Wound‐healing potential of curcumin in the carp,Labeo rohita

In this investigation, the effect of dietary administration of curcumin on the healing of skin wound in fish, Labeo rohita, has been reported. Fish were divided into three groups: control group (fish without skin wound), sham group (fish with skin wound without curcumin treatment) and curcumin‐treated group (fish with skin wound and subjected to dietary administration of 1% curcumin). Experiments were conducted for 30 days to assess the healing of skin wounds at different time intervals using scanning electron microscopy, histology, and mucopolysaccharide and enzyme histochemistry. In the curcumin‐treated group, healing of skin wounds was found to be enhanced than in the sham group as indicated by early restoration of morphology of the surface layer of epithelial cells; the density of the mucous goblet cells; the density of club cells in epidermal layer; and early granular tissue formation, collagen deposition and tissue remodelling in dermal layer. Furthermore, peroxidase and catalase enzyme activity showed increased endogenous defence system in the curcumin‐treated group compared with the sham group. It could be concluded that dietary administration of curcumin is beneficial in rapid healing of skin wounds in fish. Early healing of wounds could be considered to prevent the invasion of pathogens and to maintain the integrity of the surrounding tissue.     

Suture technics for the abdominal wall after midline laparotomy

During the last 10 years 268 laparotomies by a midline incision were performed. A high incidence of seromas, fistulas caused by suturing material and wound ruptures were observed. After finding only a small number of publications in the veterinary medicine about wound healing disturbances an analysis of the human medical literature was done. This induced the change of our surgical procedure and suturing technique: The midline incision and surgical management during the laparotomies in 14 horses suffering from colic were performed by a minimized trauma. The abdominal wall was closed layer by layer in the following manner: peritoneum by a running suture of catgut metric 5, linea alba by Sultan single sutures of polyglycolic acid material metric 5 (12 cases) or of polydioxanone metric 4 (2 cases), subcutaneous layer by a running suture of polyglycolic acid material metric 4, skin by single sutures of monofilamentous synthetic material metric 2. Thus, no complications in wound healing were observed in these 14 horses. Therefore, we recommend to prove the described surgical and suturing procedure by a larger number of horses undergoing a laparotomy by a midline incision.     

Evaluation of zinc supplementation in European sea bass (Dicentrarchus labrax) juvenile diets

This study aimed to investigate the effect of increased zinc dietary levels on the growth performance, feed utilization, immune status and induced wound healing in European sea bass (Dicentrarchus labrax). Fish weighing 10 g were fed five diets containing organic zinc at 30, 70, 110 and 150 mg kg^?1 diet and one inorganic zinc source (zinc oxide) at 150 mg kg^?1 diet for a period of 12 weeks. No significant (P>0.05) differences were found in the growth performance parameters (final weight, specific growth rate), immunological indices tested (respiratory burst activity in whole blood) and the wound‐healing process. Zinc accumulation in the skin, vertebrae and liver increased significantly (P<0.01) with increased dietary zinc levels but not in muscle. The highest zinc concentrations were obtained in skin tissue, followed by vertebrae, liver and muscle tissue. Using the concentration of zinc in skin as a response criterion, broken‐line analysis showed that the supplementation of 148 mg organic Zn kg^?1 diet seemed to be the optimum dietary zinc supplementation level for sea bass juveniles.     

Characterization of Gelatinolytic Activity in Seminal Plasma of Some Teleost Fish

Gelatin-containing SDS-PAGE combined with the incubation of gels in buffers containing protease inhibitors was performed for the visualization and characterization of proteolytic activity in teleost fish seminal plasma. To demonstrate the class of detected enzymes we used serine protease inhibitor – benzamidine or EDTA which inhibits metalloproteases activity. Additionally the effects of calcium ions on protease activity were investigated. Multiple gelatinolytic activities in seminal plasma of 10 teleost fish species from three orders (Cypriniformes, Salmoniformes, Perciformes) were found. Most proteases were either stimulated by Ca²⁺ and inhibited by EDTA or inhibited by benzamidine. This suggests that metalloproteases and serine proteases are major gelatinolytic proteases of fish seminal plasma. In cyprinid species we found a common profile of two gelatinolytic activities; the first band (60–66 kDa) belonged to metalloproteases, and the second one (76–81 kDa) belonged to serine proteases. Other bands were also visible and they represented mostly serine protease activity. Species from the Salmoniformes order showed a similarity in metalloproteases with molecular weights of about 64 and 75 kDa. Salmonid species also had similar serine proteases with molecular weights of about 102 and 165 kDa. In European grayling seminal plasma we found metalloproteases with molecular weight of 51, 57, 64, 70 kDa and two serine proteases activities of 35 and 125 kDa. Percid species had metalloproteases activities of 53 and 63 kDa and serine protease activity of 100 kDa. Protease of other, presently unknown classes were also found in seminal plasma of asp, chub, European grayling and pikeperch. The physiological role of seminal plasma proteases is still unknown.     

