Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain  that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds. Single titin molecules span entire half-sarcomeres  and contain strain-compliant elements, forming an elastic lattice within the cytoskeleton of acto-myosin motors ,6 and E3The kinase domain near the C-terminus of titin (TK) in the sarcomeric M-line plays an important role in mechanotransduction. TK binds a protein complex formed by the autophagosomal receptors nbr1 and p62, and the E3 ubiquitin ligase MuRF2 . A forcein vitro and their phosphorylation is seemingly unrelated to their function in the TK-signalosome , all three TK variants—including the inactive TKK36L—showed similar phospho-transfer activities on Tcap and were modestly stimulated by Ca2+/calmodulin (a). As an independent validation, we studied the activity of immuno-complexed wild-type TK where its active site had been blocked by a specific antibody directed against the P+1 loop . Immuno-complexed TK, non-complexed TK samples and non-treated TK controls showed similar levels of activity (b). Hence, activity data from either mutated or immuno-complexed samples together suggested that insect cell preparations catalysed Tcap phosphorylation in a TK-independent way, with kinase activity arising from other component(s) in the cell milieu.In activity assays that used ATPATP) at 30°C. Tcap, MLC-derived peptide (KKRARAATSNVFS), Tcap-derived peptide (RRSLSRSMSQEAQRG), casein and myelin basic protein were tested at 4 µg/reaction. Where indicated, the reaction mixture was supplemented with 0.5 mM CaCl2 and 0.4 µg/reaction calmodulin from bovine testis (Sigma-Aldrich). For reaction mixtures that assayed peptide substrates, an aliquot was withdrawn at indicated time points and spotted on EDTA impregnated P81 phosphocellulose paper (Whatman). The latter was washed extensively with 75 mM orthophosphoric acid and once with ethanol, dried and exposed to a phosphoscreen. Reaction mixtures with protein substrates were first separated on SDS-PAGE, the gel briefly stained, dried and exposed to the phosphoimager screen. Screens were imaged with a Fujifilm BAS 2500 phosphoimager and images processed using AIDA (Raytest).Phosphorylation assays were performed in 20 µl assay buffer  and treated with 20 mM FSBA at 30°C for 1 h. FSBA was removed by buffer exchange into phosphorylation assay buffer (above) supplemented with 5 mM DTT.5.6.g for 30 min, aliquoted, frozen in liquid nitrogen and stored at −80°C.Quadriceps tissue from adult mice was pulverized under liquid nitrogen and the powder homogenized in 20 mM Tris pH7.5, 100 mM NaCl, 2 mM β-ME and complete protease inhibitor (Roche). Extraction proceeded for 90 min on ice. The resulting extracts were clarified at 3000Y170E were immobilized on 30 µl of Glutathione-Separose beads and washed with PBS, 0.1% NP40. Extracts were thawed on ice and spun for 1 min at 21 000g. The supernatant was diluted by adding three volumes of PBS, 0.1% NP40. Next, 1 ml of diluted extract was mixed with 30 µl of beads loaded with a given titin fragment and incubated for 16 h at 4°C under light stirring. Mixtures were spun for 1 min at 1000g, supernatants were removed and the beads washed 3× with 1 ml PBS, 0.1% NP40. After a final wash with PBS, supernatants were removed and the beads resuspended in SDS sample buffer. Bound material was examined by SDS-PAGE and Western blotting using a MuRF1-specific antibody.For GST pull-downs, 50 µg each of GST, GST-A168–170, GST-A168-TK, GST-TK and GST-TK5.7.6-MuRF1-coiled-coil . To remove unreacted biotin, the sample was filtered twice through a PD-10 column  in PBS. One millimolar DTT and 50% glycerol were added and the sample frozen until further use.Purified Hisorted in ) was bio−1 in TBST/1% milk. The membrane was washed in TBST and incubated for 1 h in TBST containing 2 µg ml−1 streptavidin conjugated to alkaline phosphatase (Thermo Scientific). After washes in TBST and a final wash in TBS, the detection with NBT/BCIP was done as described by the supplier (Roche).Purified titin constructs were spotted (approx. 1 µg) on nitrocellulose membranes pre-wetted with TBS. The filter was blocked by TBST/5% milk, washed with TBST and incubated for 3 h with biotinylated MuRF1-coiled-coil at a concentration of 0.5 µg ml


That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 μM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers  after grazing tall fescue pastures for 105 days. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated . Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.Determining a grazing animal's susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids  on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of  Lolium arundinaceum (Schreb.) S. J. Darbyshire] infected with the endophytic fungus  is responsible for the production of ergot alkaloids which have a significant economic impact on beef cattle. Consumption of those mycotoxins is known to lower average daily gain, negatively impact reproductive traits, decrease parasite resistance, and reduce heat tolerance, all of which drive up the cost of production , dihydroergotamine (DHET), and ergonovine (EN) were dissolved in methanol (100%) resulting in final assay concentrations of 0, 20, and 40 μM. Enzyme activity for CYP450 was assayed using the Promega™ P450-Glo assay , multiple t-tests were used to separate means. Results are presented as a percent of CYP450 activity with zero alkaloid present.Luminescence data for each alkaloid were analyzed independently using mixed procedures. Main effects were replicate, alkaloid, and concentration; response variable was CYP450 activity as indicated by luminescence. If n = 39; 203 ± 27 kg; 216 ± 2.6 days of age) were weaned, vaccinated, and treated with anthelmintic in May. Fourteen days after weaning, steers were randomly assigned to a pasture of either Kentucky 31  or HiMag4  for a summer grazing trial. Pastures and tall fescue cultivars were as previously described . Healthy, fall-born Angus-sired crossbreed steers ] concentrations were determined by validated radioimmunoassay. At day 105, each animal was weighed and a urine sample collected. Urine samples were stored at −20°C until assayed. Total alkaloids were determined by ELISA . Inhibition of CYP450 was determined using the assay (Promega™ P450-Glo) described above using 12.5 μL of non-diluted urine in each reaction test.At weaning, approximately 10 mL of blood was collected from each steer, stored at 5°C until centrifuged at 1200 × CYP3A28 [National Center for Biotechnology Information (NCBI) gi1769423; . Steer genotypes at SNP site C994G were homozygous cytosine, homozygous guanine, and heterozygous.Steers were genotyped at CYP3A28 single nucleotide polymorphism (SNP), C994G, using methods previously described  then means were separated using multiple t-tests.Results of the CYP450 enzyme assay were reported in luminescence (arbitrary units). Pearson correlation coefficients were determined between dependent variables. Data were analyzed using mixed procedures with pasture as the experimental unit, genotype within pasture as random term, fescue cultivar as repeated, and creatinine as a covariate. If Figure r > 0.30; P < 0.05) with body weight and ADG. Although steer body weights were correlated with IGF-1, ADG was not correlated to IGF-1. Inhibition of CYP450 activity by urine was correlated  with total ergot alkaloid concentration. Inhibition of CYP450 activity by urine also tended to be correlated  with ADG.Steer weight gain 0.53 ± 0.14 kg/d) was typical for summer grazing of tall fescue with light grain supplementation. Table 4 kg/d waP > 0.1) urine inhibition of CYP450.Figure N. coenophialum is difficult. Previously, ergot alkaloids have been determined in cattle serum via HPLC  pastures infested with wild-type Stuedemann et al.  successfThe P450-Glo assay also lacks selectivity as any compound that will inhibit CYP450 enzymes will be detected. However, the antibody used for ELISA is specific for the lysergic acid moiety (Hill and Agee, P4503A28 were associated with cattle profitability traits (Sales et al., Selecting livestock that are less susceptible to ergot alkaloid poisoning has the potential to improve livestock production where tall fescue is a component of the grazing system. In reality that is what livestock producers have been doing for the last 60 years. If the primary forage is tall fescue for a producer and the livestock do not reproduce then they are culled which results in selection for animals that tolerate the ergot alkaloids. Previously, we demonstrated that mutations in bovine cytochrome P4503A28 genotype. In addition, we can envision the use of the P450-Glo-assay as a selection assay. We have not tested our hypothesis, but it is plausible that a group of replacement animals could be purposely exposed to a large concentration of ergot alkaloids and urine monitored for inhibition of the P450-Glo-assay. Presumably those animals with less inhibitory urine would be those with more tolerance of ergot alkaloids, but that would need to be tested before being recommended as a selection practice.The overall goal of our research is to develop management tools that can increase the sustainability of livestock production. In addition, we are interested in understanding the genetic and physiological mechanisms related to animal productivity and toxicology. Our results suggest that cytochrome P450 can be used as management tool for selecting animals based on their cytochrome The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, the mechanisms involved in the release of peptide hormones, GLP-1 and PYY in response to TGR5 activation by bile acids and the effect of H2S on bile acid-induced release of GLP-1 and PYY are unclear. In the present study, we have identified the signaling pathways activated by the bile acid receptor TGR5 to mediate GLP-1 and PYY release and the mechanism of inhibition of their release by H2S in enteroendocrine cells. The TGR5 ligand oleanolic acid (OA) stimulated Gαs and cAMP formation, and caused GLP-1 and PYY release. OA-induced cAMP formation and peptide release were blocked by TGR5 siRNA. OA also caused an increase in PI hydrolysis and intracellular Ca2+. Increase in PI hydrolysis was abolished in cells transfected with PLC-ε siRNA. 