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Previous studies have shown that some inflammatory cytokines promote the expression of corticotropin-releasing hormone (CRH) in trophoblasts during pregnancy and that placental CRH could induce the production of adrenocorticotropic hormone (ACTH) in humans. However, whether the same is true in rodent placenta remains unclear. In this study, we examined the effect of pro-inflammatory cytokine LIF on the induction of CRH in mouse trophoblast stem cells (mTSCs). During differentiation, the CRH levels in mTSCs gradually increased. On days 3 and 5 after LIF supplementation, Crh expression in the differentiated mTSCs was significantly increased with LIF treatment than those without LIF treatment. Moreover, the CRH concentration in the culture media increased. Thereafter, we examined the contribution of the downstream pathways of LIF to CRH induction in differentiated mTSCs. The LIF-induced upregulation of CRH was attenuated by inhibition of PI3K/AKT and MAPK phosphorylation but not by inhibition of JAK/STAT3. Therefore, in mTSCs, LIF increased Crh expression through activation of the PI3K/AKT and MAPK pathways but not by the JAK/STAT3 pathway. The present study suggests that mTSC is an ideal in vitro model for studying regulation and function of placental CRH. • Corticotropin-releasing hormone was expressed in mouse trophoblast stem cells and increased during their differentiation. • Leukemia inhibitory factor induced corticotropin-releasing hormone in mouse trophoblast stem cells. • PI3K/AKT and MAPK but not JAK/STAT3 pathways mediated LIF-induced CRH expression in mouse trophoblast stem cells. • Mouse trophoblast stem cell is an ideal in vitro model for studying regulation and function of placental CRH. [ABSTRACT FROM AUTHOR]

Hyperglycemia-induced endothelial dysfunction is generally believed to be the basis of diabetic vascular complications. Dopamine receptors is known to play an important protective role in diabetes. However, the protective effect of dopamine receptors against hyperglycemia-induced endothelial damage in diabetic rats is still unknown. In the present study, we established a cell model of hyperglycemia-induced endothelial dysfunction by treating human umbilical vein endothelial cells (HUVEC) with high glucose. MTT and lactate dehydrogenase assays results showed that high glucose treatment significantly reduced the cell viability and down-regulated dopamine D 4 receptor. Pre-treatment with PD168077, a specific D 4 receptor agonist, greatly improved endothelial cell viability and decreased apoptosis. Furthermore, pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and endothelial nitric oxide synthase (eNOS) eliminated the protective effect of D 4 receptor against endothelial injury. More importantly, the expression level of D 4 receptor was also dramatically down-regulated in the arterial endothelium of rats with streptozotocin-(STZ)-induced diabetes, and the STZ-induced impairment of acetylcholine-induced vasodilation was reversed by activation of D 4 receptor. In conclusion, our results indicated that dopamine D 4 receptor protected against hyperglycemia-induced endothelial dysfunction via the PI3K/eNOS pathway, which may provide a novel strategy in the treatment of diabetes. • Dopamine D4 receptor is down-regulation in hyperglycemia-induced endothelial dysfunction. • D4 receptor agonist greatly improved endothelial cell viability and decreased apoptosis. • Protective effect of D4 receptor against endothelial injury is via PI3K/Akt/eNOS/NO pathway. [ABSTRACT FROM AUTHOR]

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri . Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli , but exhibits a potential inhibition activity against chymotrypsin. [ABSTRACT FROM AUTHOR]

The amphibian temporins, amongst the smallest antimicrobial peptides (AMPs), are α-helical, amphipathic, hydrophobic and cationic and are active mainly against Gram-positive bacteria but inactive or weakly active against Gram-negative bacteria. Here, we report two novel members of the temporin family, named temporin-1Ee (FLPVIAGVLSKLF amide ) and temporin-1Re (FLPGLLAGLL amide ), whose biosynthetic precursor structures were deduced from clones obtained from skin secretion-derived cDNA libraries of the European edible frog, Pelophylax kl. esculentus , by ‘shotgun’ cloning. Deduction of the molecular masses of each mature processed peptide from respective cloned cDNAs was used to locate respective molecules in reverse-phase HPLC fractions of secretion. Temporin-1Ee (MIC = 10 μM) and temporin-1Re (MIC = 60 μM) were both found to be active against Gram-positive Staphylococcus aureus , but retaining a weak haemolytic activity. To our knowledge, Single-site substitutions can dramatically change the spectrum of activity of a given temporin. Compared with temporine-1Ec, just one chemically-conservative substitution (Val8 instead of Leu8), temporin-1Ee bearing a net charge of +2 displays broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC = 40 μM). These factors bode well for translating temporins to be potential drug candidates for the design of new and valuable anti-infective agents. [ABSTRACT FROM AUTHOR]

