Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog,Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (K_m = 0.098 ± 0.003 and K_m = 0.092 ± 0.008) than PK from control and anoxic conditions (K_m = 0.065 ± 0.003 and K_m = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.     

Characterization of γ-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 γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°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°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°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°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°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 γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°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 γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-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.     

Survival and metabolism of <Emphasis Type="Italic">Rana arvali</Emphasis>s during freezing

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of −3°C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and −1°C). Lactate accumulation was only observed in the liver (4.1 ± 0.8 against 16.6 ± 2.4 μmol g⁻¹ fresh weight (FW) between 4 and −1°C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4 ± 0.7 μl O₂ g⁻¹ FW h⁻¹ at −4°C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.     

Regulation of hexokinase by reversible phosphorylation in skeletal muscle of a freeze-tolerant frog

Hexokinase (HK) was isolated from hind leg skeletal muscle of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K(m) and V(max)) of HK showed significant increases in K(m) glucose (from 144 ± 4.4 to 248 ± 1 2.0 μM) and K(m) ATP (from 248 ± 8.5 to 330 ± 20.9 μM), as well as a decrease in V(max) (from 86.1 ± 0.40 to 52 ± 0.49 mUmg(-1) of protein) in frogs following freezing exposure, indicating lower affinity for HK substrates and lower enzyme activity in this state. Subsequent analyses indicated that differential phosphorylation of HK between the two states was responsible for the altered kinetic properties. HK was analyzed by SDS-PAGE; phosphoprotein staining revealed a 33% decrease in phosphate content of HK from frozen frogs but immunoblotting showed no change in total HK protein content. Muscle extracts from control and frozen frogs were incubated with ions and second messengers to stimulate the actions of protein kinases and protein phosphatases, with results indicating that HK can be phosphorylated by protein kinases A and C, and AMP-activated protein kinase, and can be dephosphorylated by protein phosphatases 1, 2A and 2C. The data indicate that in control frogs, HK is in a higher phosphate form and displays a high substrate affinity and high activity, whereas in frozen frogs HK is less phosphorylated, with lower substrate affinity and lower activity. Studies also showed that HK affinity for ATP decreases further in response to low temperature, but that high cryoprotective glucose concentrations can prevent these changes in affinity. Finally, the activity and structure of HK from frozen frogs is more sensitive to non-compatible osmolytes than the enzyme in control frogs.     

Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog

Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K _m and V _max) of G6PDH showed a significant increase in K _m G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K _m NADP⁺ (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K _m G6P of 94.1 ± 1.1 μM and K _m NADP⁺ of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K _m G6P was 172 ± 4.3 μM, K _m NADP⁺ was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.     

Effects of photon flux density and agricultural fertilizers on the development of <Emphasis Type="Italic">Sarcothalia crispata</Emphasis> tetraspores (Rhodophyta,Gigartinales) from the Strait of Magellan,Chile

Tetraspores of Sarcothalia crispata from San Juan Bay, Strait of Magellan, Chile, were cultivated under different combinations of photon flux densities and agricultural fertilizers in the laboratory. In the experiment, the S. crispata specimens were cultured in combinations of different photon flux densities (50, 100, 150 μmol photons m^-2 s^-1) and enriched seawater solutions (sodium nitrate + monocalcium phosphate, urea + monocalcium phosphate, ammonium nitrate + monocalcium phosphate), always adjusting the N and P concentrations to 10 and 3 mg L^-1, and in sea water as control. After 45 days, the tetrasporeling plants were found to be larger at photon flux densities of 50 and 100 μmol photons m^-2 s^-1 in the nutrient enrichment experiments; growth was greatest in the sea water enriched with ammonium nitrate and urea. An analysis of the combined effect of the photon flux density and nutrients revealed that the best combination for sporeling growth was the ammonium nitrate and urea solution at 50–100 μmol photons m^-2 s^-1.     

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 °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. Accepted: 2 January 1997     

Purification and Characterization of Novel α-Amylase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KIBGE HAS

Purification of extracellular α-amylase from Bacillus subtilis KIBGE HAS was carried out by ultrafiltration, ammonium sulfate precipitation and gel filtration chromatography. The enzyme was purified to homogeneity with 96.3-fold purification with specific activity of 13011 U/mg. The molecular weight of purified α-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular α-amylase showed that the enzyme had a Km and V _max value of 2.68 mg/ml and 1773 U/ml, respectively. The optimum activity was observed at pH 7.5 in 0.1 M phosphate buffer at 50°C. The amino acid composition of the enzyme showed that the enzyme is rich in neutral/non polar amino acids and less in acidic/polar and basic amino acids. The N-terminal protein sequence of 10 residues was found to be as Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly (S-S-N-K-L-T-T-S-W-G). Furthermore, the protein was not N-terminally blocked. The sequence of α-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported α-amylases from Bacillus strain.     

Creatine kinase regulation by reversible phosphorylation in frog muscle

Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent K_m creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with ³²P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of ³²P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.     

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.     

