Partial cDNA Research Articles

Glutamate synthases from conifers: gene structure and phylogenetic studies

BackgroundPlants synthesize glutamate from ammonium by the combined activity of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) through the glutamate synthase cycle. In plants, there are two forms of glutamate synthases that differ in their electron donors, NADH-GOGAT (EC 1.4.1.14) and Fd-GOGAT (EC 1.4.7.1), which have differential roles either in primary ammonia assimilation or in the reassimilation of ammonium from different catabolic processes. Glutamate synthases are complex iron-sulfur flavoproteins containing functional domains involved in the control and coordination of their catalytic activities in annual plants. In conifers, partial cDNA sequences for GOGATs have been isolated and used for gene expression studies. However, knowledge of the gene structure and of phylogenetic relationships with other plant enzymes is quite scant.ResultsTechnological advances in conifer megagenomes sequencing have made it possible to obtain full-length cDNA sequences encoding Fd- and NADH-GOGAT from maritime pine, as well as BAC clones containing sequences for NADH-GOGAT and Fd-GOGAT genes. In the current study, we studied the genomic organization of pine GOGAT genes, the size of their exons/introns, copy numbers in the pine genome and relationships with other plant genes. Phylogenetic analysis was performed, and the degree of preservation and dissimilarity of key domains for the catalytic activities of these enzymes in different taxa were determined.ConclusionsFd- and NADH-GOGAT are encoded by single-copy genes in the maritime pine genome. The Fd-GOGAT gene is extremely large spanning more than 330 kb and the presence of very long introns highlights the important contribution of LTR retrotransposons to the gene size in conifers. In contrast, the structure of the NADH-GOGAT gene is similar to the orthologous genes in angiosperms. Our phylogenetic analysis indicates that these two genes had different origins during plant evolution. The results provide new insights into the structure and molecular evolution of these essential genes.

A Highly Sensitive and Label-Free Microbead-Based ‘Turn-On’ Colorimetric Sensor for the Detection of Mercury(II) in Urine Using a Peroxidase-Like Split G-Quadruplex–Hemin DNAzyme

A highly sensitive and label-free microbead-based ‘turn-on’ assay was developed for the detection of Hg2+ in urine based on the Hg2+-mediated formation of intermolecular split G-quadruplex–hemin DNAzymes. In the presence of Hg2+, T–T mismatches between the two partial cDNA strands were stabilized by a T–Hg2+–T base pair, and can cause the G-rich sequences of the two oligonucleotides to associate to form a split G-quadruplex which is able to bind hemin to form the catalytically active G-quadruplex–hemin DNAzyme. This microbead-based ‘turn-on’ process allows the detection of Hg2+ in urine samples at concentrations as low as 0.5 pM. The relative standard deviation and recovery are 1.2–3.9 and 98.7–103.2%, respectively. The remarkable sensitivity for Hg2+ is mainly attributed to the enhanced mass transport ability that is inherent in homogeneous microbead-based assays. Compared with previous developments of intermolecular split G-quardruplex–hemin DNAzymes for the homogeneous detection of Hg2+ (the limit of detection was 19nM), a signal enhancement of ~1000 times is obtained when such an assay is performed on the surface of microbeads.

Estrogen-regulated expression of P450arom genes in the brain and ovaries of adult female Indian climbing perch, Anabas testudineus.

Cytochrome P450arom (CYP19), a product of cyp19a1 gene, catalyzes the conversion of androgens to estrogens and is essential for regulation of reproductive function in vertebrates. In the present study, we isolated partial cDNA encoding the ovarian (cyp19a1a) and brain (cyp19a1b) P450arom genes from adult female perch, Anabas testudineus and investigated their regulation by estrogen in vivo. Results demonstrated that cyp19a1a and cyp19a1b predominate in ovary and brain respectively, with quantity of both attuned to reproductive cycle. To elucidate estrogen-regulated expression of cyp19a1b in brain and cyp19a1a in ovary, dose- and time-dependent studies were conducted with estrogen in vitellogenic-stage fish in the presence or absence of specific aromatase inhibitor fadrozole. Results demonstrated that treatment of fish with 17β-estradiol (E2; 1.0 μM)) for 6 days caused significant upregulation of cyp19a1b transcripts, aromatase B protein, and aromatase activity in brain in a dose- and time-dependent manner. Ovarian cyp19a1a mRNA, aromatase protein, and aromatase activity, however, was less responsive to E2 than brain. Treatment of fish with an aromatase inhibitor fadrozole for 6 days attenuated both brain and ovarian cyp19a1 mRNAs expression and stimulatory effects of E2 was also significantly reduced. These results indicate that expression of cyp19a1b in brain and cyp19a1a in ovary of adult female A. testudineus was closely associated to plasma E2 levels and seasonal reproductive cycle. Results further show apparent differential regulation of cyp19a1a and cyp19a1b expression by E2/fadrozole manipulation.

