Characterization and expression of Cd8 molecules in mandarin fish Siniperca chuatsi

The full‐length complementary DNA (cDNA) sequences encoding cd8α and cd8β molecules were sequenced and characterized from mandarin fish Siniperca chuatsi. Conserved motifs and residues were found to be present in derived peptides of the Cd8 molecules. For example, WXR motif, DXGXYXC motif, and four cysteine residues were present in the extracellular region of the Cd8 protein. Threonine, serine and proline residues involved in multiple O‐linked glycosylation events were located in the membrane proximal hinge region. The common CPH motif in the cytoplasmic tail was detected similar to other teleost Cd8 molecules. Different from those in mammals, S. chuatsi Cd8 sequences have many extra cysteine residues (C149 in Cd8α sequence and C46, C51 and C158 in Cd8β sequence), which also exist in other teleost Cd8 molecules. Real‐time polymerase chain reaction (RT‐PCR) and Western blot analyses revealed that the thymus had the highest expression of cd8 messenger (m)RNA and protein. After stimulated with phytohaemagglutinin, polyriboinsine‐polyribocyaidylic acid and concanavalin A (ConA), the expression level of cd8 mRNA increased significantly in head‐kidney lymphocytes at 4 and 8 h, but decreased to normal level at 12 h. Similarly, stimulation with ConA in vivo also led to an increase in the cd8 mRNA level in the spleen. Immunohistochemistry analysis demonstrated that Cd8α‐positive cells can be detected in the thymus, spleen and intestine by using polyclonal anti‐Cd8α antibody.     

Derivation of the clonal‐cell lines from Siberian sturgeon (Acipenser baerii) head‐kidney cell lines and its applicability to foreign gene expression and virus culture

This study was conducted to establish and characterize the clonal‐cell lines from Siberian sturgeon Acipenser baerii head‐kidney tissues and to evaluate its applicability as a research tool. From the culture of A. baerii head‐kidney derived cells, 10 cell lines were established first and then eight clonal‐cell lines were derived from clonal growth and colony expansion of two cell lines that showed significant high colony‐forming ability. All eight clonal‐cell lines were morphologically similar and grew stably under monolayer culture but their growth rates were significantly different. They possessed diploid DNA contents, expressed epithelial cell‐related genes and showed strong anchorage dependency to substrates. When a clonal‐cell line was transfected separately with three plasmid vectors including fluorescent reporter genes driven by cytomegalovirus, marine medaka Oryzias dancena β‐actin or A. baerii β‐actin promoter, the cell lines expressed fluorescent signals regardless of promoter types. The cells harbouring foreign genes could be expanded to stable cell lines under drug selection and then they additionally could form the extensively proliferating colonies at low‐density culture. Finally, the clonal‐cell lines showed the susceptibility to viral haemorrhagic septicaemia virus (VHSV). Collectively, the clonal‐cell lines from A. baerii head kidney were established and these cell lines will be able to provide an excellent in vitro system for various biological studies in this fish species.     

Histological observations on lymphomyeloid organs of the Antarctic fish Trematomus bernacchii (Teleostei: Nototheniidae)

Lymphomyeloid organs of the Antarctic fish Trematomus bernacchii were studied with the aim of analysing some morphological aspects related to adaptation to low environmental temperature. The thymus of T. bernacchii was flattened, incompletely lobated and scarcely regionalised. It was filled by lymphoid elements intermingled with stromal elements. The head kidney appeared highly vascularised and mainly lymphopoietic. The spleen appeared mainly erythropoietic, with scarcely developed areas of white pulp. Received: 10 February 1997 / Accepted: 4 May 1997     

Ovarian cavity fluid of the viviparous surfperch Neoditrema ransonnetii suppresses the spontaneous cytotoxic activity of head‐kidney leucocytes against xenogeneic targets

In this study, the effect of ovarian cavity fluid (OCF) from a surfperch Neoditrema ransonnetii on the cytotoxic activity of leucocytes was investigated. In an assay targeting RTG‐2, a cell line derived from rainbow trout Oncorhynchus mykiss gonads, leucocytes from both the spleen and head kidney showed spontaneous killing activity. Pre‐incubation with OCF significantly suppressed the cytotoxic activity of head‐kidney leucocytes towards RTG‐2. This suppressive activity was due to the presence of low molecular‐mass materials. These results suggest that OCF plays significant roles in pregnancy by its ability to modulate cytotoxicity with maternal leucocytes.     

