MH_dev_67

DB00644 specific binding sites in uterine leiomyomata . DB00644 ( DB00644 ) analogs can cause regression of uterine leiomyomata . This effect is thought to be mediated by the inhibition of gonadotropin release and steroid synthesis . In the present study we examined the possibility that these analogs may also act directly on uterine leiomyomata . Specific binding sites for DB00644 are present in myoma membranes , as 125I - DB06719 SUB binding was displaced with equal efficiency by the superagonists , DB06719 SUB and D-Trp 6 - DB00644 , and by the antagonist Organon 30276 , but not by unrelated peptides such as thyrotropin releasing hormone and oxytocin . A nonlinear Scatchard curve obtained for DB06719 SUB specific binding suggests the presence of at least two binding sites , one of which exhibits a relatively high affinity for DB00644 analogs ( Kd of approximately 10 (-8 ) M ) . Western blotting with a specific P30968 antibody revealed the presence of a 60 kDa protein in myoma membranes . This protein has a similar molecular weight to the purified pituitary P30968 . These results indicate , for the first time , the presence of specific binding sites for DB00644 in uterine leiomyomata , suggesting a direct effect of DB00644 analogs on this tissue .

DB06287 MEN induces surfactant lipid accumulation and lung inflammation in mice . Interstitial lung disease ( ILD ) is a well-known adverse effect of mammalian target of rapamycin ( P42345 ) inhibitors . However , it remains unknown how lung toxicities are induced by P42345 inhibitors . Here , we constructed a mouse model of P42345 inhibitor-induced ILD using temsirolimus and examined the pathogenesis of the disease . Male ICR mice were treated with an intraperitoneal injection of different doses of temsirolimus ( 3 or 30 mg · kg ( - 1 ) · wk ( - 1 ) ) or vehicle . DB06287 MEN treatment increased capillary-alveolar permeability and induced neutrophil infiltration and fibrinous exudate into the alveolar space , indicating alveolar epithelial and / or endothelial injury . It also induced macrophage depletion and the accumulation of excessive surfactant phospholipids and cholesterols . Alveolar macrophage depletion is thought to cause surfactant lipid accumulation . To further examine whether temsirolimus has cytotoxic and / or cytostatic effects on alveolar macrophages and alveolar epithelial cells , we performed in vitro experiments . DB06287 MEN inhibited cell proliferation and viability in both alveolar macrophage and alveolar epithelial cells . DB06287 MEN treatment caused some signs of pulmonary inflammation , including upregulated expression of several proinflammatory cytokines in both bronchoalveolar lavage cells and lung homogenates , and an increase in lymphocytes in the bronchoalveolar lavage fluid . These findings indicate that temsirolimus has the potential to induce alveolar epithelial injury and to deplete alveolar macrophages followed by surfactant lipid accumulation , resulting in pulmonary inflammation . This is the first study to focus on the pathogenesis of P42345 inhibitor-induced ILD using an animal model .

Selective cleavage of ErbB 4 by G-protein-coupled gonadotropin-releasing hormone receptor in cultured hypothalamic neurons . DB00644 ( DB00644 ) is secreted from hypothalamic neurons ( DB00644 neurons ) . DB00644 neurons have a P30968 belonging to the G-protein-coupled receptors . The stimulation of this receptor activates extracellular signal-regulated kinase ( P29323 ) . In the present study , we found that epidermal growth factor receptor ( P00533 ) and ErbB 4 were expressed in immortalized DB00644 neurons ( GT1 - 7 cells ) . AG1478 , a relatively specific inhibitor of the ErbB family , and small interfering RNA ( siRNA ) for ErbB 4 inhibited the DB00644 - induced activation of P29323 in GT1 - 7 cells , suggesting that P00533 and ErbB 4 were necessary for the activation . In addition , DB00644 induced the cleavage of ErbB 4 and accumulation of an 80 - kDa fragment . After treatment of the cells with 50 nM DB00644 for 5 min , about 80 % of ErbB 4 was cleaved . Biotinylation of cell surface proteins revealed that more than 70 % of the cell surface ErbB 4 was cleaved by DB00644 treatment . A higher concentration and longer treatment were necessary for DB00644 to induce ErbB 4 cleavage than P29323 activation . TAPI - 2 , an inhibitor of tumor necrosis factor-α-converting enzyme ( P78536 ) , and siRNA for P78536 inhibited the cleavage of ErbB 4 , suggesting that P78536 was involved . After ErbB 4 cleavage , the activation of P29323 by neuregulin 1 was almost completely inhibited . These results suggest that the down-regulation of ErbB 4 expression is induced by G-protein-coupled receptor stimulation .

