Localization of dopamine in the freshwater hydrozoanHydra attenuata

Summary High performance liquid chromatography (HPLC), with electrochemical detection, is an analytical method sensitive enough to permit quantification of dopamine, dihydroxyphenylalanine (DOPA) and 5-S-cysteinyl DOPA in single or hemisected specimens ofHydra attenuata. Dopamine and 5-S-cysteinylDOPA appear to be the quantitatively predominant catechol compounds inH. attenuata, whereas DOPA is present in minor amounts. The presence of DOPA and 5-S-cysteinylDOPA, and the quantitative correlation between dopamine and these compounds in many specimens, suggests that dopamine inH. attenuata, as in higher animals, is formed through decarboxylation of DOPA. Contrary to the dopaminergic nerves in higher animals, DOPA inHydra seems to be oxidized and 5-S-cysteinyl DOPA is formed as a by-product. The oxidation of DOPA indicates that the hydroxylation of tyrosine into DOPA in the tissues ofH. attenuata is mediated by a tyrosinase rather than a tyrosine hydroxylase. Immunocytochemical methods demonstrate a highly variable distribution of dopamine in the tissues of different specimens ofH. attenuata. Dopamine immunoreactivity is confined to ectodermal tissue and can be found in several different cell types including nerve cells, battery cells, nematocytes, epithelial cells and interstitial undifferentiated cells. The large amounts of dopamine found in some specimens ofH. attenuata indicate some biological function, although its sporadic occurrence in neurites makes it less plausible as a generally utilized neurotransmitter in this animal.     

鳗鲡出血性开口病的病理学研究

本文报告了我国发生的一种鳗鲡病毒病──鳗鲡出血性开口病的组织病理变化：肝、肾、脾脏组织出血、细胞变性,骨质内有大县白细胞浸润,肝、肾、脾脏细胞超微结构病理变化明显,肾、脾脏造血组织和外周血细胞出现核染色质边集、奇异形核,大量髓鞘样结构、自噬体和自噬溶酶体等,并可见类似凋亡细胞及调亡小体结构和邻近细胞内吞噬体增多现象。根据骨组织中白细胞浸润及器官和血液中部分细胞结构已出现异型性特征,作者认为,鳗鲡出血性开口病可能有癌变的趋势．     

Selectivity of mixed-function oxygenase enzyme induction in flounder (Pseudopleuronectes americanus) collected at the site of the Baie Verte, Newfoundland oil spill

A marked level of mixed-function oxygenase (MFO) enzyme induction was observed in kidney but not in liver tissues of winter flounder collected at the site of a spill of No. 2 fuel oil in Newfoundland. A number of MFO-linked, electron transport components including cytochrome P450, cytochrome P450 reductase and cytochrome b5 reductase were also slightly elevated in kidney tissues of fish at the oiled site. The results obtained in this particular field trial demonstrate that reliance on the measurement of liver MFO parameters alone could lead to false negatives in biological monitoring programs. The study also indicates that the exclusive use of liver tissues to investigate the induction potential of various chemicals should be avoided.     

Gulonolactone oxidase is missing in teleost fish. The direct spectrophotometric assay

Data in the literature imply that some fish species evolved with the capacity to synthesize ascorbic acid. Gulonolactone oxidase activity has been reported in kidney and/or liver tissues. However, it is shown here that this microsomal enzyme activity is missing in common carp hepatopancreas and kidney, whereas high activity was confirmed in pigeon kidney, rat liver, bovine liver and amphibian (Xenopus) kidney tissues. A new assay using either the whole tissue homogenate or microsomes solubilized by sodium deoxycholate was developed to directly measure the formation of ascorbic acid spectrophotometrically. Identical values were found using this assay as well as the assay in which formed ascorbate was determined by the dinitrophenyl hydrazine (DNPH) method. In some experiments, these results were confirmed by polarographically measured oxygen consumption.     