Chitosan enhances resistance in rubber tree (Hevea brasiliensis), through the induction of abscisic acid (ABA)

Chitosan is considered as a natural biodegradable compound that has potential to control plant diseases. In this work, we investigated the effect of chitosan in stimulating defense responses in rubber tree against Phytophthora palmivora. Foliar spraying of either 0.2% (w/v) chitosan or 50 μM abscisic acid (ABA) were efficient in the reduction of disease severity in P. palmivora infected rubber tree. In addition, the increases of enzyme activities, i.e. catalase, peroxidase, polyphenol oxidase and phenylalanine ammonia lyase, the expression of HbPR1, HbGLU, HbASI and HbCAT genes and the deposition of callose and lignin were observed in chitosan- or ABA-treated rubber tree leaves. Besides, a partial cDNA sequence for 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme involved in ABA biosynthesis was firstly isolated from rubber tree using RT-PCR (designated HbNCED). The HbNCED gene fragment comprised of 753 bp and the deduced HbNCED protein showed 94% similarity to the NCED5 from Ricinus communis. Moreover, the up-regulation of HbNCED gene by exogenous chitosan was correlated with the induction of endogenous ABA measured by HPLC. Our results suggested that the exogenous chitosan could effectively reduce disease severity through the up-regulation of defense-related genes and ABA-biosynthesis gene, which led to the activation of defense-related proteins and ABA level in rubber tree.

Analysis of vitellogenin gene structure in Caspian roach, Rutilus caspicus (Pisces: Cyprinidae) during exposure to Atrazine

Chemical contamination of aquatic environments to EDCs has become a major focus of environmental toxicology research. The exposure of fishes to estrogenic EDCs in aquatic environments is most frequently assessed by analyzing Vitellogenin (Vg) (the egg yolk precursor protein) expression. Therefore, characterization of Vg gene is of high priority for EDCs bio-monitoring. So, we prepared liver tissue samples of Caspian roach, Rutilus caspicus for RNA extraction. Following the cDNA synthesis, specifically - designed primers were employed to amplify the Vg gene and ultimately sequence it. The evolutionary analyses of the sequence were performed using MEGA7 software. The obtained results indicated that the designed primers successfully amplified the partial cDNA sequence. Our results indicated that this sequence most probably belongs to the Vg1 form of the gene. Moreover, it was demonstrated that Caspian roach and Petroleuciscus esfahani share a common ancestor. Noteworthy, the study of Vg gene would be helpful to understand the molecular mechanisms of development and would be used to establish a bio-monitoring tool for detection of exposure to different EDCs.

Regulation of pregnane-X-receptor and microRNAs on detoxification-related genes expressions in Mugilogobius abei under the exposure to diclofenac.

Diclofenac (DCF) has been recognized as an emerging contaminant in aquatic environments. Though many studies have investigated the toxic effects of DCF in human and mammals, limited information is available for the responses of genes associated with detoxification metabolisms in non-target aquatic organisms such as fish. In the present study, a small benthic fish Mugilogobius abei, was chosen as the test organism and the effects of DCF on detoxification-related genes at transcriptional level in M.abei were investigated. Partial cDNAs of pregnane-X-receptor (pxr), cytochrome P450 3A (cyp 3a) and alpha-gst were cloned firstly. The responses of cyp 1a, cyp 3a, alpha-gst and p-gp genes and associated microRNAs expressions were measured under different concentrations of DCF exposure (0.5, 5, 50, 500μg/L) for 24h and 168h. Induction of cyp 1a, cyp 3a, alpha-gst, p-gp and pxr mRNA expressions was observed under DCF exposure for different time. Positive concentration-response relationships between DCF concentrations and cyp 1a as well as alpha-gst mRNA expression were observed under DCF exposure for 24h. The similar trend between pxr mRNA expression and cyp 3a gene expression suggested the role of pxr in regulation of its downstream detoxification genes involved in DCF detoxification in M.abei. The negative correlation between miR-27a and p-gp expression under DCF exposure for 24h indicated the role of miRNA in post transcriptional regulation on detoxification-related genes mRNAs in M.abei exposed to DCF. Overall, DCF exposure, even at environmental levels, may interrupt the responses of the detoxification genes in M.abei, which may affect the response of the exposed organism to other pollutants. This work provides implications on the bio-monitoring and risk assessment of DCF in aquatic ecosystems by using of local native fish species.