Components of the Arabidopsis mRNA decapping complex are required for early seedling development

To understand the mechanisms controlling vein patterning in Arabidopsis thaliana, we analyzed two phenotypically similar mutants, varicose (vcs) and trident (tdt). We had previously identified VCS, and recently, human VCS was shown to function in mRNA decapping. Here, we report that TDT encodes the mRNA-decapping enzyme. VCS and TDT function together in small cytoplasmic foci that appear to be processing bodies. To understand the developmental requirements for mRNA decapping, we characterized the vcs and tdt phenotypes. These mutants were small and chlorotic, with severe defects in shoot apical meristem formation and cotyledon vein patterning. Many capped mRNAs accumulated in tdt and vcs mutants, but surprisingly, some mRNAs were specifically depleted. In addition, loss of decapping arrested the decay of some mRNAs, while others showed either modest or no decay defects, suggesting that mRNAs may show specificity for particular decay pathways (3′ to 5′ and 5′ to 3′). Furthermore, the severe block to postembryonic development in vcs and tdt and the accompanying accumulation of embryonic mRNAs indicate that decapping is important for the embryo-to-seedling developmental transition.     

Effect of cerebrocrast on body and organ weights,food and water intake,and urine output of normal rats

Type 2 diabetes is associated with obesity, insulin resistance, hyperglycemia, hyperphagia, polyuria, body weight gain, excessive secretion of glucocorticoids (GCs), thymus involution, adrenal gland hypertrophy, diabetic nephropathy, etc. We examined the effect of cerebrocrast, a new antidiabetic agent (synthesized in the Latvian Institute of Organic Synthesis), on body weight, food and water intake, urine output, and on changes of organ weight: that is, kidney, thymus, adrenal gland of normal rats. Cerebrocrast was administered at doses of 0.05 and 0.5 mg kg⁻¹ per os (p.o.) once a day for three consecutive days, and its effects were observed from 3 to 27 days after the last administration. Cerebrocrast, during the experimental period, decreased body weight by an average of approximately 32.3%, food intake by about 10–15% at the beginning of the experiments and by 22.6% at the end of the experiments, especially at a dose of 0.5 mg kg⁻¹. Water intake and urine output in comparison with controls were decreased. The daily food intake decreased about 1.0 and 2.1 g by administering single cerebrocrast doses of 0.05 and 0.5 mg kg⁻¹ body weight (b.w.), respectively, but by administering for three consecutive days, food intake decreased by about 2.2 and 3.4 g, respectively. The weekly body weight gain decreased by administering a single dose of cerebrocrast by 2.61 and 2.51 g, respectively, and by triple administration it decreased by 4.36 and 3.07 g, respectively. Cerebrocrast has long‐lasting effects on these parameters and on thymus and adrenal gland weight. As cerebrocrast decreased glucose levels in normal and streptozotocin (STZ)‐induced diabetic rats, it also promoted glucose uptake by the brain, intensified insulin action and formation de novo of insulin receptors. We can conclude that cerebrocrast may regulate food intake and body weight through glucose sensing by proopiomelanocortin (POMC) neurons, that are involved in control of glucose homeostasis, stimulation of α‐melanocyte‐stimulating hormone (α‐MSH) secretion, activation of MC4‐Rs and inhibition of neuropeptide Y (NPY) in the ARC of the hypothalamus, affecting the kidney, and causing decreased urine output and water intake. Moreover, it could stimulate secretion of vasopressin. By administration of cerebrocrast thymus mass was increased, thereby preventing the action of GCs. As cerebrocrast inhibited L‐ and T‐type calcium channels, it can prevent vasoconstriction of kidney arterioles and aldosterone secretion that have significant roles in the development of hypertension and diabetic nephropathy. These properties of cerebrocrast are important for treatment of Type 2 diabetes and its consequent development of hypertension and diabetic nephropathy. Copyright © 2008 John Wiley & Sons, Ltd.     

