棘头梅童鱼肝型脂肪酸结合蛋白基因克隆及组织表达分析

为进一步了解肝型脂肪酸结合蛋白基因(L-FABP基因)在棘头梅童鱼(Collichthys lucidus)营养调控、脂肪酸代谢和免疫应答等方面的功能作用,以棘头梅童鱼全基因组组装数据为基础,获得L-FABP基因的含开放阅读框的cDNA序列,利用生物信息学方法分析编码的蛋白质结构及进化关系,采用荧光定量(qRT-PCR)技术研究了不同组织中L-FABP基因的表达情况。结果表明,L-FABP基因cDNA序列为475 bp,其中5’非翻译区为51 bp,3’非翻译区为40 bp,开放阅读框为384 bp,编码127个氨基酸。其结构由10个β-折叠链和2个α-螺旋链组成,在氨基酸3~126位点间具有保守的脂质/胞质性脂肪酸结合结构域。棘头梅童鱼L-FABP基因与大黄鱼、条纹狼鲈同源性较近,与绿河鲀、尼罗罗非鱼和斑马鱼同源性较远。L-FABP基因在棘头梅童鱼肝脏、肠道、眼、大脑、心脏、肌肉、胃、鳃、脾脏、头肾和中肾中均有表达,其中肝脏中的L-FABP相对表达量最高,显著高于其他组织(P<0.05)。     

草鱼肝微粒体的提取及CYP酶活性的测定

CYP酶代谢是药物生物转化的主要途径,其数量和活性大小直接影响药物在体内的活化与代谢。我们对草鱼肝微粒体CYP酶含量及其活性进行了初步研究,以差速离心法提取草鱼肝微粒体,以CO还原差示光谱法测得CYP酶及细胞色素b5含量分别为0.619±0.102 nmol/mg、0.264±0.042 nmol/mg。以7-乙氧异吩噁唑酮-O-脱乙基反应、苯胺-4-羟化反应、氨基比林-N-脱甲基反应作为CYP1A、CYP2E、CYP3A的探针反应,测得EROD酶活为0.043±0.004 nmol/mg/min,ANH酶活为0.028±0.002 nmol/mg/min,AMND酶活为0.207±0.035 nmol/mg/m in。结果表明草鱼肝微粒体中CYP酶发育完好,并且具有参与药物代谢的3种主要亚型活性,其含量与活性大小与其它实验动物相差较大。本实验的方法与结果为草鱼CYP酶的系统研究提供可靠手段,最终为指导水产合理用药提供理论依据。     

The cytochrome P450 system of Atlantic salmon (Salmo salar): II. Variations in hepatic catalytic activities and isozyme patterns during an annual reproductive cycle

A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5–7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17 was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b₅, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b₅, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17 levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17 as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17 independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goks\/oyr 1989).     

Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout (Oncorhynchus mykiss). II. Adaptive response of glucose 6-phosphate dehydrogenase activity to high-carbohydrate/low-protein and high-fat/non-carbohydrate diets

The influence of long-term administration of high-carbohydrate/low-protein and high-fat/non-carbohydrate diets were studied in relation to kinetic behaviour of glucose 6-phosphate dehydrogenase in liver and kidney of rainbow trout. In all cases studied, the saturation curves of these enzyme showed typical hyperbolic kinetics without evidence of sigmoidicity. After 30 days of feeding with a high-fat diet (170 g kg^?1), there was a significant decrease in V_max and specific activity (45%) as well as catalytic efficiency (39%) without changes in K_m or activity ratio of hepatic glucose 6-phosphate dehydrogenase. These changes agree more with a clearly decreased cell concentration than with an inhibition of the pre-existing enzyme. The administration of a high-carbohydrate diet (60 g kg^?1), contrary to what was previously thought, decreased V_max by 21% and specific activity and catalytic efficiency by 30%, without significant changes in the other kinetic parameters of the hepatic enzyme. The kinetic behaviour under these nutritional conditons was due to the rejection of this diet by the fish and thus could be considered a low-feeding situation. On the other hand, no variations in the kinetics of renal glucose 6-phosphate dehydrogenase were found, clearly demonstrating that in this organ, the pentosephosphate pathway showed no adaptive response related to fattyacid and other lipid synthesis. The activity of the renal enzyme was consistently half that of the hepatic enzyme.     