Localization of major yolk protein in the digestive tract of the sea urchin Strongylocentrotus intermedius

In the present study, we examined the localization of the major yolk protein (MYP) in the intestine of the sea urchin Strongylocentrotus intermedius. First, partial MYP complementary DNA was isolated from the sea urchin intestine. The expression level of MYP messenger RNA (mRNA) along the sea urchin digestive tract is highest in the intestine, so we performed in situ hybridization and immunohistochemical analysis using this tissue. No MYP mRNA was detected in the luminal epithelium, connective tissue, muscle tissue, or coelomic epithelium by in situ hybridization analysis. Positive immunohistochemical staining was observed in the luminal epithelium, inner epithelium and connective tissue, the signal being strongest in the latter. We conclude that MYP synthesized in the inner epithelial cells is moved to and stored in connective tissue and the luminal epithelium, before being secreted into the body cavity and the inner digestive cavity of the sea urchin.

Spatio-temporal expression of Mesocestoides corti McVAL2 during strobilar development

VAL proteins belong to a diverse superfamily containing the CAP domain, with members described for various eukaryotic organisms, including parasites. They are implicated in diverse biological activities and, as secreted proteins, may be related in host – parasite interactions. For this reason they have been proposed as vaccine candidates against nematode infections. However, little is known about their function in cestodes.In M. corti, four partial cDNA sequences coding for members of the CAP superfamily were previously isolated. In this work we characterize the expression of McVAL2 in the larvae and segmented worms of M. corti, describing mRNA and protein localization using fluorescent microscopy. We also optimized real time PCR analysis for quantification of mRNA expression through the different stages of strobilar development. We show that McVAL2 is differentially located, depending on the developmental stage, and can be used as a molecular marker for the neuroendocrine system in the larvae.The dynamic and stage-specific expression patterns of McVAL2, combined with the large number of VAL proteins found in the genomes of parasitic flatworms, suggest varied roles for the VAL protein family in the biology of these parasites.

Identification and characterization of the lamprey Flotillin-1 gene with a role in cell adhesion

Flotillin-1 is a kind of localize into specific cholesterol rich microdomains in cellular membranes and highly conserved lipid rafts marker protein widely distributed in animals and plants. It provides a platform for the reaction of many proteins in signal transduction, as scaffolding plays an important role in transmembrane signaling and cell adhesion. Here, Flotillin-1 protein from lamprey was identified and characterized (designated as L-Flotillin-1). After a partial cDNA sequence of L-Flotillin-1 was identified in a lamprey supraneural body cDNA library, the full-length cDNA was obtained using 3’- and 5’-rapid amplification of cDNA ends (RACE). L-Flotillin-1 encodes 424 amino acids and contains a prohibitin domain and a flotillin repetitive area. The L-Flotillin-1 protein was primarily distributed in kidney, supraneural body, gill, heart, liver and intestine via real-time PCR and immunohistochemistry assays. Immunofluorescence and western blot results showed that L-Flotillin-1 was considered to be used as a marker protein of lamprey lipid rafts and exosomes. Furthermore, overexpression of pEGFP-N1-L-Flotillin-1 induced the up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) mRNA levels. These results indicated that the L-Flotillin-1 gene encodes Flotillin-1 protein that was used as a conserved marker protein and may play an important role in cell adhesion, providing clues for understanding the universal functions of Flotillin-1 proteins in other species and suggesting that these proteins could serve as pattern recognition molecules in immunotherapy. We revealed that Flotillin-1 protein of lamprey overexpression in human cells plays a prevalent role in cell migration and provide new thought of treatment to diseases.

