The evaluation of acute toxicity,antimicrobial activity of 1‐phenyl‐5‐p‐tolyl‐1H‐1, 2, 3‐triazole,and binding to human serum albumin

1‐Phenyl‐5‐p‐tolyl‐1H‐1, 2, 3‐triazole (PPTA) was a synthesized compound. The result of acute toxicities to mice of PPTA by intragastric administration indicated that PPTA did not produce any significant acute toxic effect on Kunming strain mice. It exhibited the various potent inhibitory activities against two kinds of bananas pathogenic bacteria, black sigatoka and freckle, when compared with that of control drugs and the inhibitory rates were up to 64.14% and 43.46%, respectively, with the same concentration of 7.06 mM. The interaction of PPTA with human serum albumin (HSA) was studied using fluorescence polarization, absorption spectra, 3D fluorescence, and synchronous spectra in combination with quantum chemistry and molecular modeling. Multiple modes of interaction between PPTA and HSA were suggested to stabilize the PPTA–HSA complex, based on thermodynamic data and molecular modeling. Binding of PPTA to HSA induced perturbation in the microenvironment around HSA as well as secondary structural changes in the protein.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.     

DPP‐IV‐resistant,long‐acting oxyntomodulin derivatives

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon‐like peptide 1 (GLP‐1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose‐dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37‐amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP‐1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment‐associated nausea than GLP‐1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half‐life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP‐IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP‐IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon‐like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258–2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.     

PTRN‐1/CAMSAP promotes CYK‐1/formin‐dependent actin polymerization during endocytic recycling

Cargo sorting and membrane carrier initiation in recycling endosomes require appropriately coordinated actin dynamics. However, the mechanism underlying the regulation of actin organization during recycling transport remains elusive. Here we report that the loss of PTRN‐1/CAMSAP stalled actin exchange and diminished the cytosolic actin structures. Furthermore, we found that PTRN‐1 is required for the recycling of clathrin‐independent cargo hTAC‐GFP. The N‐terminal calponin homology (CH) domain and central coiled‐coils (CC) region of PTRN‐1 can synergistically sustain the flow of hTAC‐GFP. We identified CYK‐1/formin as a binding partner of PTRN‐1. The N‐terminal GTPase‐binding domain (GBD) of CYK‐1 serves as the binding interface for the PTRN‐1 CH domain. The presence of the PTRN‐1 CH domain promoted CYK‐1‐mediated actin polymerization, which suggests that the PTRN‐1‐CH:CYK‐1‐GBD interaction efficiently relieves autoinhibitory interactions within CYK‐1. As expected, the overexpression of the CYK‐1 formin homology domain 2 (FH2) substantially restored actin structures and partially suppressed the hTAC‐GFP overaccumulation phenotype in ptrn‐1 mutants. We conclude that the PTRN‐1 CH domain is required to stimulate CYK‐1 to facilitate actin dynamics during endocytic recycling.     

Enzyme Kinetics of an Alternative Splicing Isoform of Mitochondrial 8‐Oxoguanine DNA Glycosylase,OGG1‐1b,and Compared with the Nuclear OGG1‐1a

Eight alternatively spliced isoforms of human 8‐oxoguanine DNA glycosylase (OGG1) (OGG1‐1a to ‐1c and ‐2a to ‐2e) are registered in the National Center for Biotechnology Information. OGG1(s) in mitochondria have not yet been fully characterized biochemically. In this study, we purified mitochondrial recombinant OGG1‐1b protein and compared its activity with nuclear OGG1‐1a protein. The reaction rate constant (k_g) of the 7,8‐dihydro‐8‐oxoguanine (8‐oxoG) glycosylase activity of OGG1‐1b was 8‐oxoG:C >> 8‐oxoG:T >> 8‐oxoG:G > 8‐oxoG:A (7.96, 0.805, 0.070, and 0.015 min^?1, respectively) and that of the N‐glycosylase/DNA lyase activity (k_gl) of OGG1‐1b was 8‐oxoG:C > 8‐oxoG:T ?8‐oxoG:G >> 8‐oxoG:A (0.286, 0.079, 0.040, and negligible min^?1, respectively). These reaction rate constants were similar to those of OGG1‐1a except for k_gl against 8‐oxoG:A. APEX nuclease 1 was required to promote DNA strand breakage by OGG1‐1b. These results suggest that OGG1‐1b is associated with 8‐oxoG cleavage in human mitochondria and that the mechanism of this repair is similar to that of nuclear OGG1‐1a.     

