Biomedical subjects

P M Schulte

Publications and source records attributed to P M Schulte.

17 recordsLinked to original sources

PubMed2007

Metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to severe hypoxia.

We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 +/- 0.19 mg O(2)/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD(+)]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADP(free)], redox state, or [pyruvate]). Hypoxia exposure caused an approximately 65% decrease in gill Na(+)/K(+) ATPase activity, which was not matched by changes in Na(+)/K(+) ATPase alpha-subunit protein abundance indicating post-translational modification of Na(+)/K(+) ATPase was responsible for the decrease in activity. Despite decreases in gill Na(+)/K(+) ATPase activity, plasma [Na(+)] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na(+)/K(+) ATPase alpha-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na(+)/K(+) ATPase alpha-subunit relative to other genes.

Adaptation, Physiological↗

PubMed2006

Effects of acclimation and incubation temperature on the glutathione antioxidant system in killifish and RTH-149 cells.

Glutathione (GSH) is an important antioxidant that is involved in a multitude of cellular processes. However, in fish, GSH levels, turnover, and activity of associated enzymes are low when compared to those of mammals. To determine whether temperature influences the GSH antioxidant system in fish, and can explain the differences in GSH between fish and mammals, we examined the effects of acclimation temperature on total GSH (tGSH) levels and apparent half-life (as an estimate of turnover) in a rainbow trout hepatoma cell line (RTH-149), and GSH levels, and glutathione peroxidase (GPx) and reductase (GR) activity in the eurythermal killifish. Increasing incubation temperature decreased half-life and transiently increased levels of tGSH in RTH-149 cells. In killifish, increased acclimation temperature increased tGSH levels in the liver, brain and muscle, and increased hepatic GPx and GR activities. When the relationships between temperature and GSH half-life, levels and enzyme activity were extrapolated to 37 degrees C, temperature could only partially accounted for differences in the GSH antioxidant system in fish compared to mammals. The differences in the GSH antioxidant system between fish and mammals may not be solely due to temperature effects, but also to the increased metabolic cost of endothermy in mammals.

Acclimatization↗

PubMed2006

Reciprocal expression of gill Na+/K+-ATPase alpha-subunit isoforms alpha1a and alpha1b during seawater acclimation of three salmonid fishes that vary in their salinity tolerance.

The upregulation of gill Na+/K+-ATPase activity is considered critical for the successful acclimation of salmonid fishes to seawater. The present study examines the mRNA expression of two recently discovered alpha-subunit isoforms of Na+/K+-ATPase (alpha1a and alpha1b) in gill during the seawater acclimation of three species of anadromous salmonids, which vary in their salinity tolerance. Levels of these Na+/K+-ATPase isoforms were compared with Na+/K+-ATPase activity and protein abundance and related to the seawater tolerance of each species. Atlantic salmon (Salmo salar) quickly regulated plasma Na+, Cl- and osmolality levels within 10 days of seawater exposure, whereas rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) struggled to ionoregulate, and experienced greater perturbations in plasma ion levels for a longer period of time. In all three species, mRNA levels for the alpha1a isoform quickly decreased following seawater exposure whereas alpha1b levels increased significantly. All three species displayed similar increases in gill Na+/K+-ATPase activity during seawater acclimation, with levels rising after 10 and 30 days. Freshwater Atlantic salmon gill Na+/K+-ATPase activity and protein content was threefold higher than those of Arctic char and rainbow trout, which may explain their superior seawater tolerance. The role of the alpha1b isoform may be of particular importance during seawater acclimation of salmonid fishes. The reciprocal expression of Na+/K+-ATPase isoforms alpha1a and alpha1b during seawater acclimation suggests they may have different roles in the gills of freshwater and marine fishes; ion uptake in freshwater fish and ion secretion in marine fishes.

Acclimatization↗

PubMed2005

Ionoregulatory changes in different populations of maturing sockeye salmon Oncorhynchus nerka during ocean and river migration.

