Effects of reactive radicals and heat on <Emphasis Type="Italic">trans</Emphasis>-isomerization of eicosapentaenoic acid

Trans geometric isomers of eicosapentaenoic acid (TEPA) have been found as minor components in human platelets. However, there is little information on the mechanism of trans-isomerization of eicosapentaenoic acid (EPA) in vitro and in vivo. The effects of reactive radicals and heat on trans-isomerization of EPA were examined. Trans-isomerization occurred when EPA ethyl ester reacted with nitrogen dioxide radical (NO₂) but not with 2,2′-azobis(2,4-dimethylvaleronitrile). TEPA was also produced from EPA ethyl ester heated at 200°C for 60 h. No TEPA, however, was detected in sardines Sardinops melanostictus after boiling, roasting, or microwave heating. These results suggest that EPA is trans-isomerized by NO₂ in vivo while trans-isomerization of EPA does not occur during conventional cooking.     

Osmoregulatory actions of growth hormone and its mode of action in salmonids: A review

Osmoregulatory actions of growth hormone (GH) and its mode of action in salmonids are reviewed. We present evidence suggesting that insulin-like growth factor I (IGF-I) mediates some of the actions of GH on seawater acclimation. Plasma concentration and turnover of GH rise following exposure to seawater. Exogenous GH (in vivo) increases gill Na⁺,K⁺-ATPase activity and the number of gill chloride cells, and inhibits an increase in plasma osmolarity and ions following transfer of fish to seawater. A single class of high affinity GH receptors is present in the liver, gill, intestine, and kidney. The levels of IGF-I mRNA in the liver, gill and kidney increased after GH-injection. After transfer to seawater, IGF-I mRNA increased in the gill and kidney following the rise in plasma GH, although no significant change was seen in the liver. Injection of IGF-I improved the ability of the fish to maintain plasma sodium levels after transfer to seawater. GH treatment also sensitizes the interrenal to adrenocorticotropin (ACTH), increasing cortisol secretion. Both cortisol and IGF-I may be involved in mediating the action of GH in seawater adaptation, although studies on the effect of GH on osmoregulatory physiology of non-salmonid species are limited. An integrated model of the osmoregulatory actions of GH is presented, and areas in need of research are outlined.

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Résumé Cet article est une revue des effets osmorégulateurs de l'hormone de croissance et de son mode d'action. Nous présentons des résultats qui suggèrent que le facteur de croissance de type insuline (IGF-I) est un médiateur de certaines des actions de la GH sur l'adaptation à l'eau de mer. Les concentrations plasmatiques et le renouvellement de la GH augmentent après transfert en eau de mer. La GH exogène stimule (in vivo) l'activité Na⁺,K⁺-ATPase et le nombre de cellules à chlorure branchialeset inhibe les augmentations de l'osmolarité et des concentrations ioniques du plasma observées après transfert en eau de mer. Une seule classe de récepteurs à haute affinité pour la GH est présent dans le foie, les branchies, l'intestin et le rein. Les niveaux d'ARNm d'IGF dans le foie, les branchies et le rein augmentent après injection de la GH. Après transfert en eau de mer, les ARNm de l'IGF augmentent dans les branchies et dans le rein en suivant l'augmentation de GH plasmatique, bien qu'aucune modification ne soit observée au niveau du foie. L'injection d'IGF augmente la capacité du poisson à maintenir ses niveaux de sodium plasmatique après transfert en eau de mer. Le traitement à la GH augmente la sensibilité à l'adrenocorticotropine (ACTH) et stimule donc les niveaux de cortisol. A la fois le cortisol et l'IGF-I semblent impliqués comme médiateurs des effets de la GH dans l'adaptation à l'eau de mer, bien que les études sur les effets de la GH sur la physiologie de l'osmorégulation chez les espèces non-salmonidés restent encore limitées. Un modèle intégré des actions de la GH sur l'osmorégulation est présenté et les domaines de recherche à développer sont soulignés.

     

Toxicity of wild juvenile “komonfugu” Takifugu flavipterus in the Bay of Sanriku Coast,Tohoku Area,Northern Japan

To clarify the mechanism of tetrodotoxin (TTX) accumulation in pufferfish, we compared the toxicity of two sets of wild juvenile “komonfugu” Takifugu flavipterus. The first set was sampled from Onisawa Fishing Port (FP) located in Okirai Bay, the Pacific Coast of Sanriku, Tohoku Area, Northern Japan. The second set was collected from the Onisawa FP and reared in an outdoor laboratory tank supplied with different seawater (Yoshihama Bay). The fish were sampled regularly and on the same days. The amount of TTX (mouse unit (MU)/fish) in the fish at Onisawa FP increased until 20 days and thereafter it did not change, while the amount of TTX in the fish in the laboratory tank remained low, and the TTX concentration (MU/g fish) decreased. Next, we compared the toxicity of wild juvenile T. flavipterus collected from Okirai Bay (Onisawa FP and Okirai FP) and Yoshihama Bay (Yoshihama FP). Large differences in TTX levels were observed among the fish from the three FPs. The amounts and concentrations of TTX in the fish at Onisawa FP were higher than those in the fish from the other two FPs. These results indicate that a large variation in toxic activity exists in the juvenile T. flavipterus in the bay of the Sanriku Coast.

