Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin. (C) 2004 Elsevier B.V. All rights reserved.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25A degrees C to 35A degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages

Shell formation is one of the important events during larval development and metamorphosis in bivalves. However, the molecular mechanisms and environmental cues regulating shell initiation and growth are unclear. Here, we report that ferritin, a principal protein for biological iron storage and metabolism, might play a role in larval shell development of the bivalve mollusk Meretrix meretrix. A full-length ferritin subunit cDNA, named as MmeFer, was cloned and characterized. The MmeFer mRNA expression in different developmental stages, from trochophore to post larvae, was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). MmeFer mRNA expression in larvae of later developmental stages increased at least 8-fold following trochophores. Moreover, the temporal and spatial expressions of MmeFer mRNA were examined by whole mount in situ hybridization. In the trochophore stage, MmeFer was detectable where it was supposed to be for shell initiation. In the later developmental stages, MmeFer was found near digestive glands and mantle that secret larval shell. MmeFer expression was also detected in larvae cultured in artificial seawater with different iron concentrations ranging from 0 to 100 mu M. These results suggest that ferritin may play a role in the shell formation of mollusks. (C) 2009 Elsevier Inc. All rights reserved.

CDNA cloning and mRNA expression of the lipopolysaccharide- and beta-1,3-glucan-binding protein gene from scallop Chlamys farreri

Lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP) play a crucial role in the innate immune response of invertebrates as a pattern recognition protein (PRP). The scallop LGBP gene was obtained from Chlamys farreri challenged by Vibrio anguillarum by randomly sequencing cDNA clones from a whole body cDNA library, and by fully sequencing a clone with homology to known LGBP genes. The scallop LGBP consisted of 1876 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 440 amino acids with the estimated molecular mass of 47.16 kDa and a predicted isoelectric point of 5.095. The deduced amino acid sequence showed a high similarity to that of invertebrate recognition proteins from blue shrimp, black tiger shrimp, mosquito, freshwater crayfish, earthworms, and sea urchins, with conserved features including a potential polysaccharide-binding motif, a glucanase motif, and N-glycosylation sites. The temporal expression of LGBP genes in healthy and V. anguillarum-challenged C farreri scallop, measured by real-time semiquantitative reverse transcription polymerase chain reaction (PCR), showed that expression was up-regulated initially, followed by recovery as the stimulation cleared. Results indicated that scallop LGBP was a constitutive and inducible acute-phase protein that could play a critical role in scallop-pathogen interaction. (C) 2004 Elsevier B.V. All rights reserved.

CD4+ T cells play an important role in acute experimental pancreatitis in mice

BACKGROUND & AIMS: Few data are available on the potential role of T lymphocytes in experimental acute pancreatitis. The aim of this study was to characterize their role in the inflammatory cascade of acute pancreatitis. METHODS: To type this issue, acute pancreatitis was induced by repeated injections of cerulein in nude mice and in vivo CD4(+) or CD8(+) T cell-depleted mice. The role of T lymphocyte-costimulatory pathways was evaluated using anti-CD40 ligand or anti-B7-1 and -B7-2 monoclonal blocking antibodies. The role of Fas-Fas ligand was explored using Fas ligand-targeted mutant (generalized lymphoproliferative disease) mice. Severity of acute pancreatitis was assessed by serum hydrolase levels and histology. Intrapancreatic interleukin 12, interferon gamma, Fas ligand, and CD40 ligand messenger RNA were detected by reverse-transcription polymerase chain reaction. Intrapancreatic T lymphocytes were identified by immunohistochemistry. RESULTS: In control mice, T cells, most of them CD4(+) T cells, are present in the pancreas and are recruited during acute pancreatitis. In nude mice, histological lesions and serum hydrolase levels are significantly decreased. T-lymphocyte transfer into nude mice partially restores the severity of acute pancreatitis and intrapancreatic interferon gamma, interleukin 12, and Fas ligand gene transcription. The severity of pancreatitis is also reduced by in vivo CD4(+) (but not CD8(+)) T-cell depletion and in Fas ligand-targeted mutant mice. Blocking CD40-CD40 ligand or B7-CD28 costimulatory pathways has no effect on the severity of pancreatitis. CONCLUSIONS: T lymphocytes, particularly CD4(+) T cells, play a pivotal role in the development of tissue injury during acute experimental pancreatitis in mice.

Myocardial expression of the endothelin system in endotoxin-treated rats.

Although circulating plasma levels of endothelin (ET)-1 are elevated in endotoxemia, little is known about the myocardial expression of the ET system in endotoxic shock. We assessed the temporal mRNA expression pattern of key components of the ET system (pre-pro ET (ppET) -1, -2, ET-converting enzyme-1, ETA and ETB receptors) by reverse transcription polymerase chain reaction in a rat model of early endotoxic shock. Lipopolysaccharide (5 mg/kg, i.p.) caused a transient increase (p 12-fold increase; p

Isolation of retinal progenitor and stem cells from the porcine eye

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues. Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies. Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia. Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.

Localization of follistatin in the rat testis.

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT-PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.

