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#31830476   2019/12/09 To Up

Alanine scanning and characterization of core peptides in Scombridae fish family for construction of Kiss1 super analog.

Chronic Kiss1 administration strongly promotes gonadal development in immature chub mackerel (cm) (Scomber japonicus). Here, we performed an Alanine scanning (Ala-scanning) of Kiss1 to determine its key residues. Additionally, we examined functional peptides from 16 Scombridae species to develop maturation-inducing super-analogs that can be used universally in Scombridae species. In the Ala-scanning of Kiss1-15 (QDMSSYNFNSFGLRY), substitution of Gln and Asp did not affect agonistic activity. This suggests that peptides could be downsized. Furthermore, it is possible that Phe can be substituted by unnatural amino acids that are difficult to degrade. In molecular cloning, only Scomber showed a 16-residue form as a putative mature peptide. The other genera, did not have a His residue at the N-terminal, which indicated that the functional peptide was 15 residues and the second and third residues from the N-terminal showed variation between interspecies. Next, we examined the binding affinity of various synthetic Kiss1 core peptides in Scombridae interspecies using an SRE-Luc reporter system. We cloned Kiss1 receptors (KissR1) from bluefin tuna (bft) (Thunnus orientalis) and Japanese Spanish mackerel (jsm) (Scomberomorus niphonius) for the first time. In binding affinity with cmKissR1, bftKissR1, and jsmKissR1, the species specificity of the second residue from the N-terminus in each ligand could be ignored, but the difference in the third residue strongly affected receptor binding. Scombridae species possess the same Kiss1 system but the structure of the functional peptide might be species-specific.
Hirofumi Ohga, Fumiko Akase, Ryo Sakanoue, Ayami Matsushima, Kohei Ohta, Michiya Matsuyama

1893 related Products with: Alanine scanning and characterization of core peptides in Scombridae fish family for construction of Kiss1 super analog.

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#30849410   2019/03/05 To Up

Molecular characterization, expression analysis, and functional properties of multiple 5-hydroxytryptamine receptors in Pacific abalone (Haliotis discus hannai).

Neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT) in the central nervous system regulate diverse physiological functions, including reproduction, feeding, learning, and memory, in diverse animal phyla. 5-HT and the 5-HT1 subtype receptor play important roles in sexual maturation and in the initiation of gamete release in mollusks. However, little is known about the involvement of other 5-HT receptor subfamilies in the reproduction process. In the present study, we identified the cDNAs encoding eight subtypes of 5-HT receptors from the ganglia tissues of the Pacific abalone Haliotis discus hannai (Mollusca; Gastropoda; Haliotidae), and examined the gonadal expression of the transcripts of 5-HT receptors. A phylogenetic analysis indicated that the molluskan 5-HT receptors are largely classified into four major clades: 5-HT1/5/7, 5-HT2, 5-HT4, and 5-HT6. Among the H. discus hannai (Hdh) 5-HT1-7 transcripts, Hdh5-HT1B, 4A, 4B, and 6 were the major subtypes detected in the mature ovary. Estradiol-17β injection into the pedal sinus induced the downregulation of 5-HT4B and upregulation of 5-HT6 transcripts in the ovary of mature abalone within 72 h. In HEK293 cells overexpressing Hdh5-HT1B, forskolin-stimulated cAMP response element luciferase (CRE-Luc) reporter activity was inhibited by 5-HT in a dose-dependent manner, whereas serum response element luciferase (SRE-Luc) activity was not affected. In Hdh5-HT4A-expressing HEK293 cells, forskolin-stimulated CRE-Luc and SRE-Luc reporter activities were both marginally increased by treatment with a high dose of 5-HT. Our results provide new insights into the roles of 5-HT through diverse G protein-coupled 5-HT receptors in the reproductive process of mollusks.
Kyeong Seop Kim, Mi Ae Kim, Young Chang Sohn

1488 related Products with: Molecular characterization, expression analysis, and functional properties of multiple 5-hydroxytryptamine receptors in Pacific abalone (Haliotis discus hannai).



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#30797596   2019/02/15 To Up

In vitro effects of tongue sole LPXRFa and kisspeptin on relative abundance of pituitary hormone mRNA and inhibitory action of LPXRFa on kisspeptin activation in the PKC pathway.