8-pCPT-2′-O-Me-cAMP, a selective activator of Epac, stimulated PI hydrolysis, and GLP-1 and PYY release. L-Cysteine, which activates endogenous H2S producing enzymes cystathionine-γ-lyase and cystathionine-β-synthase, and NaHS and GYY4137, which generate H2S, inhibited PI hydrolysis and GLP-1 and PYY release in response to OA or 8-pCPT-2′-O-Me-cAMP. Propargylglycine, an inhibitor of CSE, reversed the effect of L-cysteine on PI hydrolysis and GLP-1 and PYY release. We conclude: (i) activation of Gαs-coupled TGR5 receptors causes stimulation of PI hydrolysis, and release of GLP-1 and PYY via a PKA-independent, cAMP-dependent mechanism involving Epac/PLC-ε/Ca2+ pathway, and (ii) H2S has potent inhibitory effects on GLP-1 and PYY release in response to TGR5 activation, and the mechanism involves inhibition of PLC-ε/Ca2+ pathway.Activation of plasma membrane TGR5 receptors in enteroendocrine cells by bile acids is known to regulate gastrointestinal secretion and motility and glucose homeostasis. The endocrine functions of the gut are modulated by microenvironment of the distal gut predominantly by sulfur-reducing bacteria of the microbiota that produce H Hormones released from the gastrointestinal tract play an important role in the regulation of several physiological functions including food intake, gastrointestinal motility and secretions, glucose and lipid metabolism, and energy expenditure  and cystathionine-β-synthase (CBS), and to lesser extent by 3-mercaptopyruvate sulfurtransferase (3-MST), regulate various physiological functions such as secretion, motility and visceral nociception  to determine the mechanism of GLP-1 and PYY release by TGR5 activation, and (ii) to determine the effect of endogenous H2S on TGR5-mediated GLP-1 and PYY release. We show that cAMP-dependent GLP-1 and PYY release in response to TGR5 activation reflects activation of Epac (an exchange protein activated directly by cAMP) and PLC-ε, and release of Ca2+. We also show that stimulation of endogenous H2S formation by activation of CSE inhibited TGR5-dependent GLP-1 and PYY release by blockade of the Epac/PLC-ε pathway.H35S]GTPγS, [125I]cAMP, [3H]myo-inositol were obtained from NEN Life Sciences Products . U73122 and myristoylated PKI were obtained from Calbiochem . Rabbit polyclonal antibody to TGR5 and PLC-ε were purchased from Abcam , antibodies to Gαs, Gαq, Gαi1, Gαi2, and Gαi3 were from Santa Cruz Biotechnology Inc. . Lipofectamine™ 2000 transfection reagents and SuperScript™ II Reverse Transcriptase kit were obtained from Invitrogen . All other reagents were obtained from Sigma .Oleanolic acid was obtained from Sigma Aldrich . TGR5 antibody was obtained from Abcam . GTPγS in a solution containing 10 mM HEPES (pH 7.4), 100 μM EDTA, and 10 mM MgCl2. The reaction was stopped with 10 volumes of 100 mM Tris-HCl medium (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 μM GTP and the mixture was placed in wells pre-coated with specific G protein antibodies (1:1000 dilution). Coating with G protein antibodies (1:1000) was done after the wells were first coated with anti-rabbit IgG for 2 h on ice. After incubation for 2 h on ice, the wells were washed three times with phosphate buffer solution containing 0.05% Tween 20, and the radioactivity from each cell was counted by liquid scintillation.G proteins selectively activated by the TGR5 ligand were identified as described previously  with a 40 × oil-immersion objective. Excitation at 340 and 380 nm was achieved using a 75 W Xenon arc lamp with a monochromator  controlled by MetaFluor software  and emission was recorded with a CCD camera . Background-subtracted fluorescence was normalized to a baseline average measured before application of the first test reagent and expressed as a 340/380 nm ratio, and the response to OA was defined as increase in 340/380 ratio.Cells were plated in Matrigel-coated dishes  1–3 days prior to use and loaded with fura-2 AM by incubation for 2 h in a medium containing 25 mM Hepes (pH 7.4), 120 mM NaCl, 4.0 mM KCl, 1 mM CaCl6 cells/ml) in the presence of 3-isobutyl-1-methylxanthine , and the reaction was terminated after 60 s with 6% cold trichloroacetic acid (Vol/Vol). The mixture was centrifuged at 2000 × g for 15 min at 4°C; the supernatant was extracted three times with 2 ml diethyl ether, and the samples were lyophilized. The samples were reconstituted and cAMP was measured in triplicate using 100 μl aliquots by radioimmunoassay as described before  was added to 0.5 ml of cell suspension . The sequence of TGR5 siRNA was as follows; Forward 5′-CCC AAC UUC UCC UUC CUC UTT-3′ and Reverse TGR5: 5′-AGA GGA AGG AGA AGU UGG GTT-3′. Double-stranded RNA oligoribonucleotide NNGCGCGCUUUGUAGGAUUCA (5′-3′) was used as a control siRNA. The sequence of PLC-ε siRNA was as follows; Sense: 5′ CUG AUC CUC AAG ACG UUA Att 3′ and Antisense: 5′ UUA ACG UCU UGA GGA UCA Gtt 3′. STC-1 cells were seeded on 100 mm plates and allowed to adhere overnight. At the time of transfection, cells were 80–85% confluent and the siRNAs were transfected into STC-1 cells at a final concentration of 20 μM by use of lipofectamine 2000 reagent  according to the manufacturer's instructions. Cells were kept for 48 h before use for biochemical measurements.STC-1 cells were washed three times with DMEM and incubated for 30 min at 37°C in DMEM containing various test reagents. After incubation, the conditioned medium was collected and the concentration of GLP and PYY was determined by enzyme immunoassay with a specific ELISA kits for GLP-1 and PYY   in inositol-free medium and treated with OA or 8-pCPT-2′-O-Me-cAMP (10 μM) in the presence or absence of inhibitors for 1 min in a medium containing 25 mM HEPES, 115 mM NaCl, 5.8 mM KCl, 2.1 mM KH2PO4, 2 mM CaCl2, 0.6 mM MgCl2, and 14 mM glucose. The reaction was terminated by the addition of 940 μl of chloroform-methanol-HCl (50:100:2). After extraction with 340 μl of chloroform and 340 μl of H2O, the upper aqueous phase was applied to DOWEX AG-1 columns. [3H]inositol phosphates were then eluted, and the radioactivity was determined by liquid scintillation.PI hydrolysis was measured as total inositol phosphate formation with the use of anion exchange chromatography as described previously , CBS (345 bp), and CSE (851 bp) . These results suggest that OA-induced GLP-1 and PYY release is mediated by TGR5, but not by other receptors such as nuclear receptor FXR.We initially examined whether STC-1 cells are responsive to the TGR5 selective ligand OA to release GLP-1 and PYY. The dose of OA was selected based on our previous work in pancreatic β cells and isolated smooth muscle cells  , Gαi2 (3 ± 8% increase), Gαi3 (6 ± 9% increase), or Gαq (10 ± 15% increase) antibody. These results suggest that TGR5 is selectively coupled to the activation of Gs. Consistent with activation of Gαs, OA caused an increase in cAMP formation in a concentration-dependent manner with an EC50 of 0.7 ± 0.1 μM and a maximum stimulation of 1.20 ± 0.07 pmol/mg protein above basal levels (0.054 ± 0.008 pmol/mg protein) was obtained with 10 μM OA  caused a significant increase in the binding of inositol labeled cells with OA resulted in an increase in PI hydrolysis in a concentration-dependent manner with an EC50 of 0.83 ± 0.15 μM and a maximal stimulation of 8715 ± 1024 cpm/mg protein above basal levels (1856 ± 302 cpm/mg protein) with 10 μM OA  and a selective Epac ligand, 8-pCPT-2′-O-Me-cAMP (10 μM) stimulated PI hydrolysis that was significantly inhibited (75 ± 5% inhibition) by U73122 (1 μM)  above base line .OA selectively activated GαA Figure . OA-induA Figure . These r) Figure . Stimulae Figure ; inset.The involvement of PLC-ε in OA-stimulated PI hydrolysis was examined using PLC-ε siRNA. Suppression of PLC-ε expression by transfection of PLC-ε siRNA was validated by western blot analysis Figure . Neither2+ and cAMP/PKA pathways in OA-mediated GLP-1 and PYY release was examined using selective inhibitors. OA-induced GLP-1 release was significantly inhibited by incubation of cells with U73122 or with the Ca2+ chelator, BAPTA-AM , but not with myristoylated PKI (14 ± 8% inhibition)  and PYY release (4.2 ± 0.7-fold increase) that was inhibited by U73122 (84 ± 7% inhibition in GLP-1 release and 65 ± 6% inhibition in PYY release) or BAPTA-AM (83 ± 6% inhibition in GLP-1 release and 78 ± 5% inhibition in PYY release)  Figure . Similar) Figure . 