Wheat germ agglutinin behaves as a DB00644 antagonist but induces gonadotrope desensitization . Preincubation of cultured rat pituitary cells with 10 micrograms / ml of either wheat germ agglutinin ( WGA ) or concanavalin A inhibited LH release stimulated with DB00644 ( 0.5 nM ) by 55 % and 40 % , respectively . WGA-inhibition of LH release stimulated by DB00644 was dose-dependent , reaching a plateau of 75 % inhibition at 50 micrograms / ml . Concomitantly , WGA induced a dose-dependent inhibition of 125I - DB06719 SUB specific binding to pituitary cells , with a maximal inhibition of 45 % . The inhibition of 125I - DB06719 SUB binding by WGA is due to P30968 internalization and not to persistent occupancy of the receptors . In addition to the effect of WGA on receptor internalization , WGA also induced partial desensitization of pituitary cells but was ineffective in modulating DB00644 - induced desensitization . These findings indicate that WGA has all the characteristics of a DB00644 antagonist , nevertheless , it does induce desensitization of cultured rat pituitary cells to further stimulation with DB00644 .

Stimulatory effects of 5HT1A receptor agonists on luteinizing hormone-releasing hormone release from cultured fetal rat hypothalamic cells : interactions with progesterone . Previous works have suggested an interactive stimulatory effect of progesterone ( P ) and serotonin ( 5 - HT ) on luteinizing hormone release . The purpose of the present study was to determine whether 5 - HT via P08908 receptors interacts with P in the process of luteinizing hormone-releasing hormone ( P01148 ) release . Using fetal hypothalamic neurons in primary cell cultures the first goal of this study was to determine the effects of P08908 receptor agonists on P01148 secretion . 8 - Hydroxy - 2 ( di-n-propylamino ) tetralin ( 8 - OH-DPAT ) or ipsapirone ( 10 ( - 5 ) M ) significantly stimulated P01148 release . Pharmacological studies have allowed to rule out the possible involvement of alpha 2 - or beta-adrenoreceptors , or 5 - HT uptake sites , in the stimulatory effect of 8 - OH-DPAT on P01148 release , thus demonstrating the specific involvement of P08908 receptors in the stimulation of P01148 release . The second goal was to test the ability of P to stimulate P01148 release from fetal hypothalamic neurons . P ( 10 ( - 6 ) M ) applied for 30 or 120 min significantly stimulated P01148 secretion . The maintenance of the stimulation of P01148 release by P after a cycloheximide treatment or by an impermeable analog of P , P - 3 - BSA , has suggested a nongenomic effect of P on P01148 release . The effects of a pretreatment of cells by P on 8 - OH-DPAT-induced P01148 release were tested . While 10 ( - 7 ) M P alone did not stimulate P01148 release , this concentration of steroid potentiated the P01148 response to 10 ( - 5 ) M 8 - OH-DPAT . These findings led to the conclusion that P acting at the level of the plasma membrane potentiates the stimulatory effect of P08908 receptor agonists on P01148 release .