Fluorescent aptasensor for antibiotic detection using magnetic bead composites coated with gold nanoparticles and a nicking enzyme

Antibiotic abuse has been bringing serious pollution in water, which is closely related to human health. It is desirable to develop a new strategy for antibiotic detection. To address this problem, a sensitive fluorescent aptasensor for antibiotic detection was developed by utilizing gold nanoparticles modified magnetic bead composites (AuNPs/MBs) and nicking enzyme. AuNPs/MBs were synthesized with the help of polyethylenimine (PEI). The prepared AuNPs/MBs acted as dual-functional scaffolds that owned excellent magnetic separation capacity and strong covalent bio-conjugation. The non-specifically absorbed aptamers in AuNPs/MBs were less than that in MBs. Hence, the fluorescent aptasensor based on AuNPs/MBs show a better signal to background ratio than that based on carboxyl modified magnetic beads (MBs). In this work, ampicillin was employed as a model analyte. In the presence of ampicillin, the specific binding between ampicillin and aptamer induced structure-switching that led to the release of partial complementary DNA (cDNA) of aptamer. Then, the released cDNA initiated the cycle of nicking enzyme assisted signal amplification (NEASA). Therefore, a large amount of taqman probes were cleaved and fluorescence signal was amplified. The prepared fluorescent aptasensor bring sensitive detection in range of 0.1–100 ng mL−1 with the limit of detection of 0.07 ng mL−1. Furthermore, this aptasensor was also successfully applied in real sample detection with acceptable accuracy. The fluorescent aptasensor provides a promising method for efficient, rapid and sensitive antibiotic detection.

Molecular characterization and expression analysis of three TLR genes in yellow catfish (Pelteobagrus fulvidraco): Responses to stimulation of Aeromonas hydrophila and TLR ligands

Toll-like receptors (TLRs) are one of the most extensively researched pattern recognition receptors (PRRs) and play an important role in the innate immune system. In this study, partial cDNA sequences of the Pf_TLR18 and Pf_TLR19 genes and complete cDNA sequence of the Pf_TLR21 gene were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORFs) of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes were 1956 bp, 2262 bp and 2949 bp in length, encoding 651, 753 and 982 amino acids, respectively. The Pf_TLR18 and Pf_TLR19 consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor domain, and the Pf_TLR21 only has LRRs and TIR domain. Homologous identity revealed that the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes have high nucleotide and protein sequence similarity with channel catfish, especially the TIR domains that exhibited the greatest conservation compared to channel catfish. Ontogenetic expression analyses indicated that the mRNA expressions of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes could be detected from fertilized eggs to 30 day post-hatching and they exhibited different variation trends after hatching. The three TLR genes were expressed in various tissues, but they were mostly highly expressed in the spleen. The mRNA expression levels of the three genes were up-regulated in the spleen, head kidney, trunk kidney, liver and blood after challenge of killed Aeromonas hydrophila. In addition, the expressions of the three TLR genes were induced to up-regulate in isolated peripheral blood lymphocytes of yellow catfish after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). Our findings indicate that the three TLR genes may play a potential role in the host defense against pathogenic microbes. These results will provide valuable information to better understand the function of TLR genes in the innate immune system of yellow catfish.

Effects of rapamycin on life span and on expression of TOR and S6K in Brachionus calyciflorus (Rotifera)

AB Aquatic Biology Contact the journal Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AB 26:49-56 (2017) - DOI: https://doi.org/10.3354/ab00673 Effects of rapamycin on life span and on expression of TOR and S6K in Brachionus calyciflorus (Rotifera) Huanhuan Xu, Lulu Liu, Yuqi Su, Ye Liang, Jiaxin Yang* School of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, PR China *Corresponding author: yangjx@njnu.edu.cn ABSTRACT: The mechanistic target of rapamycin (mTOR) coordinates a complex signal pathway from translation to autophagy that is a key regulator of not only growth and proliferation but also metabolism and aging. mTOR is sensitive to many environmental and endocrine stimuli. We investigated the influence of TOR signaling on aging and reproduction of the rotifer Brachionus calyciflorus using rapamycin as an exogenous inhibitor. We found that 2 and 4 µM rapamycin extended B. calyciflorus life span by 15 and 22%, respectively compared with controls (p < 0.05). The reproductive peak was significantly delayed by rapamycin at 2 and 4 µM (p < 0.01), but the pre- and post-reproduction periods were not significantly different from controls (p > 0.05). Partial cDNAs coding 375 bp for TOR and 951 bp for S6 kinase (S6K) were obtained from B. calyciflorus expressed sequence tags. The identities of the deduced amino acid sequences of B. calyciflorus cDNAs to their human orthologs were 58% for TOR and 68% for S6K. TOR and S6K mRNA expression were up- or down-regulated by different rapamycin concentrations (0.5, 1, 2, 4, 8, and 16 µM) and treatment intervals (control, 12, 24, 36, and 48 h). The results indicated that TOR inhibition acted additively to extend rotifer life span, with up- and down-regulation simultaneously impacting reproduction and gene expression. KEY WORDS: Aging · Life span · mRNA expression levels · Rapamycin · Reproduction · Rotifer Full text in pdf format PreviousNextCite this article as: Xu H, Liu L, Su Y, Liang Y, Yang J (2017) Effects of rapamycin on life span and on expression of TOR and S6K in Brachionus calyciflorus (Rotifera). Aquat Biol 26:49-56. https://doi.org/10.3354/ab00673 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AB Vol. 26. Online publication date: May 15, 2017 Print ISSN: 1864-7782; Online ISSN: 1864-7790 Copyright © 2017 Inter-Research.