8-pCPT- Figures . These r Figures .2S, cells were incubated with L-cysteine, an activator of endogenous enzymes CSE and CBS, for 10 min and then GLP-1 and PYY release in response to OA was measured. L-cysteine inhibited both GLP-1 and PYY release in a dose-dependent manner , a selective inhibitor of CSE was used    and the increase was not significantly affected by L-cysteine, NaHS or GYY4137  and the inhibition was significantly reversed by PPG (28 ± 4% inhibition) , NaHS (72 ± 7% inhibition), or GYY4137 (53 ± 3% inhibition)  Figure . OA-indu) Figure . Stimula) Figure . These rs/cAMP/Epac/PLC-ε/Ca2+ pathway and stimulation of GLP-1 and PYY release. These conclusions are reached through several lines of evidence. (1) Specific involvement of TGR5 in GLP-1 and PYY release is supported by the finding that suppression of TGR5 by siRNA greatly attenuated the increase in cAMP formation and GLP-1 and PYY release in response to OA. (2) Selective activation of Gs and stimulation of adenylyl cyclase activity and PI hydrolysis, and Ca2+ release occurs in response to OA. (3) OA-induced PI hydrolysis and GLP-1 and PYY release was blocked by suppression of PLC-ε and PI hydrolysis inhibitor (U73122), but not by a PKA inhibitor (myristoylated PKI). (4) Suppression of PLC-ε expression blocked the increase in PI hydrolysis in response to OA. These data, in conjunction with the data using a cAMP analog that selectively activated Epac and which also caused stimulation of PI hydrolysis and GLP-1 and PYY release, provided further confirmation of the key role of the Epac/PI-PLC/Ca2+ pathway in GLP-1 and PYY release. It is noteworthy that in the present study we used OA, a selective agonist of the TGR5 receptor, rather than native bile salts, to examine the regulation of PYY and GLP-1 release  and regulates many physiological functions via activation of monomeric G protein Rap1 , or the exogenous H2S donors NaHS and GYY4137 inhibited OA-induced GLP-1 and PYY release, and (ii) inhibition of release is due to suppression of the Epac/PLC-ε/Ca2+ pathway downstream of cAMP pathway. Our results also demonstrated that when CSE was inhibited by the specific inhibitor DL-propargylglycine (PPG), inhibition of GLP-1 and PYY release was greatly attenuated suggesting a role for CSE in endogenous H2S generation and of H2S in regulating the TGR5-mediated release of GLP-1 and PYY. Similar levels of inhibition of OA-induced GLP-1 and PYY release with the rapid releasing donor, NaHS, and the slow releasing donor, GYY4137, suggest that the rate of H2S generation is not an important factor in the inhibitory action. The contractile effect of oxytocin in rat and human myometrium was also inhibited by both NaHS and GYY4137 in the same manner (Robinson and Wray, 2S which could be toxic and is not physiological. The slow release of H2S generated by GYY4137, is certainly more physiological in time course, and is comparable to the effect of endogenous H2S generated from CSE and CBS activity (Robinson and Wray, 2S on insulin secretion and smooth muscle contraction was demonstrated largely to be due to the opening of KATP channels (Yang et al., ATP channels in mediating the effects of H2S on GLP-1 and PYY release induced by activation of TGR5 receptors was not evident in the present study. In contrast this study indicates that H2S acts downstream of the cAMP generation induced by TGR5 activation, most likely by inhibiting PI hydrolysis and the subsequent increase in intracellular calcium needed for vesicle release from the enteroendocrine cells.Other important results of the present study demonstrate that TGR5-induced release of GLP-1 and PYY from enteroendocrine cells is modulated by endogenous agents produced within the wall of the gut such as H2+ pathway and both endogenous and exogenous H2S inhibit TGR5-mediated GLP-1 and PYY release. Inhibition of TGR5 mediated effects by H2S is due to inhibition of PI hydrolysis (Figure 2S in the lumen where it has immediate access to enteroendocrine cells. The endogenous generation of H2S by CBS and CSE in mammalian cells in the gut wall such as smooth muscle cells in the adjacent muscle likely plays an important role in the regulation of gastrointestinal functions under physiological and pathophysiological conditions. To understand the physiological and pathophysiological significance of H2S, further studies are warranted to elucidate the mechanisms involved in the activation of endogenous H2S generating enzymes and identify the cellular targets of H2S which regulate GLP-1 and PYY release.In conclusion, we have provided evidence indicating that GLP-1 and PYY release in response to TGR5 activation is mediated via the Gs/cAMP/PLC/Cas Figure . Sulfur-The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Poecilia mexicana, which has received considerable attention as a model organism to examine evolutionary adaptations to extreme environmental conditions. Nonetheless, basic ecological data on the endemic cave molly population are still missing; here we aim to provide data on population densities, size class compositions and use of different microhabitats. We found high overall densities in the cave and highest densities at the middle part of the cave with more than 200 individuals per square meter. These sites have lower H2S concentrations compared to the inner parts where most large sulfide sources are located, but they are annually exposed to a religious harvesting ceremony of local Zoque people called La Pesca. We found a marked shift in size/age compositions towards an overabundance of smaller, juvenile fish at those sites. We discuss these findings in relation to several environmental gradients within the cave , but we also tentatively argue that the annual fish harvest during a religious ceremony (La Pesca) locally diminishes competition , which is followed by a phase of overcompensation of fish densities.The Cueva del Azufre in Tabasco, Mexico, is a nutrient-rich cave and its inhabitants need to cope with high levels of dissolved hydrogen sulfide and extreme hypoxia. One of the successful colonizers of this cave is the poeciliid fish  Astyanax mexicanus is a model organism for EvoDevo studies of cave evolution , Astyanax aeneus (Characidae) as well as ‘Cichlasoma’ salvini and Thorichtys helleri (Cichlidae) occur  occur . In surre) occur . In the er caves  and otheer caves .P. mexicana inhabiting the Cueva del Azufre. Today, the system is increasingly influenced by a growing number of visitors which reach their peak during a traditional annual ceremony of the local indigenous Zoque people named ‘La Pesca’. The Cueva del Azufre is sacred to the Zoque people, and once a year, on the first Sunday of Easter week, the Zoque enter the cave and introduce rotenone- and deguelin-containing barbasco roots  into the water. Rotenone is an inhibitor of the mitochondrial complex-I of the respiratory chain, causing reduced cellular respiration , we combined our assessment of fish densities with an investigation of microhabitat use by different size classes of cave mollies.In the present study, we provide first data of local densities within different chambers of the Cueva del Azufre and adjacent El Azufre and discuss our findings with regard to environmental conditions and annual harvesting of cave mollies. We used a non-invasive technique to repeatedly assess fish densities and size-distribution patterns (as a proxy for age) inside the Cueva del Azufre (up- and downstream of the barbasco-release site) and in the sulfidic creek leaving the cave (El Azufre). Moreover, given the high structural heterogeneity of the water course inside the Cueva del Azufre with respect to water depth and flow velocity , and becP. mexicana (2S) into the creek draining the cave  and sites directly downstream of the release point of barbasco that are strongly affected by the annual ceremony . At each of the eight sample sites, we defined sampling grids consisting of 50 × 50 cm quadrants with wooden sticks fixed in the ground (or stones where a grid angle fell on the shore). The number of quadrants was mostly 25 per sampling grid . In the narrow non-sulfidic surface creek (CC), however, the arrangement of quadrants was more longitudinal (4 × 7 = 28 quadrants), and in chamber V, where a particularly high degree of structural heterogeneity precluded defining larger grids, one sampling site of 5 × 5 quadrants and two smaller ones  were defined . The griDaily measurements took place between 11:00 a.m. and 4:30 p.m. Each site was visited at least 5 times (mean ± SD = 6.25 ± 1.16) on consecutive days. During the counts, we slowly approached a site while trying to avoid any movements that would cause the resident fish to flee, and we counted juveniles (<10 mm standard length (SL)), sub-adults (10–30 mm) and adults (>30 mm) in each quadrant. The observer was standing motionless at least 1.5 m downstream from the respective quadrant. Sizes were estimated qualitatively, aided by a prior training session that used wooden sticks of known size as a reference. Our definition of adults roughly followed −1. Research followed the authorizations from CONAPESCA-DGOPA.09004.041111.3088 and Tacotalpa, Tabasco municipality.Habitat parameters were assessed after the last fish count. For each quadrant, we determined water depth using a wooden ruler stuck vertically into the water at five random locations and calculating the mean from those five measurements. Flow velocity was measured on the water surface by scoring the time a small wooden stick of about 3 cm length took to float through the whole length of a quadrant (measurement was repeated five times per quadrant and averaged across the five observations per quadrant). Mean surface flow velocity was then expressed as cm*sF1,162 = 0.12, P = 0.98) and ‘mean flow rate’  as covariates, but removed them from the final analysis since neither had a significant effect . We used Fisher’s LSD tests for pairwise post hoc comparisons among sites.Our first question was whether population densities differed