[ Serotonin transporter gene and stress reactivity in unipolar depression . Role of the Q9Y251 system as endophenotype of the P31645 gene ] . BACKGROUND : A length polymorphism in the promoter region of the serotonin transporter gene ( 5 - HTTLPR ) is associated with both depression and hypothalamic-pituitary-adrenal ( Q9Y251 ) system activity . A dysregulation of the Q9Y251 system is considered to be a candidate endophenotype of depression . The objective of the present study was an investigation of a possible gene-endophenotype-interaction between 5 - HTTLPR and Q9Y251 system activity in a sample of inpatients with major depression . MATERIALS AND METHODS : A total of 237 inpatients with major depression were genotyped for 5 - HTTLPR and participated in a combined dexamethasone-corticotropin-releasing hormone test ( DB00514 MEN - P06850 test ) as well as using the Hamilton score ( Hamilton rating scale for depression ) to determine the severity of the psychopathology . RESULTS : Patients with the ss-genotype showed a significantly higher Q9Y251 - system activity in comparison to patients with the lI-genotype , but no association between 5 - HTTLPR and the severity of psychopathology could be detected . CONCLUSIONS : The results of the current study demonstrate an influence of 5 - HTTLPR on dysregulation of the Q9Y251 system in patients with major depression and support the hypothesis that 5 - HTTLPR - and Q9Y251 - system-interaction constitutes an important component in the pathogenesis of depression .

Production and characterization of antibodies to gonadotropin-releasing hormone receptor . Antibodies to the gonadotropin-releasing hormone ( DB00644 ) receptor of bovine pituitary membranes have been raised in rabbits by immunization with affinity-purified receptor preparations . These antibodies did not compete with 125I - labeled DB00644 analog ( DB06719 SUB ) for binding to the receptors but did precipitate rat and bovine solubilized receptors labeled with 125I - DB06719 SUB . Binding of the antibodies to the receptors was also demonstrated by immunoprecipitation of 125I - labeled purified receptors and photoaffinity-labeled receptors . The antibodies did not have a DB00644 - like activity but rather inhibited , in a dose-dependent manner , DB00644 - stimulated luteinizing hormone release from cultured rat pituitary cells . In addition , the antibodies did not inhibit luteinizing hormone release stimulated by high K + concentration . This suggests that the antibodies recognize domains of the receptor other than the binding site of the hormone and thereby inhibit the biological response . These P30968 antibodies provide a useful tool for studying P30968 structure , function , localization , and biosynthesis .

Hormonal regulation of human trophoblast differentiation : a possible role for 17beta - estradiol and DB00644 . We have examined the role of 17beta - estradiol and gonadotropin releasing hormone ( DB00644 ) in the regulation of functional differentiation in human trophoblasts . In contrast to its recognized functions as a proliferation-promoting hormone in a variety of cell types , we found that 17beta - estradiol induced terminal differentiation in human trophoblastic cells , and that this event was estrogen-receptor-mediated . This process involved a loss in expression of Cyclins A2 and E , and a coincident increase in p27 ( Kip 1 ) . The anti-proliferative effects of 17beta - estradiol were annulled by specific transforming growth factor-beta 1 ( TGFbeta 1 ) - neutralizing antibody , suggesting that 17beta - estradiol may mediate its growth-inhibitory actions , through TGFbeta 1 activity . Following exposure to DB06719 SUB , cultured human trophoblastic cells stopped proliferating and formed functionally mature syncytiotrophoblasts . This differentiation event , that involved a drastic loss in expression of proliferating-cell-nuclear-antigen , could be blocked by DB00050 , suggesting the involvement of functional DB00644 receptors . Preliminary studies on the characterization of the human placental P30968 , indicate the presence of multiple receptor isoforms across human gestation .