CDNA cloning, molecular modeling and docking calculations of L-type lectins from Swartzia simplex var. grandiflora (Leguminosae, Papilionoideae), a member of the tribe Swartzieae

The genus Swartzia is a member of the tribe Swartzieae, whose genera constitute the living descendants of one of the early branches of the papilionoid legumes. Legume lectins comprise one of the main families of structurally and evolutionarily related carbohydrate-binding proteins of plant origin. However, these proteins have been poorly investigated in Swartzia and to date, only the lectin from S. laevicarpa seeds (SLL) has been purified. Moreover, no sequence information is known from lectins of any member of the tribe Swartzieae. In the present study, partial cDNA sequences encoding L-type lectins were obtained from developing seeds of S. simplex var. grandiflora. The amino acid sequences of the S. simplex grandiflora lectins (SSGLs) were only averagely related to the known primary structures of legume lectins, with sequence identities not greater than 50–52%. The SSGL sequences were more related to amino acid sequences of papilionoid lectins from members of the tribes Sophoreae and Dalbergieae and from the Cladratis and Vataireoid clades, which constitute with other taxa, the first branching lineages of the subfamily Papilionoideae. The three-dimensional structures of 2 representative SSGLs (SSGL-A and SSGL-E) were predicted by homology modeling using templates that exhibit the characteristic β-sandwich fold of the L-type lectins. Molecular docking calculations predicted that SSGL-A is able to interact with D-galactose, N-acetyl-D-galactosamine and α-lactose, whereas SSGL-E is probably a non-functional lectin due to 2 mutations in the carbohydrate-binding site. Using molecular dynamics simulations followed by density functional theory calculations, the binding free energies of the interaction of SSGL-A with GalNAc and α-lactose were estimated as −31.7 and −47.5 kcal/mol, respectively. These findings gave insights about the carbohydrate-binding specificity of SLL, which binds to immobilized lactose but is not retained in a matrix containing D-GalNAc as ligand.

Identification of suitable reference genes for quantitative real-time PCR normalization in blotched snakehead Channa maculata.

A systematic study was conducted to identify reliable reference genes for normalization of gene expression analysis in the blotched snakehead Channa maculata under normal physiological conditions. Firstly, the partial complementary (c)DNA of nine candidate reference genes (actb, tmem104, ube2l3, ef1α, churc1, tmem256, rpl13a, sep15 and g6pd) were cloned from C. maculata. The expression levels of these genes were then assessed in embryos of different developmental stages and various tissue types of adult fish using quantitative real-time (qrt-)PCR. RefFinder algorithm was used to evaluate the expression stability of these genes based on their cycle-threshold (Ct ) values in the qrt-PCR analysis. Results showed that there was no single best reference gene for all stages of embryos and adult tissues tested. Furthermore, it was found that, among the nine candidate genes tested, actb and tmem104 were the most stable reference genes across adult tissue types, while sep15 and tmem256 were the most stable ones across developmental stages of embryos. These stable reference genes are recommended for normalization of gene expression analysis in C. maculata.

Molecular cloning and characterization of rosmarinic acid biosynthetic genes and rosmarinic acid accumulation in Ocimum basilicum L.