栎金钱菌活性提取物抗衰老研究

用栎金钱菌活性提取物灌胃衰老模型小鼠,观察其对小鼠血清SOD活性、肝组织MDA和脂褐素的含量、脾指数、胸腺指数和肾指数的影响。结果表明,栎金钱菌活性提取物能使雄性小鼠血清SOD活性提高40.16%,肝MDA含量减少47.12%,肝脂褐素含量减少89.48%,脾指数增加35.13%,胸腺指数增加48.92%,肾指数增加11.26%,表明栎金钱菌活性提取物具有良好的抗衰老作用。栎金钱菌活性提取物800mg/kg·d的剂量具有最好的抗衰老效果,且具有性别的差异,对雄性小鼠的抗衰老作用略优于对雌性小鼠的。     

栎金钱菌活性提取物抗衰老研究

用栎金钱菌活性提取物灌胃衰老模型小鼠,观察其对小鼠血清SOD活性、肝组织MDA和脂褐素的含量、脾指数、胸腺指数和肾指数的影响。结果表明,栎金钱菌活性提取物能使雄性小鼠血清SOD活性提高40.16%,肝MDA含量减少47.12%,肝脂褐素含量减少89.48%,脾指数增加35.13%,胸腺指数增加48.92%,肾指数增加11.26%,表明栎金钱菌活性提取物具有良好的抗衰老作用。栎金钱菌活性提取物800mg/kg·d的剂量具有最好的抗衰老效果,且具有性别的差异,对雄性小鼠的抗衰老作用略优于对雌性小鼠的。     

Ontogeny of lymphoid organs and development of IgM-bearing cells in Atlantic halibut (Hippoglossus hippoglossus L.)

In teleost fish, the head kidney, thymus, and spleen are generally regarded as important immune organs. In this study, the ontogeny of these organs was studied in Atlantic halibut (Hippoglossus hippoglossus), larvae at various stages of development. We observed that the kidney was present at hatching, the thymus at 33 days post hatch (dph), while the spleen was the last organ to be detected at 49 dph. All three lymphoid organs were morphologically well developed during late metamorphic stages. Real time RT-PCR analysis showed that IgM mRNA expression could be observed at 66 dph and later, which correlates well with in situ hybridisation data showing that a few IgM positive cells could be detected in the anterior kidney and spleen from 66 dph. Our data also showed that the highest levels of IgM mRNA could be detected in halibut spleen. Immunostaining using a monoclonal antibody against halibut IgM detected IgM positive cells at 94 dph in both the head kidney and the spleen, which is much later than the IgM mRNA. Numerous cells expressing both IgM mRNA and protein could be detected in the spleen and anterior kidney and also to some extent in thymus specimens from adult halibut.     

Lymphomyeloid organs of the Antarctic fish Trematomus nicolai and Chionodraco hamatus (Teleostei: Notothenioidea): a comparative histological study

Lymphomyeloid organs of two common species of Antarctic fish, Trematomus nicolai and Chionodraco hamatus, were studied with the aim of analysing some morphological aspects of these organs in relation to adaptation to low environmental temperature. The thymuses of T. nicolai and C. hamatus were flattened, incompletely lobated, with numerous Hassall-like bodies, which were mainly located in the central part of the organ in C. hamatus. In T. nicolai, thymocytes, erythroid and reticular epithelial cells filled the organ. In C. hamatus, the thymocytes intermingled with reticular epithelial cells were often close to groups of melano-macrophages. In both species, the thymus did not show distinct compartmentalisation; however, the thymocytes had significantly different sizes in the outer and inner portions of the thymus. The head kidney of both species was completely filled by haematopoietic tissue, highly vascularised and mainly lymphopoietic in T. nicolai, while both erythropoietic and lymphopoietic in C. hamatus. The spleen appeared mainly erythropoietic in T. nicolai and mainly lymphopoietic in C. hamatus. Solitary melano-macrophages in T. nicolai were close to numerous small vascular ellipsoids where erythroid and lymphoid cells were intermingled without the formation of red and white pulp areas. In C. hamatus, large lymphoid areas were organised around the capillaries. The possible adaptation of lymphoid organs to the low temperature of polar water is discussed. Accepted: 8 November 1999     

Identification and expression analysis of irak1 gene in common carp Cyprinus carpio L.: indications for a role of antibacterial and antiviral immunity

