%), liver gamma-GTPase (by 71%), and red muscle glutaredoxin (GRN) (by 56%). By contrast, anoxia exposure increased the activities of GR in liver and red muscle (by 52 and 80%), glutathione synthetase (GS) in white muscle (by 300%), and GRN in white muscle (by 400%). During aerobic recovery after anoxia, GST activity decreased in red muscle, kidney, and brain (by 72, 56, and 39%); GR decreased in liver and red muscle (by 52 and 80%); and GRN fell in red muscle (by 56%). Other activities rose during recovery: GR in heart (by 64%), GS in heart and brain (by 200%), and gamma-GTPase in brain (by 63%). Tissue pools of total glutathione were high in comparison with other ectotherms. Levels decreased during anoxia in four organs to 49-67% of control values. During aerobic recovery the reduced glutathione-to-oxidized glutathione ratio (GSH/GSSG) increased in heart, kidney, and brain, indicating that oxidative stress did not occur in these organs. Rather than maintaining high levels of glutathione in tissues to prevent oxidative stress during aerobic recovery, turtles sustain high GSH/GSSG by regulating the activities of glutathione-using enzymes.

Acclimatization↗

PubMed1996

Anoxia-induced gene expression in turtle heart. Upregulation of mitochondrial genes for NADH-ubiquinone oxidoreductase subunit 5 and cytochrome c oxidase subunit 1.

A cDNA library constructed from heart of anoxia-exposed adult turtles (Trachemys scripta elegans) was differentially screened with 32P-labeled single-stranded cDNA probes from heart of control versus anoxic animals to clone genes induced by anoxia stress. Four cDNA clones, pBTaR20, pBTaR34, pBTaR63 and pBTaR914 were obtained and confirmed to be upregulated in response to anoxic submergence (20 h in N2-bubbled water at 7 degrees C). Two clones, pBTaR20 and pBTaR63, were characterized by sequence analysis and in vivo expression. The clone pBTaR20 had a 1597-bp cDNA sequence and pBTaR63 contained a 1837-bp sequence. The pBTaR20 sequence contained a single open reading frame that was very close to full length and could potentially encode a polypeptide with 508 amino acids. The deduced polypeptide sequence showed approximately 83% of the residues identical with the sequence of cytochrome c oxidase subunit 1 (CO1) that is encoded by a mtDNA gene Cox1. The clone pBTaR63 contained a single potentially full-length open reading frame that could encode a polypeptide of 591 residues. This was similar to another mitochondrial protein, NADH-ubiquinone oxidoreductase subunit 5 (ND5), which is encoded by mtDNA gene Nad5. Analysis of the time course of expression of Cox1 and Nad5 by northern hybridization analysis showed that mRNA transcripts for both accumulated rapidly (within 1 h) in response to anoxia exposure. Both showed similar increases in their transcript content after 1 h of anoxia but with longer anoxia exposures (5 or 20 h) Nad5 mRNA levels remained high whereas Cox1 mRNA content declined somewhat. Northern-blot hybridization also revealed differential expression of these two genes in five other organs of T. s. elegans during anoxia exposure (brain, kidney, liver, red and white skeletal muscle), with a particularly large increase in mRNA transcript levels of both genes in anoxic red muscle. Organ-specific analysis of these genes in a freeze-tolerant turtle species (Chrysemys picta marginata) also showed that differential expression of these genes occurred in response to the ischemia induced by plasma freezing.

Amino Acid Sequence↗

PubMed1996

Protein kinase involvement in land snail aestivation and anoxia: protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism.

In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors affecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP3 concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg2+ ion but was inhibited at Mg2+ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I50 = 39 mM) was much lower than that of the neutral salts (I50 approximately 240 mM). The enzyme exhibited a broad pH optimum between pH 6.5-8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Q10 value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.

Animals↗

PubMed1996

Characterization of gamma-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica.

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of gamma-glutamyltranspeptidase in the liver. On exposure to -2.5 degrees C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4 degrees C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at -2.5 degrees C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at -2.5 degrees C on cooling; a 10.5-fold increase after 12 h at -2.5 degrees C on the initiation of freezing; and a 22-fold increase after 24 h at -2.5 degrees C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1.8-fold in the thawed state, while plasma ketone levels were increased 1.8-fold in the frozen state and 1.5-fold in the thawed state. Plasma total T3 levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of gamma-glutamyltrans-peptidase was found to be elevated at all stages of the freeze-thaw process. After 1, 12 and 24 h at -2.5 degrees C, activities were increased 2.5-, 2.3-, 2.4-fold respectively in the homogenates and 2.5-, 2.2-, 2.4-fold respectively in the plasma membranes. After thawing, activities were still increased 1.9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of gamma-glutamyltranspeptidase was increased 1.4-fold after 1 h at -2.5 degrees C after which it returned to normal. The role of thyroid hormone in producing the increase in gamma-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.

Adaptation, Physiological↗

PubMed1996

Metabolic adaptations supporting anoxia tolerance in reptiles: recent advances.

Animal survival during severe hypoxia and/or anoxia is enhanced by a variety of biochemical adaptations including adaptations of fermentative pathways of energy production and, most importantly, the ability to sharply reduce metabolic rate by 5-20 fold and enter a hypometabolic state. The biochemical regulation of metabolic arrest is proving to have common molecular principles that extend across phylogenetic lines and that are conserved in different types of arrested states (not only anaerobiosis but also estivation, hibernation, etc.). Our new studies with anoxia-tolerant vertebrates have identified a variety of regulatory mechanisms involved in both metabolic rate depression and in the aerobic recovery process using as models the freshwater turtle Trachemys scripta elegans and garter snakes Thamnophis sirtalis parietalis. Mechanisms include: 1) post-translational modification of cellular and functional proteins by reversible phosphorylation and changes in protein kinase (PKA, PKC) and/or phosphatase activities to regulate this, 2) reversible enzyme binding associations with subcellular structural elements, 3) differential gene expression and/or mRNA translation producing new mRNA variants and new protein products, 4) changes in protease activity, particularly the multicatalytic proteinase complex, and 5) both constitutive and anoxia-induced modifications to cellular antioxidant systems to deal with oxidative stress during the anoxic-aerobic transition of recovery.

Adaptation, Physiological↗