Heat stress‐induced alterations of antioxidants in the freshwater fish Channa punctata Bloch

The effect of elevated temperature on the antioxidants in the freshwater fish Channa punctata was investigated. Fish stressed with an elevated temperature of 12° C, range ± 1° C over the ambient temperature for 3 h showed a significant ( P  < 0·05–0·01) reduction in the levels of antioxidants: reduced glutathione (GSH) and non‐protein thiols. Activity of glutathione reductase was also reduced in all the tissues (liver, kidney and gills) after 3 h of heat stress and 24 h recovery. Catalase (CAT) showed enhanced activity in liver in both the conditions while gills and kidney showed a decreased CAT activity. Glutathione S‐transferase (GST) activity in kidney and liver decreased significantly ( P  < 0·05–0·01) after 3 h of heat stress. At 24 h GST activity showed a tendency to normalize in all the tissues along with a concomitant increase in the GSH level in the kidney. Total and protein thiols in heat stressed fish when matched with controls, showed significant ( P  < 0·05) reduction in the kidney only with a transient increase in liver and gills. Heat shock also induced lipid peroxidation in 3 h heat‐treated and recovery groups when compared with controls. Elevated temperature therefore resulted in tissue specific and time‐dependent alterations of antioxidants in the fish. It also induced lipid peroxidation in various tissues.     

Physiological and metabolic adjustments of Hoplosternum littorale (Teleostei,Callichthyidae) during starvation

All animals face the possibility of limitations in food resources that could ultimately lead to mortality caused by starvation. The primary goal of this study was to characterize the various physiological strategies that allow fish to survive starvation. A multiparametric approach, including morphological biomarkers, blood plasma metabolites, oxidative stress and energy reserves, was used to assess starvation effects on the fish Hoplosternum littorale. Adult specimens were maintained at four experimental groups: control (fed ad libitum), and starved (not fed) fish for 7 and 28 days. Significant changes were observed not only after 28 days, but also after 7 days of starvation. In the shorter period, the hepatosomatic index as well as plasma triglycerides and glucose were significantly lower in starved fish than in the control ones. These results were accompanied by reduced lipid, glycogen and protein reserves in liver and diminished glycogen content in muscle, suggesting the need of these macromolecules as fuel sources. In addition, increased antioxidant enzyme activities were observed in gills, without evidence of oxidative stress in any of the evaluated tissues. Most significant differences were found in 28-days starved fish: total body weight together with the hepatosomatic index was lower when compared to control fish. The plasmatic metabolites tested (glucose, triglyceride, cholesterol and protein), all energy reserves in liver and glycogen content in muscle decreased in 28-days starved fish. Lipid oxidative damage was reported in liver, kidney and brain, and antioxidant enzymes (GST, GR, GPx and CAT) were activated in gills. According to the multivariate analysis, oxidative stress markers and metabolic parameters were key biomarkers that contributed in separating starved from fed fish. Our study allowed an integrated assessment of the fish response to this particular condition.     

Hepatic and nonhepatic sterol synthesis and tissue distribution following administration of a liver selective HMG-CoA reductase inhibitor, CI-981: comparison with selected HMG-CoA reductase inhibitors.

Since cholesterol biosynthesis is an integral part of cellular metabolism, several HMG-CoA reductase inhibitors were systematically analyzed in in vitro, ex vivo and in vivo sterol synthesis assays using [14C]acetate incorporation into digitonin precipitable sterols as a marker of cholesterol synthesis. Tissue distribution of radiolabeled CI-981 and lovastatin was also performed. In vitro, CI-981 and PD134967-15 were equipotent in liver, spleen, testis and adrenal, lovastatin was more potent in extrahepatic tissues than liver and BMY21950, pravastatin and PD135023-15 were more potent in liver than peripheral tissues. In ex vivo assays, all inhibitors except lovastatin preferentially inhibited liver sterol synthesis; however, pravastatin and BMY22089 were strikingly less potent in the liver. CI-981 inhibited sterol synthesis in vivo in the liver, spleen and adrenal while not affecting the testis, kidney, muscle and brain. Lovastatin inhibited sterol synthesis to a greater extent than CI-981 in the spleen, adrenal and kidney while pravastatin and BMY22089 primarily affected liver and kidney. The tissue distribution of radiolabeled CI-981 and lovastatin support the changes observed in tissue sterol synthesis. Thus, we conclude that a spectrum of liver selective HMG-CoA reductase inhibitors exist and that categorizing agents as liver selective is highly dependent upon method of analysis.     