among sampling sites. We used data for the different quadrants per site (averaged from the repeated measurements) and expressed density as total numbers of individuals per square meter. Density estimates per quadrant were used as the dependent variable in a univariate general linear model (GLM) with ‘sampling site’ as a fixed factor. We initially entered ‘mean water depth’ (A further question was whether size-class compositions differed among sample sites and whether distribution patterns would be stable among repeated sampling days. We used the Bray-Curtis index  to estima) fish densities were sufficiently high and (b) sufficient variability of those environmental variables was found to allow for a meaningful analysis. All other sites were excluded from this analysis. For each site, fish density per quadrant was entered as the dependent variable in repeated measures (rm) GLMs with ‘size class’ (three levels) as the repeated measurement. We grouped water depth  and flow velocity  into three classes each and used these habitat parameters as fixed factors. However, neither the main factor ‘flow velocity’ nor any interaction term involving ‘flow velocity’ had a significant effect in any of the three site-specific models , and so we subsequently removed this term from all models.Our first analysis detected pronounced variation in population densities and size distributions (see results) and thus, we decided to analyze potential effects of water depth and flow velocity  in a site-wise fashion. We focused on sites inside the Cueva del Azufre  where  towards the cave . Also, sites downstream of the barbasco release-site  had considerably higher densities than the site in chamber X that lies upstream of the release-site .When comparing mean densities per quadrant across sites we detected a significant difference among sampling sites  are found in parts of chamber X, where most large sulfide sources are located, while concentrations in chamber V are lower (2–32 µM), as H2S is increasingly bound with oxygen with increasing distance from the sulfide sources  compared to chamber X (site 8) if fish moved between cave chambers but were less likely to return to dark sites, but this line of argumentation is not compatible with the observation of small-scale genetic structure among different cave chambers  bacteria and aquatic invertebrates  was followed by a biomass overcompensation of the juvenile stage as a result of increased adult mortality. Age-specific adult fecundity and body mass of one- and two year old perch increased after the disease outbreak, suggesting that increased adult mortality released perch from competition and cannibalism, thereby increasing somatic and reproductive growth . In parecundity , which mecundity . This idty rates . Anothere growth . In the e growth . Hence, hamber X , confirm2 in some parts of the cave. While other selective forces certainly also need to be considered, we argue that the annual La Pesca has major effects on the population ecology and evolutionary trajectory of cave mollies. We are aware of potential caveats of this line of argument, as not all differences reported here may be due to the annual La Pesca ceremony. Nevertheless, from a conservational point of view, knowledge about whether and how human activities affect teleost populations is especially pertinent in the case of locally adapted populations that are endemic to a small area. Therefore, we recommend a repeated sampling before and after La Pesca in order to demonstrate the influence of the ritual. While human influences on highly endemic, locally adapted populations (or, in terms of conservation biology, evolutionary significant units; In summary, we found remarkable fish densities of more than 200 individuals per m10.7717/peerj.490/supp-1Supplemental Information 1Click here for additional data file.


Schizosaccharomyces pombe and Schizosaccharomyces japonicus [S. pombe. In contrast, Ned1 is not regulated during division in S. japonicus, thus limiting membrane availability and necessitating NE breakage. Interspecies gene swaps result in phenotypically normal divisions with the S. japonicus lipin acquiring an S. pombe-like mitotic phosphorylation pattern. Our results provide experimental evidence for the mitotic regulation of phosphatidic acid flux and suggest that the regulatory networks governing lipin activity diverged in evolution to give rise to strikingly dissimilar mitotic programs.Eukaryotes remodel the nucleus during mitosis using a variety of mechanisms that differ in the timing and the extent of nuclear envelope (NE) breakdown. Here, we probe the principles enabling this functional diversity by exploiting the natural divergence in NE management strategies between the related fission yeasts aponicus , 2, 3. W •S. pombeLipin phosphorylation by CDK drives mitotic nuclear envelope expansion in •CDK-dependent lipin phosphorylation is required for closed mitosis•S. japonicusLipin is not regulated by mitotic CDK in the related species •Interspecies gene swaps reveal species-specific trans-regulation of lipin activity Using closely related yeasts, Makarova et al. uncover a molecular basis for variability in nuclear envelope expansion during mitosis. They show that cells undergoing closed mitosis expand their nuclear envelope prior to division by entraining inactivation of the phosphatidic acid flux regulator lipin to high CDK activity. S. pombe and Saccharomyces cerevisiae solve this problem through nuclear envelope (NE) expansion at mitotic entry [S. japonicus, an S. pombe relative, does not expand its NE and instead relies on NE breakdown during anaphase to allow chromosome segregation [The surface area of a mother nucleus undergoing closed mitosis must increase to allow intranuclear mitotic spindle elongation and formation of the daughter nuclei. The model yeasts ic entry , 7, 8, 9regation , 3.Abnormal NE proliferation in interphase has been linked to changes in phosphatidic acid metabolism , 11, 12,S. pombe and S. japonicus, as visualized by the luminal ER marker GFP-ADEL  showed that, unlike in budding yeast [D383E/D385E in S. pombe and Ned1D422E/D424E in S. japonicus) exhibited comparable ER expansion and formation of NE flares . We identified two clusters of phosphorylation in S. pombe Ned1 located in its C terminus—S587/S597/T598/S599 and S627/S629. Notably, S627/S629 represents a repeated relaxed S-P consensus motif for phosphorylation by the cyclin-dependent kinase 1 (CDK1). Analysis of Ned1 of S. japonicus revealed two possible phosphorylation sites—S235 located between the N-lipin and catalytic domains and S651 at its C terminus  virtually abolished CDK phosphorylation in vitro , suggesting that the mutant protein did not support growth. To investigate this possibility, we constructed a diploid strain where one of the wild-type copies of ned1 was replaced by the ned1S627A/S629A allele tagged with the selectable auxotrophic marker ura4+. The growth rates of diploid WT/WT and WT/ned1S627A/S629A cells at 30°C were comparable although the WT/ned1S627A/S629A diploids exhibited pronounced lag phase at lower cell densities  did not visibly alter NE and ER morphology, with cells undergoing normal mitotic divisions and maintaining viability  of S. pombe and S. japonicus, leaving the host-specific untranslated gene regions intact. Interestingly, germination of S. pombe spores carrying the ned1S. japonicus::ura4+ (ned1S.j.) allele as its sole ned1 copy yielded healthy cells exhibiting normal chromosome partitioning as judged by staining the DNA with DAPI  did not lead to obvious differences in mitotic nuclear dynamics . The fact that the two lipins could substitute for each other when expressed in a heterologous system indicates that the regulatory modes rather than the lipin protein properties diverged between the two fission yeasts.Indeed, the population-doubling times were comparable between the lipin-swapped and the wild-type cultures (2.34 ± 0.18 versus 2.31 ± 0.11 hr for S.j.-GFP were markedly different in G2-arrested and mitotic S. pombe cells, indicating that the transplanted Ned1 protein could be subject to S. pombe-specific mitotic phosphorylation events (S. japonicus could be phosphorylated by Cdk1 in vitro (S. japonicus has several putative CDK phosphorylation sites, including five at its C terminus, S499, S538, T620, T638, and S651, suggesting that similar phosphoregulation could occur in S. pombe cells. Replacement of these serine and threonine residues with non-phosphorylatable alanines rendered the transplanted Ned1S.j. defective in supporting mitotic division in S. pombe cells. Similar to the phenotype observed in S. pombe ned1S627A/S629A mutant (ned1S.j.-5A::ura4+ allele failed to properly partition chromosomes and divide the nucleus in the first mitotic division (S.j.