Accumulating evidence has indicated that natural killer cells (NK cells) play an important role in immune responses generated in the liver. However, the underlying molecular basis for local immune regulation is poorly understood. Mice were intraperitoneally injected with polyinosinic-polycytidylic acid (PolyI:C) at a dose of 20 mg/kg body wt. The percentage and absolute number of NK cells in the liver were analysed with flow cytometry. LSECtin knockout mice and LSECtin cDNA plasmids were used for analyze the role of LSECtin in hepatic NK cell regulation in vivo. Here, we show that the C-type lectin LSECtin, a member of the DC-SIGN family, is a novel liver regulator for NK cells. LSECtin could bind to NK cells in a carbohydrate-dependent manner and could regulate the number of hepatic NK cells. In the NK cell-mediated acute liver injury model induced with PolyI:C, the exogenous expression of LSECtin accelerated NK cell-induced liver injury, whereas the absence of LSECtin ameliorated this condition. Our results reveal that LSECtin is a novel, liver-specific NK cell regulator that may be a target for the treatment of inflammatory diseases in the liver. [ABSTRACT FROM AUTHOR]

Abstract: The proprotein convertases subtilisin kexin 9 (PCSK9) binds to the epidermal growth factor domain A (EGF-A) of low-density lipoprotein receptor (LDLR) and leads to its destruction. However, the intracellular processes leading to LDLR degradation have not been fully delineated. In this report, we show that PCSK9 treatment can lead to ubiquitination of LDLR, which was enhanced in the presence of proteasome inhibitor MG132. Furthermore, LDLR protein carrying mutations in the C-terminal ubiquitination sites was resistant to PCSK9-mediated degradation. Our data suggest that the ubiquitination system is involved in PCSK9-induced LDLR degradation. [Copyright &y& Elsevier]

Abstract: A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific “on-chip” captured proteins have been obtained that were pure enough to do “on-chip” digestion and the subsequent identification of the “on-chip” bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays. [Copyright &y& Elsevier]

This study examined whether retarded angiogenesis in a hypertension animal model was associated with impaired VEGF signaling. Furthermore, we sought to determine whether this impairment could be overcome by VEGF addition. Using a rat sponge implantation model, we confirmed impaired angiogenesis in spontaneous hypertensive rats (SHRs). Fourteen days after sponge implantation, the level of angiogenesis in SHRs was approximately half of those in age-matched normotensive Wistar–Kyoto or Sprague–Dawley rats. Significantly, expression of kinase-insert domain-containing receptor (KDR) and membrane type 1 matrix metalloproteinase (MT1-MMP) was reduced in SHRs compared to controls. Immunohistological analysis indicated endothelial proliferation was decreased in SHRs. Gene transfer of human VEGF121 increased KDR and MT1-MMP expression in SHRs. VEGF121 also up-regulated endothelial proliferation and angiogenesis. Our results indicate down-regulated KDR and MT1-MMP expression is associated with an impaired angiogenesis in SHRs. VEGF gene transfer is effective in ameliorating the impaired angiogenesis in SHRs. [Copyright &y& Elsevier]

Abstract: Casitas B-lineage lymphoma b (Cblb) is a negative regulator of T-cell activation and dysfunction of Cblb in rats and mice results in autoimmunity. In particular, a nonsense mutation in Cblb has been identified in a rat model of autoimmune type 1 diabetes. To clarify the possible involvement of CBLB mutation in type 1 diabetes in humans, we performed mutation screening of CBLB and characterized functional properties of the mutations in Japanese subjects. Six missense mutations (A155V, F328L, N466D, K837R, T882A, and R968L) were identified in one diabetic subject each, excepting N466D. Of these mutations, F328L showed impaired suppression of T-cell activation and was a loss-of-function mutation. These data suggest that the F328L mutation is involved in the development of autoimmune diseases including type 1 diabetes, and also provide insight into the structure–function relationship of CBLB protein. [Copyright &y& Elsevier]

