Postprandial hepatic protein expression in trout Oncorhynchus mykiss a proteomics examination

Following a meal, a series of physiological changes occurs in animals as they digest, absorb and assimilate ingested nutrients, the kinetics of these responses depends on metabolic rate and nutrient quality. Here we investigated the hepatic proteome in the ectothermic teleost, the rainbow trout, following a single meal to define the postprandial expression of hepatic proteins. The fish were fed a high marine fishmeal/fish oil single meal following a period of 24 h without feeding. Liver protein profiles were examined by 2D gel electrophoresis just before feeding (time 0 h) and at 6 and 12 h after feeding. Of a total of 588 protein spots analysed in a temporal fashion, 49 differed significantly in abundance between the three time groups (ANOVA, p<0.05), before and after feeding, 15 were increased and 34 were decreased in abundance after feeding. Amino acid metabolism-regulated proteins such as phenylalanine-4-hydroxylase and proliferating cell antigen were increased in abundance 12 and 6 h following the meal, suggesting by this time that the fish were increasing their protein turnover to utilize efficiently their dietary protein consumption. Overall, these results highlight some specificity of the trout metabolism and identify postprandial response of metabolism-related proteins 6–12 h after feeding a single meal.     

Uptake of tritiated thymidine in muscle of juvenile carp

In juvenile carp (4.5–6 cm s.l .) labelled myosatellite cell nuclei are found 24 h after injection of tritiated thymidine, but labelled myonuclei after 48 h, indicating that fish myonuclei do not proliferate but originate from undifferentiated myogenic cells. The time pattern accords with the estimated cell-cycle time of adult myogenic cells of carp.     

Influence of fish size on proliferation and differentiation of cultured myosatellite cells of white axial muscle of carp (Cyprinus carpio L.)

Abstract. The in vitro proliferation [uptake of 5-bromo-2'-deoxyuridine (BrdU)] and the degree of differentiation (presence of desmin) of myosatellite cells isolated from white axial muscle of carp between 3 cm and 27 cm standard length (SL) were examined 17 h after isolation. The fraction of the myosatellite cells that were both desmin positive and BrdU positive never exceeded 2% of the total number of isolated myosatellite cells, irrespective of the standard length of the donor(s). This indicates that, for carp, the temporal relationship between replication and desmin expression of myosatellite cells is different from that described for myogenic cells of mammals and birds. The percentage of BrdU positive myosatellite cells was significantly correlated with standard length: it increased from 10% for carp of about 5 cm SL to 40–50% for carp between 20 cm and 27 cm SL. The percentage of desmin positive myosatellite cells was about 50–60%; it was not significantly correlated with standard length. The percentage of myosatellite cells that were both BrdU negative and desmin negative showed a stepwise difference in this percentage with increasing length. Fish smaller than 10 cm SL, had more of these cells (10–40%), than larger fish (which had 0–12%). So, apparently the composition of the myosatellite cell population changes during growth. The low percentage of proliferating cells, and the relatively high percentage of differentiated (desmin positive) myosatellite cells obtained from 3–6 cm large carp, suggests that, in these small fish, muscle growth strongly depends on the use of a pool of myogenic cells that has been formed at an earlier stage of their development.     

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.     

Antigen inhalation induces mast cells and eosinophils infiltration in the guinea pig esophageal epithelium involving histamine-mediated pathway

Antigen challenge in sensitized guinea pig esophagus in vitro induces mast cell degranulation and histamine release. This study tests the hypothesis that antigen inhalation in vivo induces infiltration of the esophageal epithelium by mast cells and eosinophils via a histamine pathway. Actively sensitized guinea pigs were exposed to inhaled 0.1% ovalbumin. One or 24 h after inhalation exposure, the esophagus was processed for immunofluorescent staining of mast cell tryptase and eosinophil major basic protein (MBP). Additional animals were pretreated with thioperamide, a histamine H4/H3 receptor antagonist. Total tryptase- and MBP-labeled cells and percent of positive cells in the epithelial layer were counted. The total number of mast cells was unchanged after inhalation challenge, but the percentage in the epithelium increased 1 h after challenge. The total number of eosinophils increased 1 h after challenge, and the percentage migrating to the epithelium increased by 24 h after challenge. Mast cell migration into the mucosal epithelium preceded that of eosinophils. Thioperamide inhibited mast cell and eosinophil migration. In conclusion, antigen inhalation in sensitized animals induces mast cells and eosinophils to infiltrate in the esophageal epithelium via histamine-mediated mechanism.     

Energetic costs at different levels of feeding in juvenile cod, Gadus morhua L.

