Biomedical subjects

G K Iwama

Publications and source records attributed to G K Iwama.

At least 19 recordsLinked to original sources

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

Changes in free and total plasma cortisol levels in juvenile haddock (Melanogrammus aeglefinus) exposed to long-term handling stress.

We measured changes in free and total plasma cortisol levels, plasma glucose, gill hsp70 levels, and growth in haddock (Melanogrammus aeglefinus) subjected to a long-term handling stress (15 s out of water, each day, for 4 weeks), and the effect of this long-term stress on the ability of haddock to respond to an acute stressor. The acute stressor was a single handling stress, and fish were sampled at 1, 6, and 12 h post-stress. During the long-term stress study, free and total plasma cortisol levels increased significantly (10-fold) in the stressed group after the second week. However, the percentage of free cortisol was already significantly elevated by the first week (control 17%, stressed 55%), and remained high during the second week (control 35% and stressed 65%). After 3 and 4 weeks of handling, both free and total cortisol declined in stressed fish to levels that were not significantly different from pre-stress values. Control fish grew significantly more than stressed fish (by 32% and 18%, respectively) over the 4 week study, and condition factor only increased in control fish. Although fish from the control group showed elevated total plasma cortisol levels (to 47 ng mL(-1)) 1 h after the acute stress, and the levels in stressed fish were comparable to those for the control fish, no significant increase in plasma cortisol was measured in the group subjected to the long-term stress. Free plasma cortisol levels did not increase significantly in either group following the acute stress. However, free plasma cortisol levels were significantly higher in long-term stress group, as compared with the control group, at 6 h post-stress. Plasma glucose and gill hsp70 levels were not altered by either the long-term stress or acute stressor. Our data indicate that cortisol (free and total), but not glucose or hsp70, appears to be adequate to assess short- and long-term stress in haddock.

Animals↗

PubMed2004

Biochemical responses of two erythrinidae fish to environmental ammonia.

The non-ionized form of ammonia is very toxic to many aquatic species. It is especially important in several aspects of fish biology. A large range of organismal strategies for coping with environmental stressors is usually observed in living organisms. Among those, the responses for managing chemical stressors are well studied. The present work compares biochemical responses of two evolutionarily close species, Hoplias malabaricus and Hoplerythrinus unitaeniatus, exposed to environmental ammonia. Adult fish were submitted to 1.0 mg/L of ammonium chloride for 24 hours, and plasma ammonia and urea levels were determined. The activities of OUC enzymes OCT and ARG, and the accessory enzyme GS, were quantified in liver extract and are expressed below in mumol/min/mg of wet tissue. Increases in OUC enzymes (GS from 1.14 to 2.43, OCT from 0.81 to 1.72, and ARG from 3.15 to 4.23), plasma ammonia (from 0.95 to 1.42 mmol/L), and plasma urea (from 0.82 to 1.53 mmol/L) were observed (p < 0.05) in H. malabaricus exposed to 1 mg/L of ammonia chloride. The GS in H. unitaeniatus increased from 1.43 to 1.84, however the OCT, ARG, and plasma urea from H. unitaeniatus did not change. These data indicate that each species responds differently to the same environmental stressor.

Adaptation, Physiological↗

PubMed2004

Observations on the inherent variability of measuring lysozyme activity in coho salmon (Oncorhynchus kisutch).

Lysozyme activity is a common measurement of innate immunity. It has also been used to investigate genetic variation and an animal's responses to factors such as stress, infections and variations in diet. This research demonstrates the inherent variation in lysozyme activity in unstimulated coho salmon (Oncorhynchus kisutch). The role of maternal contribution, early life stage development and fish mass are considered. Genetic variation within and between strains of coho was found to be significant at selected life stages. Our results indicate that strain differences in lysozyme activity are more accurately measured by comparing the genetic variation after the eyed stage, when maternal effects are reduced. A positive correlation between plasma/serum lysozyme activity and fish mass is reported here. In summary, this study shows the role of maternal, developmental stage and size in lysozyme activity in fish, and emphasizes the importance of considering such variables when measuring the variability of lysozyme activity in fish.