Extrapituitary gonadotropin-releasing hormone (GnRH) binding sites in goldfish

In teleosts, as in other vertebrates, the secretion of pituitary gonadotropin (GTH) is mediated by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). Recent findings in teleosts indicate that GnRH receptors are not restricted to the pituitary gonadotropes and are also associated with somatotropes as well as being present in a number of other tissues. In the present study, we provide novel information on GnRH binding in a number of extrapituitary tissues in goldfish. However, we do not intend to provide full characterization of GnRH binding sites in various extrapituitary tissues in goldfish as this would clearly be outside the scope of this paper. In this study we examined GnRH binding in a number of extrapituitary tissues in goldfish and observed specific binding in ovary, testis, brain, liver and kidney. No specific GnRH binding was observed in muscle, skin, gut, gill and heart. In general, the present findings together with the results of other studies carried out in our laboratory demonstrate that mature goldfish ovary and testis contain two classes of GnRH binding sites, high affinity/low capacity and low affinity/high capacity sites with binding characteristics similar to those of the pituitary GnRH receptors. The brain of goldfish was also found to contain two classes of GnRH binding sites, a super-high affinity/low capacity and a low affinity/high capacity sites. Furthermore, study of goldfish liver and kidney demonstrated the presence of a single class of GnRH binding sites with characteristics different from those of pituitary, ovary, testis and brain. Overall, it is evident that goldfish contains a family of GnRH binding sites which can be classified into four groups based on binding affinities: 1) A class of high affinity binding sites present in the pituitary, ovary and testis, 2) a class of super high affinity sites so far only detected in the brain, 3) a class of intermediate-affinity GnRH binding sites in the liver and kidney, and 4) a class of low affinity binding sites present in all the tissues containing specific GnRH binding sites except for liver and kidney.     

Hepatocellular adaptation to extreme nutritional conditions in ide,Leuciscus idus melanotus L. (Cyprinidae). A morphofunctional analysis

The adaptive response of the omnivorous ide,Leuciscus idus melanotus, to drastic metabolic conditions was analyzed on different levels of organisation investigating a variety of parameters: Organism (condition factor, liver-somatic index), organ (liver structure), cellular and subcellular level (hepatocyte structure, glycogen and lipid storage, contents and distribution of nucleic acids, enzyme alterations).During starvation, ide were able to maintain liver integrity in a biphasic process: after an initial phase of disturbance, ide established a new structural and metabolic homeostasis. Recovery from starvation was possible only with a complete diet but not with a sucrose diet. Carbohydrate overload, as evoked by sucrose refeeding, did not result in liver or carcass fattening as known from mammals.To the best of our knowledge, the present study is the first to use enzyme histochemistry in fish nutrition research. In mammals, histochemistry is particularly helpful for understanding processes of hepatic metabolic adaptation. In fish, however, on the basis of our results, enzyme histochemical studies appear to be of limited value, as long as no further data are available on a zonal distribution of enzyme activities in teleost liver parenchyma. Instead, the histochemical detection of the distribution of hepatic storage products and RNA-positive material yielded important information on liver adaptive processes.Abbreviations H starvation - SU sucrose - AD artificial diet - AcP acid phosphatase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GLU -glucuronidase - ME malic enzyme - PHO glycogen phosphorylase - UE unspecific esterase - RER rough endoplasmic reticulum     

Interacting effects of diet and environmental temperature on biochemical parameters in the liver ofLeuciscus idus melanotus (Cyprinidae: Teleostei)

One year old golden ide (Leuciscus idus melanotus) were starved for 2 weeks at 20°C (time zero) and then they were fed either freeze-dried mosquito larvae (natural diet) or a commercial fish chow (artificial diet) at an ambient temperature of 14°C and 20°C, respectively. Growth parameters and biochemical data in the liver were measured at time zero and after 3 and 7 weeks of specific regimen. If compared to natural food, the artificial diet caused transient increase in anabolic activity of the liver, but prevented long-lasting hepatocyte proliferation (increase of total liver DNA). After 3 weeks on artificial diet, the body weight was significantly higher and the liver mass doubled compared to ide kept on the natural regimen; tissue DNA content indicated that the hepatocyte volume increased mainly by increased protein content and corresponding uptake of water; lipid and glycogen contents were increased by a factor of 3–6, and the RNADNA ratio was increased accordingly. The growth of the ide, as reflected by the condition factor and hepatosomatic index (HSI), was virtually stopped when the artificial regimen was maintained for 7 weeks, whereas on natural food the condition factor was increased and liver weight and DNA were doubled. Growth of the fish maintained at 14°C was significantly lower than that of fish maintained at 20°C, though the total liver DNA was still increased on the natural regimen during the experiment. Less lipid was stored by fish maintained at 14°C, whereas protein and glycogen deposits were enlarged, if compared to fish maintained at 20°C. The specific activity of cytochrome oxidase in liver mitochondria and of NADPH cytochrome c reductase in the microsomal fraction were found independent of diet and ambient temperature. Oxidative capacity of hepatocytes (mitochondrial protein/mg DNA) remained unchanged, and microsomal protein/cell appeared reduced in response to the artificial food. However, reduction of cell number on this diet resulted in less total mitochondrial and microsomal protein in the organ. Golden ide liver is found to be a sensitive experimental model to characterize the mutual influence of diet and temperature on fish; the results are discussed with regard to the usage of golden ide as test fish for water pollution.     