Intracellular signaling pathways activated by kisspeptins through Q969F8 : do multiple signals underlie function diversity ? Kisspeptins , a family of peptide products derived from the KiSS - 1 gene , activate their cognate receptor Q969F8 in various target tissues to exert disparate functions , including inhibition of tumor metastasis and control of reproductive function . In contrast to the plethora of studies that have analyzed in recent years the regulatory functions of the KiSS - 1 / Q969F8 system , only a limited number of reports have been primarily focused on delineating the intracellular signaling pathways involved . Nevertheless , there is solid evidence indicating that kisspeptin can activate a wide variety of signals via Q969F8 . These include typical G-protein ( Galphaq / 11 ) - coupled cascades , such as activation of phospholipase C ( P98160 ) , and subsequent accumulation of inositol - ( 1,4 , 5 ) - triphosphate ( IP3 ) , intracellular Ca ( 2 + ) mobilization , and activation of protein kinase C . However , kisspeptin also activates pathways related to mitogen activated protein kinases ( MAPK ) , especially P27361 / 2 , and p38 and phosphatidylinositol - 3 - kinase ( PI3K ) / Akt . Additionally , the kisspeptin / Q969F8 pair can also influence cell signaling by interacting with other receptors , such as chemokine receptor P61073 , and P30968 . Kisspeptin can also affect other signaling events , like expression of matrix metalloproteinase 9 ( via NFkappaB ) , and that of calcineurin . The information gathered hitherto clearly indicates that activation of a specific set of interconnected signals is selectively triggered by kisspeptin via Q969F8 in a cell type-dependent manner to precisely regulate functions as distinct as hormone release and cell migration . In this scenario , it will be important to decipher kisspeptin / Q969F8 signaling mechanisms in reproductive and non-reproductive tissues by studying additional models , especially on natural kisspeptin targets expressing endogenous Q969F8 .

Rotenone remarkably attenuates oxidative stress , inflammation , and fibrosis in chronic obstructive uropathy . Mitochondrial abnormality has been shown in many kidney disease models . However , its role in the pathogenesis of chronic kidney diseases ( CKDs ) is still uncertain . In present study , a mitochondrial complex I inhibitor rotenone was applied to the mice subjected to unilateral ureteral obstruction ( UUO ) . Following 7 - days rotenone treatment , a remarkable attenuation of tubular injury was detected by DB00233 staining . In line with the improvement of kidney morphology , rotenone remarkably blunted fibrotic response as shown by downregulation of fibronectin ( FN ) , plasminogen activator inhibitor - 1 ( P05121 ) , collagen I , collagen III , and α-SMA , paralleled with a substantial decrease of TGF-β 1 . Meanwhile , the oxidative stress markers thiobarbituric acid-reactive substances ( TBARS ) and heme oxygenase 1 ( P09601 ) and inflammatory markers P01375 - α , IL - 1β , and P05362 were markedly decreased . More importantly , the reduction of mitochondrial DNA copy number and mitochondrial P03886 ( mtND 1 ) expression in obstructed kidneys was moderately but significantly restored by rotenone , suggesting an amelioration of mitochondrial injury . Collectively , mitochondrial complex I inhibitor rotenone protected kidneys against obstructive injury possibly via inhibition of mitochondrial oxidative stress , inflammation , and fibrosis , suggesting an important role of mitochondrial dysfunction in the pathogenesis of obstructive kidney disease .

Bresol inhibits phosphodiesterase 4 gene expression and modulates the levels of select mediators of inflammation in human monocytic cells . Bresol-a poly-herbal formulation , has been reported to be effective against bronchial asthma and allergic rhinitis in children . In vivo studies have supported the anti-histaminic and anti-anaphylactic action of bresol . However , the mechanism of action of bresol in modulation of inflammation has not been studied at the cellular and molecular level . The present study was aimed to elucidate the mechanism ( s ) of action of bresol at the cellular and molecular levels , using human monocyte leukemia cells . The effects of bresol on phosphodiesterase 4B ( Q07343 ) gene expression were analyzed using human monocytic U937 leukemia cells . The ability of bresol to stimulate DB02527 formation in these cells , as well as its effects on mediators of inflammation like tumor necrosis factor-α ( TNFα ) , nitric oxide ( NO ) , and cycloxygenase - 2 ( P35354 ) in lipopolysaccharide ( LPS ) - stimulated U937 cells , were also studied . The results here indicated that bresol exhibited potential anti-inflammatory properties by inhibiting LPS-induced Q07343 gene expression in the cells . Bresol also dose dependently activated DB02527 formation , and inhibited TNFα , NO , as well as P35354 formation in the LPS-stimulated cells . Based upon the results , we concluded that the reported anti-inflammatory activity of bresol might be attributed to its abilities to inhibit Q07343 and thus elevate DB02527 levels in human monocytes . The anti-inflammatory effects of bresol might also be a result of the capacity of bresol to modulate the formation of TNFα , NO , and P35354 in monocytes .

