A complete biosynthetic pathway of the long-chain polyunsaturated fatty acids in an amphidromous fish, ayu sweetfish Plecoglossus altivelis (Stomiati; Osmeriformes).

The biosynthetic capability of the long-chain polyunsaturated fatty acids (LC-PUFA) in teleosts are highly diversified due to evolutionary events such as gene loss and subsequent neo- and/or sub-functionalisation of enzymes encoded by existing genes. In the present study, we have comprehensively characterised genes potentially involved in LC-PUFA biosynthesis, namely one front-end desaturase (fads2) and eight fatty acid elongases (elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl7, elovl8a and elovl8b) from an amphidromous teleost, Ayu sweetfish, Plecoglossus altivelis. Functional analysis confirmed Fads2 with Δ6, Δ5 and Δ8 desaturase activities towards multiple PUFA substrates and several Elovl enzymes exhibited elongation capacities towards C18-20 or C18-22 PUFA substrates. Consequently, P. altivelis possesses a complete enzymatic capability to synthesise physiologically important LC-PUFA including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) from their C18 precursors. Interestingly, the loss of elovl2 gene in P. altivelis was corroborated by genomic and phylogenetic analyses. However, this constraint would possibly be overcome by the function of alternative Elovl enzymes, such as Elovl1b, which has not hitherto been functionally characterised in teleosts. The present study contributes novel insights into LC-PUFA biosynthesis in the relatively understudied teleost group, Osmeriformes (Stomiati), thereby enhancing our understanding of the complement of LC-PUFA biosynthetic genes within teleosts.

Computed tomography findings of intersigmoid hernia.

Purpose: To evaluate the computed tomography findings of intersigmoid hernias. Material and methods: Between April 2010 and March 2018, 7 patients who were surgically diagnosed with intersigmoid hernia in 3 institutions were enrolled in this study. Two radiologists evaluated imaging findings for the herniated small bowel, the distance between the occlusion point and bifurcation of the left common iliac artery, and the anatomic relationship with adjacent organs. Results: All patients were male, and their mean age (standard deviation, range) was 61.0 (13.5, 36-85) years. The mean size of the bowel loops was 5.2 (1.3, 4.0-8.3) cm in the caudal direction, 3.6 (0.8, 2.5-5.1) cm in the lateral, and 3.4 (0.6, 2.5-4.7) cm in the anterior-posterior direction. The volume was 37.9 (27.8, 15.6-103.0) cm3 approximated by an ellipse, and 24.0 (17.7, 9.9-65.6) cm3 approximated by a truncated cone. The obstruction point was located 3.6 (0.6, 2.8-4.7) cm inferior to the bifurcation of the left common iliac artery. In all cases, the small bowel ran under the point at which the inferior mesenteric vessels bifurcated to the superior rectal vessels and the sigmoid vessels and formed a sac-like appearance between the left psoas muscle and the sigmoid colon. The ureter ran dorsal to the point of the bowel stenosis, and the left gonadal vein ran outside the small bowel loops. Conclusions: All cases showed common imaging findings, which may be characteristic of men's intersigmoid hernia. In addition, the fossa's position was lower, and the size was larger than in the previous study, which may be a risk factor.

Determination of dietary essential fatty acids in a deep-sea fish, the splendid alfonsino Beryx splendens: functional characterization of enzymes involved in long-chain polyunsaturated fatty acid biosynthesis.

The splendid alfonsino Beryx splendens is a commercially important deep-sea fish in East Asian countries. Because the wild stock of this species has been declining, there is an urgent need to develop aquaculture systems. In the present study, we investigated the long-chain polyunsaturated fatty acid (LC-PUFA) requirements of B. splendens, which are known as essential dietary components in many carnivorous marine fish species. The fatty acid profiles of the muscles, liver, and stomach contents of B. splendens suggested that it acquires substantial levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from its natural diet. The functional characterization of a fatty acid desaturase (Fads2) and three elongases (Elovl5, Elovl4a, and Elovl4b) from B. splendens confirmed their enzymatic capabilities in LC-PUFA biosynthesis. Fads2 showed Δ6 and Δ8 bifunctional desaturase activities. Elovl5 showed preferential elongase activities toward C18 and C20 PUFA substrates, whereas Elovl4a and Elovl4b showed activities toward various C18-22 substrates. Given that Fads2 showed no Δ5 desaturase activity and no other fads-like sequence was found in the B. splendens genome, EPA and arachidonic acid cannot be synthesized from C18 precursors; hence, they can be categorized as dietary essential fatty acids in B. splendens. EPA can be converted into DHA in B. splendens via the so-called Sprecher pathway. However, given that fads2 is only expressed in the brain, it is unlikely that the capacity of B. splendens to biosynthesize DHA from EPA can fulfill its physiological requirements. These results will be useful to researchers developing B. splendens aquaculture methods.

