Two rounds of genome duplication (GD) in the ancestor of vertebrates, followed by additional GD d... more Two rounds of genome duplication (GD) in the ancestor of vertebrates, followed by additional GD during the evolution of ray-finned fishes (Actinopterygii), expanded certain gene families, including those encoding the hypoxia inducible transcription factor (HIF). The present study analyzed Actinopterygian genomes for duplicates of HIFα, the subunit that confers oxygen-dependent gene regulation. In contrast to tetrapod vertebrates that retain three HIFα genes from the ancestral vertebrate GD, four HIFα forms were found in the genomes of primitive Actinopterygians (spotted gar and Asian arowana). All four forms have been retained in zebrafish and related species (Otocephala) and salmonids and their sister taxa (northern pike) but one of them (HIF4α) was lost during the evolution of more derived fishes (Neoteleostei). In addition, the current analyses confirm that Otocephala retain duplicates of HIF1α and HIF2α from the teleost-specific GD, provide new evidence of salmonid-specific dupl...
Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the lan... more Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the land snail Oreohelix spp. decreased by 83% compared to standard non-estivating rates. During both non-estivating and estivating conditions, the quantity of heat dissipated per mole of O2 consumed was indicative of a completely aerobic metabolism. This calorimetric-respirometric (C/R) ratio was −461±12 kJ mol−1O2 (S.E.M., N=5) under standard non-estivating conditions and −464±26 kJ mol−1O2 (N=4) during estivation. Respiratory exchange ratios reflected a primary dependence upon carbohydrate as a metabolic substrate during both states. Carbon dioxide retention occurred during the first 36h of estivation, resulting in an increase in hemolymph PCOCO2 and a decrease in pH. The respiratory acidosis during short-term estivation was not compensated by elevation of hemolymph [HCO3−] above levels predicted from the in vitro nonbicarbonate buffer value of hemolymph. A brief period of rapid CO2 release,...
ABSTRACTStandard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS)... more ABSTRACTStandard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were determined for the Gulf killifish, Fundulus grandis, an ecologically dominant estuarine fish, acclimated to lowered salinity, elevated temperature, and lowered oxygen concentration. Acclimation to low salinity resulted in a small, but significant, elevation of Pcrit; acclimation to elevated temperature increased SMR, MMR, AAS, and Pcrit; acclimation to low oxygen led to a small increase in SMR, but substantial decreases in MMR, AAS, and Pcrit. Variation in these metabolic traits among individuals was consistent and repeatable when measured during multiple control exposures over seven months. Trait repeatability was unaffected by acclimation condition suggesting that repeatability of these traits is not context dependent. There were significant phenotypic correlations between specific metabolic traits: SMR was positively correlated with MMR and P...
For most fishes, there is an oxygen level, the critical oxygen tension (Pcrit), below which oxyge... more For most fishes, there is an oxygen level, the critical oxygen tension (Pcrit), below which oxygen consumption (MO2) becomes dependent upon ambient oxygen partial pressure (PO2). We compare multiple curve-fitting approaches to estimate Pcrit of the Gulf killifish, Fundulus grandis, during closed and intermittent-flow respirometry. The traditional approach fitting two line segments of MO2 versus PO2 produced high and variable estimates of Pcrit. Nonlinear regression using hyperbolic or Weibull functions resulted in either variable Pcrit estimates or, in some cases, failed to converge upon meaningful solutions. Pcrit determined as the PO2 when MO2 equals standard metabolic rate (SMR) based upon a linear relationship of MO2 and PO2 at low PO2 were consistent across fish and experimental trials. Therefore, we recommend that Pcrit specifically refer to the PO2 below which SMR cannot be maintained. Its determination, therefore, requires accurate measurement of SMR.
Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the lan... more Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the land snail Oreohelix spp. decreased by 83 % compared to standard non-estivating rates. During both non-estivating and estivating conditions, the quantity of heat dissipated per mole of O2 consumed was indicative of a completely aerobic metabolism. This calorimetric-respirometric (C/R) ratio was — 461 ± 12 kJ
Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition ... more Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Kis = 0.86 mM, Kii = 1.0 mM, Kid = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propo...
SUMMARYThe goal of the current study was to generate a comprehensive, multi-tissue perspective of... more SUMMARYThe goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l-1 dissolved oxygen (∼3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis(in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and...