The energetic costs associated with feeding by juvenile cod were determined by means of an open-circuit respirometer. Fish acclimated to several temperatures (7, 10, 15 and 18°C) were kept at natural lighting levels, and fed inside their individual respirometers. They consumed a diet compounded from natural foods, at five different ration levels, their oxygen consumption being monitored continually over an 11–16 day period.
After each meal the rate of oxygen consumption increased to above the pre-feeding level, reaching a peak 8–10 h later. With each successive meal the oxygen consumption showed a cumulative increase, reaching a maximum usually after the last meal.
The elevation in metabolic rate associated with feeding was dependent upon ration size, increasing linearly as the food intake increased. The effect was also dependent upon temperature; for fish fed to satiation the total energy cost was equivalent to 11.9, 10.9, 16.4 and 17.1% of the ingested energy at 7, 10, 15 and 18°C respectively. For resting satiated fish the rate of oxygen consumption was close to the maximum rate for active fish.     

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines. Intrinsic differences in growth characteristics and insulin response

Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant (P less than 0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10(-10)-10(-9) M. Slow cultures bound more [125I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.     

Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata)

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.     

Response of hexokinase enzymes and the insulin system to dietary carbohydrates in the common carp, Cyprinus carpio

The response of the common carp to diets with varying amounts of digestible starch, provided either as pea meal (LP, HP, 30 and 46% peas, respectively) or as cereal (LW, HW, 30 and 46% wheat, respectively), was studied and compared with the response to a carbohydrate-free protein-rich diet (CF). Here we focused on the utilisation of dietary carbohydrates by examining the relationship between dietary starch intake, hepatic hexokinase activities, circulating insulin and muscle insulin receptor system. Plasma glucose concentration and hepatic high Km hexokinase (glucokinase, GK) activity were not affected by the content of digestible starch, but 6 h after feeding enzyme activity was higher in the fish fed carbohydrate diets. Similarly, low Km hexokinase (HK) activity was also higher in the fish 24 h after feeding. Fat gain and protein retention were significantly improved by increased digestible starch intake, especially in the HP group, which in turn, presented the highest plasma insulin levels. Glycogen stores were moderately increased by the ingestion of digestible starch. The number of insulin receptors was greater in the CF group than in fish on carbohydrates, except the HP group. Our results confirmed that the common carp uses dietary carbohydrates efficiently, especially when there are provided by peas. This efficiency might be related to the enhanced response of postprandial insulin observed in the HP group.     

Proliferative potential of larval cells of the mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> and their capacity to differentiate into myogenic cells in culture

Cell differentiation and proliferation can be regulated by the extracellular matrix. To compare cell proliferation and myogenic differentiation in cultivated Mytilus larval cells on different substrates (collagen I and fibronectin), a double immunostaining with subsequent confocal microscopy were used for simultaneous detection of dividing and muscle cells. The proliferative activity was monitored using two markers, proliferating cell nuclear antigen (PCNA) and phospho-histone H3. The maximum number of mitotic (phosphohistone H3-positive) cells was observed after 4 h of cultivation (approximately 3%), but later, after 48 h, it decreased to 0.5%. Most of these cells formed small aggregates on all substrates tested. After 24 h of cultivation, the number of mitotic cells was approximately 5–7 times lower than that of S-phase (PCNA-positive) cells. The decrease in cell proliferation was accompanied by intensification of myogenic differentiation. First muscle cells were detected after 6 h of cultivation on fibronectin; numerous contracting muscle cells were observed after 24 h of cultivation. In contrast, the cells cultivated on collagen had mostly a rounded shape, did not spread, and showed a contracting activity only in rare cases. A small number of muscle cells with PCNA-positive nuclei were detected after 3-day-cultivation on fibronectin. We suggest that at early stages of cultivation, muscle precursor cells, or myoblasts, are able to synthesize DNA but lose this ability later.     

Induction of rat jejunal epithelial cell expression of sucrase-isomaltase by glucocorticoids in primary cell culture and in vivo