Impact of purine nucleotide on fatty acid metabolism and expression of lipid metabolism-related gene in the liver cell of rainbow trout Oncorhynchus mykiss.

Studies have suggested that dietary purine nucleotides (NT) affect the muscle and liver fatty acid composition of rainbow trout. To examine the direct regulation of liver fatty acid metabolism by purine NT in rainbow trout, the liver cells were cultured in the presence of 500 µmol/L inosine, adenosine, or guanosine monophosphate (IMP, AMP, or GMP). The expression of pparα was significantly decreased in the liver cells cultured with purine NT for 24 h, whereas the expression of fads2 (Δ5) was increased. Docosahexaenoic acid (DHA) content in the liver cells was significantly higher after culturing with GMP. To determine the dose-dependent effects of NT, 50, 100, and 500 µmol/L GMP was added to the liver cells cultured in L-15 medium. At 48 h, 20:4n - 6, 22:5n - 3, 22:6n - 3, Æ© PUFA, and Æ© n - 3 PUFA content in the 50 µM GMP-containing medium was significantly higher compared with the other medium. The expression of Δ5 fads2, elovl2, and elovl5 in the liver cells was significantly higher in the 500 µmol/L GMP-containing medium at 48 h along with increased srebp-1 expression. These results suggest that purine NT directly affect fatty acid composition through modification of fatty acid metabolism-related genes in the liver of rainbow trout.

A complete enzymatic capacity for biosynthesis of docosahexaenoic acid (DHA, 22 : 6n-3) exists in the marine Harpacticoida copepod Tigriopus californicus.

The long-standing paradigm establishing that global production of Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) derived almost exclusively from marine single-cell organisms, was recently challenged by the discovery that multiple invertebrates possess methyl-end (or ωx) desaturases, critical enzymes enabling the biosynthesis of n-3 LC-PUFA. However, the question of whether animals with ωx desaturases have complete n-3 LC-PUFA biosynthetic pathways and hence can contribute to the production of these compounds in marine ecosystems remained unanswered. In the present study, we investigated the complete enzymatic complement involved in the n-3 LC-PUFA biosynthesis in Tigriopus californicus, an intertidal harpacticoid copepod. A total of two ωx desaturases, five front-end desaturases and six fatty acyl elongases were successfully isolated and functionally characterized. The T. californicus ωx desaturases enable the de novo biosynthesis of C18 PUFA such as linoleic and α-linolenic acids, as well as several n-3 LC-PUFA from n-6 substrates. Functions demonstrated in front-end desaturases and fatty acyl elongases unveiled various routes through which T. californicus can biosynthesize the physiologically important arachidonic and eicosapentaenoic acids. Moreover, T. californicus possess a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid via the 'Δ4 pathway'. In conclusion, harpacticoid copepods such as T. californicus have complete n-3 LC-PUFA biosynthetic pathways and such capacity illustrates major roles of these invertebrates in the provision of essential fatty acids to upper trophic levels.

Flatfishes colonised freshwater environments by acquisition of various DHA biosynthetic pathways.

The colonisation of freshwater environments by marine fishes has historically been considered a result of adaptation to low osmolality. However, most marine fishes cannot synthesise the physiologically indispensable fatty acid, docosahexaenoic acid (DHA), due to incomplete DHA biosynthetic pathways, which must be adapted to survive in freshwater environments where DHA is poor relative to marine environments. By analysing DHA biosynthetic pathways of one marine and three freshwater-dependent species from the flatfish family Achiridae, we revealed that functions of fatty acid metabolising enzymes have uniquely and independently evolved by multi-functionalisation or neofunctionalisation in each freshwater species, such that every functional combination of the enzymes has converged to generate complete and functional DHA biosynthetic pathways. Our results demonstrate the elaborate patchwork of fatty acid metabolism and the importance of acquiring DHA biosynthetic function in order for fish to cross the nutritional barrier at the mouth of rivers and colonise freshwater environments.

Cloning and functional characterization of fads2 desaturase and elovl5 elongase from Japanese flounder Paralichthys olivaceus.

