Biomedical subjects

K B Storey

Publications and source records attributed to K B Storey.

At least 37 records · Page 2Linked to original sources

PubMed2001

Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel.

The effects of short-term burst (5 min at 1.8 m/s) swimming and long-term cruiser (60 min at 1.2 m/s) swimming on maximal enzyme activities and enzyme distribution between free and bound states were assessed for nine glycolytic and associated enzymes in tissues of horse mackerel, Trachurus mediterraneus ponticus. The effects of exercise were greatest in white muscle. The activities of phosphofructokinase (PFK), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase), and phosphoglucomutase (PGM) all decreased to 47, 37, 37 and 67%, respectively, during 60-min exercise and all enzymes except phosphoglucoisomerase (PGI) and PGM showed a change in the extent of binding to subcellular particulate fractions during exercise. In red muscle, exercise affected the activities of PGI, FBPase, PFK, and lactate dehydrogenase (LDH) and altered percent binding of only PK and LDH. In liver, exercise increased the PK activity 2.3-fold and reduced PGI 1.7-fold only after 5 min of exercise but altered the percent binding of seven enzymes. Fewer effects were seen in brain, with changes in the activities of aldolase and PGM and in percent binding of hexokinase, PFK and PK. Changes in enzyme activities and in binding interactions with subcellular particulate matter appear to support the altered demands of tissue energy metabolism during exercise.

Animals↗

PubMed2001

Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: evidence for two distinct enzyme forms.

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37 degrees C body temperature) and hibernating (torpid, 5 degrees C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5+/-2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335+/-5 kDa for euthermic and 320+/-5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.

Animals↗

PubMed2000

Mitogen-activated protein kinases and anoxia tolerance in turtles.

The response of two vertebrate mitogen-activated protein kinase (MAPK) family members, the extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs), to anoxia exposure in vivo was examined in organs (liver, heart, kidney, brain, spleen) of the anoxia-tolerant adult turtle, Trachemys scripta elegans. ERKs activities rose during anoxia only in spleen (a 2.8-fold increase). JNK activity showed a significant increase only in liver (4-fold increase) after 5 hr of anoxic submergence but declined thereafter. Levels of the transcription factor c-Fos were strongly suppressed in liver, heart, and kidney of anoxia-exposed turtles, whereas levels increased 2-fold in anoxic brain. The effect of anoxia on c-Myc was organ-specific and variable with 2- and 1.5-fold increases in protein expression in kidney and brain, respectively, and a 60% decrease in anoxic spleen. These results for an anoxia-tolerant animal suggest the potential importance of the MAPKs and of the immediate-early genes (c-fos, c-myc) in mediating adaptive responses to oxygen deprivation.

Adaptation, Biological↗

PubMed2000

Induction of synthesis of an antimicrobial peptide in the skin of the freeze-tolerant frog, Rana sylvatica, in response to environmental stimuli.

An extract of skin taken from specimens of the freeze-tolerant wood frog, Rana sylvatica, that were collected from cold (<7 degrees C) ponds and maintained at 5 degrees C lacked detectable antimicrobial activity. In contrast, an extract of skin taken from specimens maintained at 30 degrees C for 3 weeks under laboratory conditions contained a high concentration (approximately 4 nmol/g) of a single antimicrobial peptide of the brevinin-1 family (FLPVVAGLAAKVLPSIICAVTKKC). The peptide inhibited growth of Escherichia coli (minimum inhibitory concentration 45 microM) and Staphylococcus aureus (minimum inhibitory concentration 7 microM). The data suggest that synthesis of the peptide is induced when the animal is in an environment that promotes the growth of microorganisms consistent with a role in the animal's defense strategy.

Adaptation, Physiological↗

PubMed2000

Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus.

AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge.

AMP Deaminase↗

PubMed2000

Hepatic changes in the freeze-tolerant turtle Chrysemys picta marginata in response to freezing and thawing.

