用粉浆废液提取蛋白的研究

本文研究了由粉浆废液中提取粗蛋白的方法，测定了粗蛋白中蛋白质、氨基酸、脂肪等成份含量。按本文所述方法处理粉浆废液，不仅可以提高产品的附加值，而且可降低污染，利于环保。     

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)。     

Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss)

Uncoupling proteins are mitochondrial anions transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition, and fatty acid metabolism. The mRNA expression of rainbow trout UCP2 genes (UCP2A and UCP2B) was monitored during embryogenesis and early larval development. Both genes were recruited early and displayed similar steadily decreasing patterns from fertilization until hatching. The expression of UCP2A and UCP2B appeared significantly differentiated after hatching and during the yolk sac absorption, with UCP2A displaying higher expression. We suggest that UCP2 expression profiles in the rainbow trout embryo could be associated with the utilization of lipids as a source of energy during development.     

The effects of elevated winter temperature and sub-lethal pollutants (low pH, elevated ammonia) on protein turnover in the gill and liver of rainbow trout (Oncorhynchus mykiss)

Appetite, growth, and protein turnover (synthesis, growth and degradation) of liver and gills were measured in juvenile rainbow trout (Oncorhynchus mykiss) fed to satiation, and exposed for 90 days to elevated winter temperatures (+2 °C above ambient) and either low pH (5.2) in softwater or 70 M total ammonia (TAmm) in hardwater. All fish increased in weight during the experiments, but those exposed to +2°C grew significantly more than those at ambient temperature due to a stimulation of appetite. During the relatively constant temperature of the first 75 days, +2 °C caused a significant increase in the rates of protein synthesis and degradation in the liver of hardwater-acclimated fish, as a result of an increase in RNA translational efficiency (KRNA). The elevated temperature also induced an increase in gill protein synthesis in softwater-acclimated fish but in this case the underlying mechanism was an increase in Cs, the capacity for protein synthesis (RNA:protein) rather than in KRNA. The addition of 70 M TAmm had no effect on protein turnover in either liver or gills of hardwater-acclimated fish. Low pH inhibited protein growth in the liver of softwater-acclimated fish at day 90 under both temperature regimes. This inhibition was effected via a decrease in protein synthesis at control temperature but via an increase in protein degradation when the fish were exposed to both low pH and +2 °C. From these results we conclude that a simulated global warming scenario has potentially beneficial rather than detrimental effects on protein turnover and growth of freshwater fish during winter.     

Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen.     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

Polka dot grouper Cromileptes altivelis fingerlings require high protein and moderate lipid diets for optimal growth and nutrient retention

An 8‐week comparative slaughter experiment was carried out to determine the effect of dietary protein and lipid on growth, apparent digestibility (AD) and nutrient retention of polka dot grouper Cromileptes altivelis. Fingerlings were fed diets that varied in crude protein (CP) at 55 g kg^?1 increments between 410 and 630 g kg^?1 dry matter (DM) and at either a moderate (150 g kg^?1 DM) or high (240 g kg^?1DM) lipid concentration. Each diet was fed to satiety twice daily to four replicate tanks (110 L) of fish. One replicate block of tanks comprised 150 fish of mean (±SD) initial weight of 9.6 ± 0.29 g, which were distributed equally to 10 tanks. The other three replicate blocks of tanks comprised 300 fish of 12.6 ± 0.45 g, which were distributed equally to 30 tanks. Tanks were provided with filtered and heated (29 ± 0.5 °C) seawater in a flow‐through system within a laboratory where photoperiod was maintained at 12 : 12 h light–dark cycle. Voluntary food intake was not significantly affected by either the CP or lipid concentration of the diet (mean ± SD of 1.93 ± 0.146 g week^?1) but there was a trend for intake to be higher on the moderate compared with the high lipid diets (mean ± SEM of 1.97 versus 1.89 ± 0.033 gweek^?1, respectively). Daily growth coefficient (DGC) and food conversion ratio (FCR) improved linearly (P < 0.01) with increasing dietary CP (from 0.94 to 1.35% day^?1 for DGC and 1.58 to 1.00 g DM g^?1 wet gain for FCR) and these responses were almost coincident for each of the lipid series. The AD of CP increased linearly with increasing dietary CP (from 46.8 to 74.1%) and was independent of dietary lipid. Apparent digestibility of energy increased curvilinearly with increasing dietary CP, with the quadratic component being more prominent for the high‐lipid series. Increasing the amount of lipid in the diet markedly increased the lipid content of the fish from an initial composition (mean ± SD) of 173 ± 7.3 g kg^?1 to a final composition (mean ± SEM) of either 217 or 250 ± 5.9 g kg^?1 for moderate and high‐lipid series, respectively. Total body lipid content tended to increase linearly with increasing dietary CP for the high‐lipid series but with an opposite effect for the moderate‐lipid series. The retention of digestible nitrogen decreased linearly with increasing dietary CP but at a steeper rate for the moderate, compared with the high, lipid series (from 62.7 to 35.7%, slope ?0.115 for moderate‐lipid and 54.6 to 41.9%, slope ?0.050 for high‐lipid). A quadratic function of dietary CP concentration best explained the retention of digestible energy with the curvilinearity being more marked for the high, compared with the moderate, lipid diet series. While there was some indication that ingested lipid spared dietary protein, the results showed a far greater propensity of polka dot grouper fingerlings to use protein as the prime dietary energy source. Diets for juvenile polka dot grouper should contain not less than 440 g digestible protein kg^?1 DM and at least 150 g lipid kg^?1 DM.     