Reliable proteomic analysis of biological tissues requires sampling approaches that preserve prot... more Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one-dimensional (1-D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap-freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlater(®). In liver and heart, the protein profiles in 1-D gels were better preserved by snap-freezing, while in gill, the 1-D protein profile was better preserved by immersion in RNAlater(®). In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC-MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.
Patterns of protein expression were examined in white skeletal muscle from adult zebrafish (Danio... more Patterns of protein expression were examined in white skeletal muscle from adult zebrafish (Danio rerio). High resolution two-dimensional gel electrophoresis resolved between 300 and 400 spots with molecular masses between 20 and 120 kDa and isoelectric points between about 5 and 8. Forty spots, representing a range of protein size, charge, and abundance were excised, digested with trypsin, and subjected to matrix-assisted laser-desorption/ionisation-time of flight mass spectrometry for protein identification. Twenty-nine spots were identified, including enzymes of energy metabolism, contractile proteins, an iron transport protein, and a heat shock protein. In addition, several spots matched theoretical proteins predicted from genome sequencing. These theoretical proteins were tentatively identified by similarity to known proteins. Patterns of muscle protein expression were then measured after zebrafish were exposed to low oxygen (16 torr) for 48 h, an exposure previously shown to increase the survival of zebrafish at more severe reductions in oxygen. Exposure to low oxygen (hypoxia) did not change the general pattern of protein expression but did affect the amounts of six low abundance proteins. The relatively subtle effects of hypoxia on patterns of muscle protein expression contrasts the widespread changes previously documented in mRNA levels in this and other species of fish during hypoxic stress. The difference between protein and mRNA expression illustrates the need to integrate both measures for a more complete understanding of gene expression in fish during hypoxic exposure.
We examined whether western fence lizards Sceloporus occidentalis occurring in thermally divergen... more We examined whether western fence lizards Sceloporus occidentalis occurring in thermally divergent environments display differential responses to high temperature in locomotor performance and heat-shock protein (Hsp) expression. We measured maximum sprint speed in S. occidentalis from four populations at paired latitudes and elevations before and after exposure to an experimental heat treatment and then quantified hind-limb muscle Hsp70 expression. Lizards collected from northern or high-elevation collection sites suffered a greater reduction in sprint speed after heat exposure than lizards collected from southern or low-elevation sites. In addition, lizards from northern collection sites also exhibited an increase in Hsp70 expression after heat exposure, whereas there was no effect of heat exposure on Hsp70 expression in lizards from southern collection sites. Across all groups, there was a negative relationship between Hsp70 expression and sprint speed after thermal stress. This result is significant because (a) it suggests that an increase in Hsp70 alone cannot compensate for the immediate negative effects of high-temperature exposure on sprint speed and (b) it demonstrates a novel correlation between an emergent property at the intersection of several physiological systems (locomotion) and a cellular response (Hsp70 expression). Ultimately, geographic variation in the effects of heat on sprint speed may translate into differential fitness and population viability during future increases in global air temperatures.
In this study, patterns of protein expression in zebrafish (Danio rerio) white skeletal muscle af... more In this study, patterns of protein expression in zebrafish (Danio rerio) white skeletal muscle after 48 h exposure to hypoxia (P(O2)=1.9 kPa) or normoxia (P(O2)=18.6 kPa) were evaluated using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Proteins were separated over two pH ranges in the first dimension (pH 4-7 and pH 7-11) prior to separation in the second dimension, resolving a total of 821 protein spots. Of these, 77 spots (9.4%) differed between hypoxia and normoxia (p ≤ 0.01), with approximately twice as many proteins being higher during hypoxia (56) compared to the number found to be higher in normoxic fish (26). Thirty-one protein spots were identified by MALDI-TOF/TOF mass spectrometry. The expression of several glycolytic enzymes was greater in hypoxia than in normoxia, whereas enzymes associated with mitochondrial ATP synthesis were lower during hypoxia. Among the more highly up-regulated proteins during hypoxia were two variants of hemoglobin α subunit. These patterns of protein expression are consistent with a hypoxic response that enhances anaerobic metabolism and O(2) transport to tissues, with a concomitant suppression of mitochondrial metabolism. These proteomic changes may contribute to the acclimation of zebrafish to hypoxia, thereby increasing their tolerance of low oxygen concentrations.
