The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri

Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. (c) 2005 Published by Elsevier Ltd.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDS1KK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F chinensis. (c) 2006 Elsevier B.V. All rights reserved.

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis.

Interleukin (IL)-10, a potent anti-inflammatory cytokine, limits the severity of acute pancreatitis and downregulates transforming growth factor (TGF)-beta release by inflammatory cells on stimulation. Proinflammatory mediators, reactive oxygen species, and TGF-beta can activate pancreatic stellate cells and their synthesis of collagen I and III. This study evaluates the role of endogenous IL-10 in the modulation of the regeneration phase following acute pancreatitis and in the development of pancreatic fibrosis. IL-10 knockout (KO) mice and their C57BL/6 controls were submitted to repeated courses (3/wk, during 6 wk, followed by 1 wk of recovery) of cerulein-induced acute pancreatitis. TGF-beta(1) release was measured on plasma, and its pancreatic expression was assessed by quantitative RT-PCR and immunohistochemistry. Intrapancreatic IL-10 gene expression was assessed by semiquantitative RT-PCR, and intrapancreatic collagen content was assessed by picrosirius staining. Activated stellate cells were detected by immunohistochemistry. S phase intrapancreatic cells were marked using tritiated thymidine labeling. After repeated acute pancreatitis, IL-10 KO mice had more severe histological lesions and fibrosis (intrapancreatic collagen content) than controls. TGF-beta(1) plasma levels, intrapancreatic transcription, and expression by ductal and interstitial cells, as well as the number of activated stellate cells, were significantly higher. IL-10 KO mice disclosed significantly fewer acinar cells in S phase, whereas the opposite was observed for pseudotubular cells. Endogenous IL-10 controls the regeneration phase and limits the severity of fibrosis and glandular atrophy induced by repeated episodes of acute pancreatitis in mice.

Effects of Aging and Moderate Calorie Restriction on the Reproductive Axis of the Male Rhesus Macaque (Macaca mulatta)

Calorie restriction (CR) has been established as the only non-genetic method of altering longevity and attenuating biological changes associated with aging. This nutritional paradigm has been effective in nematodes, flies, rodents, dogs and possibly non-human primates. Its long history notwithstanding, little is known regarding the exact mechanism(s) of CR action or its potential impact on the hypothalamic-pituitary-gonadal (HPG) axis. The objectives of this project were to: 1) analyze neuroendocrine changes to the HPG axis that occur with aging and 2) evaluate the effects of moderate CR on reproductive function in male rhesus macaques. Pituitary gene expression profiling, semi-quantitative RT-PCR (sqRT-PCR) and immunohistochemistry showed circadian clock mechanism components present in three age categories of macaques, demonstrated age differences in expression for Per2, indicated differential expression of Per2 and Bmal1 at opposing time points and revealed daily rhythmic expression of REV-ERBα protein. These data indicate the ability of the macaque pituitary to express core-clock genes, their protein products, and to do so in a 24-hour rhythm. Young Adult CON and CR pituitary gene expression profiles detected potential differential expression in <150 probesets. A decline in>TSHR and CGA was detected in CR macaques as measured by sqRT-PCR. Other genes investigated showed no diet-induced changes. Young Adult CON and CR testicular gene expression profiles detected potential differential expression in <300 probesets although mRNA expression was not altered based on sqRT-PCR and real-time RT-PCR. Age-related>and/or diet-induced changes in HSD17β3, INSL3, CSNK1E and CGA were observed in a separate experiment with CGA in Old Adult CR subjects returning to youthful levels. Semen samples were collected from Young Adult CON and CR macaques. Normal spermiogram measures, ZP-binding, AR assay and SCSA^® were conducted and indicated no differences between CON and CR-treated animals. Both groups exhibited similar daily testosterone profiles with no differences in mean or maximum levels; however, daily minimum testosterone levels were lower in CON animals. It appears that moderate CR had limited impact on neuroendocrine or reproductive function in male rhesus macaques based on our selected endpoints. Thus, advantageous CR health benefits can be achieved without obvious negative consequences to the HPG axis.

MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

BACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

Gene expression disruptions of organism versus organ in Drosophila species hybrids.

Hybrid dysfunctions, such as sterility, may result in part from disruptions in the regulation of gene expression. Studies of hybrids within the Drosophila simulans clade have reported genes expressed above or below the expression observed in their parent species, and such misexpression is associated with male sterility in multigenerational backcross hybrids. However, these studies often examined whole bodies rather than testes or had limited replication using less-sensitive but global techniques. Here, we use a new RNA isolation technique to re-examine hybrid gene expression disruptions in both testes and whole bodies from single Drosophila males by real-time quantitative RT-PCR. We find two early-spermatogenesis transcripts are underexpressed in hybrid whole-bodies but not in assays of testes alone, while two late-spermatogenesis transcripts seem to be underexpressed in both whole-bodies and testes alone. Although the number of transcripts surveyed is limited, these results provide some support for a previous hypothesis that the spermatogenesis pathway in these sterile hybrids may be disrupted sometime after the expression of the early meiotic arrest genes.

Advanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic rats

Advanced glycation end products (AGEs) have been implicated in the progressive vascular dysfunction which occurs during diabetic retinopathy. In the current study we have examined the role of these adducts in blood-retinal barrier (BRB) breakdown and investigated expression of the vasopermeabilizing agent vascular endothelial growth factor (VEGF) in the retina. When normoglycemic rats were injected with AGE-modified albumin daily for up to 10 days there was widespread leakage of FITC-dextran and serum albumin from the retinal vasculature when compared to control animals treated with nonmodified albumin. Ultrastructural examination of the vasculature revealed areas of attenuation of the retinal vascular endothelium and increased vesicular organelles only in the AGE-exposed rats. Quantitative RT-PCR and in situ hybridization demonstrated a significant increase in retinal VEGF mRNA expression (P <0.05). These results suggest that AGEs can initiate BRB dysfunction in nondiabetic rats and a concomitant increase in retinal VEGF expression. These findings may have implications for the role of AGEs in the pathogenesis of diabetic retinopathy.

Regulation of FOS by different compartmental stresses induced by low levels of ionizing radiation

We irradiated different cellular compartments and measured changes in expression of the FOS gene at the mRNA and protein levels. [H-3]Thymidine and tritiated water were used to irradiate the nucleus and the whole cell, respectively. I-125-Concanavalin A binding was used to irradiate the cell membrane differentially. Changes in FOS mRNA and protein levels were measured using semi-quantitative RT-PCR and SDS-PAGE Western blotting, respectively, Irradiation of the nucleus or the whole cell at a dose rate of 0.075 Gy/h caused no change in the level of FOS mRNA expression, but modestly (1.5-fold) induced FOS protein after 0.5 h, Irradiation of the nucleus at a dose rate of 0.43 Gy/h induced FOS mRNA by 1.5-fold after 0.5 h, but there was no significant effect after whole-cell irradiation. FOS protein was transiently induced 2.5-fold above control levels 0.5 h after a 0.43-Gy/h exposure of the nucleus or the whole cell. Irradiation of the cell membrane at a dose rate of 1.8 Gy/h for up to 2 h caused no change in the levels of expression of FOS mRNA or protein, but a dose rate of 6.8 Gy/h transiently increased the level of FOS mRNA S-fold after 0.5 h, These data demonstrate the complexity of the cellular response to radiation-induced damage at low doses. The lack of quantitative agreement between the transcript and protein levels for FOS suggests a role for posttranscriptional regulation. (C) 2000 by Radiation Research Society.

Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE)

Aims/hypothesis: Up-regulation of the receptor for AGEs (RAGE) and its ligands in diabetes has been observed in various tissues. Here, we sought to determine levels of RAGE and one of its most important ligands, S100B, in diabetic retina, and to investigate the regulatory role of S100B and RAGE in Müller glia.

Methods: Streptozotocin-diabetes was induced in Sprague-Dawley rats. RAGE, S100B and glial fibrillary acidic protein (GFAP) were detected in retinal cryosections. In parallel, the human retinal Müller cell line, MIO-M1, was maintained in normal glucose (5.5 mmol/l) or high glucose (25 mmol/l). RAGE knockdown was achieved using small interfering RNA (siRNA), while soluble RAGE was used as a competitive inhibitor of RAGE ligand binding. RAGE, S100B and cytokines were detected using quantitative RT-PCR, western blotting, cytokine protein arrays or ELISA. Activation of mitogen-activated protein kinase (MAPK) by RAGE was determined by western blotting.

Results: Compared with non-diabetic controls, RAGE and S100B were significantly elevated in the diabetic retina with apparent localisation in the Müller glia, occurring concomitantly with upregulation of GFAP. Exposure of MIO-M1 cells to high glucose induced increased production of RAGE and S100B. RAGE signalling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevented cytokine responses induced by high glucose and S100B in Müller glia.

Conclusions/interpretation: Hyperglycaemia in vivo and in vitro exposure to high glucose induce upregulation of RAGE and its ligands, leading to RAGE signalling, which links to pro-inflammatory responses by retinal Müller glia. These data shed light on the potential clinical application of RAGE blockade to inhibit the progression of diabetic retinopathy.

Differences in the Endocannabinoid System of Sperm from Fertile and Infertile Men

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB(1), CB(2)) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB(1), CB(2) and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB(1) and CB(2) receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems. 

Histone deacetylase 7 controls endothelial cell growth through modulation of beta-catenin

Rationale: Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function.

Objective: This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism.

Methods and Results: Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of ß-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G1 phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other ß-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased ß-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced ß-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to ß-catenin and forms a complex with 14-3-3 e, ?, and ? proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLC?-IP3K (phospholipase C?–inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with ß-catenin, disrupting the complex and releasing ß-catenin to translocate into the nucleus.

Conclusions: These findings demonstrate that HDAC7 interacts with ß-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth

Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.