In this study, the full‐length complementary (c)DNA of interleukin‐1 receptor‐associated kinase 1 gene (irak1) was cloned from common carp Cyprinus carpio. The complete open reading frame of irak1 contained 2109 bp encoding a protein of 702 amino acid residues that comprised a death domain, a ProST region, a serine–threonine‐specific protein kinase catalytic domain and a C‐terminal domain. The amino‐acid sequence of C. carpio Irak1 protein shared sequence homology with grass carp Ctenopharyngodon idellus (84·5%). The phylogenetic tree of IRAKs separated the polypeptides into four clades, comprising IRAK1s, IRAK2s, IRAK3s and IRAK4s. Cyprinus carpio Irak1 fell into the cluster with previously reported IRAK1s including teleost Irak1s. The irak1 gene was highly expressed in gills, followed by brain, skin, hindgut, buccal epithelium, spleen, foregut, head kidney and liver, and was expressed at lowest levels in gonad and muscle. The irak1 messenger (m)RNA expression was up‐regulated in liver, spleen, head kidney, foregut, hindgut, gills and skin after stimulation with Vibrio anguillarum and poly(I:C), and significantly high up‐regulated expression was observed in liver and spleen. These results implied that irak1 might participate in antibacterial and antiviral innate immunity. These findings gave the indications that irak1 may participate in antibacterial and antiviral immunity.     

Molecular cloning and characterization of a C‐type lectin in yellow catfish Tachysurus fulvidraco

This study represents the first report of a C‐type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino‐acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real‐time (qrt)‐PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration‐dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram‐positive and Gram‐negative bacteria in a calcium‐dependent manner. These results suggested that Ctl might be a C‐type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR).     

Tissue expression and bioinformatics analysis of the vitellogenin gene of Asian arowana (Scleropages formosus)

The RACE technique was used to clone the full‐length vitellogenin (VTG) cDNA sequence of Asian arowana (Scleropages formosus). The full‐length sequence was 5,550 bp with an open reading frame of 5,238 bp, encoding 1,745 amino acids, and 5′ and 3′ UTRs (untranslated regions) of 45 bp and 267 bp, respectively. Phylogenetic analysis showed that S. formosus and silver arowana (Osteoglossum bicirrhosum) share a close evolutionary relationship (bootstrap 100%). The quantitative real‐time PCR results showed that vtg expression was significantly higher in liver and gonads of male and female fish compared with its expression in the other tissues tested (p < 0.01). The relative expression levels of vtg in liver, gland, kidney, heart, head kidney, and brain of female fish were significantly higher than in the corresponding tissues of male fish (p < 0.05).     

Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes: Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post‐hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse‐like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood–thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.     

The primary localization of ascorbate and its synthesis in the kidneys of acipenserid (Chondrostei) and teleost (Teleostei) fishes

The kidneys of the rainbow trout Oncorhynchus mykiss, the channel catfish Ictalurus punctatus (both Teleostei), and the white sturgeon, Acipenser transmontanus (Chondrostei) displayed similar profiles of ascorbate distribution irrespective of the capability of synthesizing ascorbic acid. The head kidney was found to be the richest in ascorbate, whereas the trunk kidney showed significantly lower ascorbate levels in all three species. The head kidney richness in ascorbate was correlated with the localization of the cortical and chromaffin tissues known to accumulate ascorbate in some fish and mammals. Based on ascorbate concentration, it was possible to distinguish the head from the trunk kidney in salmonids and sturgeons which have an antero-posterior-fused kidney. The absence of l-gulonolactone oxidase activity in the kidneys of the channel catfish and the rainbow trout was asserted biochemically. We also confirmed that the ascorbic acid-synthesizing enzyme exists in white sturgeon kidney, and found that the enzyme distribution was inversely correlated with ascorbate concentrations. An active transport of ascorbate might exist in the head kidney of both acipenserids and the teleosts in order to maintain this vitamin at high concentrations. This report suggests a link between ascorbate concentration and its physiological functions in kidneys of lower vertebrates.Abbreviations AA ascorbic acid - TAA total ascorbic acid - DHA dehydroascorbic acid - DCIP dichloroindophenol - EDTA ethylenediaminetetra-acetic acid - GLO l-gulonolactone oxidase