Animals↗

PubMed2003

The effects of stress on the association between hsp70 and the glucocorticoid receptor in rainbow trout.

The purpose of this study was to characterize the association between hepatic heat shock protein 70 (hsp70) and the glucocorticoid receptor in rainbow trout that were exposed to heat stress, cortisol, and beta-naphthoflavone. This study is the first to document that the glucocorticoid receptor complex in rainbow trout hepatic tissues contains hsp70. Heat stress significantly increased levels of total cellular hsp70, and by discerning the association of hsp70 with the glucocorticoid receptor, we demonstrated that heat stress significantly increased the amount of hsp70 not bound to the glucocorticoid receptor, while significantly decreasing the amount of hsp70 bound to the glucocorticoid receptor. By calculating the ratio of hsp70 bound to the glucocorticoid receptor, to the total number of glucocorticoid receptors, stress (heat stress and cortisol-treatment) promoted the association of hsp70 with the glucocorticoid receptor. These findings demonstrate a functional and structural link between hsp70 and the glucocorticoid receptor in rainbow trout, and raise questions regarding the existence of a complex, interrelated stress response that spans all levels of biological organization within the whole animal.

Animals↗

PubMed2001

Coho salmon Oncorhynchus kisutch strain differences in disease resistance and non-specific immunity, following immersion challenges with Vibrio anguillarum.

Two strains of freshwater-reared coho salmon Oncorhynchus kisutch were compared for differences in the activity of selected non-specific immune factors before and after lethal and non-lethal immersion challenges with the marine bacterial pathogen Vibrio anguillarum (Vang). Two disease challenge experiments were performed. The first experimental challenge resulted in no mortality; however, significant strain and challenge treatment effects were detected at Day 16 post-challenge. Strain differences in plasma lysozyme activity were found in pre-challenge samples. The second challenge experiment compared the same strains of coho salmon following immersion challenges in different doses of Vang. The fish were sampled at Days 0, 2, 7, and 18 post-challenge and mortality, plasma lysozyme, and anterior kidney phagocyte respiratory burst activity were compared. There were significant strain differences in mortality in the high dose group. The more disease-resistant strain was found to have higher levels of plasma lysozyme and anterior kidney phagocyte respiratory burst activity. These strain differences were detected at various times in the lethal (high dose) and non-lethal challenge groups. There was a clear relationship between the enhanced survival of the more disease-resistant strain and a more sustained, elevated non-specific immune response following the experimental disease challenges. The results of this study suggest that the basis for strain differences in innate disease resistance is related to the ability of the fish to respond quickly to the initial infection and to maintain the response until the infection is quelled.

Animals↗

PubMed2001

The effects of cortisol on heat shock protein 70 levels in two fish species.

We studied the relationship between heat stress (2 h, +12 degrees ) and increased levels of circulating cortisol (50 microg cortisol/g body weight) on heat shock protein 70 (hsp70) levels in liver and gill tissues of rainbow trout (Oncorhynchus mykiss) and tilapia (Oreochromis mossambicus). The administration of cortisol by intraperitoneal injection (no heat stress) did not alter tissue hsp70 levels compared to sham implanted (no heat stress) trout and tilapia. We found elevated levels of cortisol significantly suppressed the heat stress-induced levels of hepatic hsp70 by 34.2% and 31.0%, 3 and 24 h post-heat stress, respectively, compared to sham implanted trout. Additionally, elevated levels of cortisol significantly suppressed the heat stress-induced levels of gill hsp70 by 66.2% in trout (3 h post stress) and 26.7% in tilapia (4 h post stress), compared to sham implanted fish. These results suggest that cortisol may be mediating hsp70 levels in fish tissues following times of physiological stress, and that the neuroendocrine and cellular stress responses may be functionally related in these two different species of fish.

Animals↗

PubMed2001

Synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin.