-5E caused a loss-of-function phenotype in S. pombe, with mutant cells exhibiting constitutive ER and nuclear membrane expansion (S. japonicus, the same phosphomimetic mutant triggered ER-NE expansion during interphase and decreased lipid droplet formation (S. japonicus did not cause mitotic abnormalities but produced cells with more lipid droplets (ned1S235A/S651A genetic background (The gel migration patterns of Ned1in vitro D, and NeA mutant D, many gxpansion E. When mormation F and S4Gdroplets F and S4Gckground E.S. pombe in the presence of Phos-tag in S. pombe and S. japonicus asynchronous wild-type and spo7Δ cultures. When expressed in its native wild-type environment, Ned1S. pombe migrated as multiple phospho-forms, suggesting potential phosphoregulation by multiple inputs (S. japonicus cells, Ned1S. pombe acquired a distinct electrophoretic mobility pattern, consistent with the pronounced presence of a dephosphorylated form. Introducing the transplanted protein into the spo7Δ background led to increased phosphorylation (S. pombe lipin, normally a highly phosphorylated protein, is subject to massive dephosphorylation by Spo7-Nem1 phosphatase in S. japonicus.To address the potential contribution of Spo7-Nem1 phosphatase to lipin regulation in the two species, we analyzed the electrophoretic migration of Ned1e inputs E. Strikirylation E. This sS. japonicus lipin in both yeasts. We consistently detected a major phospho-form together with a dephosphorylated species of lipin in S. japonicus wild-type cells. As expected, the lack of Spo7 led to hyperphosphorylation of Ned1 (S. pombe, Ned1S. japonicus migrated differently than it did in S. japonicus, consistent with differential phosphoregulation in this species (In a reciprocal experiment, we analyzed the phospho-state of the  of Ned1 F. When t species F.S. pombe allows this organism to inactivate the bulk of the enzyme specifically in mitosis. Lipin inactivation presumably triggers increased phospholipid production required for massive NE expansion during “closed” nuclear division. On the other hand, overall phosphorylation status of lipin in S. japonicus remains constant throughout the cell cycle (S. pombe could be due to a disruption in this balance, for instance because of concurrent inactivation of the Spo7-Nem1 phosphatase. The fact that the transplanted S. pombe lipin is more dephosphorylated in S. japonicus suggests a powerful contribution of phosphatase in this species (Taken together, our results suggest that the fundamental differences between mitotic strategies of the two fission yeasts might depend on divergence of the regulatory networks controlling lipin activity. Phosphorylation of Ned1 by CDK1 in ll cycle G. This d species E. AlternS. pombe, lipin deficiency in S. japonicus produces considerably stronger expansion of the NE-ER system and has a more deleterious impact on cellular growth rate (S. japonicus cells. Work in budding yeast uncovered crosstalk between lipin function and transcriptional control of a cohort of inositol-responsive lipid biosynthesis genes [S. pombe and S. japonicus and deducing species-specific response patterns to genetic perturbations of the lipin-centered circuitry will be important to illuminate possible physiological and metabolic foundations for the distinct lipin network topologies within the clade.As compared to wth rate  and S1. is genes , but thiis genes . DetermiMetazoan lipins and the Spo7-Nem1-related phosphatases are important for NE/ER biogenesis and mitotic remodeling , 36, 37 S. pombe and S. japonicus strains used in this study and their genotypes are listed in S. japonicus strains including several fluorescent tags, a conditional cdc25 mutant, and a ned1 replacement strain. J.-S.C. performed in vitro kinase assays and mass spectrometry. J.R.B. analyzed mass spectrometry data and edited the manuscript. K.L.G. provided input into the design and interpretation of experiments and edited the manuscript. S.O. guided the project and co-wrote the manuscript.M.M. did in vivo experiments and co-wrote the manuscript. Y.G. generated a number of


Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in   •Sequences flanking the Par3 CR3 consensus PKC site cooperate to inhibit aPKC•A Par3 CR3 inhibitory arm disrupts aPKC P-loop/αB/αC contacts and αC-helix position•Mutating either CR3 arm switches Par3 into an efficient aPKC substrate in vitro•Equivalent Bazooka substitutions alter its apical localization to AJs in vivo Par3 is required for aPKC membrane recruitment, yet it polarizes to adherens junctions upon phosphorylation. Soriano et al. show that Par3 antagonizes active aPKC kinase by separating crucial N-lobe contacts. Disrupting high-affinity Par3 contacts switches it to an efficient aPKC substrate and polarizes Par3/Bazooka from apical domains to adherens junctions. Drosophila or PKCι/PKCζ isozymes in mammals), its binding partner Par6, and the small guanosine triphosphatase Cdc42 are three essential determinants of apical membrane identity in both Drosophila and mammals (Drosophila) (known as the Par complex) at the apical membrane  . The pla mammals . The aPKmembrane . Associamembrane .Drosophila to be dependent on aPKC phosphorylating Par3/Baz on serine 980 in vivo   . Like Pabs (Crb)  and aPKCbs (Crb) . Par3/Baosophila . Similarosophila . The preosophila . Howeverosophila . These fhttps://www.kinexus.ca). In these contexts aPKC phosphorylation inactivates substrates with basophilic membrane-binding motifs with embedded phosphorylation sites such that they are displaced from membranes  is able to bind to PKCι (KD-2P) and a partially primed low-activity form (referred to as PKCιKD-1P), referring to the status of the two “priming” phosphorylation sites at pT412 and pT564  of 0.45 ± 0.18 μM. In contrast, peptides from other known aPKC substrates such as Par1 were efficiently phosphorylated and were unable to inhibit (CR3 binds to PKCιKD-2P with submicromolar affinity (KD of 0.47 ± 0.09 μM), as does an S827A mutant (KD of 0.97 ± 0.07 μM) . In contrast, PKCιKD-1P was not inhibited to the same extent and had a much lower affinity for Par3CR3  E. PKCιKDCR3 could inhibit PKCιKD-2P, we determined the 2.0-Å crystal structure of a longer Par3 peptide (residues 816–841) bound to PKCιKD-2P and Mg-AMPPNP (adenylyl imidodiphosphate) , across the G helix (site 2) through to the activation loop, αB and αC helices of the PKCιKD-2P N lobe (site 3)  forms a salt bridge with the conserved αC-helix glutamate (E302PKCι) side chain found in many active kinase conformers (To understand how Par3osphate) . The Paren bonds B. It eng(site 3) A and 2B.cleotide C. The conformers . The ternformers . A magnenformers . The Mg1CR3 binds to site 1 (PKCιKD-2P kinase C lobe) through an F-X-R motif at positions −9 (F−9) and −7 (R−7) defined relative to the phospho-acceptor (P site) at serine 0 . F−9 lies deep within a hydrophobic cleft formed by M341PKCι, M344PKCι, and L381PKCι beneath the nucleotide pocket . The phospho-acceptor serine-0 hydroxyl hydrogen bonds to side chains of D378PKCι, K380PKCι, and T416PKCι, preventing a catalytically competent orientation for nucleophilic attack on the ATP γ-phosphate. Glycine-rich loop residues S264PKCι and Y265PKCι side chains contact the CR3 main-chain atoms near the P site, as does the activation-loop main chain, to orient the Par3CR3 peptide and position the M+1 side chain into the known P+1 AGC kinase hydrophobic pocket . Residue K+4 directly contacts pT412PKCι of the activation loop enhancing the recognition of mature, primed PKC ιKD-2P, but importantly not a partially primed PKCιKD-1P. Crucially, the R+5 side chain is buried within a hydrophobic pocket beneath the regulatory αC helix. The pocket is lined by side chains from Y265PKCι on the glycine loop and W298PKCι of the αC helix, each making π-stacking interactions with the guanidino group of R+5  lies adjacent to an acidic patch within the αB helix making side-chain and main-chain contacts to D295PKCι and a Mg ion   suggests that the R+5 side-chain guanidine group lies close to the Mg2 ion of an active kinase conformation , which makes direct contact with D295PKCι, is a phospho-acceptor site targeted by the ROCK kinase, leading to a disruption of PKCι interaction with Par3   exhibitend 4DC2) C and 3D.nd 4DC2) . This anαC helix B. Other αC helix . Structuormation , indicatith Par3 . This woCR3-PKCιKD-2P inhibitory complex differs significantly from a previous structure of an ATP-binding deficient and partially primed PKCι K283R mutant  bound to Par3CR3   as the F−9 and R−7 contacts used by Par3CR3, despite their different position in the primary sequence  A–S4E. Thpeptide) . We there analog E. Mangansequence F. MoreovCR3 structure, it is evident that the F-X-Rshort peptide does not inhibit PKCι because it lacks a C-terminal inhibitory motif  suggest that these substitutions uncouple the ability of Par3CR3 to inhibit PKCιKD-2P, resulting in access to the PKC consensus site at S827Par3 and efficient phosphorylation by PKCιKD-2P. The magnitude of the increased kcat values allowed the measurement of a KM for the A-X-A Par3CR3 substrate (KM of 39 μM), which was not possible for wild-type Par3CR3. Combining both site 1 and site 3 mutations (A-X-A + A-A-T) within Par3CR3 generated a very poor substrate that was not detectably phosphorylated and had no measurable interaction with PKCιKD-2P (data not shown). These data indicate that while mutating either the anchoring arm or inhibitory arm switches Par3CR3 to an efficient aPKC substrate, the remaining arm must contribute sufficient binding affinity (both are basophilic) as mutating both arms generates a Par3CR3 that is neither a substrate nor an inhibitor. These striking results are also consistent with the notion that tight inhibitory binding of Par3CR3 to PKCιKD-2P must prevent its phosphorylation while weaker binding without the inhibitory interactions exposes its PKC site, switching it to a highly efficient in vitro PKCι substrate.Surprisingly, the in vitro kinase assay demonstrated that either an A-X-A or A-A-T mutation gave a substantial increase in Par3t values A and 5C.PKCι/D382PKCι (GFP-PKCι-D/D) that directly contact the R−7 side chain of Par3 also markedly reduced the Par3-PKCι interaction  characterized in vitro should affect the localization of Par3/Baz in vivo. To test this