Two rounds of genome duplication (GD) in the ancestor of vertebrates, followed by additional GD d... more Two rounds of genome duplication (GD) in the ancestor of vertebrates, followed by additional GD during the evolution of ray-finned fishes (Actinopterygii), expanded certain gene families, including those encoding the hypoxia inducible transcription factor (HIF). The present study analyzed Actinopterygian genomes for duplicates of HIFα, the subunit that confers oxygen-dependent gene regulation. In contrast to tetrapod vertebrates that retain three HIFα genes from the ancestral vertebrate GD, four HIFα forms were found in the genomes of primitive Actinopterygians (spotted gar and Asian arowana). All four forms have been retained in zebrafish and related species (Otocephala) and salmonids and their sister taxa (northern pike) but one of them (HIF4α) was lost during the evolution of more derived fishes (Neoteleostei). In addition, the current analyses confirm that Otocephala retain duplicates of HIF1α and HIF2α from the teleost-specific GD, provide new evidence of salmonid-specific dupl...
Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the lan... more Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the land snail Oreohelix spp. decreased by 83% compared to standard non-estivating rates. During both non-estivating and estivating conditions, the quantity of heat dissipated per mole of O2 consumed was indicative of a completely aerobic metabolism. This calorimetric-respirometric (C/R) ratio was −461±12 kJ mol−1O2 (S.E.M., N=5) under standard non-estivating conditions and −464±26 kJ mol−1O2 (N=4) during estivation. Respiratory exchange ratios reflected a primary dependence upon carbohydrate as a metabolic substrate during both states. Carbon dioxide retention occurred during the first 36h of estivation, resulting in an increase in hemolymph PCOCO2 and a decrease in pH. The respiratory acidosis during short-term estivation was not compensated by elevation of hemolymph [HCO3−] above levels predicted from the in vitro nonbicarbonate buffer value of hemolymph. A brief period of rapid CO2 release,...
ABSTRACTStandard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS)... more ABSTRACTStandard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were determined for the Gulf killifish, Fundulus grandis, an ecologically dominant estuarine fish, acclimated to lowered salinity, elevated temperature, and lowered oxygen concentration. Acclimation to low salinity resulted in a small, but significant, elevation of Pcrit; acclimation to elevated temperature increased SMR, MMR, AAS, and Pcrit; acclimation to low oxygen led to a small increase in SMR, but substantial decreases in MMR, AAS, and Pcrit. Variation in these metabolic traits among individuals was consistent and repeatable when measured during multiple control exposures over seven months. Trait repeatability was unaffected by acclimation condition suggesting that repeatability of these traits is not context dependent. There were significant phenotypic correlations between specific metabolic traits: SMR was positively correlated with MMR and P...
For most fishes, there is an oxygen level, the critical oxygen tension (Pcrit), below which oxyge... more For most fishes, there is an oxygen level, the critical oxygen tension (Pcrit), below which oxygen consumption (MO2) becomes dependent upon ambient oxygen partial pressure (PO2). We compare multiple curve-fitting approaches to estimate Pcrit of the Gulf killifish, Fundulus grandis, during closed and intermittent-flow respirometry. The traditional approach fitting two line segments of MO2 versus PO2 produced high and variable estimates of Pcrit. Nonlinear regression using hyperbolic or Weibull functions resulted in either variable Pcrit estimates or, in some cases, failed to converge upon meaningful solutions. Pcrit determined as the PO2 when MO2 equals standard metabolic rate (SMR) based upon a linear relationship of MO2 and PO2 at low PO2 were consistent across fish and experimental trials. Therefore, we recommend that Pcrit specifically refer to the PO2 below which SMR cannot be maintained. Its determination, therefore, requires accurate measurement of SMR.
Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the lan... more Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the land snail Oreohelix spp. decreased by 83 % compared to standard non-estivating rates. During both non-estivating and estivating conditions, the quantity of heat dissipated per mole of O2 consumed was indicative of a completely aerobic metabolism. This calorimetric-respirometric (C/R) ratio was — 461 ± 12 kJ
Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition ... more Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Kis = 0.86 mM, Kii = 1.0 mM, Kid = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propo...
SUMMARYThe goal of the current study was to generate a comprehensive, multi-tissue perspective of... more SUMMARYThe goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l-1 dissolved oxygen (∼3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis(in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and...