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 °C for 60 days, to compare LCD-incidence. In the fish reared at 20 °C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10⁶ PCR-U mg⁻¹ tissue, were detected in the fins and skin of LCD-affected fish at 20 °C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 °C and 30 °C; however, a low level of LCDV (10³ PCR-U mg⁻¹ tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 °C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 °C.     

Microsatellite markers derived from bay scallop <Emphasis Type="Italic">Argopecten irradians</Emphasis> expressed sequence tags

ABSTRACT:   When data mining was performed on the National Center for Biotechnology Information database, a total of 2038 sequences from five different expressed sequence tag libraries were registered. Eighty sequences (3.9%) were found to contain 91 microsatellites. Clustering analysis indicated that 23 sequences of these expressed sequence tags fell into five clusters and that the remaining 57 sequences were independent. The di- and tri-nucleotide repeat motifs accounted for approximately 62.1% of the total microsatellites. The most abundant dinucleotide microsatellite was TA, followed by GA and CA, and the trinucleotide microsatellites GAT and GGT showed a high abundance. Nineteen sequences representing di-, tri-, tetra- and penta-nucleotides motifs were chosen for the design of polymerase chain reaction (PCR) primers. Of primer pairs, 16 successfully amplified scorable PCR products and 11 revealed polymorphism, with the average polymorphic information content value of 0.5082 and 3.1 alleles per locus. A transferability analysis on three other related scallop species, Chlamys farreri, Chlamys nobilis and Patinopecten yessoenssis , showed that only 1 of 16 primer pairs could amplify PCR products with the expected size in Chlamys nobilis .     

Organic components from scallop shell increase expression of keratinocyte growth factor in human skin fibroblast

We observed previously that scallop shells contain organic components that have various useful applications for skin. The organic components from scallop shell (scallop shell extract) enhance the turnover rate of the rat skin epidermal layer and increase the rate of recovery of UVB-induced injury to rat dorsal skin. We investigated the effect of scallop shell extract on the expression of keratinocyte growth factor (KGF) from human skin fibroblasts because it has been reported that KGF promotes wound healing and reduces the generation of UVB-induced free radicals in skin. Scallop shell extract increased the mRNA expression level of KGF in skin fibroblasts and the secretion of KGF from the fibroblasts at the protein level. These results suggest that the scallop shell extract may promote recovery from UVB-induced injury in skin through an increase in the secretion of KGF from the fibroblast cell.     

Scallop shell extract promotes recovery from UV-B-induced damage in rat skin epidermal layer

ABSTRACT:   It has been previously reported that scallop shell extract can protect keratinocyte cells against UV-B-induced damage in vitro by acting as an antioxidant and a growth promoter. To establish the growth promoting activity of scallop shell extract, the effect was investigated in vivo. Exposure of rat dorsal skin to UV-B caused the formation of erythema and eschar. Either the scallop shell extract or vehicle (water) was applied once a day for 5 days to the injured dorsal area and the area of the erythema and eschar was estimated using National Institute of Health (NIH) image software. Wound healing of the erythema and eschar was clearly promoted after application of scallop shell extract when compared to application of the vehicle control. Histological studies indicated that scallop shell extract promoted the recovery of the epidermal layer. These results suggest that scallop shell extract activates rat keratinocyte cells and promotes the turnover of skin stratum corneum. This conclusion was also supported by measurement of the turnover rate of the stratum corneum. Therefore, scallop shell extract may be effective in protecting skin against UV-B.     

Expression of a recombinant crustacean hyperglycemic hormone of the kuruma prawn Penaeus japonicus in methylotrophic yeast Pichia pastoris

ABSTRACT:   Until now, six crustacean hyperglycemic hormones (CHH) designated Pej-SGP-I, -II, -III, -V, -VI and -VII have been characterized in the kuruma prawn Penaeus japonicus . All CHH consist of 72 amino acid residues and have an amidated carboxyl (C)-terminus. In the present study, we expressed Pej-SGP-III in methylotrophic yeast Pichia pastoris in order to obtain a large quantity of recombinant CHH possessing biological activity. A cDNA encoding Pej-SGP-III that had been previously cloned was processed by polymerase chain reaction (PCR) and the resulting product was ligated into an expression vector. Pichia pastoris was transformed with this vector after which a recombinant Pej-SGP-III was expressed having an additional amino acid residue (glycine) at the C-terminus (rPej-SGP-III-Gly), a form considered to be a putative precursor of this hormone. rPej-SGP-III-Gly secreted into the culture medium was purified by reversed-phase HPLC, and amidated using a peptidylglycine alpha-amidating enzyme. The amidated rPej-SGP-III (rPej-SGP-III) showed hyperglycemic activity in in vivo bioassay almost comparable to that of the natural Pej-SGP-III. rPej-SGP-III thus obtained will be a useful tool not only for its physiological study but also for the determination of its 3-D structure.     