prediction, we mutated the CR3 region of full-length GFP-tagged Drosophila Baz in the F-X-R motif to A-X-A or the K-H-T motif to A-A-T and examined their apical domain or AJ localization in vivo. In the follicular epithelium, GFP-tagged wild-type Baz (GFP-Baz) co-localizes with aPKC at the apical membrane and also localizes to AJs . Efforts to raise a Baz phospho-antibody against S980 peptides excluding the F-X-R motif were not successful. Therefore, we verified that the A-X-A Baz mutant was phosphorylated in Drosophila cells, by preparing transfected S2 cell extracts containing wild-type or A-X-A mutant Baz and probed S980Baz phosphorylation status using dimethyl labeling and mass spectrometry. Previous efforts to identify the BazCR3 phospho-site in wild-type and A-X-A mutant contexts using trypsin digest were unsuccessful due to cleavage at R979 and K984, yielding very short peptides. Therefore, BazCR3 samples were first treated by in-gel reductive dimethylation, to generate the BazCR3 peptide spanning the sequence (phospho)SISE(me2K)HHAALDAR. The dimethylation reaction modifies lysine ɛ-amino groups, thereby greatly reducing the ability of trypsin to cleave after lysines. This allowed capture of the phospho-BazCR3 peptide, facilitated quantification of chromatographic peak areas, and identified phospho-peptides. The forward sample reaction used heavy (CD2O with wild-type Baz mutant) or light (CH2O with A-X-A Baz) reagents, resulting in a mass difference of 12 Da and an m/z difference of 4 for the triply charged target peptide (2O with A-X-A Baz mutant) or light (CH2O with wild-type Baz) reagents, and two control peptides were also used to assess any differences in peptide recovery from the SDS-PAGE gel , or even regulation of the aPKC activation loop (by PDK1 or by dephosphorylation by an unknown A-loop phosphatase). One or more of these factors, depending on the physiopathological status of the epithelium, could affect the Par3-binding mode. We do note that Kibra (and its homolog WWC2) contains an F-X-R motif and a K-R-T hook flanking its known aPKC phosphorylation site (between residues 911 and 978), suggesting that it too could act as dual-action inhibitor/substrate, consistent with biochemical data . One possible explanation for this open issue is that discrete functional states of the Par complex (Par6-aPKC-Cdc42-Par3) may exist. Par3-dependent recruitment of aPKC to apical membranes may evoke a higher-order oligomer consistent with known Par3 CR1-dependent oligomers  provoke Par3 phosphorylation by overcoming CR3 antagonism.Our findings suggest that aPKC is inhibited by Par3/Baz within the Par complex, yet it is known that the Par complex contains active aPKC kinase and can phosphorylate many substrates  was prepared using a baculovirus encoding residues 248–596 (GenBank: NM_002740.5), fused to a glutathione S-transferase (GST) tag as described previously .Mammalian plasmids pEGFP-PKCι-WT, pEGFP-PKCι-DD/AA (D339A/D382A), pK-myc-Par3-WT , pK-myc-Par3-AXA (F818A/R820A), and pK-myc-Par3-A (S827A) included human PKCι and Par3 cDNAs. Mutagenesis of PKCι and Par3 was performed using QuikChange (Stratagene). Recombinant human PKC-iota kinase domain . The kinetic constants were determined by fitting the data to the Michaelis-Menten equation. Data are represented as mean ± SEM. Fluorescence anisotropy assays were performed to determine the KD for each peptide labeled with a fluorescein tag following standard protocols using a Safire2 plate reader (Tecan). The anisotropy values were normalized and the KD was determined using non-linear regression. All experiments were performed in triplicate and for at least three independent protein preparations.The ADP Quest kit (DiscoveRx) was used to determine the kKD-2P was incubated with a 3-molar excess of nucleotide or analog with either Mg2+ or Mn2+ (see ∗Trek (3A8W). Refinement was carried out in Phenix  were transfected (10-μg portion of DNA or 5-μg + 5-μg portions of DNA for co-transfections) using Fugene HD (Roche) according to the manufacturer's instructions. The cells were then grown in normal medium for 36 hr.10), rabbit polyclonal anti-Par3 (Millipore), and rabbit polyclonal anti-GFP antibody (Santa Cruz Biotechnology). Anti-phospho-S827 Par3 antibody was raised in-house using an antigen lacking the F-X-R site of Par3CR3. Immunoprecipitation and immunoblotting was carried out as described in The following antibodies were used for immunoblotting: mouse monoclonal anti-PKCι (recognition for human PKCι), mouse monoclonal anti-Myc . A targeted scan was performed for the S980-containing peptides and this was alternated with a top-10 data-dependent acquisition scan. Mascot-generated DAT files were converted to Skyline-compatible biblio.spec libraries, and heavy and light peak areas were extracted by Skyline software version 2.5.0.6079  vector for transgenesis (Bestgene). The UAS.GFP-BazS980E line was a gift from F. Pichaud. Primers used for mutagenesis are described in Expression of Ovaries were dissected in PBS, fixed for 20 min in 4% paraformaldehyde in PBS, washed for 30 min in PBS/0.1% Triton X-100 (PBST), and blocked for 15 min in 5% normal goat serum/PBST (PBST/NGS). Primary antibodies were diluted in PBST/NGS and samples were incubated overnight at 4°C. Either optical cross-sections through the middle of egg chambers or apical sections of the follicular epithelium are shown. Primary antibodies used are described in CR3-PKCιKD-2P peptide structure determination and refinement. A.P. refined the Mn-ADP-PKCιKD-FXRshort peptide structure. M.I. purified, crystallized, and refined the AMPPCP-complexed PKCιKD-2P structure. E.V.S. and M.I. carried out kinetic assays and fluorescence anisotropy measurements. P.K. and S.K. assisted with protein production and virus preparation. A.E. prepared S2 cell extracts containing wild-type and mutant Baz protein. K.B. and B.S. performed the dimethyl-labeling and mass spectrometry analyses. B.K. prepared and characterized recombinant PKCιKD and PKCζKD proteins. P.S. prepared constructs for full-length human Par3 and mutants. N.O’R. purified all peptides used in this study. P.R. conducted co-immunoprecipitations using full-length Par3 with full-length PKCι and raised the anti-phospho-Par3 antibody. M.L. prepared the PKCι D/D mutant. G.F. and B.J.T. carried out all the Drosophila experiments. M.I. prepared the figures. N.Q.M. and B.J.T. planned the project, designed experiments, and wrote the paper.E.V.S. performed all stages of the Mg-AMPPNP-Par3


Fusarium is related to the presence of nanometre-scale particles which are far more numerous, and therefore potentially far more important for cloud glaciation than whole intact spores or hyphae. In addition, we quantify the ice-nucleating activity of nano-ice nucleating particles (nano-INPs) washed off pollen and also show that nano-INPs are present in a soil sample. Based on these results, we suggest that there is a reservoir of biological nano-INPs present in the environment which may, for example, become aerosolised in association with fertile soil dust particles.Most studies of the role of biological entities as atmospheric ice-nucleating particles have focused on relatively rare supermicron particles such as bacterial cells, fungal spores and pollen grains. However, it is not clear that there are sufficient numbers of these particles in the atmosphere to strongly influence clouds. Here we show that the ice-nucleating activity of a fungus from the ubiquitous genus  The formation of ice in mixed-phase clouds, where both supercooled water droplets and ice coexist, impacts upon precipitation, cloud lifetimes and radiative propertiesWhile inorganic INPs are thought to be of critical importance in the initiation of ice at temperatures below approximately −20°C, these species  are unlikely to be sufficiently active above about −15°C to influence clouds36et al. have estimated that for birch pollen, a single grain can possess tens of thousands of ice-nucleating nano-scale particles, each of which can be readily extracted from the parent grain by contact with water14Mortierella alpina are also nanometer in scaleIt is known that the ice-nucleating sites on intact biogenic INPs are far smaller than the intact cells, grains or spores themselves, and in some cases can be entirely harboured on separable fragments91011In this paper we hypothesise that biological, nanometer scaled INPs which are bound to other atmospheric particulates are important for cloud glaciation; this hypothesis is illustrated in 17181921Fusarium, birch (Betula pendula) pollen and a soil sample. Through a combination of filtration and size exclusion chromatography we demonstrate that the entities responsible for nucleation in Fusarium avenaceum are nano-scale species which are readily extractable into water. In addition it is shown that individual pollen grains can harbour on the order of 104 distinct ice-nucleating nano-particles capable of inducing nucleation at relatively warm temperatures (T > −18°C). We also demonstrate that nano-INPs can also be found in soils, and exhibit comparable thermal instabilities to known proteinaceous INPs, such as those we find in Fusarium avenaceum.To explore this hypothesis, we examine the ice-nucleating activities of nano-particles associated with biological materials including a soil-borne plant pathogenic fungus from the widely distributed genus F. avenaceum) mycelium in microlitre-sized droplets and cooling them while monitoring for freezing events, we determined the cumulative number of active nucleation sites as a