The effect of consuming different proportions of hummer fish on biochemical and histopathological changes of hyperglycemic rats

Hammour fish (grouper fish) are known to be of great nutritional value for human consumption, as their protein has a high biological value and contains all the essential amino acids. Grouper fish are also a good source of minerals, vitamins, and fats that contain essential fatty acids. Thus, the current study aims to know the effect of different proportion of hummer fish on biochemical and histopathological changes of hyperglycemic rats. Twenty-four (24) Sprague Dawley-strain male albino rats, which weighed 150 ± 10 g, were divided into four groups. One group served as the negative control (normal), while the others were rendered diabetic using alloxan. One of the diabetic groups was considered the positive control and fed a standard diet, whereas the remaining two groups were fed with a 20% and 25% hammour fish diet for 28 days. At the end of the experiment, blood samples were taken from all the rats, and their organs were removed and subjected to biochemical analysis. The results indicated that the group fed with the 25% hammour fish diet exhibited significantly lower levels of liver, kidney, and heart damage, along with lower levels of serum glucose, total cholesterol, triglycerides, LDL, GOT, GPT and ALP, as compared to the positive control. The urea and creatinine levels were significantly higher for the rats that were fed the 20% hammour fish diet than for those in the positive control. The histopathological study of the heart showed a slight improvement of the heart tissues with the increase of hammour fish intake compare to the positive control, while kidney of rat from group 4, which were fed 25% hammour fish, showed granularity of epithelial lining glomerular tufts.     

嘉陵江8种鱼类不同组织微量元素含量分析

测定了黄颡鱼（Pelteobagrus fulvidraco）、中华倒刺钯（Spinibarbus sinensis）、大鳍鲠（Mystus macropterus）、长吻鲍（Leiocassis longirostris）、大眼鳜（Siniperca kneri）、小口鲇（Silurus asotus）、岩原鲤（Procypris rabaudi）和圆口铜鱼（Coreius guichenoti）8种嘉陵江名贵鱼不同组织微量元素含量及肌肉和脑中Ca、P含量,结果表明,鱼脑中各类矿物质元素显著高于肌肉;肌肉5种微量元素总量由低到高依次为：圆口铜鱼、中华倒刺鳃、长吻鲍、大鳍鲮、黄颡鱼、大眼鳜、岩原鲤、小口鲇;而脑由低到高依次为：大眼鳜、岩原鲤、小口鲇、圆口铜鱼、大鳍鲢、中华倒刺鳃、长吻鲍、黄颡鱼。微量元素在各内脏器官的分布：铁和铜以肝（胰）脏最高,肾脏其次,胃肠最低;锰和锌的含量肾脏〉肝（胰）脏〉胃肠;镁的分布各种鱼差异较大。     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

ATP and ADP hydrolysis in the kidney and liver of fish, chickens and rats

We investigated NTPDase-like activity [ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases)] in liver and kidney membrane from silver catfish (Rhamdia quelen), chicken (Gallus gallus) and rat (Rattus norvegicus) under different conditions and in the presence of several inhibitors. The cation concentration required for maximal activity was 0.5, 1.5 and 2.0 mM for fish, chicken and rat liver, respectively (with ATP and ADP as substrates). The maximal activity in the kidney was observed at calcium concentrations of 0.5, 2.0, 1.5 mM (ATP) and 0.5, 1.5, 1.0 (ADP) for fish, chickens and rats, respectively. The results showed that the pH optimum for all animals and for the two tissues was close to 8.0. The temperature chosen was 25 °C for fish and 36 °C for chicken and rat preparations. Ouabain had no effect on the NTPDase-like activity of fish, chickens or rats. NTPDase activity was decreased in the presence of lanthanum in the chicken (ADP) and rat (ATP and ADP) liver. In the kidney, lanthanum inhibited fish ATP and rat ATP and ADP (0.2 mM) hydrolysis. N-ethylmaleimide (NEM) had an inhibitory effect on the kidney of all species at the concentration of 3.0 mM (ADP). Orthovanadate only inhibited fish membrane NTPDase; azide only inhibited the preparation at high concentrations (10 mM) and fluoride inhibited it at 10 mM (fish and chicken) and 5 mM (rat). Trifluoperazine (0.05–0.2 mM) and suramin (0.03–0.3 mM) inhibited NTPDase at all concentrations tested. These results suggest that NTPDase-like activity shows a different behavior among the vertebrate species and tissues studied. Additionally, we propose that NTPDase1 is the main enzyme present in this preparation.     