We have aimed to investigate the expression of genes related to rosmarinic acid (RA) synthesis and rosmarinic acid content in 2 Ocimum basilicum cultivars, green (cinnamon) and purple (red rubin) basil. Specifically, genes related to rosmarinic acid biosynthesis were cloned and characterized for O. basilicum. We obtained partial cDNAs of tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate reductase (HPPR), which were of 323 bp and 616 bp in size, respectively. The transcription levels of most genes related to rosmarinic acid synthesis were higher in green basil compared to purple basil, except for ObPAL and Ob4CL in the root. The highest expression was obtained in the leaves of green basil for all genes and the roots of purple basil for all genes, except for TAT. The highest rosmarinic acid content was obtained in the leaves of both cultivars, with higher RA accumulating in green basil compared to purple basil. The leaves had the highest RA content out of all plant organs, with the RA accumulation in the leaves of green basil being 1.64 times higher compared to purple basil. Further study is required to investigate whether a similar trend is observed across O. basilicum cultivars of different color types.

Uninhibited biosynthesis and release of phytosiderophores in the presence of heavy metal (HM) favors HM remediation.

Production of phytosiderophore (PS) has been causally related to iron-deficiency tolerance in cereals. However, PS can also chelate heavy metal and thus may represent a viable phytoextraction strategy on contaminated soils. Two separate experiments were conducted to assess the affect of heavy metal on phytosiderophore biosynthesis and their release in the rhizosphere of wheat. Root exudates were collected from 10-day-old wheat seedlings raised on Fe-deficient nutrient solution in the presence of 2.5, 5.0, and 10mM concentration of different heavy metals (Cd, Pb, and Ni) for 3-day period, for the phytosiderophore and the heavy metal analysis. Plant uptake of respective heavy metal was positively correlated with the heavy metal concentration of the nutrient solution. Phytosiderophore release was positively influenced in the presence of the heavy metal. Increasing concentration of Cd, Pb, and Ni showed positive correlation with the PS release until 5mM concentration followed by a decline at 10mM. However, a higher induction of PS release was measured in wheat seedlings treated with Cd and Pb than Ni. Further, transcript expression analysis of nicotianamine synthase (NAS) and nicotianamine amino transferase (NAAT), involved in phytosiderophore biosynthesis, was done in roots of 10-day-old Fe-deficient wheat subjected to 2.5, 5.0, and 10mM of Cd, Pb, and Ni. Both NAS and NAAT were expressed not only under Fe deficiency but also in the presence of Cd, Pb, and Ni. Sequencing of partial cDNA of NAS revealed a nucleotide length of 998bp, while multiple sequence alignment of NAS with HvNAS revealed 92% sequence similarity. This study irrevocably shows that phytosiderophore biosynthesis and release are not impaired in the presence of heavy metal and that phytosiderophore mediates the uptake of toxic heavy metal.

Irisin in goldfish (Carassius auratus): Effects of irisin injections on feeding behavior and expression of appetite regulators, uncoupling proteins and lipoprotein lipase, and fasting-induced changes in FNDC5 expression

Irisin is a peptide cleaved from the fibronectin type III domain containing protein 5 (FNDC5) gene that is secreted predominantly by muscle cells but also by other tissues including brain and intestine. In mammals, irisin has been shown to have thermogenic actions via the modulation of uncoupling proteins (UCPs) and to affect feeding and energy homeostasis via actions in brain, adipose tissue, liver, muscle and gastrointestinal tract. To examine the role of irisin on feeding and metabolism in fish, the effects of peripheral (intraperitoneal) injections of irisin on feeding behavior, glucose levels and the mRNA expressions of appetite regulators (cocaine and amphetamine regulated transcript CART, agouti related protein AgRP, orexin), UCPs and lipoprotein lipase LPL and brain factors (brain-derived neurotrophic factor , BDNF and tyrosine hydroxylase TH) were assessed in brain, white muscle and intestine. Irisin injections (100ng/g) induced a decrease in food intake and increases in brain orexin, CART1 and CART2, UCP2, BDNF, muscle UCP2 and intestine LPL mRNA expressions but did not affect blood glucose levels, brain AgRP, TH, UCP1, UCP3 and LPL or muscle UCP1, UCP3 and LPL expressions. A partial goldfish FNDC5 cDNA was isolated and the expressions of FDNC5, UCPs, LPL and BDNF were also compared between fed and fasted fish. Fasting induced decreases FNDC5 mRNA expression in the brain and intestine, but not in muscle. Fasting also induced increases in brain BDNF and LPL expressions and increases in UCP1, UCP2, UCP3 and LPL expressions in muscle. Our result suggest that irisin is an anorexigenic factor in fish and its actions might be in part mediated by appetite-regulating factors such as CART and orexins as well as UCP2 and brain factors such as BDNF.