Urea is not a universal cryoprotectant among hibernating anurans: Evidence from the freeze-tolerant boreal chorus frog (Pseudacris maculata)

Freeze-tolerant organisms accumulate a diversity of low molecular weight compounds to combat negative effects of ice formation. Previous studies of anuran freeze tolerance have implicated urea as a cryoprotectant in the wood frog (Lithobates sylvatica). However, a cryoprotective role for urea has been identified only for wood frogs, though urea accumulation is an evolutionarily conserved mechanism for coping with osmotic stress in amphibians. To identify whether multiple solutes are involved in freezing tolerance in the boreal chorus frog (Pseudacris maculata), we examined seasonal and freezing-induced variation in several potential cryoprotectants. We further tested for a cryoprotective role for urea by comparing survival and recovery from freezing in control and urea-loaded chorus frogs. Tissue levels of glucose, urea, and glycerol did not vary significantly among seasons for heart, liver, or leg muscle. Furthermore, no changes in urea or glycerol levels were detected with exposure to freezing temperatures in these tissues. Urea-loading increased tissue urea concentrations, but failed to enhance freezing survival or facilitate recovery from freezing in chorus frogs in this study, suggesting little role for urea as a natural cryoprotectant in this species. These data suggest that urea may not universally serve as a primary cryoprotectant among freeze-tolerant, terrestrially hibernating anurans.     

Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C, and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization, but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells. Accepted: 28 April 1999     

Field ecology of freezing: Linking microhabitat use with freezing tolerance in Litoria ewingii

A small number of vertebrate species, including some frogs, are freezing tolerant and survive ice forming in their bodies under ecologically relevant conditions. Habitat use information is critical for interpreting laboratory studies of freezing tolerance, but there is often little known about the winter habitat and behaviours of the species under study. This work describes microhabitats used by the freezing‐tolerant frog Litoria ewingii Duméril and Bibron 1841 and their temperature characteristics. In winter, L. ewingii used microhabitats with wood, located further away from water than in summer. Microhabitat temperature records showed that frog microhabitats regularly fell below the temperature at which frog body fluids freeze (?1°C), and cooled substantially more slowly than did the air temperature. Temperatures were highly variable between microhabitats, seasons and years, with a minimum of ?2.4°C and a maximum cooling rate of 0.77°C h^?1. Frozen frogs were observed to recover in the field, demonstrating freezing tolerance. Both the characteristics of microhabitats and their selection are important in ensuring freezing survival.     

Freezing tolerance of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) gametophyte assessed by chlorophyll fluorescence

In January and February 2010, heavy sea ice formed along the coast of the Bohai Sea and the northern Yellow Sea, China. Intertidal organisms were subjected to serious freezing stress. In this study, we investigated the freezing tolerance of the upper intertidal economic seaweed Porphyra yezoensis. The maximum photochemical efficiency of PS II (F _v/F _m) in undehydrated thalli remained high after 24 h at −2°C and that in dehydrated thalli decreased in a proportion to thallial water loss. F _v/F _m dropped sharply after 24 h at −20°C, regardless of absolute cellular water content (AWC). The F _v/F _m in frozen thalli recovered rapidly at 0–20°C. A wide range of water loss in the thalli enhanced their tolerance to freezing. F _v/F _m values in undehydrated thalli dropped sharply after 3 d at −2°C or 10 d at −20°C while those in dehydrated thalli (20–53% AWCs) remained at high levels after 9 d at −2°C or 30 d at −20°C. These results indicate that P. yezoensis has high freezing tolerance by means of dehydration during the ebb tide and rapid recovery of F _v/F _m from freezing. A strategy of P. yezoensis industry to avoid heavy loss during freezing season is discussed based on these findings.     

Stress-induced antioxidant defense and protein chaperone response in the freeze-tolerant wood frog <Emphasis Type="Italic">Rana sylvatica</Emphasis>

Freeze tolerance is an adaptive response utilized by the wood frog Rana sylvatica to endure the sub-zero temperatures of winter. Survival of whole body freezing requires wood frogs to trigger cryoprotective mechanisms to deal with potential injuries associated with conversion of 65–70% of total body water into ice, including multiple consequences of ice formation such as cessation of blood flow and cell dehydration caused by water loss into ice masses. To understand how wood frogs defend against these stressors, we measured the expression of proteins known to be involved in the antioxidant defense and protein chaperone stress responses in brain and heart of wood frogs comparing freezing, anoxia, and dehydration stress. Our results showed that most stress proteins were regulated in a tissue- and stress-specific manner. Notably, protein levels of the cytosolic superoxide dismutase (SOD1) were upregulated by 1.37?±?0.11-fold in frozen brain, whereas the mitochondrial SOD2 isoform rose by 1.38?±?0.37-fold in the heart during freezing. Catalase protein levels were upregulated by 3.01?±?0.47-fold in the brain under anoxia stress, but remained unchanged in the heart. Similar context-specific regulatory patterns were also observed for the heat shock protein (Hsp) molecular chaperones. Hsp27 protein was down-regulated in the brain across the three stress conditions, whereas the mitochondrial Hsp60 was upregulated in anoxic brain by 1.73?±?0.38-fold and by 2.13?±?0.57-fold in the frozen heart. Overall, our study provides a snapshot of the regulatory expression of stress proteins in wood frogs under harsh environment conditions and shows that they are controlled in a tissue- and stress-specific manner.