Recent research has identified endogenous cationic antimicrobial peptides as important factors in the innate immunity of many organisms, including fish. It is known that antimicrobial activity, as well as lysozyme activity, can be induced in coho salmon (Oncorhynchus kisutch) mucus after exposure of the fish to infectious agents. Since lysozyme alone does not have antimicrobial activity against Vibrio anguillarum and Aeromonas salmonicida, a four-step protein purification protocol was used to isolate and identify antibacterial fractions from bacterially challenged coho salmon mucus and blood. The purification consisted of extraction with hot acetic acid, extraction and concentration on a C(18) cartridge, gel filtration, and reverse-phase chromatography on a C(18) column. N-terminal amino acid sequence analyses revealed that both the blood and the mucus antimicrobial fractions demonstrated identity with the N terminus of trout H1 histone. Mass spectroscopic analysis indicated the presence of the entire histone, as well as fragments thereof, including a 26-amino-acid N-terminal segment. These fractions inhibited the growth of antibiotic-supersuscptible Salmonella enterica serovar Typhimurium, as well as A. salmonicida and V. anguillarum. Synthetic peptides identical to the N-terminally acetylated or C-terminally amidated 26-amino-acid fragment were inactive in antimicrobial assays, but they potentiated the antimicrobial activities of the flounder peptide pleurocidin, lysozyme, and crude lysozyme-containing extracts from coho salmon. The peptides bound specifically to anionic lipid monolayers. However, synergy with pleurocidin did not appear to occur at the cell membrane level. The synergistic activities of inducible histone peptides indicate that they play an important role in the first line of salmon defenses against infectious pathogens and that while some histone fragments may have direct antimicrobial effects, others improve existing defenses.

Aeromonas↗

PubMed2000

Antimicrobial peptides protect coho salmon from Vibrio anguillarum infections.

Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.

Amino Acid Sequence↗

PubMed1999

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and ovulation rate in female coho salmon, Oncorhynchus kisutch, close to final maturation.

Plasma levels of 17beta-estradiol, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), and testosterone were measured in adult female coho salmon in late vitellogenesis, approximately 1.5 months before spawning and just before and following intraperitoneal injection with the aromatase inhibitor (AI) Fadrozole. Injection at dosages of 0.1, 1.0, and 10.0 mg AI/kg body wt caused a significant drop in plasma 17beta-estradiol levels relative to preinjection values within 3 or 6 h. Injection of 10 mg AI/kg body wt caused a significant increase in plasma 17alpha-20beta-P levels within 3 h. Ten days after injection 67% of the fish treated with 10 mg AI/kg body wt had ovulated in contrast with 0% in the group injected with 0.1 mg AI/kg body wt. The fertilization rate of the eggs varied between 96% in the control group and 85% in the groups injected with AI. We conclude that the shift from 17beta-estradiol to 17alpha,20beta-P biosynthesis, which is characteristic of maturing Oncorhynchus sp., was advanced significantly by treatment with AI and that Fadrozole can be used as a tool to investigate periovulatory endocrine changes in salmon.

Animals↗

PubMed1999

Energy cost of NaCl transport in isolated gills of cutthroat trout.

Few studies have made direct estimates of the energy required for ion transport in gills of freshwater (FW) and seawater (SW) fish. Oxygen consumption was measured in excised gill tissue of FW-adapted cutthroat trout (Oncorhynchus clarki clarki) to estimate the energy cost of NaCl transport in that osmoregulatory organ. Ouabain (0.5 mM) and bafilomycin A1 (1 microM) were used to inhibit the Na+-K+ and H+ pumps, respectively. Both inhibitors significantly decreased gill tissue oxygen consumption, accounting for 37% of total tissue respiration. On a whole mass basis, the cost of NaCl uptake in the FW trout gill was estimated to be 1.8% of whole animal oxygen uptake. An isolated, saline-perfused gill arch preparation was also used to compare gill energetics in FW- and SW-adapted trout. The oxygen consumption of FW gills was significantly (33%) higher than SW gills. On a whole animal basis, total gill oxygen consumption in FW and SW trout accounted for 3.9 and 2.4% of resting metabolic rate, respectively. The results of both experiments suggest that the energy cost of NaCl transport in FW and SW trout gills represents a relatively small (<4%) portion of the animal's total energy budget.