Results of previous studies indicated the existence of LPXRFa, the piscine ortholog of gonadotropin-inhibitory hormone (GnIH), and kisspeptin (Kiss2) in tongue sole (Cynoglossus semilaevis), and that LPXRFa exerts an inhibitory effect on Kiss2 activation in the protein kinase A (PKA) pathway. The functions in the control of reproduction and whether LPXRFa antagonizes the action of Kiss2 by inhibiting the protein kinase C (PKC) pathway, however, are still unknown. In the present study, there was an initial investigation of the direct effects of LPXRFa and Kiss2 on relative abundance of pituitary hormone mRNA transcripts using a whole pituitary culture system. Results indicated that LPXRFa-1 specifically functioned to increase relative abundance of lhβ mRNA when there were comparisons with the control, without any effect on relative abundance of gh, gthα and fshβ mRNA. Treatment with LPXRFa-2 resulted in a reduction in relative abundance of gthα and lhβ mRNA, and did not alter relative abundance of fshβ mRNA. Treatment of LPXRFa-2 resulted in a greater relative abundance of gh mRNA. Treatment with Kiss2, however, resulted in an increase in relative abundance of gthα and fshβ mRNA transcripts, without altering relative abundances of gh and lhβ mRNA. Subsequently, there was valuation of the potential interaction between LPXRFa and kisspeptin in COS-7 cells transfected with the cognate receptors. Both LPXRFa-1 and LPXRFa-2 suppressed serum responsive element-dependent luciferase (SRE-luc) activity when compared to stimulation with Kiss2 alone, indicating an inhibitory effect of LPXRFa on kisspeptin activation on the PKC pathway. Overall, data from the present study provide novel evidence for differential actions of LPXRFa and kisspeptin on pituitary hormone synthesis as well as for the interaction between LPXRFa and kisspeptin systems in teleosts.
Bin Wang, Guokun Yang, Yongjiang Xu, Yaxing Zhang, Xuezhou Liu

2075 related Products with: In vitro effects of tongue sole LPXRFa and kisspeptin on relative abundance of pituitary hormone mRNA and inhibitory action of LPXRFa on kisspeptin activation in the PKC pathway.

100ug1mg10mg2 Pieces/Box100ug1mg96T2 Pieces/Box96 tests2 Pieces/Box100μg2 Pieces/Box

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#29746909   2018/05/07 To Up

Characterization of LPXRFa receptor in the half-smooth tongue sole (Cynoglossus semilaevis): Molecular cloning, expression profiles, and differential activation of signaling pathways by LPXRFa peptides.

Gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide, serves as a key player in the regulation of reproduction across vertebrates, acting on the brain and pituitary to modulate reproductive physiology and behavior. However, little information is available in teleosts regarding the intracellular signal transduction pathway in response to GnIH. To this end, we first cloned the gene of LPXRFa (the piscine ortholog of GnIH) receptor in the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. The full-length cDNA of LPXRFa receptor was 2201 bp in size with an open reading frame (ORF) of 1365 bp that encoded 454 amino acids. Tissue distribution showed that LPXRFa receptor transcripts could be detected at high levels in the brain, to a lesser extent in the pituitary, and at low levels in the ovary and other peripheral tissues. In vitro functional analysis revealed that putative tongue sole LPXRFa-1 and LPXRFa-2 peptides significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity in COS-7 cells transfected with the novel receptor, and these stimulatory effects were evidently reduced by two inhibitors of the PLC/PKC pathway. In addition, neither LPXRFa-1 nor LPXRFa-2 altered the cAMP-responsive element (CRE)-luc activity, but only LPXRFa-2 could markedly decrease forskolin-induced CRE-luc activity in COS-7 cells expressing its cognate receptor. Taken together, our results encompass the first study reporting the existence of LPXRFa receptor in the order Pleuronectiformes and provide novel evidence of differential activation of signaling pathways by LPXRFa peptides in fish.
Bin Wang, Guokun Yang, Quan Liu, Jingkai Qin, Yongjiang Xu, Wensheng Li, Xuezhou Liu, Bao Shi

2279 related Products with: Characterization of LPXRFa receptor in the half-smooth tongue sole (Cynoglossus semilaevis): Molecular cloning, expression profiles, and differential activation of signaling pathways by LPXRFa peptides.

2 Pieces/Box2 Pieces/Box100ug2 Pieces/Box100ug100ug Lyophilized 100ul100.00 ul100μg100ug

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#28754347   2017/07/25 To Up

Inhibitory action of tongue sole LPXRFa, the piscine ortholog of gonadotropin-inhibitory hormone, on the signaling pathway induced by tongue sole kisspeptin in COS-7 cells transfected with their cognate receptors.

Kisspeptin (Kiss) acts as a positive regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the intricate web of intracellular signal transduction pathways in response to Kiss is still far from being fully understood in teleosts. Accordingly, we investigated the molecular mechanism of Kiss action and its possible interaction with LPXRFa signaling in this study. In vitro functional analysis revealed that synthetic tongue sole Kiss2 decapeptide increased the cAMP responsive element-dependent luciferase (CRE-luc) activity in COS-7 cells transfected with its cognate receptor, while this stimulatory effect was markedly reduced by two inhibitors of the adenylate cyclase (AC)/protein kinase A (PKA) pathway. Similarly, Kiss2 also significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity, whereas this stimulatory effect was evidently attenuated by two inhibitors of the phospholipase C (PLC)/protein kinase C (PKC) pathway. In addition, LPXRFa-2 suppressed Kiss2-elicited CRE-luc activity in a dose-dependent manner. Taken together, Kiss2 utilizes both AC/PKA and PLC/PKC pathways to exert its functions via its cognate receptor and LPXRFa may antagonize the action of Kiss2 by inhibiting kisspeptin signaling. As far as we know, this study is the first to characterize the half-smooth tongue sole kisspeptin and LPXRFa signaling pathway in COS-7 cells transfected with their cognate receptors and provides novel information on the interaction between LPXRFa system and kisspeptin system in teleosts.
Bin Wang, Guokun Yang, Quan Liu, Jingkai Qin, Yongjiang Xu, Wensheng Li, Xuezhou Liu, Bao Shi

2091 related Products with: Inhibitory action of tongue sole LPXRFa, the piscine ortholog of gonadotropin-inhibitory hormone, on the signaling pathway induced by tongue sole kisspeptin in COS-7 cells transfected with their cognate receptors.