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na⁺,K⁺)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na⁺,K⁺)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na⁺,K⁺)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na⁺,K⁺)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change. Accepted: 21 January 1997     

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.     

Interactive effects of high stocking density and food deprivation on carbohydrate metabolism in several tissues of gilthead sea bream Sparus auratus

The influence of high stocking density (HSD) and food deprivation was assessed on carbohydrate metabolism of several tissues of gilthead sea bream Sparus auratus for 14 days. Fish were randomly assigned to one of four treatments: (1) fed fish under normal stocking density (NSD) (4 kg m(-3)); (2) fed fish under HSD (70 kg m(-3)); (3) food-deprived fish under NSD; and (4) food-deprived fish under HSD. After 14 days, samples were taken from the plasma, liver, gills, kidney and brain for the assessment of plasma cortisol, levels of metabolites and the activity of several enzymes involved in carbohydrate metabolism. HSD conditions alone elicited important changes in energy metabolism of several tissues that in some cases were confirmatory (5-fold increase in plama cortisol, 20% increase in plasma glucose, 60% decrease in liver glycogen and 20% increase in gluconeogenic potential in the liver) whereas in others provided new information regarding metabolic adjustments to cope with HSD in the liver (100% increase in glucose phosphorylating capacity), gills (30% decrease in capacity for phosphorylating glucose), kidney (80% increase in the capacity of phosphorylating glucose) and brain (2.5-fold increase in ATP levels). On the other hand, food deprivation alone resulted in increased plasma cortisol, and metabolic changes in the liver (enhanced gluconeogenic and glycogenolytic potential of 13% and 18%, respectively) and brain (10% increase in glycolytic capacity), confirmatory of previous studies, whereas new information regarding metabolic adjustments during food deprivation was obtained in the gills and kidney (decreased lactate levels in both tissues of 45% and 55%, respectively). Furthermore, the results obtained provided, for the first time in fish, information indicating that food deprivation increased the sensitivity of gilthead sea bream to the stress induced by HSD compared with the fed controls, as demonstrated by increased plasma cortisol levels (50% increase vs. fed fish) and a further increase in the capacity to export glucose mobilized from liver glycogen stores (70% decrease vs. fed fish). These results lend support for a cumulative effect of both stressors on plasma cortisol and parameters assessed on carbohydrate metabolism in the present experiments, and provide information regarding reallocation of metabolic energy to cope with simultaneous stressors in fish.     

Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure

Enzymatic and non-enzymatic antioxidants serve as an important biological defense against environmental oxidative stress. Information on antioxidant defense in fish is meager despite that fish are constantly exposed to a myriad of environmental stress including the oxidants. This study, therefore, assesses the activities of antioxidant enzymes viz., glutathione peroxidase, catalase and glutathione S-transferase and the non-enzymatic antioxidants viz., glutathione and metallothionein in various tissues of freshwater fish Channa punctatus (Bloch), in response to short-term and long-term exposures to paper mill effluent. The fish were exposed to the effluent at a concentration of 1.0% (v/v) for 15, 30, 60 and 90 days. The exposure caused a time-dependent increase in glutathione level (P < 0.001), activities of glutathione peroxidase (P < 0.05 to P < 0.001), glutathione S-transferase (P < 0.001) and a marginal initial decrease in catalase activity in the liver (P < 0.01 to P < 0.001). Metallothionein was induced in liver after 60 days of exposure. Two isoforms of metallothionein were detected. Catalase activity also increased 60 days afterwards. Antioxidant pattern was different in gill and kidney showing that liver was more resistant to oxidative damage as compared to gills and kidney. Our results demonstrate a pollutant-induced adaptive response in fish. In addition, levels of enzymatic and non-enzymatic tissue antioxidants may serve as surrogate markers of exposure to oxidant pollutants in fish.     

Acylase distribution in animal and human tissues

Distribution of acylase in different tissues of nine species of animals was studied. The following types of nitrogen elimination were distinguished: ammoniatelic (fish), uricotelic (birds) and uriotelic (amphibians, mammalians). The enzymic activity was estimated in the tissues of the brain, lung, muscle, liver, kidney, spleen, pancreas, small intestine and blood serum. The acylase activity was found in the kidney, liver and pancreas. Its level in the kidney increases with the animal weight growth, the enzyme activity being observed only in the cortical layer.