We present the first data on changes in ionoregulatory physiology of maturing, migratory adult sockeye salmon Oncorhynchus nerka. Fraser River sockeye were intercepted in the ocean as far away as the Queen Charlotte Islands (approximately 850 km from the Fraser River) and during freshwater migration to the spawning grounds; for some populations this was a distance of over 700 km. Sockeye migrating in seawater toward the mouth of the Fraser River and upriver to spawning grounds showed a decline in gill Na+,K+-ATPase activity. As a result, gill Na+,K+-ATPase activity of fish arriving at the spawning grounds was significantly lower than values obtained from fish captured before entry into freshwater. Plasma osmolality and chloride levels also showed significant decreases from seawater values during the freshwater migration to spawning areas. Movement from seawater to freshwater increased mRNA expression of a freshwater-specific Na+,K+-ATPase isoform (alpha1a) while having no effect on the seawater-specific isoform (alpha1b). In addition, gill Na+,K+-ATPase activity generally increased in active spawners compared with unspawned fish on the spawning grounds and this was associated with a marked increase in Na+,K+-ATPase alpha1b mRNA. Increases in gill Na+,K+-ATPase activities observed in spawners suggests that the fish may be attempting to compensate for the osmotic perturbation associated with the decline in plasma chloride concentration and osmolality.

Analysis of Variance↗

PubMed2004

Prior PCB exposure suppresses hypoxia-induced up-regulation of glycolytic enzymes in Fundulus heteroclitus.

Increased activity of the glycolytic enzymes is a conserved feature of the cellular response to hypoxia, and may represent a protective mechanism by which cells can survive short-term hypoxic exposure. Gene induction by hypoxia involves a dimer of the hypoxia inducible factor (HIF)-1 alpha and the nuclear cofactor HIF-1 beta, also called the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also involved in induction of genes in response to aryl hydrocarbon exposure. To assess the possibility of interaction between these pathways, we examined changes in the activity of the glycolytic enzymes in response to hypoxia and polychlorinated biphenyl (PCB) exposure in the liver of a teleost fish, Fundulus heteroclitus. After 3 days of hypoxic exposure (dissolved oxygen levels between 1.5 and 2.0 mg/L), there were significant increases in the activity of six glycolytic enzymes (PGI, ALD, TPI, PGK, PGM and LDH). In contrast, intraperitoneal injection of 1 microg/g body weight of PCB #77 (3,3',4,4'-tetrachlorobiphenyl) caused significant decreases in glycolytic enzyme activity after 7 days of exposure. When fish were injected with PCB #77 and then (4 days later) exposed to hypoxia for 3 days as before, we observed no induction of the glycolytic enzymes. This suggests that there is an antagonistic interaction between exposure to PCBs and hypoxia in F. heteroclitus. Prior PCB exposure could make these fish less tolerant of environmental hypoxia.

Animals↗

PubMed2004

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss).

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.

Animals↗

PubMed2002

Heat shock protein genes and their functional significance in fish.

Despite decades of intensive investigation, important questions remain regarding the functional, ecological, and evolutionary roles of heat shock proteins. In this paper, we discuss the utility of fish as a model system to address these questions, and review the relevant studies of heat shock protein genes and the regulation of their expression in fish. Although molecular studies of the heat shock proteins in fish are still in their early descriptive phase, data are rapidly being collected. More is known about the biotic and abiotic factors regulating heat shock proteins. We briefly review these studies and focus on the role of heat shock proteins in development, their regulation by the endocrine system, and their importance in fish in nature. Functional genomics approaches will provide the tools necessary to gain a comprehensive understanding of the significance of heat shock proteins in the cellular stress response, in the physiological processes at higher levels of organization, and in the whole animal in its natural environment.

Adaptation, Physiological↗

PubMed2001

Environmental adaptations as windows on molecular evolution.