The cell cycle phase that mediates the induction of intestinal sucrase-isomaltase (SI) expression by glucocorticoids was investigated by measuring migration rates of 3H-DNA-labeled and of SI-containing epithelial cells by autoradiography and indirect immunofluorescent staining after simultaneous administration of [3H]thymidine and cortisone to 12-d-old rat pups. By 24 and 48 h, lead 3H-DNA-labeled cells had migrated 7.8 and 12.4 cell positions higher on the villus than lead cells expressing SI. Cell migration rates from 12 to 24 h and 24 to 48 h were 0.68 and 0.97 cell position/h. Thus, commitment to SI expression occurred in cells 11.5-12.8 h after the S phase, which is calculated to be in the G1 phase. To determine whether committed cells need to replicate to express SI, cell differentiation was examined in primary cultures of crypt cells originating from corticosterone-treated rats. About two-thirds of cultured cells were retarded in the S phase after plating, as judged by no increase of DNA labeling indices, no change in epithelial cell number, and the absence of mitosis (less than 0.01%). The proportion of cells expressing SI increased from 0 to 6-8% between 12 and 24 h, and reached 48% 48 h after plating on collagen-coated dishes. SI expression did not occur in cells plated on glass or plastic surfaces. Pulse labeling with [35S]methionine confirmed that de novo synthesis of SI occurred in cell cultures. Thus, additional cell cycling of committed cells occurring in vivo is not obligatory for the expression of SI.     

饲料中豆粕替代鱼粉蛋白对异育银鲫生长、代谢及免疫功能的影响

本实验评价了饲料中豆粕替代鱼粉蛋白后对异育银鲫的生长、饲料利用、氮代谢和鱼体免疫力等的影响。实验设计4种等氮等能的饲料,每种3个重复,分别以豆粕替代饲料中鱼粉蛋白的0%（对照,D1）、20%（D2）、80%（D3）和100%（D4）。实验在半循环水养殖系统持续16周,鱼的初重约2.32g,实验期间水温23－30℃。结果表明,随着饲料中豆粕含量的升高,摄食率显著升高(P＜0.05),特定生长率、饲料转化效率、蛋白沉积率和能量沉积率显著降低(P＜0.05);蛋白表观消化率显著升高,干物质和能量表观消化率则显著降低(P＜0.05);总氮摄入量、表观氮摄入量、粪氮排出量、非粪氮排泄量、总氮沉积率均随着饲料中豆粕含量的升高呈显著降低到的趋势(P< 0.05),生产每千克鱼的氮排放量则随着饲料中豆粕含量的升高显著升高(P< 0.05);血清葡萄糖和甘油三酯的含量显著升高,而胆固醇的含量显著降低(P＜0.05);血清的溶菌酶显著降低,超氧化物岐化酶逐渐升高(P＜0.05)。     

Compensatory feeding in response to variable food quality by Melanoplus differentialis

Abstract. The behavioural mechanisms driving compensatory food consumption in response to dietary dilution as well as the relationship between feeding time and food residence time (i.e. digesta retention time in the gut) were studied using the non-diapausing strain of the grasshopper Melanoplus differentialis (Thomas) (Orthoptera: Acrididae). 3-day-old, sixth-instar nymphs and 3-day-old adults were fed artificial diets containing 1%, 3% and 5% total nitrogen (N) at 30C, LD 14:10h; the feeding behaviour was recorded using electronic monitoring devices connected to microcomputers for 24 h. The percentage of time spent feeding increased linearly as diets were diluted using non-digestible cellulose from 5% to 1% N. This response was due to an increase in the number of meals while the meal duration of a feeding bout was unaltered. Sixth-instar nymphs spent about 40% more time feeding than the larger adults. The increased feeding time in nymphs resulted from both more frequent feeding bouts and longer meal duration. Feeding time and food residence time were highly negatively related.     

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines

Abstract. Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant ( P <0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10⁻¹⁰−10⁻⁹ M . Slow cultures bound more [¹²⁵I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.     

Hematological responses of the Neotropical teleost matrinxã (Brycon cephalus) to environmental nitrite

Environmental increase in nitrite impairs the function of several aquatic species, including fishes. Nitrite reacts with hemoglobin yielding the non-functional methemoglobin (metHb), and many physiological disturbances can arise. The physiological mechanisms to cope with nitrite are still unclear in fish. Hematological parameters, the role of NADH-methemoglobin reductase system and the electrolytic balance were studied in the freshwater teleost Brycon cephalus (matrinx?) exposed to 0.2, 0.4 and 0.6 mg/L of nitrite N-NO(2) for 24 and 96 h. Hematocrit, total hemoglobin and the red blood cell (RBC) number decreased. Methemoglobin content increased from 1% to 69% for 24 h of exposure and drastically from 5-6% to 90% for 96 h. The activity of NADH-methemoglobin reductase system displayed a tendency of increase in response to nitrite concentration or time of exposure. In the plasma, nitrite was accumulated to values 30-fold higher than the environmental concentration. The plasma K(+) concentration increased only in fish exposed to NO(2) for 24 h. No changes in plasma protein and Na(+) were observed during nitrite exposure but Cl-presented a punctual increase at 0.2 mg/L N-NO(2)-96 h. The hematological data suggest that nitrite caused functional and hemolytic anemia. Furthermore, the electrolytic balance was relatively undisturbed, and the nitrite clearance in matrinx? is likely depending on other factors than NADH-methemoglobin reductase system.     

Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate

Using agrichemicals to control unwanted species has become a necessary and common worldwide practice to improve crop production. Although most currently used agrichemicals are considered relatively safe, continuous usage contributes for soil and water contamination and collateral toxic effects on aquatic species. Few studies correlated the presence of agrichemicals on fish blood cells and natural immune system. Thus, in this study, silver catfish (Rhamdia quelen) were exposed to sublethal concentrations (10% of the LC(50-96 h)) of a glyphosate based herbicide and hematological and natural immune system parameters were evaluated. Silver catfish fingerlings exposed to glyphosate for 96 h had a significant reduction on blood erythrocytes, thrombocytes, lymphocytes and total leukocytes in contrast to a significant increase in the number of immature circulating cells. The effect of glyphosate on natural immune system was evaluated after 24h or 10 days exposure by measuring the phagocytic index of coelomic cells, and lysozyme, total peroxidase, bacteria agglutination, bactericidal activity and natural complement hemolytic activity in the serum of fingerlings. A significant reduction on phagocytic index, serum bacteria agglutination and total peroxidase was observed only after 24h exposure to glyphosate. In contrast, fingerlings exposed to glyphosate for 10 days had a significant lower serum bacteria agglutination and lysozyme activity. Glyphosate had no effect on serum bactericidal and complement natural hemolytic activity after 24h or 10 days exposure. Nonetheless, the information obtained in this study indicates that glyphosate contaminated water contributes to alter blood cells parameters and to reduce the activity of natural immune components important to mediate fish resistance to infecting microorganisms.     

Changes in tissue adenylates and water content of bluegill, Lepomis macrochirus, exposed to copper

Bluegill were exposed to copper (2–0 mg/1⁻¹ unfiltered) for 24, 48, 72 or 96 h at 24° C. This concentration approximated the 96 h LC20. Water content of liver increased 4–8% after 24 h exposure and muscle 4–6% by 48 h. These changes persisted throughout the 96 h experiments. Copper exposed fish exhibited decreases in muscle ATP, ADP, total adenylates, and energy charge by 48 h. There was a trend toward recovery of ATP with further exposure. Liver ATP of exposed fish was lower than controls at all intervals with the greatest difference evident at 48 h. No significant changes in brain adenylates were observed. Muscle and liver lactic acid was unchanged at 48 h exposure, therefore tissue hypoxia was not the cause of the adenylate changes. It was concluded that copper causes decreases in muscle and liver ATP several days before probable death of copper exposed fish and the changes seen are the result of dilution by increased tissue water, and the copper acting on certain cellular enzymes involved in detoxification and energy metabolism.     

Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.     

Hepatocyte growth factor is necessary for efficient outgrowth of injured peripheral axons in in vitro culture system and in vivo nerve crush mouse model

Hepatocyte growth factor (HGF) is a neurotrophic factor and its role in peripheral nerves has been relatively unknown. In this study, biological functions of HGF and its receptor c-met have been investigated in the context of regeneration of damaged peripheral nerves. Axotomy of the peripheral branch of sensory neurons from embryonic dorsal root ganglia (DRG) resulted in the increased protein levels of HGF and phosphorylated c-met. When the neuronal cultures were treated with a pharmacological inhibitor of c-met, PHA665752, the length of axotomy-induced outgrowth of neurite was significantly reduced. On the other hand, the addition of recombinant HGF proteins to the neuronal culture facilitated axon outgrowth. In the nerve crush mouse model, the protein level of HGF was increased around the injury site by almost 5.5-fold at 24 h post injury compared to control mice and was maintained at elevated levels for another 6 days. The amount of phosphorylated c-met receptor in sciatic nerve was also observed to be higher than control mice. When PHA665752 was locally applied to the injury site of sciatic nerve, axon outgrowth and injury mediated induction of cJun protein were effectively inhibited, indicating the functional involvement of HGF/c-met pathway in the nerve regeneration process. When extra HGF was exogenously provided by intramuscular injection of plasmid DNA expressing HGF, axon outgrowth from damaged sciatic nerve and cJun expression level were enhanced. Taken together, these results suggested that HGF/c-met pathway plays important roles in axon outgrowth by directly interacting with sensory neurons and thus HGF might be a useful tool for developing therapeutics for peripheral neuropathy.