Induction of (<Emphasis Type="Italic">Z</Emphasis>)-8-heptadecene in marine green alga <Emphasis Type="Italic">Bryopsis maxima</Emphasis> by mechanical wounding

ABSTRACT:   After mechanical wounding of the marine green alga Bryopsis maxima in a phosphate buffer, one main organic component was released to the solution. The organic compound was characterized by a molecular ion at 238  m/z by gas chromatography–mass spectrometry (GC-MS) and was deduced as 8-heptadecene by the fragment pattern. To identify the stereochemistry of 8-heptadecene, synthetic ( Z / E )-8-heptadecene was coinjected to the solution of wounded B. maxima . Comparing retention times and mass spectra with the synthetic sample, the target hydrocarbon obtained from wounded B. maxima was coincident with ( Z )-8-heptadecene.     

The influence of ultraviolet-B radiation on the mechanism of wound repair in the skin of the Atlantic salmon, Salmo salar L.

Abstract. Exposure of surgically damaged skin of Atlantic salmon, Salmo salar L., to low levels of ultraviolet-B radiation in clear water showed that the basic mechanism of superficial wound healing, characterized by epidermal migration, was unaffected. However, the migrating cells were more vulnerable to the necrotizing effects of the irradiation than the surrounding donor epithelium and longer standing effects such as wound contracturc and resolution of epidermal structure failed to occur.     

Tissue localization of Aeromonas salmonicida in Atlantic salmon, Salmo salar L., following experimental challenge

Three groups of Atlantic salmon, Salmo salar L., were exposed to live, colony-forming, radiolabelled Aeromonas salmonicida bacteria in a bath challenge: (1) fish with artificial wounds; (2) fish with a reduced epidermal mucus layer caused by removal of the mucus layer on two occasions by a swabbing procedure; and (3) a control group of untreated fish. Fish were killed 2, 6 and 24 h after challenge, and radioactivity (cpm g^–1) was measured in the blood, mucus, skin, wound area, gills, anterior kidney, posterior kidney, spleen, midgut and hindgut. The highest levels of radioactivity were measured in the wound areas and in the gills. There was a significant positive correlation between the levels of radioactivity in the gills and blood, and between the mucus and skin at 2 h post-challenge. Two hours after the bath challenge, live A. salmonicida bacteria were found in the blood of fish in the 'swabbed' and 'artificial wound' groups, and not in the control group. Twenty-four hours after the bath challenge, the kidney of fish from all groups contained viable bacteria, whereas the blood was negative.     

Hemagglutinating activity and electrophoretic pattern of hemolymph serum proteins of Penaeus monodon and Macrobrachium rosenbergii to white spot syndrome virus injections

The giant fresh water prawn Macrobrachium rosenbergii is known to be highly tolerant to white spot syndrome virus (WSSV) infections when compared to the widely cultured marine tiger shrimp Penaeus monodon. At present, the exact mechanism of tolerance by M. rosenbergii to WSSV is not known. In this study, we attempt to study the effect of WSSV injections on the hemagglutination activity of the hemolymph serum of both P. monodon and M. rosenbergii and look for changes if any, on their hemolymph serum protein electrophoretic patterns. Our results show that M. rosenbergii had significantly (p < 0.05) higher hemagglutinating activity against mouse erythrocytes when compared to P. monodon. As the infection progressed to 48 h there was a further increase (p < 0.05) in the hemagglutination activity in M. rosenbergii, while it decreased in P. monodon. 12% SDS-PAGE analysis of the hemolymph serum of M. rosenbergii infected with WSSV did not show any new protein bands, whereas few bands with decreased intensity was observed in moribund P. monodon where the hemagglutinating activity was also observed to be decreased. The results indicate that hemolymph hemagglutinin levels are modulated in crustaceans as a response to viral infections.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).     