Select hepatic changes in the freeze-tolerant hatchling turtle, Chrysemys picta marginata, were studied in response to freezing at -2.5 degrees C and thawing. Upon freezing, a small, selective increase in the liver weight with no increase in body weight was seen suggestive of an hepatic capacitance response. In all turtles studies, lobular differences in the hepatic content of glycogen were evident: the smaller lobe contained twice as much glycogen as the larger lobe. The response to freezing and thawing was comparable. Total hepatic glycogen levels of turtles were reduced approximately 60 per cent from control levels in the frozen state and recovered to >80 per cent of control levels in the thawed state. Compared to the control state, turtle blood glucose levels were: unchanged after 12 h in the cool state; reduced 28 per cent after 24 h and increased two-fold after 48 h in the frozen state; and increased 4.5-fold in the thawed state. Thus, changes in hepatic glycogen metabolism occur without large changes in blood glucose levels. In turtle liver plasma membranes, the hepatic alpha(1)-adrenergic receptor was barely detectable and did not change. The beta(2)-adrenergic receptor was expressed at high levels and, compared to control levels, was: unchanged after 12 h in the cool state; reduced 20 per cent after 24 h and 40 per cent after 48 h in the frozen state. On thawing, this receptor was 50 per cent of control levels. While catecholamines working through the beta(2)-adrenergic receptor may effect early hepatic glycogen breakdown in response to freezing, other factors must be involved to complete the process. The plasma membrane-bound enzyme gamma-glutamyltranspeptidase displayed a different pattern of changes indicative of selective modulation: it was increased 2.7-fold over control levels in the cool state; unchanged in the frozen state; and increased 1.8-fold in the thawed state. The activity of the kidney enzyme was decreased in the cool state and slightly increased in the frozen and thawed states emphasizing the tissue-specific nature of the changes in the activity of gamma-glutamyltranspeptidase in response to freezing and thawing. The similarities and differences of the hepatic changes in response to freezing and thawing in the freeze-tolerant hatchling turtle to those we have previously reported for the freeze-tolerant frog are discussed.

Animals↗

PubMed2000

Purification and characterization of protein kinase A from liver of the freeze-tolerant wood frog: role in glycogenolysis during freezing.

Freeze tolerance by various amphibians includes cryoprotectant production in the form of glucose. Activation of the catalytic subunit of liver cAMP-dependent protein kinase (PKAc) facilitates activation of glycogenolysis, a critical biochemical process necessary for production of glucose. Here, we purified PKAc from Rana sylvatica liver to determine the extent to which cold temperature, which stimulates cryoprotectant production, affected PKAc activity and function. PKAc was purified to greater than 95% homogeneity, with a final specific activity of 71 nmol phosphate transferred/min/mg protein. The molecular weight of frog liver PKAc was 47.6 +/- 1.1 kDa and K(m) values for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 microM at 22 degrees C, respectively. K(m) values for both substrates dropped significantly at 5 degrees C. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA(i), H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at low temperature. The effect of temperature on enzyme activity indicated a conformational change in PKAc at 10 +/- 2 degrees C, with calculated activation energies of 51 +/- 4 kJ/mol at temperatures above 10 degrees C and 110 +/- 9 kJ/mol below 10 degrees C. PKAc in wood frog liver plays a crucial role in mediating the freeze-induced glycogenolysis that is responsible for the production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on enzyme function, increased substrate affinity and reduced ion inhibition, appear to be central to this role.

Acclimatization↗

PubMed2000

Gene up-regulation in heart during mammalian hibernation.

A cDNA library prepared from heart of hibernating golden-mantled ground squirrels, Spermophilus lateralis, was differentially screened to clone genes that were up-regulated during hibernation. Two differentially expressed clones were found after three rounds of screening and were confirmed as up-regulated by Northern blotting. Clone Ang6 encoded a polypeptide with 116 amino acids that was identified as the ventricular isoform of myosin light chain 1 (MLC1(v)). Clone Ang19 coded for 274 amino acid residues of the mitochondrially encoded protein subunit 2 of NADH-ubiquinone oxidoreductase (ND2). Both proteins showed high amino acid sequence identity with their human counterparts, 97.5% for MLC1(v) and 66% for ND2. Northern blot hybridization revealed differential expression of these genes in multiple organs during hibernation. Transcript levels of both were approximately twofold higher in heart and three- to fourfold higher in skeletal muscle of hibernating, versus euthermic, animals. ND2 was also up-regulated in hibernator liver. Hibernation-induced up-regulation of MLC1(v) suggests that a restructuring of myosin subunit composition could contribute to changes in muscle contractility needed for hypothermic function, whereas changes in ND subunit composition may affect the function of the electron transport chain during hibernation.