Minimising aerobic respiratory demands could form the basis to sub-lethal copper tolerance by rainbow trout gill epithelial cells in vitro

Mechanisms of Cu tolerance were investigated in respiratory epithelial cell cultures, from rainbow trout gills, by studying O₂ consumption and protein synthesis rates, intracellular Na concentration and TER. The lowest concentration found to reduce O₂ consumption was 25 M Cu. This did not affect either protein synthesis rate or intracellular Na concentration and was interpreted in terms of copper tolerance; i.e., how these two energetically demanding processes are maintained despite a reduction in aerobic ATP supply. The relationship between protein synthesis rate and synthesis cost is exponential and the cost of protein synthesis in gill cells was found to be minimal (i.e., this cell occupies a position on the asymptotic section of the protein synthesis rate/synthesis cost model) and unaffected by 25 M Cu. Thus protein synthesis rates could be maintained since any reduction would represent an insignificant energy saving. Intracellular Na concentrations and O₂ consumption rates were linearly correlated suggesting reducing intracellular maintenance costs would have a greater significance in terms of overall energetic conservation. Intracellular Na maintenance costs, calculated from O₂ consumption rates and intracellular Na concentrations, were found to decline after exposure to 25 M Cu. Since TER was unaffected this implied the reduced costs arose from membrane `channel arrest'. Thus the Na/K ATPase energy demands, associated with maintaining intracellular Na concentration, could be reduced by decoupling metabolic demand and membrane function. Therefore this study may demonstrate how the flexibility of cellular energetics enables gill epithelial cells to tolerate sub-lethal Cu.     

Characterization of a cDNA encoding a homolog of actin-related protein 4 from a marine red alga Porphyra yezoensis

ABSTRACT:   A cDNA ( PyARP4 ) containing an open reading frame for a protein of 573 amino acids was identified in the marine red alga Porphyra yezoensis . The conceptual PyARP4 protein exhibits significant similarity to actin-related protein (ARP) 4 in the terrestrial plant Arabidopsis . Comparison of the deduced amino acid sequence showed moderate sequence identity (30%) to a conventional actin in P. yezoensis , as seen in comparisons between ARP and conventional actins of other organisms. A putative bipartite nuclear localization signal and an actin motif were found within the PyARP4 amino acid sequence. In a phylogenetic analysis, the PyARP4 was found to cluster with the ARP4 of other organisms. The expression level of PyARP4 did not change significantly among four developmental stages of life cycle and was lower than that of a conventional actin. This cDNA therefore may serve as a useful internal standard in gene expression analyses of differentially expressed genes in P. yezoensis .     

Sunflower seed meal as a protein source in diets for Tilapia rendalli (Boulanger, 1896) fingerlings

This study tested the use of sunflower seed meal as a dietary protein replacement for fishmeal in practical diets for tilapia fingerlings. In order to determine the optimum substitution level for this species, sunflower seed meal was used to replace 10%, 20%, 30%, 40% and 50% of the fishmeal protein in diets containing 40% protein overall. During a 70‐day feeding period, diets containing 10% and 20% plant protein provided the best growth and feeding efficiency results, with values statistically similar to those of the control diet based on fishmeal as the sole protein. The unitary feeding cost index showed the highest profit with diets containing 20% sunflower seed meal. Growth depletion observed with diets containing higher sunflower seed meal percentages is explained by increasing Phe and Met deficiencies, and high non‐digestible fibre content. It was concluded that sunflower seed meal is a suitable feed ingredient for tilapia complete diets when it constitutes up to 20% of the dietary protein.