Japanese flounder Paralichthys olivaceus has an essential requirement for long-chain polyunsaturated fatty acids (LC-PUFA), particularly docosahexaenoic acid and eicosapentaenoic acid, but the enzymes involved in LC-PUFA biosynthesis are thought to be absent or to have low activity. Teleost fish, in particular, have quite diversified substrate preference of these enzymes even among closely related species, implying that each species could have different LC-PUFA biosynthetic capabilities. Therefore, in the present study, we characterized Japanese flounder fatty acid desaturase 2 (Fads2) and elongation of very long-chain fatty acids protein 5 (Elovl5) in order to precisely characterize the LC-PUFA biosynthesis pathway. Fads2 has Δ6 and Δ8 desaturase activity and Elovl5 has elongase activity toward C18 and C20 PUFA, suggesting that Japanese flounder is capable of synthesizing 20:4n-3 and 20:3n-6 from 18:3n-3 and 18:2n-6, respectively. Expression analysis showed that the fads2 was highly expressed in the brain and eye, while the elovl5 was highly expressed in the eye and pyloric caeca. This information will be beneficial for developing an ideal feed to support the aquaculture of Japanese flounder.

Polyunsaturated fatty acid metabolism in a marine teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2 desaturase and Elovl5 and Elovl4 elongases.

To reduce the requirement for fish oil in marine aquaculture, it would be advantageous to endow marine fish species with the capability for the endogenous biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). For this purpose, we have previously produced transgenic Nibe croaker (Nibea mitsukurii) carrying an elongase of very-long-chain fatty acids 2 (elovl2) gene isolated from Masu salmon (Oncorhynchus masou). However, fatty acid analysis revealed that 24:5n-3 accumulated in the liver of the transgenic fish, whereas the DHA level did not differ between non-transgenic and transgenic fish. Therefore, to select more effective enzymes for successful transgenic synthesis of DHA, understanding the endogenous DHA biosynthetic pathway in the Nibe croaker is considered to be important. The present study aimed to investigate the biochemical functions of the Elovl5, Elovl4 and Fads2 enzymes involved in the DHA biosynthetic pathway in the Nibe croaker. The results showed that both Elovl5 and Elovl4 were able to elongate C18 fatty acids to C22 fatty acids and that Fads2 had Δ6 desaturase activity toward C18 fatty acids and weak Δ8 desaturase activity toward C20 fatty acids. On the other hand, Fads2 was found to lack the ability to convert 24:5n-3 to 24:6n-3, a fatty acid that can directly be converted to DHA via ß-oxidation.

Effects of organic acids on growth and phosphorus utilization in rainbow trout, Oncorhynchus mykiss. .

The present study aimed to investigate the effect of phosphorus and various organic acids supplementation on growth, nutrient retention and loading in rainbow trout fed on low fishmeal based diets. Five experimental diets were formulated, Diet D1 (0.5P) was positive control with 0.5% inorganic phosphorus addition and D2 (0P, negative control) without addition of inorganic phosphorus. D3, D4 and D5 were supplemented with 1% fumaric (FuA), formic (FoA) and acetic (AA) acids, respectively. All the diets were fed until satiation to duplicate groups of 25 fish for 12 weeks. Feed conversion ratio (FCR) was recorded best with FoA diet and absorption of phosphorus was significantly higher (P < 0.05) in all the diets supplemented with organic acids and was similar to positive control. The phosphorus and nitrogen retention with FoA diet was significantly high (P < 0.05) as compared to 0P diet, in turn reducing their excretion. Hence, the present study demonstrated that, without additional phosphorus in the low fish meal-based diets, FoA supplement improved growth performance, absorption and retention of phosphorus and nitrogen and reducing excretion; thus it can be better incorporated to develop environment-friendly feed for rainbow trout.

Modification of the n-3 HUFA biosynthetic pathway by transgenesis in a marine teleost, nibe croaker.