Amino Acid Sequence↗

PubMed2000

Seasonal change and prolonged anoxia affect the kinetic properties of phosphofructokinase and pyruvate kinase in oysters.

The effects of seasonal change, November versus July, and prolonged anoxia (96 h under N2 gas) on the properties of phosphofructokinase and pyruvate kinase from five tissues (gill, mantle, hepatopancreas, phasic adductor, catch adductor) of the oyster, Crassostrea virginica were investigated. Both enzymes showed tissue-specific and season-specific changes in kinetic properties; for pyruvate kinase this correlated with seasonal differences in enzyme elution patterns on hydroxylapatite chromatography. Kinetic properties of both enzymes in winter were consistent with primarily catabolic roles in glycolysis with responsiveness to cellular energy demands, whereas in summer these enzymes may be more closely regulated with respect to the biosynthetic and gluconeogenic functions of the tissues. Anoxia-induced changes in phosphofructokinase properties were relatively minor but anoxia stimulated changes in pyruvate kinase properties and elution profiles on hydroxylapatite in all tissues except mantle, with much greater effects seen for the enzyme from winter versus summer animals. For example, anoxia-induced changes in pyruvate kinase from winter gill included a fourfold rise in the substrate affinity constant for phosphoenolpyruvate, a sevenfold increase in the concentration of fructose-1,6-bisphosphate needed to activate the enzyme by 50%, and a 50% decrease in the concentration of L-alanine that inhibits activity by 50%. Changes in pyruvate kinase kinetics and hydroxylapatite elution patterns during prolonged anoxia are consistent with covalent modification of pyruvate kinase but contrary to results for many other mollusc species, anoxia exposure appears to induce a dephosphorylation of the enzyme.

Adaptation, Physiological↗

PubMed2000

The muscle fatty acid binding protein of spadefoot toad (Scaphiopus couchii).

Fatty acid binding protein was purified from skeletal muscle of the spadefoot toad (Scaphiopus couchii), an estivating species. While estivating, this animal relies on the fatty acid oxidation for energy. Hence we were interested in the behaviour of fatty acid binding protein under conditions of elevated urea (up to 200 mM) and potassium chloride such as exist during estivation. Also we examined whether there were interactions between glycolytic intermediates and the binding ability of the protein. The amount of bound fatty acid (a fluorescence assay using cis-parinarate) was not affected (P < 0.05) by glucose, fructose 6-phosphate or phosphoenolpyruvate at physiological concentrations. By contrast, glucose 6-phosphate increased the amount of bound cis-parinarate but the apparent dissociation constant was not different from the control. Fructose 1,6-bisphosphate but not fructose 2,6-phosphate decreased cis-parinarate binding by 40%, commensurate with doubling the apparent dissociation constant (1.15-2.62 microM). Urea, guanidinium and trimethylamine N-oxide at 200 mM increased cis-parinarate binding 60% over controls. Urea (1 M) and KCl (200 mM) did not affect cis-parinarate binding compared to controls. The interaction of this fatty acid transporter with fructose 1,6-bisphosphate is discussed in terms of reciprocal interaction with phosphofructokinase since fatty acid is also an inhibitor of phosphofructokinase.

Animals↗

PubMed2000

Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog.

The responses of mitogen-activated protein kinase (MAPK) family members, including ERK (extracellular signal-regulated kinase), JNK (c-Jun NH2-terminal kinase), and p38, in the metabolic responses to whole animal freezing (up to 24 h frozen at -2.5 degrees C) and thawing (up to 4 h at 5 degrees C after a 12 h freeze) were examined in four organs (liver, kidney, heart, brain) of the freeze-tolerant wood frog Rana sylvatica. Levels of the active phosphorylated form of p38 increased within 20 min as an early response to freezing in liver and kidney but rose later (after 12 h) in heart. Both JNK and p38 were activated during thawing in liver, kidney and heart with temporally-distinct patterns in each organ. The only MAPK response to freeze/thaw in frog brain was a transient elevation of p38 after 90 min thawing. ERK activity did not respond to freeze/thaw in any organ. The levels of c-Fos increased during freezing in kidney and brain whereas c-Jun was unaffected by freeze/thaw. Organ-specific responses by MAPKs, particularly p38, suggest that these may have roles in regulating metabolic or gene expression responses that may be adaptive in dealing with freezing stress or metabolic recovery during thawing.