Changes in gene regulation may play an important role in adaptive evolution, particularly during adaptation to a changing environment. However, little is known about the molecular mechanisms underlying adaptively significant variation in gene regulation. To address this question, we are using environmental adaptations in populations of a fish, Fundulus heteroclitus as a window into the molecular evolution of gene regulation. F. heteroclitus are found along the East Coast of North America, with populations distributed along a steep thermal gradient. At the extremes of the species range, populations have undergone local adaptation to their habitat temperatures. A variety of genes differ in their regulation between these populations. We have determined the mechanism responsible for changes in lactate dehydrogenase-B (Ldh-B) gene regulation. A limited number of mutations in the regulatory sequence of this gene result in changes in its expression. Both the phenotypic (increased LDH activity) and genotypic (changes in Ldh-B regulatory sequences) differences between populations have been shown to be affected by natural selection, rather than genetic drift. Therefore, even a small number of mutations within important regulatory sequences can provide evolutionarily significant variation and have an impact on environmental adaptation.

Adaptation, Physiological↗

PubMed2001

Structure and sequence conservation of a putative hypoxia response element in the lactate dehydrogenase-B gene of Fundulus.

Many aquatic habitats are characterized by periodic or sustained episodes of low oxygen concentration, or hypoxia, and organisms that survive in these habitats do so by utilizing a suite of behavioral, physiological and biochemical adjustments to low oxygen (1-3). In the killifish Fundulus heteroclitus, one response to prolonged exposure to hypoxia is an increase in the activity of lactate dehydrogenase-B (LDH-B), the terminal enzyme of anaerobic glycolysis, in liver tissue (4). An increase in glycolytic enzyme activity also occurs in mammalian cells during hypoxia, a process due, in part, to increased rates of gene transcription mediated by the hypoxia-inducible transcription factor, HIF-1 (5). Given that a homolog of HIF-1 has been identified in fish (6), we hypothesized that HIF might be involved in the observed up-regulation of LDH-B in F. heteroclitus. Herein, we describe the presence of DNA elements in intron 2 of the Ldh-B gene from F. heteroclitus that resemble hypoxia response elements (HRE) describedfor mammalian genes (7-10). Specifically, over a region of approximately 50 base pairs we identified two consensus HIF-1 binding sites, as well as DNA elements that may bind other transcription factors (e.g., cyclic AMP response elements; CRE). We found that these sites were perfectly conserved among geographically diverse populations of F. heteroclitus, as well as being highly conserved among multiple species in the genus Fundulus. The spacing, orientation, and sequence conservation of these putative regulatory elements suggest that they may be functionally involved in the hypoxic regulation of Ldh-B in these fish.

Animals↗

PubMed2000

Adaptive variation in lactate dehydrogenase-B gene expression: role of a stress-responsive regulatory element.

Although changes in gene regulation may play an important role in adaptive evolution, there have been few attempts to investigate the molecular mechanisms responsible for adaptively significant variation in gene expression. Here we describe the mechanism underlying an adaptive difference in the expression of the lactate dehydrogenase-B gene (Ldh-B) between northern and southern populations of the fish Fundulus heteroclitus. Ldh-B regulatory sequences from northern and southern individuals, coupled to a luciferase reporter gene, were introduced into the livers of live fish. Deletion studies indicated that sequence changes between 400 and 500 bp upstream of the transcription start site resulted in a 2-fold difference in reporter gene transcription. These sequence changes can account for the previously observed 2-fold difference in Ldh-B transcription between populations. Variation in transcription factors did not play an important role. Sequences within the functionally important region resemble a mammary tumor virus glucocorticoid responsive element (MTV-GRE) in southern alleles, whereas northern alleles differ from the consensus by 1 bp. To test the hypothesis that this element is involved in the variation between populations of F. heteroclitus, we exposed transiently transgenic fish containing Ldh-B regulatory sequence/reporter gene constructs to handling stress or injected cortisol. Both treatments increased reporter gene transcription driven by southern alleles but not northern alleles, as expected if an MTV-GRE sequence were involved. This finding suggests that sequence variation in a GRE is the cause of the adaptive differences in Ldh-B gene expression between populations and demonstrates that small changes in gene regulatory sequences can have important evolutionary consequences.

Adaptation, Physiological↗

PubMed1998

Efficient gene transfer into Xiphophorus muscle and melanoma by injection of supercoiled plasmid DNA.