Molecular cloning and tissue expression pattern of partial hepatic growth factor cdna in arabian camel (Camelus Dromedarius)

The present study was performed to clone hepatic growth factor (HGF) in Arabian camel. RT-PCR was conducted on RNA from skeletal muscle using primers designed from the conserved regions in different animal species. The resultant PCR amplicon was subjected to sequencing and bioinformatics analysis. The results revealed that the obtained sequence belongs to HGF gene family. The nucleotides sequence was deposited in the GenBank with accession number KU736793. Furthermore, the data showed base frequencies of A = 32.77%, C = 21.67%, G = 22.06% and T = 23.5%. The nucleotide sequence alignment revealed that Camels dromedarius HGF showed 99% identity with C. bactrianus and C. ferus HGF, while it showed 97% identity with those of either Bos taurus, Capra hircus, Ovisaries and Equus caballus. Of the 766 nucleotides analysed, 36 substitutions varied from transitions and transversion were detected. The translated amino acids showed 2 non-synonymous substitutions discriminating camelids from other species; serine (S46) into alanine (A46), alanine (A46) in other species at T136→G136. The results showed a clear expression of HGF mRNA in a wide variety of the tested tissues; skeletal muscle, spleen, testes, liver, kidney and heart. The obtained results could be useful for more understanding of the structural-function relationship of HGF in Arabian camel and addressing the genetic diversity of the Arabian camel.

CDNA Cloning and Distribution of Neuromedin U and its Receptor in the Rabbit

Neuromedin U (NMU) is a kind of peptides that was initially purified from the porcine spinal cord. There are two peptides NMU-25 and NMU-8 with similar biological functions. NMU is found in peripheral organs such as gastrointestinal (GI) tract and the central nervous system (CNS). NMU has two receptors NMU-R1 and NMU-R2.NMU-R1 is expressed particularly in the various peripheral tissues, whereas NMU-R2 is expressed in the central nervous system. However, the expression and function of NMU in the rabbit remained unclear. In this study, a novel partial cDNA cloning and expression of NMU, NMU-R1 and NMU-R2 mRNAs in 30 rabbit tissueswere investigated using semi-quantitative RT-PCR. The specific fragments were amplified and target cDNA fragment lengths were 443, 366 and 203 bp, respectively consistent with the expected size of the fragment, and clear bands. The cDNA cloned of rabbit NMU, NMU-R1 and NMU-R2 sequences were found to be 84%, 84% and 87% identical to those of the corresponding human homologues, respectively. The rabbit NMU, NMU-R1 and NMU-R2 cDNAs were encoded as 147,122 and 67 amino acid sequences, respectively. Meanwhile, the rabbit NMU-R1 and NMU-R2 amino acid sequence were found to have a typical transmembrane structure of GPCR.The results of RT-PCR analysis showed that the expression of NMU, NMU-R1 and NMU-R2 mRNAs was found in the central nervous system and in the peripheral tissues, whereas NMU mRNAs were more abundant in the peripheral tissues. These results revealed that the NMU, NMU-R1 and NMU-R2 mRNAs were expressed in rabbit tissues in various distribution patterns. The study provided the experimental data for further research on the physiological function of NMU and its mechanisms of action in rabbits.

Identification and characterization of host cell proteins interacting with Scylla serrata reovirus non-structural protein p35.

We have previously shown that non-structural protein p35, encoded by Scylla serrata reovirus (SsRV) S10, may act as a viroporin. To characterize the role of p35 protein in the modulation of cellular function, a yeast two-hybrid system was used to screen a cDNA library derived from S. serrata to find its interacting partner. Protein interactions were confirmed in vitro by GST pull-down. Full cDNAs of p35 interactors were cloned by the rapid amplification of cDNA ends. After two-hybrid library screening, we isolated partial cDNAs encoding hemocyanin, cryptocyanin, and TAX1BP1. Interaction of p35 with each of hemocyanin, cryptocyanin, and TAX1BP1 was confirmed by GST pull-down. The full-length cDNA fragments of hemocyanin, cryptocyanin, and TAX1BP1 were 2287, 2422, and 3437bp, respectively, and they encoded three putative proteins with molecular masses of ~76.9, ~79.2, and ~107.2kDa, respectively. This study casts new light on the function and physiological significance of p35 during the SsRV replication cycle.