Acclimatization↗

PubMed1999

Gene expression during estivation in spadefoot toads, Scaphiopus couchii: upregulation of riboflavin binding protein in liver.

A cDNA library constructed from liver of 2-month estivating female spadefoot toads, Scaphiopus couchii, was differentially screened to reveal genes that were induced or upregulated during estivation. After two rounds of screening a clone was isolated that showed 60% higher expression in liver of estivating, versus control, toads. The clone possessed a 1.0 kb insert which annealed to a single 0.7 kb band on Northern blots. Sequencing revealed a 1053 nucleotide full-length cDNA; the largest potential open reading frame was 708 nucleotides which encoded a protein of 235 amino acids. A homology search in Genbank indicated that the protein was a riboflavin binding protein (RfBP), a monomeric phosphoglycoprotein produced by the liver of female birds, reptiles, and mammals that functions to bind plasma riboflavin and load the vitamin into eggs or fetus. To our knowledge, this is the first demonstration that RfBP is also present in amphibians. Toad RfBP showed 50% of residues identical with the chicken or turtle liver proteins and many essential structural features were conserved in the toad protein including 18 cysteine residues, two asparagine glycosylation sites, and 6 tryptophan residues. However, a region with eight phosphoserines in the chicken or turtle proteins that functions in RfBP binding to the oocyte membrane contained only three serine residues in toad RfBP, suggesting that recognition and binding to oocyte receptors must be different in toads. Northern hybridization showed that toad RfBP was largely liver-specific; no mRNA transcripts were detected in brain, gut, heart, or kidney but low message levels occurred in hind leg skeletal muscle of estivating, but not control, toads. Upregulation of RfBP in liver of estivating toads may be linked with maturation of eggs in preparation for the explosive breeding that occurs immediately upon emergence from estivation but might also have a role for the adult in "caching" riboflavin to maintain an endogenous vitamin pool over the 9-10 months of each year that toads are dormant.

Amino Acid Sequence↗

PubMed1999

Regulation of ground squirrel Na+K+-ATPase activity by reversible phosphorylation during hibernation.

Maintenance of skeletal muscle energy status during hibernation in ground squirrels, Spermophilus lateralis, was accompanied by a decrease in Na+K+-ATPase pump activity. Energy charge was maintained (0.89) during hibernation at the expense of total adenylates (decreased by 41%). Muscle Na+K+-ATPase activity was 9.1 U/mg protein in euthermic controls but decreased by 60% during hibernation. Enzyme activity was similarly suppressed in vitro when extracts of control muscle were incubated with ATP plus second messengers of protein kinases A, G or C whereas stimulation of protein phosphatases in muscle extracts from hibernators increased Na+K+-ATPase activity. Additional studies confirmed that suppression and reactivation of the enzyme in euthermic muscle extracts can be achieved with protein kinase A and alkaline phosphatase treatments, respectively, and indicated that phosphorylation changes the ATP dependency of the enzyme. Thus, hibernation-induced suppression of Na+K+-ATPase activity in muscle and other organs of ground squirrels, which is a key part of the overall suppression of metabolic rate that constitutes torpor, appears to be regulated via reversible protein phosphorylation.

Adenine Nucleotides↗

PubMed1999

Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. I. Experimental measures.