Muscle and melanoma tissue of fish in the genus Xiphophorus were examined for their ability to take up and express foreign DNA. Supercoiled plasmid DNA containing a firefly luciferase reporter gene with expression driven by the cytomegalovirus enhancer and thymidylate kinase promoter was directly injected into the muscle or melanoma of individual Xiphophorus. Expression levels gradually increased to a maximum at 6 days after injection in both tissues, and this level was maintained for at least 10 days after injection. In both muscle and melanoma, there was a clear relationship between dose injected and reporter gene activity, with maximal expression at a dose of 20 microg of plasmid injected. At higher doses expression levels declined, suggesting the possibility that the uptake mechanism can be inhibited by high concentrations of DNA. Histochemical localization using a beta-galactosidase construct revealed high expression of the enzyme in isolated muscle fibers. The activity of a second coinjected reporter gene, sea pansy (Renilla reniformis) luciferase, was highly correlated with the activity of the firefly luciferase reporter gene in both tissues (R2 >.940), suggesting that the majority of variation between samples results from variation in overall DNA uptake between individuals. When firefly luciferase activity is expressed as a function of activity of the coinjected reporter, the variation between samples is greatly reduced. As a result, small differences in activity between constructs can be detected. This demonstrates the usefulness of the system for gene expression analysis in vivo.

Animals↗

PubMed1997

Structural and functional differences in the promoter and 5' flanking region of Ldh-B within and between populations of the teleost Fundulus heteroclitus.

We have investigated the mechanisms underlying differences in the transcriptional regulation of lactate dehydrogenase-B (Ldh-B) between northern and southern populations of a teleost fish, Fundulus heteroclitus. A 1-kb region immediately 5' of the gene was sequenced from populations throughout the species range. There were two major allele classes in the sample, one containing alleles from Maine and another containing those from Florida. Populations from intermediate localities contained both allele classes. Some individuals from Georgia had sequences intermediate between the two classes, representing either ancestral alleles or recombinants. Tests of neutrality were applied to determine whether observed variation was consistent with neutral expectations. Significant deviations from neutral expectations were detected for the 5' flanking region, but not for other loci. The functional consequences of flanking sequence variation were assessed by transfection of reporter gene constructs into cultured cells and injection into living fish. Consistent with observed variation in Ldh-B transcription rate between populations, significant differences in reporter gene activity were driven by flanking regions from northern and southern populations both in cell culture and in vivo. This functional differentiation, coupled with departures from neutral expectations, suggests that selection may have acted on the regulation of Ldh-B in F. heteroclitus.

Animals↗

PubMed1996

Descriptive and functional characterization of variation in the Fundulus heteroclitus Ldh-B proximal promoter.

Variation in enzyme expression may be an important mechanism for physiological and evolutionary adaptation. The Ldh-B locus in the teleost fish Fundulus heteroclitus is one of a very few loci for which an evolutionary difference in transcription rate between populations has been demonstrated. To begin to understand the molecular modifications that are responsible for altering transcription, we have characterized the Ldh-B proximal promoter using a combination of sequence analysis, transient transfection, and in vivo footprinting. The Ldh-B gene has several transcription start sites and a TATA-less, Inr (initiator of transcription motif) containing promoter with multiple Sp1-like motifs. Transfection experiments reveal that Sp1 sites, TCC repeats, and Inrs are functional components of the proximal promoter. We find substantial sequence variation between populations within the proximal promoter (250 bp from the transcription start sites) and footprinting analysis indicates that some of this sequence variation is associated with differential protein binding to the apparent TFIID binding site and Sp1 sites. Together, these data suggest that variation in the Ldh-B proximal promoter may play a role in the observed difference in transcription rates between northern and southern populations of F. heteroclitus.

Adaptation, Physiological↗

PubMed1995

Rapid in vivo footprinting method for the detection of DNA-protein interactions in isolated nuclei.