Prostate cancer (PCa) is a common malignancy in male urinary system. Cell division cycle-associated protein 1 (CDCA1) is expressed highly in many cancer cells. Yet, whether CDCA1 play an important role in PCa progression is uncertain. pH low insertion peptide (pHLIP), a PH-induced transmembrane structure, can pass through the cell membrane into intracellular in an acidic environment. In this study, we try to confirm the expression status of CDCA1 in the PCa patients' tissues and PCa cell line. In addition, to make the CDCA1-siRNA efficiently targeting the PCa cells, pHLIP and CDCA1-siRNA were combined with disulfide bond to become effector molecules. By the characteristics of the pHLIP allosteric occurring in cancer tissue acidic microenvironment, CDCA1-siRNA may be transported specificity into prostatic cancer cells and released in the cytoplasm. The interference effect of the effector molecules on the CDCA1 was detected in vitro and in vivo. The results showed that CDCA1 was highly expressed in PCa cell line and human PCa clinical samples. Knock down CDCA1 significantly inhibit the growth and promote the apoptosis of prostatic cancer cells. In the intracellular translocation experiment, CDCA1-siRNA could be delivered into cytoplasma at pH 6.2, but not at pH 7.4. In the in vivo test, the tumor size was reduced obviously in the NOD/SCID mice treated with pHLIP-CDCA1-siRNA compared to the CDCA1-siRNA and the bioluminescent signal of Cy5-pHLIP-CDCA1-siRNA was focused detected in the tumor site. Our findings indicated that CDCA1 might be a very key molecule regulating survival and proliferation of PCa. pHLIP-CDCA1-siRNA might be a promising targeting therapy for PCa. [ABSTRACT FROM AUTHOR]

Abstract: The free fatty acid receptor, GPR40, is implicated in the pathophysiology of type 2 diabetes, and is a new potential drug target for the treatment of type 2 diabetes. Its antagonist is thought to be not only a useful chemical probe for further exploring the function of GPR40 but also a lead structure for drug development. With virtual screening based on a homology model followed by a cell-based calcium mobilization assay, we found that sulfonamides are a new class of small organic antagonists for GPR40. One of the compounds, DC260126, dose-dependently inhibited GPR40-mediated Ca2+ elevations stimulated by linoleic acid, oleic acid, palmitoleic acid and lauric acid (IC50: 6.28±1.14, 5.96±1.12, 7.07±1.42, 4.58±1.14μM, respectively), reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells. [Copyright &y& Elsevier]

Abstract: ADAMTS-4 (aggrecanase-1) is a metalloprotease that plays a role in aggrecan degradation in the cartilage extracellular matrix. In order to understand the regulation of ADAMTS-4 gene expression we have cloned and characterized a functional 4.5kb human ADAMTS-4 promoter. Sequence analysis of the promoter revealed the presence of putative binding sites for nuclear factor of activated T cells (NFAT) and Runx family of transcription factors that are known to regulate chondrocyte maturation and differentiation. Using promoter–reporter assays and mRNA analysis we have analyzed the role of chondrocyte-expressed transcription factors NFATp and Runx2 and have shown that ADAMTS-4 is a potential downstream target of these two factors. Our results suggest that inhibition of the expression/function of NFATp and/or Runx2 may enable us to modulate aggrecan degradation in normal physiology and/or in degenerative joint diseases. The ADAMTS-4 promoter would serve as a valuable mechanistic tool to better understand the regulation of ADAMTS-4 expression by signaling pathways that modulate cartilage matrix breakdown. [Copyright &y& Elsevier]

Abstract: A G-protein coupled receptor to niacin (nicotinic acid) was identified recently but the physiological/pharmacological role of the receptor remains poorly defined. We present our studies to demonstrate that HM74A, but not HM74, binds niacin at high affinities and effectively mediates Gi signaling events in human embryonic kidney HEK293 cells as well as in 3T3L1 adipocytes expressing HM74A. Furthermore, HM74A, but not HM74, expressed in differentiated 3T3L1 adipocytes effectively mediated inhibition of lipolysis by niacin. Our results provided direct evidence indicating that HM74A, but not HM74, was sufficient to mediate anti-lipolytic effect of niacin in adipose tissue. [Copyright &y& Elsevier]