Reliable proteomic analysis of biological tissues requires sampling approaches that preserve prot... more Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one-dimensional (1-D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap-freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlater(®). In liver and heart, the protein profiles in 1-D gels were better preserved by snap-freezing, while in gill, the 1-D protein profile was better preserved by immersion in RNAlater(®). In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC-MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.
Patterns of protein expression were examined in white skeletal muscle from adult zebrafish (Danio... more Patterns of protein expression were examined in white skeletal muscle from adult zebrafish (Danio rerio). High resolution two-dimensional gel electrophoresis resolved between 300 and 400 spots with molecular masses between 20 and 120 kDa and isoelectric points between about 5 and 8. Forty spots, representing a range of protein size, charge, and abundance were excised, digested with trypsin, and subjected to matrix-assisted laser-desorption/ionisation-time of flight mass spectrometry for protein identification. Twenty-nine spots were identified, including enzymes of energy metabolism, contractile proteins, an iron transport protein, and a heat shock protein. In addition, several spots matched theoretical proteins predicted from genome sequencing. These theoretical proteins were tentatively identified by similarity to known proteins. Patterns of muscle protein expression were then measured after zebrafish were exposed to low oxygen (16 torr) for 48 h, an exposure previously shown to increase the survival of zebrafish at more severe reductions in oxygen. Exposure to low oxygen (hypoxia) did not change the general pattern of protein expression but did affect the amounts of six low abundance proteins. The relatively subtle effects of hypoxia on patterns of muscle protein expression contrasts the widespread changes previously documented in mRNA levels in this and other species of fish during hypoxic stress. The difference between protein and mRNA expression illustrates the need to integrate both measures for a more complete understanding of gene expression in fish during hypoxic exposure.
We examined whether western fence lizards Sceloporus occidentalis occurring in thermally divergen... more We examined whether western fence lizards Sceloporus occidentalis occurring in thermally divergent environments display differential responses to high temperature in locomotor performance and heat-shock protein (Hsp) expression. We measured maximum sprint speed in S. occidentalis from four populations at paired latitudes and elevations before and after exposure to an experimental heat treatment and then quantified hind-limb muscle Hsp70 expression. Lizards collected from northern or high-elevation collection sites suffered a greater reduction in sprint speed after heat exposure than lizards collected from southern or low-elevation sites. In addition, lizards from northern collection sites also exhibited an increase in Hsp70 expression after heat exposure, whereas there was no effect of heat exposure on Hsp70 expression in lizards from southern collection sites. Across all groups, there was a negative relationship between Hsp70 expression and sprint speed after thermal stress. This result is significant because (a) it suggests that an increase in Hsp70 alone cannot compensate for the immediate negative effects of high-temperature exposure on sprint speed and (b) it demonstrates a novel correlation between an emergent property at the intersection of several physiological systems (locomotion) and a cellular response (Hsp70 expression). Ultimately, geographic variation in the effects of heat on sprint speed may translate into differential fitness and population viability during future increases in global air temperatures.
In this study, patterns of protein expression in zebrafish (Danio rerio) white skeletal muscle af... more In this study, patterns of protein expression in zebrafish (Danio rerio) white skeletal muscle after 48 h exposure to hypoxia (P(O2)=1.9 kPa) or normoxia (P(O2)=18.6 kPa) were evaluated using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Proteins were separated over two pH ranges in the first dimension (pH 4-7 and pH 7-11) prior to separation in the second dimension, resolving a total of 821 protein spots. Of these, 77 spots (9.4%) differed between hypoxia and normoxia (p ≤ 0.01), with approximately twice as many proteins being higher during hypoxia (56) compared to the number found to be higher in normoxic fish (26). Thirty-one protein spots were identified by MALDI-TOF/TOF mass spectrometry. The expression of several glycolytic enzymes was greater in hypoxia than in normoxia, whereas enzymes associated with mitochondrial ATP synthesis were lower during hypoxia. Among the more highly up-regulated proteins during hypoxia were two variants of hemoglobin α subunit. These patterns of protein expression are consistent with a hypoxic response that enhances anaerobic metabolism and O(2) transport to tissues, with a concomitant suppression of mitochondrial metabolism. These proteomic changes may contribute to the acclimation of zebrafish to hypoxia, thereby increasing their tolerance of low oxygen concentrations.
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