Here we report a simple method of in vivo footprinting for the detection of DNA-protein interactions in the liver of a small teleost fish, Fundulus heteroclitus. This method allows the determination of these interactions in nuclei isolated from intact liver, obviating the need for cell culture. Cells in culture often do not respond to environmental cues in the same way as do tissues within the intact organism and therefore may be inappropriate for the study of certain adaptive responses. Furthermore, cell lines are available for only a small number of marine organisms. This technique may therefore be of general utility for the study of gene regulation in a wide variety of marine organisms.

Animals↗

PubMed1992

Recovery metabolism of trout white muscle: role of mitochondria.

Recovery from burst exercise in fish is very slow. Lactate conversion to glycogen occurs primarily within white muscle and must be fueled by mitochondrially produced ATP. In a parallel study we characterized the changes in tissue metabolites associated with burst exercise and recovery in rainbow trout (Oncorhynchus mykiss) white muscle. The present study examines whether the mitochondrial capacity to produce ATP may limit the rate of recovery of trout white muscle. The cost (ATP.min-1.g-1) of glycogen resynthesis (0.05 mumol lactate converted.min-1.g tissue-1) was compared with the mitochondrial capacity to produce ATP. The cost of recovery can be met by only 3.5% of the maximal mitochondrial capacity. In fact, during recovery trout white muscle mitochondria operate at a small fraction of their in vitro maximum. This capacity is suppressed in vivo by highly inhibitory ATP/ADP and limiting phosphate. The primary signal for increased ATP synthesis associated with recovery is not a change in ATP/ADP but probably phosphate, elevated because of phosphocreatine hydrolysis and adenylate catabolism in the purine nucleotide cycle. At low ADP availability and suboptimal phosphate (less than 5 mM), acidosis enhances respiration. At high respiratory rates mitochondrial pyruvate oxidation is sensitive to pyruvate concentration over the physiological range (apparent Michaelis constant = 35-40 microM). This sensitivity is lost at the low rates that approximate in vivo respiration. Changes in lactate do not affect the kinetics of pyruvate oxidation. Fatty acid oxidation may spare pyruvate and lactate for use in glyconeogenesis, primarily through allosteric inhibition of pyruvate dehydrogenase rather than covalent modification.

Adenosine Diphosphate↗

PubMed1992

Integrating metabolic pathways in post-exercise recovery of white muscle.

Purine nucleotides (ATP, ADP, AMP, IMP), creatine, phosphocreatine, lactate, pyruvate and glycogen were measured in rainbow trout (Oncorhynchus mykiss) white muscle following exercise to exhaustion. Estimates of intracellular pH permitted calculation of free concentrations of nucleotides ([nucleotide]f) required for most models of control of energy metabolism. Creatine charge, [PCr]/([PCr]+[Cr]), fell from 0.49 +/- 0.05 (mean +/- S.E.M.) to 0.08 +/- 0.02 with exercise but recovered completely by the first sample (2 h). Although [ATP] declined to 24% of resting levels and recovered very slowly, RATP, [ATP]/([ATP]+[ADP]f+[AMP]f), and energy charge, EC, ([ATP]+0.5[ADP]f)/([ATP]+[ADP]f+[AMP]f), recovered as quickly as creatine charge. Changes in [IMP] mirrored those in [ATP], suggesting that AMP deaminase is responsible for maintaining RATP and EC. Recovery of carbon status was much slower than recovery of energy status. Lactate increased from 4 mumol g-1 at rest to 40 mumol g-1 at exhaustion and did not recover for more than 8 h. Glycogen depletion and resynthesis followed a similar time course. During the early stages of recovery, calculated [ADP]f declined by more than 10-fold relative to the resting values. The resulting high [ATP]/[ADP]f ratios may limit the rate at which white muscle mitochondria can produce ATP to fuel glycogenesis in situ. It is postulated that the high [ATP]/[ADP]f ratios are required to drive pyruvate kinase in the reverse direction for glyconeogenesis in recovery.

Adenine Nucleotides↗

PubMedDate not supplied

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: a multidisciplinary approach to address the million-year saga of a small fish.

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, DNase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation.

Adaptation, Physiological↗