Animals↗

PubMed1998

beta-Naphthoflavone abolishes interrenal sensitivity to ACTH stimulation in rainbow trout.

We report for the first time that beta-naphthoflavone (BNF) abolishes ACTH stimulation of cortisol production in rainbow trout (Oncorhynchus mykiss). There was significantly higher hepatic cytochrome P450 content and ethoxyresorufin O-de-ethylase and uridine-5'-diphosphoglucuronic acid transferase activities in BNF-treated fish than in sham-treated controls. BNF did not significantly affect either plasma turnover or tissue distribution of [3H]cortisol-derived radioactivity. Hepatic membrane fluidity and hepatocyte capacity for cortisol uptake were not altered by BNF as compared with the sham-treated fish. These results taken together suggest that BNF does not affect cortisol-clearance mechanisms in trout. A 3 min handling disturbance period elicited a plasma cortisol response in the sham-treated fish; however, the response in the BNF-treated fish was muted and significantly lower than in the sham fish. This in vivo response corroborates the lack of interrenal sensitivity to ACTH in vitro in the BNF-treated fish, suggesting that BNF affects the ACTH pathway in trout. Our results suggest the possibility that cytochrome P450-inducing compounds may affect cortisol dynamics by decreasing interrenal responsiveness to ACTH stimulation in fish, thereby impairing the physiological responses that are necessary for the animal to cope with the stressor.

Adrenocorticotropic Hormone↗

PubMed1998

Increased heat shock protein expression after stress in Japanese quail.

Heat shock proteins (HSPs) have been shown to provide information on the biological impact of environmental stress to organisms, yet none have investigated the HSP response to stress in birds. Japanese quail were exposed to seven different stressors (mild restraint, loud noise, inescapable irritation, cold temperature, isolation in darkness, and two stressful social situations) and expression of HSP30, 60, 70, and 90 in heart, liver, lung, kidney and gonads was examined. Tonic Immobility (TI) tests were also conducted to assess whether the stressors increased fear response. Increased expression of HSP70 was found in the myocardial tissue of birds exposed to loud noise, inescapable irritation, cold temperature, and isolation in darkness. Increased expression of other HSPs was not apparent in the heart or any of the other all tissues examined. Longer TI was observed only in birds exposed to the noise stress. Evidence is presented that a fairly wide range of stressors caused increased expression of HSP70 in the Japanese quail myocardial tissue and that HSPs may provide useful biomarkers for the study of environmental stress in birds.

Acoustic Stimulation↗

PubMed1997

Immunolocalization of proton-ATPase in the gills of the elasmobranch, Squalus acanthias.

Proton-ATPase was localized to mitochondria-rich cells in the interlamellar region of the gills of the elasmobranch, Squalus acanthias. Localization was accomplished using a polyclonal antibody specific for the 70 kDa subunit of the (V-type) proton-ATPase as confirmed by Western blot analysis. In addition, significant levels of N-ethymaleimide sensitive ATPase activity (0.116 +/- 0.026 mumol Pi.mg-1 protein.h-1) were also measured in crude gill membrane preparations. These data provide, for the first time, direct evidence of the localization of elements possibly involved in branchial acid-base (or ionic) regulation in elasmobranchs.

Adenosine Triphosphate↗

PubMed1997

The effect of the aromatase inhibitor fadrozole and two polynuclear aromatic hydrocarbons on sex steroid secretion by ovarian follicles of coho salmon.

A variety of endogenous and exogenous factors can influence sex steroid production by salmon ovarian follicles and ultimately impact reproductive development. We examined the effect of an aromatase inhibitor, fadrozole, and common environmental contaminants (PAHs) on sex steroid secretion by ovarian follicles. Ovarian follicles of coho salmon were incubated in vitro with various concentrations of testosterone (0.10-0.40 microM) and fadrozole (10 and 100 microM), or with varying doses (between 0.05 and 5.0 microM) of the PAHs beta-naphthoflavone (BNF) and 20-methylcholanthrene (20-MC). 17 beta-Estradiol secretion was significantly reduced when follicles were incubated in the presence of fadrozole, BNF, or 20-MC. In contrast, 17 beta-estradiol production by ovarian follicles increased in a dose-dependent manner when incubated with increasing doses of the aromatizable androgen testosterone. Although increasing doses of PAHs significantly reduced follicular 17 beta-estradiol production no effect on testosterone secretion was observed. Hence, both fadrozole and PAHs can significantly reduce 17 beta-estradiol secretion by salmon ovarian follicles and may affect female sexual development.