CD151 mediates netrin‐1‐induced angiogenesis through the Src‐FAK‐Paxillin pathway

Crosstalk between the nervous and vascular systems is important during development and in response to injury, and the laminin‐like axonal guidance protein netrin‐1 has been studied for its involvement in angiogenesis and vascular remodelling. In this study, we examined the role of netrin‐1 in angiogenesis and explored the underlying mechanisms. The effect of netrin‐1 on brain tissues and endothelial cells was examined by immunohistochemistry and western blotting in a middle cerebral artery occlusion model and in human umbilical vein endothelial cells. Cell proliferation and cell cycle progression were assessed by the MTT assay and flow cytometry, and the Transwell and tube formation assays were used to examine endothelial cell motility and function. Netrin‐1 up‐regulated CD151 and VEGF concomitant with the activation of focal adhesion kinase (FAK), Src and Paxillin in vitro and in vivo and the induction of cell proliferation, migration and tube formation in vitro. Silencing of CD151 abolished the effects of netrin‐1 on promoting cell migration and tube formation mediated by the activation of FAK/Src signalling. Netrin‐1 promoted angiogenesis in vitro and in vivo by activating the FAK/Src/Paxillin signalling pathway through a mechanism dependent on the expression of the CD151 tetraspanin, suggesting the existence of a netrin‐1/FAK/Src/CD151 signalling axis involved in the modulation of angiogenesis.     

Growth cone neuropilin‐1 mediates collapsin‐1/sema III facilitation of antero‐ and retrograde axoplasmic transport

Collapsin‐1/SemaIII, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin‐1 exerts its action are not fully understood. Collapsin‐1 induces growth cone collapse via a pathway which may include neuropilin‐1, a cell‐surface collapsin‐1 binding protein, as well as intracellular CRMP‐62 and heterotrimeric G proteins. We previously identified a second action of collapsin‐1, the facilitation of antero‐ and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin‐1 in the action of collapsin‐1 on axoplasmic transport, we produced a soluble neuropilin‐1 (sNP‐1) lacking the transmembrane and intracellular region. sNP‐1 progressively displaced the dose–response curve for collapsin‐1 to induce growth cone collapse to higher concentrations. sNP‐1 also inhibited collapsin‐1‐induced augmentation of both antero‐ and retrograde axoplasmic transport. Furthermore, an anti‐neuropilin‐1 antibody blocked the collapsin‐induced axoplasmic transport. These results together indicate that neuropilin‐1 mediates collapsin‐1 action on axoplasmic transport. To visualize collapsin‐1 binding to endogenous neuropilin‐1, we used a truncated collapsin‐1–alkaline phosphatase fusion protein (CAP‐4). CAP‐4 stains the growth cone, neurite, and cell body. However, local application of collapsin‐1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP‐1 mediates the facilitatory action of collapsin‐1 on antero‐ and retrograde axoplasmic transport. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 579–589, 1999     

Enhancement of the anti‐inflammatory activity of temporin‐1Tl‐derived antimicrobial peptides by tryptophan,arginine and lysine substitutions

Temporin‐1Tl (TL) is a 13‐residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti‐inflammatory activity. To develop novel AMP with improved anti‐inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin‐resistant Staphylococcus aureus strains compared with TL. TL‐1 and TL‐4 showed a little increase in antimicrobial selectivity, while TL‐2 and TL‐3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti‐inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor‐α (TNF‐α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF‐α in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti‐inflammatory activity is as follows: TL‐2 ≈ TL‐3 ≈ TL‐4 > TL‐1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti‐inflammatory activity. These results apparently suggest that the anti‐inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti‐inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram‐negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.