5ug2 Pieces/Box250 TESTS100uLInhibitors96 tests2 Pieces/Box1mg2 Pieces/Box2 Pieces/Box

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#28623279   2017/06/16 To Up

Divergent Regulation of Actin Dynamics and Megakaryoblastic Leukemia-1 and -2 (Mkl1/2) by cAMP in Endothelial and Smooth Muscle Cells.

Proliferation and migration of vascular smooth muscle cells (VSMCs) or endothelial cell (ECs) promote or inhibit, respectively, restenosis after angioplasty, vein graft intimal thickening and atherogenesis. Here we investigated the effects of cAMP-induced cytoskeletal remodelling on the serum response factor (SRF) co-factors Megakaryoblastic Leukemia-1 and -2 (MKL1 and MKL2) and their role in controlling VSMC and EC proliferation and migration. Elevation of cAMP using forskolin, dibutyryl-cAMP (db-cAMP), BAY60-6583 or Cicaprost induced rapid cytoskeleton remodelling and inhibited proliferation and migration in VSMCs but not EC. Furthermore, elevated cAMP inhibited mitogen-induced nuclear-translocation of MKL1 and MKL2 in VSMCs but not ECs. Forskolin also significantly inhibited serum response factor (SRF)-dependent reporter gene (SRE-LUC) activity and mRNA expression of pro-proliferative and pro-migratory MKL1/2 target genes in VSMCs but not in ECs. In ECs, MKL1 was constitutively nuclear and MKL2 cytoplasmic, irrespective of mitogens or cAMP. Pharmacological or siRNA inhibition of MKL1 significantly inhibited the proliferation and migration of VSMC and EC. Our new data identifies and important contribution of MKL1/2 to explaining the strikingly different response of VSMCs and ECs to cAMP elevation. Elucidation of these pathways promises to identify targets for specific inhibition of VSMC migration and proliferation.
Madeleine C Smith, Claire A Hudson, Tomomi E Kimura, Stephen J White, Graciela B Sala-Newby, Andrew C Newby, Mark Bond

2627 related Products with: Divergent Regulation of Actin Dynamics and Megakaryoblastic Leukemia-1 and -2 (Mkl1/2) by cAMP in Endothelial and Smooth Muscle Cells.

200ug200ul1000 TESTS/0.65ml100ul1000 tests100ug100ul200ug 100ul100ug100ul200ul

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#27094476   2016/04/19 To Up

Prevalence of KISS1 Receptor mutations in a series of 603 patients with normosmic congenital hypogonadotrophic hypogonadism and characterization of novel mutations: a single-centre study.

What is the exact prevalence of Kisspeptin Receptor (KISS1R) mutations in the population of patients with normosmic congenital hypogonadotrophic hypogonadism (nCHH) by comparison with other genes, involved in gonadotrophin-releasing hormone (GnRH) release or action?
Bruno Francou, Charlotte Paul, Larbi Amazit, Alejandra Cartes, Claire Bouvattier, Frédérique Albarel, Dominique Maiter, Philippe Chanson, Séverine Trabado, Sylvie Brailly-Tabard, Thierry Brue, Anne Guiochon-Mantel, Jacques Young, Jérôme Bouligand

1619 related Products with: Prevalence of KISS1 Receptor mutations in a series of 603 patients with normosmic congenital hypogonadotrophic hypogonadism and characterization of novel mutations: a single-centre study.

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#26883686   2016/02/13 To Up

Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans.
Joseph Aizen, Jennifer C Chandler, Quinn P Fitzgibbon, Amir Sagi, Stephen C Battaglene, Abigail Elizur, Tomer Ventura

2669 related Products with: Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

2 Pieces/Box2 Pieces/Box2 Pieces/Box100ug100ul100ug Lyophilized100ug100ug Lyophilized100ug Lyophilized100ug100ug Lyophilized100ug

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#26075265   2015/05/17 To Up

Cloning and characterization of spliced variants of the porcine G protein coupled receptor 120.

The polyunsaturated fatty acids (PUFAs) receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120) has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa) wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform) was detected in spleen, while the transcript 2 (coding 310-aa isoform) was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891) and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.
Tongxing Song, Jie Peng, Jiao Ren, Hong-Kui Wei, Jian Peng

2204 related Products with: Cloning and characterization of spliced variants of the porcine G protein coupled receptor 120.

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