Imidazole‐dicarboxylic Acid Nickel Complex as a Novel Grafting Agent for Pearl Oyster,Pinctada martensii

In the pearl cultivation industry, the implantation of the nucleus is a critical step in the production process. The surgical insertion of the nucleus and the mantle results in a wound and stress that affect not only the survival chances of the pearl oysters but also the quality of the pearls produced. This study proposes a synthesized imidazole‐dicarboxylic acid nickel complex that may hold potential in assisting the wound healing and nacre formation processes. The acute toxicity of the complex to pearl oysters, Pinctada martensii, was examined in a dose–response study over a 90‐h exposure. The safe concentrations of the nickel complex determined using probit analysis were 0.916 mg/L and 0.471 mg/L for 72‐h and 96‐h treatments, respectively. The reliability of the regression was verified using the chi‐square test. The alkaline phosphatase (ALP) activity in different regions of the treated and control oysters, along with relevant blood biochemical indices, were measured over a 90‐d period to predict the potential effects of the nickel complex on wound healing and nacre formation. A significant increase in ALP activity in the mantle, together with elevated blood levels of Mg (II) and globulin, and reduced blood levels of Zn (II) and blood urea nitrogen were observed. These results indicate that the prepared nickel complex may hold promise in helping wound healing post mantle implantation, promote the secretion of nacre, and enhance the immunity of the oysters, thus ultimately improving the yield and quality of the pearls.     

尼罗罗非鱼补体C9基因的克隆和组织表达分析

为了探究补体C9在尼罗罗非鱼(Oreochromis niloticus)免疫中发挥的作用,本研究克隆并分析了尼罗罗非鱼补体C9基因(OnC9)。结果显示:OnC9的cDNA序列全长2 502 bp,包含1 761 bp的开放阅读框(ORF),编码586个氨基酸。氨基酸序列同源性分析表明,OnC9与牙鲆(Paralichthys olivaceus)补体C9氨基酸相似性最高,达73.0%,与其他鱼类补体C9的相似性介于49.3%~71.4%之间。实时荧光定量PCR检测结果表明,OnC9基因在所检测的各个组织或器官中表达水平有明显差异,在肝脏中表达量最高,其次是肠道、后肾、皮肤、肌肉、鳃,在脑、脾脏、心脏、头肾、胸腺和血液中表达量最低。在无乳链球菌(Streptococcus agalactiae)感染鱼体后,肝脏、后肾等组织中OnC9表达量表现为在感染12 h、48 h(或36 h)、120 h先后出现三个峰值的波动表达的规律。说明OnC9在无乳链球菌感染尼罗罗非鱼后的免疫应答中发挥作用。     

In vivo fish models for visualizing Aeromonas hydrophila invasion pathway using GFP as a biomarker

The invasion pathway of Aeromonas hydrophila in vivo was studied in Crucian carp (Carassius auratus gibelio) with a virulent strain A. hydrophila J-1 transformed with a plasmid encoding green fluorescent protein (pGFPuv) (A. h J-1^GFP), which had similar virulence characteristics (haemolysin, extracellular proteases production, toxicity for EPC cells, survival in fish serum and LD₅₀ value) as the parent strain. Fish were divided into four experimental groups: (1) normal fish; (2) bacteria bath challenged, unwounded fish; (3) skin artificially wounded by scalpel, bacterial bath challenged fish; and (4) skin mucus layer partially removed by paper towel, bacterial bath challenged fish. The number of bacteria from blood, gills, kidney, muscle, liver and intestine were detected at 2, 4, 8, 12, and 24 h post-challenge. High bacterial numbers were observed in the muscle of the artificial wound group, in the kidney of the mucus removed group and in the gills of all groups. In conclusion, the gills and damaged skin are likely to be the main routes of entry for A. hydrophila, and GFP can be used as a real-time biomarker to study intimate host–pathogen interaction in fish.     

多巴色素异构酶基因对瓯江彩鲤黑斑体色的影响

多巴色素异构酶（dopachrome tautomerase，Dct）是酪氨酸酶家族的重要成员之一，在黑色素合成过程中具有重要作用。为了解多巴色素异构酶基因（）的“粉花”体色类型为研究对象，在对该基因进行克隆和表达分析的基础上，应用CRISPR/Cas9基因编辑技术研究了该基因对瓯江彩鲤黑斑体色的影响。结果发现，瓯江彩鲤存在2个Dct1与在“粉花”体色瓯江彩鲤的黑斑皮肤中的表达量显著高于白色皮肤，而Dct1和基因突变对瓯江彩鲤黑斑体色存在重要影响，与在鲤的黑斑体色形成中发挥着更大的作用。     

Effects of dichlorvos and formalin on fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells in primary culture

The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-¹⁴C]Stearic (18:0), [1-¹⁴C]lin 18:2n-6) and [1-¹⁴C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of sphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial 9, 6 and 5 fatty acyl desaturase activities. Although, as expected, the cells were m active towards [1-¹⁴C]18:3n-3, the cells were unusually active towards [1-¹⁴C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit 9 and 6 desaturation. Changes in the distribution of radioactivity between individual spholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor ish and considering the importance of eicosanoids in the biochemistry of skin, it is suggested that these agents may have direct effects on fish skin that could have important consequences for fish health in general.     

Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2–9.0) and 55 °C. CaCl₂ did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated ( P <0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.