Role of molecular species catabolism in the temperature-induced restructuring of phosphatidylcholines in liver microsomes of thermally-acclimated rainbow trout (Oncorhynchus mykiss)

Most previous studies of the temperature-induced restructuring of phospholipid molecular species composition have examined steps in the biosynthesis of phospholipids to explain the accumulation of unsaturated fatty acids in membranes of cold-acclimated poikilotherms. In contrast, the present study explores the role of phospholipases in this restructuring process by determining the rates of degradation of specific molecular species of phosphatidylcholine, using enzymes (microsomes) freshly isolated from the liver of rainbow trout. (Oncorhynchus mykiss) acclimated to either 5° or 20°C. The substrate preparation employed to assay phospholipase activity possessed a range of molecular species, all radiolabeled with 1-¹⁴C-palmitic acid at thesn-1 position, similar to that present in native trout liver microsomes. After defined periods of incubation (120 and 240 min at 5°C; 60 and 120 min at 20°C), phospholipids were extracted from the reaction mixture and the distribution of radioactivity among the molecular species of phosphatidylcholine was determined by HPLC/liquid scintillation counting. In general, molecular species catabolism was not significantly influenced by either assay or acclimation temperature. Only in 20°C-acclimated fish did a reduction in assay temperature (from to 20 to 5°C) result in significantly increased proportions of radioactivity being recovered in one polyunsaturated fatty acid-containing species (16:0/22:6-PC). It is concluded: 1) that phospholipase specificity, assayed under conditions approximating thosein situ, is not significantly influenced by temperature; and 2), that the increased proportions of unsaturated fatty acid-containing molecular species of phosphatidylcholine observed at low temperatures must reflect the specificity of biosynthetic rather than degradative processes.     

Feed deprivation of channel catfish, Ictalurus punctatus (Rafinesque), influences organosomatic indices, chemical composition and susceptibility to Flavobacterium columnare

Withholding feed has been suggested as a strategy to manage infectious disease of channel catfish, Ictalurus punctatus (Rafinesque). In a previous study, we demonstrated that deprivation of feed for as little as 7 days reduced innate resistance of catfish to Flavobacterium columnare. This study was conducted to evaluate the effect of feeding regimens [no feeding (NF), fed once every other day to satiation (FEOD) and fed once daily to satiation (FD)] on organosomatic indices, physiological changes and susceptibility of channel catfish to F. columnare. Fish that were not fed for 2 and 4 weeks had a significant increase (P < 0.05) in gutted weight:-wet weight ratio and decrease in other organosomatic indices [gut index (GI), mesenteric fat index (MFI) and hepatosomatic index (HSI)]. Haematology was not effected by feeding regimen except at week 4, when a significantly higher haemoglobin level was observed in the NF fish. Serum protein did not differ at week 2, but the level at week 4 of the NF fish (35.91 mg mL(-1)) was significantly lower than that of the FD fish (41.77 mg mL(-1)). Significantly lower (P < 0.05) blood glucose (39.5 and 40.3 mg dL(-1)) and liver glycogen (1.7 and 1.8 mg g(-1)) were seen in the NF fish at weeks 2 and 4, respectively, as compared with blood glucose and liver glycogen levels of FD fish (67.5 and 92.8 mg dL(-1) and 46.5 and 52.6 mg g(-1) at weeks 2 and 4, respectively) and FEOD (82.8 and 85.5 mg dL(-1) and 45.1 and 51.4 mg g(-1) at weeks 2 and 4, respectively). Mortality in the NF fish caused by F. columnare (78%) was significantly higher (P < 0.05) than mortality in the FD and FEOD treatments (0.0 and 1.7%, respectively). Blood glucose and liver glycogen showed the same trend of low values for NF fish following challenge (week 6). Blood glucose, liver glycogen, GI and HSI are sensitive indicators for channel catfish deprived of feed (NF) for 4 weeks. Blood glucose and liver glycogen levels around 40 mg dL(-1) and 2 mg g(-1), respectively, are indicative of starvation in juvenile channel catfish. Moreover, NF fish were susceptible to F. columnare infection. Thus, it is suggested that in the absence of natural food, juvenile channel catfish should be fed at least once every other day to apparent satiation to maintain normal physiological function and improve resistance to F. columnare.     

Studies on the green liver in cultured red sea bream fed low level and non-fish meal diets: Relationship between hepatic taurine and biliverdin levels

SUMMARY: The aim of this study was to investigate the cause of green liver in red sea bream fed substitute protein diets. Red sea bream Pagrus major was given either of the following diets for 28 weeks: (1) control diet (50% fish meal), (2) low level fish meal diet (15% fish meal), and (3) non-fish meal diet (0% fish meal). The green liver was observed in all groups tested, but the incidence was much higher in the experimental diet groups. The feeding of substitute protein diets reduced plasma triglyceride and cholesterol levels. However, there was no significant difference in plasma hepatic enzyme activities and plasma bile salts concentration among the treatments. Fish fed the substitute protein diets showed low hepatic taurine levels with an appearance of a biliverdin in the liver. Moreover, the proportion of ditaurobilirubin to total biliary bile pigments was significantly lower in fish fed the substitute protein diets. These data indicate that feeding of substitute protein diets did not induce any cholestatic hepatobiliary obstructions and that the low hepatic taurine level was one of the probable factors responsible for the occurrence of green liver in red sea bream fed substitute protein diets.