function of temperature (referred to as a cumulative nucleus spectrum). The number of active sites is reported per unit mass of the mycelium suspended in the droplets (see methods). To facilitate observation of ice nucleation at colder temperatures, experiments were also performed on suspensions which were serially diluted by factors of 100 and the results are shown in F. avenaceum contains highly active INPs, consistent with previous reports10et al.dsmooth sphere) of 13 nm for the average density of a typical protein (we take a protein density of 1.37 g cm−3 from Ericksondsmooth sphere ~6 nm), the ice-nucleating activity was dramatically reduced, falling below the limit of detection for the assay determined by background freezing of the Milli-Q® purified water due to impurities (see methods). These results show that the INPs produced by F. avenaceum can function independently of fungal cells, are easy to remove from the parent fungus and are nanometre in scale.By suspending known quantities of fungal  is larger than the nominal molecular weight cut-off of the filter applied prior to size exclusion chromatography, this is consistent with a finite distribution of pore sizes as is found in ultrafiltration membranesTo further examine the size of the ice-nucleating particles generated by tography , left. Ttography , right, F. avenaceum and those from the well-known ice-nucleating γ−proteobacteria, such as Pseudomonas syringae. For instance, the activities of both are comparable. Furthermore, both are thought to contain proteins in the ice-nucleating active sites and are sensitive to heat treatment and enzymatic digestion with proteases102326There are several similarities between the nano-INPs produced by Fusarium fungus studied here, contact with water is sufficient to separate a large proportion of the nano-INPs from the parent grains. Shown in Another class of biogenic nano-INPs are derived from pollen98 of the original 2 wt. % concentration) used to assess nucleation in this regime. From this, the number of nano-INPs active at temperatures greater than −18°C (i.e. before they are removed by dilution) can be estimated as <1012 per gram from 8 grains per gram for birch pollen13313 INPs per grain, similar to the 2 × 104 estimated by Augustin et al.Betula pendula) using a different approach.At temperatures below −18°C the characteristically sharp slope of the nucleation spectrum for pollen-derived INPs levels off . This beF. avenaceum and for birch pollen, nano-INPs were also observed to stimulate ice nucleation in aqueous soil suspensions 3462122The results presented here suggest that in order to increase the accuracy of our current model simulations of atmospheric ice nucleation processes, biogenic species other than supermicron-sized particles should be considered. However, deficiencies currently exist in the basic knowledge required to accurately assess the impacts of biological nano-INPs on cloud ice formation. These deficiencies include our knowledge of the source strengths and global distribution of nano-INPs. There are also unresolved questions on the stability of biogenic nano-INPs towards chemical and physical processing in soils and when suspended in the atmosphere.While producing inventories of the species responsible for ice-nucleating activity in complex mixtures such as soil and constraining the impacts of such particles on atmospheric ice nucleation processes is likely to be a complex undertaking, there are several factors which suggest that such a task is achievable. Relatively simple tests such as filtration, heat treatment and enzymatic digestion can be used for the rapid screening of biogenic nano-INPs in natural aerosol samples. In such a manner, datasets of the abundance of biogenic ice nucleators can be established, and their regional importance quantified. Links between the net amount of biogenic nano-INPs and governing variables  can then be examined, allowing for the global evaluation of biogenically derived nano-INPs.F. avenaceum were obtained from the Commonwealth Agricultural Bureaux International (CABI). The F. avenaceum were plated on potato dextrose sucrose agar and incubated for 28 days in the dark at 20°C. The colonies produced were ca. 4 cm in diameter, and varied in colour from white to pink. 1 mg of the mycelium was then subsequently scraped off the agar and resuspended in 10 ml of Milli-Q® grade water. No other steps to induce cell lysis were taken. The suspension was then filtered first through a 0.2 μm cellulose acetate filter , followed by a polyethersulfone centrifuge filter  and regenerated cellulose filter . The 1000 kDa filtrate was further fractionated by size exclusion chromatography using a Superose 6 10/300 GL column capable of separating proteins from 5,000–5,000,000 kDa in size. The fractionation was conducted in pure water (not in buffer) to allow for ice nucleation measurements. Protein elution was monitored via UV absorbance of the eluate at 280 nm.Freeze-dried cultures of Betula pendula) trees growing in South Moravia, Czech Republic, and was supplied by Pharmallerga®. The batch (BETP.1310) was certified to contain <0.2% spores, <0.1% other foreign pollen and the pollen was not defatted. To extract the nano-INPs, 1 g of the dried pollen was suspended in 50 ml water (2 wt. %), shaken and allowed to settle overnight in a refrigerator. After 12 hours, the pollen solution was shaken again, and then filtered, first through a 11 μm Nylon net filter (Millipore) and then through a 0.2 μm cellulose acetate filter. To obtain the 1000 kDa ultrafiltrate, Sartorius Vivaspin Ultrafilter devices as described above were employed.The pollen used in this study was collected from wild Silver birch . 10 g of the soil (<2 mm) was suspended in 100 ml of water, and sonicated for 3 minutes to disperse the sample. A 45 ml aliquot of the sample was then poured into a polypropylene centrifuge tube, and left to settle for 60 s to allow larger particles to settle out. The supernatant was withdrawn from the top 4 cm of the vial for filtration. For the 11 μm filtration, the sample was vacuum filtered through a hydrophilic nylon net filter . Filtration to 0.2 μm was conducted using a cellulose acetate filter. Ultrafiltration to yield 1000 kDa and 100 kDa fractions was conducted using Millipore Amicon Ultra and Sartorius Vivaspin centrifuge filters as outlined above. Heat treatment of the soil samples to test for thermally-labile INPs was conducted by heating a sealed Eppendorf tube containing 1 ml of the suspension to 95°C for 45 minutes. This test, which is commonly used as a qualitative assay for the presence of proteinaceous INPs1833343 min−1 of nitrogen gas to prevent unwanted condensation. The cooling rate employed for all experiments was 1 K min−1. Freezing events were monitored using a digital camera and later the video was analysed frame by frame to determine the onset of nucleation. The temperature error associated with the thermocouple was estimated as ±0.4 K. All filters employed were tested for the presence of heterogeneous INPs which could interfere with the measurements. In each case, the freezing profile indicated that droplet nucleation took place at temperatures lower than was found for Milli-Q® purified water , the cumulative number of nucleation sites active at a given temperature per unit volume of water, K(T), can be determined by:V is the droplet volume in cm3. The factor d is the dilution of the droplets relative to the initial undiluted suspension. Accordingly, by diluting the suspension, the ice-nucleating activity can be probed at lower temperatures than is observable in droplets of the initial concentration. For a known amount of nucleant per unit volume, the cumulative nucleation spectrum can be expressed relative to the mass of material suspended in the droplet via:C is the concentration of the parent particles in g/cm3. Uncertainties in the calculation of the number of nucleation sites are calculated by propagating those stemming from the concentration and droplet volume together with uncertainties which arise due to background freezing of the Milli-Q® water. In all experiments on Milli-Q® water where no nuclei were added, freezing occurred at temperatures higher than expected due to homogeneous nucleationK(T) obtained from the Milli-Q® purified water, was used to estimate background nuclei concentrations and was substracted from the cumulative nucleus spectra obtained later when nucleants were added. 68% confidence intervals constructed about this line were used to estimate uncertainties due to the variation in the number of these background nuclei, and this was propagated into the uncertainty associated with the cumulative nuclei spectra of the added nucleants.Using a time-independent (singular) analysis of the fraction of droplets frozen at a particular temperature3D.O.S. conducted the ice nucleation experiments, analysed the data and was authored the manuscript. B.J.M. and M.E.W. oversaw the project and helped to write the manuscript. J.F.R. performed and analysed data from the size exclusion chromatography. T.F.W. and N.S.U. provided data for the background ice nucleating activities of Milli-Q water. H.C.P. and J.D.A. aided with the error analysis of the data and assisted with drafting the manuscript.Supplementary Info