In this study, two methods are used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a "two-step" low-temperature microscopy (equilibrium and dynamic) freezing method and the second is a differential scanning calorimeter (DSC) technique. These two techniques were used to study (i) the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic Krogh cylinder dimensions; (ii) the "equilibrium" (infinitely slow) cooling behavior and the osmotically inactive cell volume (V(b)) of R. sylvatica liver cells; and (iii) the dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) at a cooling rate of 5 degrees C/min. Stereological analysis of the slam frozen (>1000 degrees C/min) micrographs led to the determination that 74% of the liver tissue in control frogs was cellular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Model 1) yielded distance between adjacent sinusoid centers, DeltaX = 64 microm; original sinusoid (vascular) radius, r(vo) = 18.4 microm; and length of the Krogh cylinder, L = 0.71 microm (based on an isolated frog hepatocyte cell diameter of 16 microm). A significant observation was that approximately 24% of the frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell-cell contact in the frog liver architecture a modified Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2) a second radius, r(2) = 28.7 microm, was defined (in addition to the original sinusoid radius, r(vo) = 18.4 microm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). By plotting the two-step equilibrium cooling results on a Boyle-van't Hoff plot, the osmotically inactive cell volume, V(b) was obtained as 0.4. V(o) (where V(o) is the isotonic cell volume). The two-step dynamic micrographs and the heat release measurements from the DSC were used to obtain water transport data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min.

Animals↗

PubMed1999

Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. II. Mathematical modeling.

The "two-step" low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L(pg) = 1.76 microm/min-atm and E(L(p)) = 75.5 kcal/mol for control liver cells and L(pg)[cpa] = 1.18 microm/min-atm and E(L(p))[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5 degrees C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (>/=5 degrees C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5 degrees C/min. A modified Krogh model (Model 2) was constructed to account for the cell-cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1-2 degrees C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (>/=-8 degrees C).

Animals↗

PubMed1999

Freeze-induced alterations of translatable mRNA populations in wood frog organs.

To investigate the roles that gene expression and new protein synthesis play in freezing survival by the wood frog, Rana sylvatica, we compared the in vitro translation products made from mRNA isolated from six tissues (liver, brain, heart, muscle, kidney, gut) of control (5 degrees C), frozen (24 h at -2.5 degrees C), and thawed (24 h at 5 degrees C after 24 h frozen) frogs. [(35)S]Methionine-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and located by fluorography. Results indicated specific changes in the translatable populations of mRNA in tissues of freezing-exposed frogs that were largely reversed upon thawing. Differential protein expression was greatest in the comparison of liver from control versus frozen frogs with proteins ranging from 45 to 14.8 kDa identified as enhanced or unique to the frozen state. One unique protein appeared in skeletal muscle (116 kDa) of freeze-exposed frogs while another (52.5 kDa) was enhanced. Analysis of brain and heart each revealed the presence of one protein unique to the frozen state in each (58.9 and 5.9 kDa, respectively) whereas no change in the pattern of in vitro translation products was seen in gut (stomach + intestine combined) or kidney between the three experimental states. These freeze-induced alterations in the populations of translatable mRNA suggest that changes in the complement of specific proteins underlie various adaptive responses that contribute to the freezing survival of this amphibian.

Adaptation, Biological↗

PubMed1999

Temperature regulation of glucose metabolism in red blood cells of the freeze-tolerant wood frog.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.

Animals↗

PubMed1999

Discordant responses of mitogen-activated protein kinases to anoxia and freezing exposures in hatchling turtles.

The role of two vertebrate mitogen-activated protein kinases (MAPKs) in mediating responses to in vivo anoxia or freezing exposures was examined in four organs (liver, heart, kidney and brain) of hatchling red-eared turtles, Trachemys scripta elegans, which are naturally tolerant of these stresses. The extracellular signal-regulated kinases were not stress-activated except in brain of frozen turtles. The c-Jun NH2-terminal kinases (JNKs) were transiently activated by anoxia exposure in all four organs (after 1 h in brain or 5 h in other organs) but activity was suppressed during freezing except in brain which showed a transient activation of JNK after 1 h. Changes in the concentrations of the transcription factors, c-Fos and c-Myc, were also stress- and organ-specific. The patterns of MAPK activation in a stress-tolerant animal suggest the relative importance of these kinase pathways in cellular adaptation to oxygen deprivation or freezing and identify novel natural activators of MAPKs in vivo. The specificity of the signaling pathways is also emphasized here as the general whole-body stresses, anoxia and freezing, activated individual MAPKs in a tissue-, time-, and stress-dependent manner.

Animals↗