Marine fishes are generally unable to produce sufficient quantities of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) for their normal growth and survival, as the key fatty acid-metabolizing enzymes in the EPA and DHA biosynthetic pathway are limited. It is therefore necessary to supplement cultured marine fish species diets with fish oils in order to supply EPA and DHA. Given that freshwater fishes are capable of synthesizing both EPA and DHA, they presumably express all of the enzymes required for this biosynthetic pathway. Thus, we hypothesize that transgenic marine species carrying these fatty acid-metabolizing enzymes could be reared without the dietary supplementation of fish oil. As the first step toward this goal, we used marine fish, nibe croaker to produce a transgenic line carrying the elongase gene isolated from masu salmon. Fatty acid analysis revealed that the liver EPA (20:5n-3) content in the transgenic fish was lower (3.3% vs. 7.7%). However, docosapentaenoic acid (22:5n-3) content in the transgenic fish was 2.28-fold (4.1% vs. 1.8%) higher than in non-transgenic fish. Further, tetracosapentaenoic acid (24:5n-3) was specifically detected in the transgenic fish. We therefore conclude that the development of transgenic fish lines with these fatty acid-metabolizing enzymes could be a powerful tool for manipulating fatty acid metabolic pathways in fish.

Microarray analysis of hepatic gene expression in juvenile Japanese flounder Paralichthys olivaceus fed diets supplemented with fish or vegetable oils.

Gene expression profiling was performed in Japanese flounder Paralichthys olivaceus fed diets supplemented with fish oil (FO), linseed oil (LO), or olive oil (OO) for 6 weeks. The LO and OO groups showed significantly retarded growth, lower feed intake, lower protein efficiency ratio, and lower hepatosomatic index (P < 0.05). Liver fatty acid composition reflected the dietary fatty acid composition. Microarray analysis revealed that dietary n - 3 highly unsaturated fatty acid (HUFA) deficiency affected 169 transcripts. In the LO group, 57 genes were up-regulated and 38 genes were down-regulated, whereas in the OO group nine genes were up-regulated and 87 genes were down-regulated. Analysis of the functional annotations suggested that dietary n - 3 HUFA affected genes involved in signal transduction (23.2 %), cellular processes (21.1 %), metabolism (including glucose, lipid, and nucleobase; 15.5 %), transport (11.3 %), regulation of transcription (10.5 %), and immune response (4.2 %). Several genes encoding serine/threonine kinases such as protein kinase C and calmodulin-dependent kinase and nuclear hormone receptors such as vitamin D receptor, retinoic acid receptor, and receptors for cytokines (bone morphogenic protein and transforming growth factor ß) were affected. Among 169 transcripts, 22 genes were affected in both LO and OO groups. The present study identified several genes involved in n - 3 HUFA deficiency-sensitive pathways, which will be useful for selective breeding of flounder strains able to adapt to n - 3 HUFA deficiency.

Expression of masu salmon delta5-desaturase-like gene elevated EPA and DHA biosynthesis in zebrafish.

Farmed fish could substitute for marine capture fish as a source of fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) beneficial for human health; however, they require these compounds in their diets. In the present study on a model fish species, we modified the EPA/DHA biosynthesis pathway by overexpression of masu salmon Delta5-desaturase-like gene in zebrafish to increase its ability to synthesize EPA and DHA. Expression of this gene in transgenic fish fed a commercial diet and Artemia helped to improve their EPA content by 1.21-fold and DHA by 1.24-fold. In similar fish that were fed only Artemia the increments were 1.14-fold for EPA and 1.13-fold for DHA, compared with nontransgenic fish. In contrast, eicosatetraenoic acid content decreased, as it is a substrate of Delta5-desaturase, while the total lipid remained constant. The results demonstrated that masu salmon Delta5-desaturase is functional in zebrafish and can modify its fatty acid metabolic pathway. The technique could be applied to farmed fish to generate a nutritionally richer product for human consumption.

Th1 response during ribavirin and interferon-alpha combination therapy in chronic hepatitis C.

Ribavirin and interferon-alpha induce Th1 polarization of human CD4+ T cells. The study was conducted to investigate the whether cellular immune response during ribavirin/interferon-alpha therapy is associated with viral eradication by examining mRNA expression of molecules relevant to Th1 and Th2 polarization in CD4+ cells of 13 patients with chronic hepatitis C (seven patients with sustained viral response and six with transient response). Peripheral CD4+ T lymphocytes at 0, 4 and 24 weeks of treatment were tested. There were no significant differences in the mRNA levels at each point of time of the treatment between patients with sustained viral response and those with transient response. The percent increase in mRNA level of the IL-12R beta2 chain from the baseline to the end of the treatment was significantly higher in patients with sustained viral response (15.3+/-6.1%) than in those with transient response (-1.6+/-4.7%, p<0.05). There was no significant difference in percent changes in level of IL-12R beta1 chain mRNA between the two groups. In conclusion, the results of this study indicate that the increase of Th1 response is related to the inflammatory activity in the liver and possibly to ribavirin and interferon-alpha therapy. It is also suggested that the measurement of Th1 response has the potential to distinguish patients with relapse from those with sustained virus response.