The diffusion-limited reaction rate of a uniform spherical reactant is generalized to anisotropic reactivity. Previous work has shown that the protein model of a uniform sphere is unsatisfactory in many cases. Competition of ligands binding to two active sites, on a spherical enzyme or cell is studied analytically.The reaction rate constant is given for two sites at opposite ends of the species of interest. This is compared with twice the reaction rate for a single site. It is found that the competition between sites lowers the reaction rate over what is expected for two sites individually. Competition between sites does not show up, until the site half angle is greater than 30 degrees.Competition between sites is negligible until the site size becomes large. The competitive effect grows as theta becomes large. The maximum effect is given for theta = pi/2. The purpose of this article is to generalize the Smoluchowski  calculatWe want to solvewhere c gives the ligand concentration. Figure We also havewhere c˳ is the bulk concentration of ligand. The reactive boundary conditions are given byandAs done previously ,9, equatwhere Q is evaluated, by requiring the average concentration of ligand to vanish over the active site. , by considering a reactive disk in an insulating plane. For this problem, the exact solution is known . It is kd yields  kDC = 3.The solution of equation (1) is given byPm (x) are Legendre polynomials of order m. Application of the boundary condition given by equation (2) yieldswhere the Equation (3) is satisfied with α = c˳ and with the radial functions2m in equation (10) are found using equations (5) and (6). Details, parallel a previous derivation [The coefficients a (5) and . DetailsIn this section we present the results and discuss how the method represents a step forward in the field of diffusion-controlled kinetics and biophysics in general.The results are given by the solution of the model in the preceding section, and by comparing it to twice the rate limited constant for the one site problem . The difThe diffusion limited rate constant for the two site problem iswhere 4πDR is the rate constant for a uniformly reacting sphere. The reciprocal of our rate constant isThe reciprocal of the rate constant for one site is given byk2 (θ˳) (the rate constant for 2 sites), twok1 (θ˳) (twice the rate constant for 1 site) and k1 (θ˳) . As θ˳ grows above 30 degrees, the curves grow apart. The two site curve, being less than 2 × the one site curve.Figure one site ) versus The difference between the curves is a measure of the competition between the two sites. The competition effect does not show up till around 30 degrees. For This paper represents progress in the field, by presenting a new model for the interpretation of experimental data. This is for macromolecule-ligand binding reactions that fall in the diffusion-controlled regime. Previously, only one site models were available for modeling proteins. Proteins with multiple binding sites can now be studied.The analytical expression for the diffusion-limited rate constant to two active sites on a sphere has been given. The result was used to study the competitive effects between the two sites. The effect doesn’t show up until the site half-angles is greater than 30 degrees. The competitive effect grows until its maximum value is reached at θ˳ = π/2.There are no competing interests for this paper.


Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus. The pathogenesis of atherosclerosis is multifactorial, including vasoconstriction, thromboembolism, and vascular smooth muscle cell (VSMC) proliferation and migration . Among vGlucagon-like peptide 1(GLP-1) is a 30-amino acid peptide hormone that is secreted from gut endocrine cells and is stimulated by nutrient ingestion inducing glucose-dependent insulin secretion through the activation of G-protein-coupled GLP-1 receptors on pancreatic islet cells  8] 9]. [9].8] [. [9].8] In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC) to explore the vascular protective effect of GLP-1 receptor agonist. The effect of exendin-4 on changes in intracellular signaling by Ang II was also examined to provide a possible mechanism by which exendin-4 may be used as a pharmacotherapeutic agent for the prevention of hypertension and atherosclerosis independent of its blood glucose lowering effect.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Nara Medical University’s Committee on the Ethics of Animal Experiments (Permit Number: 11011). All surgery was performed under the inhalation of isoflurane anesthesia, and all efforts were made to minimize suffering.2 in a humidified incubator. After reaching confluence in three 35-mm dishes, the cells were harvested by brief trypsinization and grown in T-75 flasks  (passage 1). RASMC were used for experiments between the third and sixth passages. When the cell confluency in culture was estimated to be 50% to over 90%, the medium was replaced with unsupplemented DMEM. The cells were further cultured for 48 h and then subjected to each experiment. In some experiments, RASMC were pre-incubated with exendin-4 for 5 min to 1h and MAP kinase inhibitors  for 1 h prior to stimulation with Ang II.RASMC were isolated from the thoracic aorta of 8-week-old male Sprague–Dawley rats by enzymatic digestion, as previously described . Cells wA cell counting assay was performed essentially according to the manufacturer’s protocol as described below  23]. In. In23]. We performed a wound healing assay to evaluate cell migration, especially transverse displacement. After culturing in serum-free DMEM for 24 h, the cells were incubated in the absence or presence of exendin-4 or MAP kinase inhibitors for 1 h. Then, the cells were treated with Ang II, wounded using a yellow pipette tip, and cultured for 24 h. Later, the wounded cultures were stained with Diff-Quick  and photographed. Cells that had migrated from the rim of the dishes were counted in 10 microscope fields per dish.For western blot analysis, RASMC were lysed immediately after each experiment and lysate proteins were collected in the manner described earlier . The celMaterials were purchased from Wako (Kyoto) or Nacalai Tesque (Kyoto) unless stated otherwise. Exendin-4 was procured from Sigma-Aldrich . The antibodies used for the western blot analyses were purchased as follows: anti SM22α, anti phospho-Yes-associated protein 1 (Yap1), anti-phospho-ERK1/2 antibody and anti-phospho-SAPK/JNK antibodies were purchased from Cell Signaling Technology, while ECL and ECL plus systems were purchased from GE Healthcare. Collagen I was purchased from Nippon Meat Packers, Inc. (Osaka). All chemical compounds were dissolved in dimethyl sulfoxide (DMSO) at final concentration of less than 1% except where specially noted.P value <0.05 was considered to be significant.All experimental values were expressed as mean ± standard error of the mean. Non-parametric ANOVA analysis with Kruskal-Wallis and Dunn’s correction was used to determine significant differences in multiple comparisons. A We firstly examined the phenotypic character of RASMC by western blot analysis using SM22α antibody, a marker for contractile phenotype, and phospho-Yap1 antibody, a marker for synthetic proliferative phenotype . As showTo evaluate the relative magnitude of Ang II-induced VSMC proliferation, growth-arrested RASMC were exposed to various concentrations of Ang II. Ang II-induced cell proliferation was determined as described under Materials and Methods. As shown in Next, to examine the concentration-response for the Ang II-induced VSMC migration, growth-arrested RASMC were exposed to various concentrations of Ang II. Ang II-induced cell migration was measured as described under Materials and Methods. As shown in In order to clarify the intracellular signaling mechanisms associated with exendin-4 inhibition of Ang II-induced RASMC proliferation and migration, the following experiments were undertaken. The effects of Ang II on the phosphorylation of ERK1/2 and JNK were assessed by western blot analysis as described under Materials and Methods. As shown in To confirm the possible involvement of ERK1/2 and JNK on Ang II-induced RASMC proliferation and migration, we examined the effects of U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) on Ang II-induced RASMC proliferation and migration. As shown in The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by pre-treatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was significantly inhibited by pre-treatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) as well as exendin-4, ERK1/2 kinase and JNK inhibitors also inhibited both RASMC proliferation and migration induced by Ang II stimulation.Ang II is known to cause hypertension through type 1 receptor mediated vasoconstriction and stimulation of aldosterone secretion from the adrenal cortex, which are associated with vascular remodeling, inflammation and oxidative stresses . The maiPreviously, we reported that Ang II stimulated RASMC migration through the activation of intracellular ERK1/2 and JNK  29]. It. It29]. In conclusion, exendin-4 inhibited RASMC proliferation and migration induced by Ang II stimulation. ERK1/2 and JNK in RASMC were phosphorylated by Ang II; however, these activations were inhibited by pretreatment with exendin-4. Similar to exendin-4, pharmacological inhibition of ERK1/2 and JNK activation by Ang II suppressed Ang II-induced RASMC proliferation and migration. The mechanism for progression of atherosclerosis will need to be clarified by further study on the fate of VSMC influenced by circulatory factors including Ang II. We hope our present findings shed light on the mechanisms of the cardioprotective effect of GLP-1 receptor agonists beyond its blood glucose lowering effect.S1 Text(DOCX)Click here for additional data file.