The beta-(1--

The stimulatory effects of the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond on the mouse spleen were studied for elucidation of the mechanism of their antitumor activity, and their stimulatory effects were compared with Lentinan. The mouse spleen's weight was increased after the intraperitoneal (i.p.) injection of the oligosaccharides compared with the saline group. In addition, routinely hematoxylin and eosin (HE)-stained spleen sections showed that the injection also changed the spleen's histopathology. RNA samples were isolated from splenocytes of oligosaccharides, Lentinan or saline-injected mice. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot showed that the administration of the oligosaccharides or Lentinan enhanced mouse spleen mRNA production of TNF-alpha but not IL-2. The injection also enhanced Concanavalin A (Con A)-induced mouse splenocytes proliferation, but the in vitro administration of the oligosaccharides did not have the proliferation-enhancing effect. Taken together, these results suggest that the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond have similar stimulatory effects as Lentinan. Additionally, they may exert their antitumor effects through the induction of splenocytes mediated immune responses.

C/EBP alpha Is Up-regulated in a Subset of Hepatocellular Carcinomas and Plays a Role in Cell Growth and Proliferation

BACKGROUND & AIMS: C/EBP alpha (cebpa) is a putative tumor suppressor. However, initial results indicated that cebpa was up-regulated in a subset of human hepatocellular carcinomas (HCCs). The regulation and function of C/EBP alpha was investigated in HCC cell lines to clarify its role in liver carcinogenesis. METHODS: The regulation of C/EBP alpha expression was studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. C/EBP alpha expression was knocked-down by small interfering RNA or short hairpin RNA. Functional assays included colony formation, methylthiotetrazole, bromodeoxyuridine incorporation, and luciferase-reporter assays. RESULTS: Cebpa was up-regulated at least 2-fold in a subset (approximately 55%) of human HCCs compared with adjacent non tumor tissues. None of the up-regulated samples were positive for hepatitis C infection. The HCC cell lines Hep3B and Huh7 expressed high, PLC/PRF/5 intermediate, HepG2 and HCC-M low levels of C/EBP alpha, recapitulating the pattern of expression observed in HCCs. No mutations were detected in the CEBP alpha gene in HCCs and cell lines. C/EBP alpha was localized to the nucleus and functional in Hep3B and Huh7 cells; knocking-down its expression reduced target-gene expression, colony formation, and cell growth, associated with a decrease in cyclin A and CDK4 concentrations and E2F transcriptional activity. Epigenetic mechanisms including DNA methylation, and the binding of acetylated histone H3 to the CEBP alpha promoter-regulated cebpa expression in the HCC cells. CONCLUSIONS: C/EBP alpha is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBP alpha may be amenable to epigenetic regulation to improve treatment outcomes.

Contractile and vasorelaxant effects of hydrogen sulfide and its biosynthesis in the human internal mammary artery

This study aimed to test these hypotheses: cystathionine gamma-lyase (CSE) is expressed in a human artery, it generates hydrogen sulfide (H2S), and H2S relaxes a human artery. H2S is produced endogenously in rat arteries from cysteine by CSE. Endogenously produced H2S dilates rat resistance arteries. Although CSE is expressed in rat arteries, its presence in human blood vessels has not been described. In this study, we showed that both CSE mRNA, determined by reverse transcription-polymerase chain reaction, and CSE protein, determined by Western blotting, apparently occur in the human internal mammary artery (internal thoracic artery). Artery homogenates converted cysteine to H2S, and the H2S production was inhibited by DL-propargylglycine, an inhibitor of CSE. We also showed that H2S relaxes phenylephrine-precontracted human internal mammary artery at higher concentrations but produces contraction at low concentrations. The latter contractions are stronger in acetylcholine-prerelaxed arteries, suggesting inhibition of nitric oxide action. The relaxation is partially blocked by glibenclamide, an inhibitor of K-ATP channels. The present results indicate that CSE protein is expressed in human arteries, that human arteries synthesize H2S, and that higher concentrations of H2S relax human arteries, in part by opening K-ATP channels. Low concentrations of H2S contract the human internal mammary artery, possibly by reacting with nitric oxide to form an inactive nitrosothiol. The possibility that CSE, and the H2S it generates, together play a physiological role in regulating the diameter of arteries in humans, as has been demonstrated in rats, should be considered.

Functional effect of the C242T polymorphism in the NAD(P)H oxidase p22phox gene on vascular superoxide production in atherosclerosis.

BACKGROUND:

Increased superoxide anion production increases oxidative stress and reduces nitric oxide bioactivity in vascular disease states. NAD(P)H oxidase is an important source of superoxide in human blood vessels, and some studies suggest a possible association between polymorphisms in the NAD(P)H oxidase CYBA gene and atherosclerosis; however, no functional data address this hypothesis. We examined the relationships between the CYBA C242T polymorphism and direct measurements of superoxide production in human blood vessels.

METHODS AND RESULTS:

Vascular NAD(P)H oxidase activity was determined in human saphenous veins obtained from 110 patients with coronary artery disease and identified risk factors. Immunoblotting, reverse-transcription polymerase chain reaction, and DNA sequencing showed that p22phox protein, mRNA, and 242C/T allelic variants are expressed in human blood vessels. Vascular superoxide production, both basal and NADH-stimulated, was highly variable between patients, but the presence of the CYBA 242T allele was associated with significantly reduced vascular NAD(P)H oxidase activity, independent of other clinical risk factors for atherosclerosis.

CONCLUSIONS:

Association of the CYBA 242T allele with reduced NAD(P)H oxidase activity in human blood vessels suggests that genetic variation in NAD(P)H oxidase components may play a significant role in modulating superoxide production in human atherosclerosis.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression

PURPOSE. Vascular endothelial growth factor (VEGF)-A and placental growth factor (PIGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PIGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS. C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PIGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS. Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PIGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PIGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PIGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS. Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.