Development of functional fish feed with natural ingredients to control heavy metals.

The effects of two natural ingredients, Chinese parsley (CP) and chitosan (CT), on growth, accumulation, and excretion of cadmium in fish body and preservation of essential trace metals in the body were investigated by using rainbow trout that had been fed cadmium-added diet, low and high concentration, for 3 weeks. This pretest confirmed that cadmium was accumulated in the liver, kidney, and intestine of the test fish. The cadmium level of the fish, fed diet with CP or CT, was decreased by 18% and 24%, respectively, compared to that of the fish given the control diet. But CP and CT did not have an influence on normal growth of test fish and the levels of essential trace metals in the body. In addition, the level of cadmium was higher in liver than kidney in the high-cadmium dietary group, indicating the Cd level in kidney follows that of liver as kidney lies in the final stage of metabolism. The cadmium accumulation in the fish body was supposed to be reduced, by giving CP to increase the solubility of Cd to body fluid by conjugation into metallothioneins (MTs), while CT was supposed to be responsible for the physical adsorption of cadmium ions by glucosamine groups.

Enhancement of EPA and DHA biosynthesis by over-expression of masu salmon delta6-desaturase-like gene in zebrafish.

The n - 3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have important nutritional benefits in humans. Farmed fish could serve as promising sources of EPA/DHA, but they need these fatty acids or their precursors in their diets. Here we transferred masu salmon delta6-desaturase-like gene in zebrafish to increase its ability for synthesizing EPA and DHA. Expression of this gene in transgenic fish elevated their EPA content by 1.4-fold and DHA by 2.1-fold. On the other hand, the alpha-linolenic acid (ALA) content decreased, it being a substrate of delta6-desaturase, while the total lipid remained constant. This achievement demonstrates that fatty acid metabolic pathway in fish can be modified by the transgenic technique, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food.

CT of internal hernias.

Computed tomography (CT) plays an important role in diagnosis of acute intestinal obstruction and planning of surgical treatment. Although internal hernias are uncommon, they may be included in the differential diagnosis in cases of intestinal obstruction, especially in the absence of a history of abdominal surgery or trauma. CT findings of internal hernias include evidence of small bowel obstruction (SBO); the most common manifestation of internal hernias is strangulating SBO, which occurs after closed-loop obstruction. Therefore, in patients suspected to have internal hernias, early surgical intervention may be indicated to reduce the high morbidity and mortality rates. In a study of 13 cases of internal hernias, nine different types of internal hernias were found and the surgical and radiologic findings were correlated. The following factors may be helpful in preoperative diagnosis of internal hernias with CT: (a) knowledge of the normal anatomy of the peritoneal cavity and the characteristic anatomic location of each type of internal hernia; (b) observation of a saclike mass or cluster of dilated small bowel loops at an abnormal anatomic location in the presence of SBO; and (c) observation of an engorged, stretched, and displaced mesenteric vascular pedicle and of converging vessels at the hernial orifice.

Antioxidant defense of rainbow trout (Oncorhynchus mykiss) in relation to dietary n-3 highly unsaturated fatty acids and vitamin E contents.

This study examined the modulation of the antioxidant status and related physiological changes in rainbow trout Oncorhynchus mykiss under different levels of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) and vitamin E. Six diets containing 0, 100 or 1000 mg alpha-tocopheryl acetate kg(-1) diet and 20% or 48% n-3 HUFA provided by normal fish oil or DHA concentrated fish oil, respectively, were fed to 100 g size fish for 15 weeks. Growth of fish fed vitamin E deficient diets under both levels of n-3 HUFA were slightly retarded, accompanied by a reduction of hematocrit values, an enlargement of liver and spleen, an elevation of lipid hydroperoxide in red blood cell and the antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase). Supplementation of vitamin E could protect the fish from these adverse effects; however the higher dose was no better compared to the moderate dose. The modulations were clearly seen in fish fed high n-3 HUFA (48%) since they were under greater oxidative stress as indicated by the markers, lipid hydroperoxide and 8-isoprostane. The increased activity of enzymes corresponds to physiological mechanisms combating the elevation of free radicals under oxidative stress and a dietary fatty acid profile-dependent moderate dose of vitamin E is all that is required to function as an effective antioxidant.