     

Involvement of Gonadotropin-Releasing Hormone in Thyroxine Release in Three different forms of Teleost Fish: Barfin Founder, Masu Salmon and Goldfish

Effects of gonadotropin-releasing hormone (GnRH) on thyroxine (T₄) release in vivo and in vitro were studied in barfin flounder Verasper moseri, masu salmon Oncorhynchus masou and goldfish Carassius auratus. Seabream GnRH (sbGnRH) at a dose of 200 ng/50 g body weight (BW) significantly increased plasma T₄ levels 1 h after the in vivo injection in the barfin flounder, but thereafter the levels normalized. Salmon GnRH (sGnRH) significantly increased plasma T₄ levels l h after the injection with a significant return to initial levels in male masu salmon and male goldfish. In contrast, sGnRH and cGnRH-II in barfin flounder, and cGnRH-II in male masu salmon and male goldfish were not effective in stimulating T₄ release. To clarify direct involvement of GnRH in T₄ release, dissected lower jaw including scattered thyroid follicles was incubated with sbGnRH (1 μg/well) in barfin flounder, and with two doses (0.1 and 1 μg/well) of sGnRH in masu salmon and goldfish in vitro. T₄ concentrations of control were stable during 24 h. Incubation of lower jaw with high dose (1 μg/well) of GnRH significantly (P<0.05) increased T₄ concentrations of incubation medium at 1 h in all experimental fishes. These results indicate that direct stimulation of T₄ secretion by GnRH occurs widely in teleost fish.     

Influence of Water Temperature on Morphological Deformities in Cultured Larvae of Japanese Eel,Anguilla japonica,at Completion of Yolk Resorption

The occurrence of morphological deformities under different rearing water temperatures (18, 20, 22, 24, 26, 28, and 30 C) was examined in Japanese eel larvae. The rates of hatching and survival until yolk resorption at 22–26 C were higher than those at other water temperatures. Fertilized eggs never hatched at 18 and 30 C. The rates of occurrence of abnormal larvae reared at the water temperatures 24–28 C were lower than those at 20 or 22 C. Pericardial edema and lower jaw deformities occurred most frequently at lower temperatures (20 and 22 C). In contrast, the incubation temperature did not significantly affect the relative frequency of some neurocranial deformities and of spinal curvature. These results imply that the optimal temperatures for rearing Japanese eel eggs and embryos are 24–26 C from the viewpoints of survival and deformity.     

Flowering behaviors of the inflorescences of an alien plant (Plantago asiatica), an alpine plant (Plantago hakusanensis), and their hybrids on Mt. Hakusan,Japan

In the subalpine zone on Mt. Hakusan, Japan, Plantago asiatica, an alien plant, and Plantago hakusanensis, a native alpine species, grow sympatrically along with their putative hybrids. Here, their flowering behavior, which affects the frequency of hybridization and the colonizing ability of P. asiatica and its hybrids, is described. The flowering behavior of each species and of two F₁ hybrids from different seed parents was determined based on the position of the flower in the inflorescence by using a generalized linear mixed model. The percentage fruit set of individually bagged inflorescences was calculated to corroborate the assumptions of the opportunities for self‐pollination. All the flowers were protogynous; however, many P. asiatica anthers dehisced before browning of the stigma in the flower and the sex presentations in the inflorescence were asynchronous. The percentage of fruit set was high. Consequently, P. asiatica has the opportunity for self‐pollination within the flower and in the inflorescence. In contrast, the P. hakusanensis anthers dehisced after browning of the stigma in the flower; their sex presentation was synchronous in the inflorescence, showing negligible opportunities for self‐pollination, and the fruit set was low. Accordingly, in the field, P. hakusanensis might require pollination among the inflorescences for seed production and be actively outcrossed, while P. asiatica is able to outcross in the early flowering phase. Therefore, P. asiatica and P. hakusanensis have opportunities for hybridization. The F₁ hybrids exhibited intermediate flowering behavior and produced fruits, demonstrating the potential to reproduce by selfing.     

Comparison of PCR assays for detection and quantification of Phomopsis sclerotioides in plant and soil

We developed a real-time PCR assay using a TaqMan probe (TM-qPCR) for specific detection and quantification of Phomopsis sclerotioides, causal agent of black root rot of cucurbit crops. The design of the primer sets and hybridization probe was based on the internal transcribed spacer region of the ribosomal DNA. The TM-qPCR assay was compared with a conventional, standard PCR (sPCR) assay and on a quantitative real-time PCR (SG-qPCR) assay based on SYBR Green I. The TM-qPCR assay had a detection limit of ca. 0.4 fg of P. sclerotioides DNA, which was approximately 100 times more sensitive than the sPCR assay and almost equivalent to the SG-qPCR assay. The TM-qPCR and SG-qPCR assays both were able to detect various quantities of P. sclerotioides DNA from diseased plants and infested soils, including DNA levels that were not detectable by the sPCR assay. However, the TM-qPCR was advantageous for samples containing PCR-inhibiting substances because its multiplex real-time PCR function allows the adjustment of cycle threshold values with an internal control. Based on the high specificity and sensitivity required for analyzing DNA in natural samples, the newly developed TM-qPCR assay was the most reliable tool for rapidly detecting and quantifying P. sclerotioides in plant and soil samples.     

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga Tricleocarpa jejuensis

Our previous studies have found that (±)-(E)-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga Tricleocarpa jejuensis showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.