Animals↗

PubMed1997

Handling stress does not affect the expression of hepatic heat shock protein 70 and conjugation enzymes in rainbow trout treated with beta-naphthoflavone.

A response in heat shock protein 70 (hsp 70) expression in the beta-naphthoflavone (BNF) treated rainbow trout (Oncorhynchus mykiss) corresponded to altered metabolic status of the liver as evidenced by the lower phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase and 3-hydroxyacylcoA dehydrogenase activities. The BNF-induced increase in hsp70 levels and conjugation enzyme activities (phase I and phase II) were not modified by handling stress. Indeed handling stress did not affect either hsp 70 levels or conjugation enzyme activities in trout liver. The decrease in hepatic PEPCK activity in the BNF group may be responsible for the attenuation of the increase in liver glucose concentration after a 3 min handling stress in this species, suggesting that BNF affects liver gluconeogenic capacity in this species. Handling stress elicited a plasma cortisol and glucose response in both the sham and BNF group, however, the cortisol response with BNF was erratic compared with the sham, implying alterations in the cortisol dynamics post-stress. These results show for the first time that BNF affects cellular metabolic responses to stress and suggests the possibility of using hsp 70 as a biomarker for toxic effects in trout.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1995

Haemocyte changes in the Pacific oyster, Crassostrea gigas, caused by exposure to domoic acid in the diatom Pseudonitzschia pungens f. multiseries.

The physiological effects of domoic acid on the immune system of marine invertebrates, which are known to accumulate this neurotoxin, have not been investigated previously. Changes in the number and relative phagocytic activity of circulating haemocytes in Crassostrea gigas exposed to domoic acid in the diatom Pseudonitzschia pungens f. multiseries for 48 hr, followed by clearance for 240 hr, were investigated using chemiluminescence. An initial stress response to the toxin assimilation was characterized by a marked increase in number and activity of haemocytes with 4 hr of exposure to the algae. Although the toxin level in the oyster increased during the 48 hr exposure, both number and activity of haemocytes declined from the 4 hr peak values to those significantly lower than control values after 24 hr clearance. This suppression in number and activity of circulating haemocytes following the initial toxin response was rectified only after 48 hr clearance, when domoic acid levels in the oyster tissue had declined to trace levels, allowing blood cells to regain their normal characteristics.

Analysis of Variance↗

PubMed1994

DNA fingerprinting used to test for family effects on precocious sexual maturation in two populations of Oncorhynchus tshawytscha (Chinook salmon).

Two single locus Variable Number Tandem Repeat (VNTR) DNA probes were used to test for differences in allele distribution between precociously mature male and immature chinook salmon, Oncorhynchus tshawytscha. Two populations were examined: Robertson Creek (RC) adult salmon, and Nicola River (NR) freshwater juveniles, or parr. Genomic DNA was extracted from 74 RC precociously mature adult males ('jacks') and 94 RC immature adults of the same age and from 45 NR precociously mature parr and 51 NR nonmaturing parr. The genomic DNA was hybridized with a single locus VNTR probe developed for chinook salmon (OtSL1), as well as one developed for Atlantic salmon, Salmo salar (Ssa1). The allele frequency distributions at both loci were significantly different for the RC jacks and immature fish, indicating a family effect on the incidence of precocious maturation in that population. No difference was found between the allele frequency distribution of the NR precocious and immature parr. A bin width sensitivity analysis showed that the comparisons of the allele frequency distributions were insensitive to the choice of bin size. No differences in heterozygosity were found between mature and immature fish at either locus for both stocks. Preliminary testing for family effects on phenotypes of interest, such as alternative life history strategies, can be performed using hypervariable VNTR DNA probes, prior to implementing costly and involved breeding programmes.

Alleles↗