The P08908 - receptor agonist flibanserin reduces drug-induced dyskinesia in O75916 - deficient mice . Drug-induced dyskinesia is a major complication of dopamine replacement therapy in advanced Parkinson ' s disease consisting of dystonia , chorea and athetosis . Agonists at P08908 - receptors attenuate levodopa-induced motor complications in non-human primates . Mice with increased dopamine D2 receptor ( P14416 ) signalling due to the lack of expression of the regulator of G-protein signalling 9 ( O75916 ) also develop dyskinesia following levodopa treatment . We investigated whether the P08908 - receptor agonist flibanserin compared with buspirone reduces motor abnormalities induced by levodopa or quinelorane , a selective dopamine D2 - receptor agonist . Following dopamine depletion via reserpine , 40 mice ( 20 wild-type and 20 O75916 knock-out ) were treated with flibanserin or buspirone in combination with levodopa or quinelorane . Motor behaviour was analysed using open field analysis . O75916 knock-out mice displayed significantly more drug-induced dystonia ( p < 0.04 ; t test ) than wild type . In quinelorane-treated wild-type mice flibanserin as well as buspirone significantly reduced dystonia ( p < 0.05 ) . In O75916 knock-out animals again both reduced quinelorane-induced dystonia . However , flibanserin was significantly more effective ( p = 0.003 ) . Following reserpine pretreatment and administration of levodopa wild-type and Q99697 9 knock-out mice showed mild to moderate dystonia . Surprisingly , 10 mg / kg buspirone increased dystonia in both animal groups , whereas it was decreased by 10 mg / kg flibanserin . However , compared with levodopa alone only the increase of dystonia by buspirone was significant ( p < 0.04 ) . DB04908 MEN showed promising antidyskinetic effects in a model of drug-induced dyskinesia . Our data underline the possible benefit of P08908 agonists in drug-induced dyskinesia .

Biphasic action of cyclic adenosine 3 ' , 5 ' - monophosphate in gonadotropin-releasing hormone ( DB00644 ) analog-stimulated hormone release from GH3 cells stably transfected with P30968 complementary deoxyribonucleic acid . GH3 cells are a PRL-secreting adenoma cell line derived from pituitary lactotropes . These cells have been stably transfected with rat P30968 complementary DNA to produce four cell lines : Q92820 ( 3 ) 1 ' , Q92820 ( 3 ) 2 ' , Q92820 ( 3 ) 6 ' , and Q92820 ( 3 ) 12 ' . In response to either DB00644 or DB06719 SUB ( a metabolically stable DB00644 agonist ) , these cell lines synthesize PRL in a DB02527 - dependent manner . Only Q92820 ( 3 ) 6 ' cells desensitize in response to persistent treatment with 10 ( - 7 ) g / ml DB06719 SUB . Q92820 ( 3 ) 1 ' , Q92820 ( 3 ) 2 ' , and Q92820 ( 3 ) 12 ' cells , however , can be made refractory to DB06719 SUB stimulation by raising DB02527 levels either by the addition of ( Bu ) 2cAMP to the medium or by treatment with cholera toxin . In Q92820 ( 3 ) cells , low levels of DB02527 fulfill the requirements for a second messenger , whereas higher levels appear to mediate the development of desensitization . The observation that in Q92820 ( 3 ) 6 ' cells , DB02527 production persists after the onset of desensitization is consistent with the view that the mechanism responsible for desensitization is distal to the production of DB02527 . Moreover , the absence of any significant difference in the amount of DB02527 produced per cell in Q92820 ( 3 ) 2 ' , Q92820 ( 3 ) 6 ' , or Q92820 ( 3 ) 12 ' cells suggests that elevated DB02527 production per cell does not explain the development of desensitization in Q92820 ( 3 ) 6 ' cells . We suggest that DB06719 SUB - stimulated PRL synthesis in Q92820 ( 3 ) 6 ' cells is mediated by a different DB02527 - dependent protein kinase pool ( s ) than that in nondesensitizing Q92820 ( 3 ) cells . Such a protein kinase A pool ( s ) may be more susceptible to degradation via DB02527 - mediated mechanisms than the protein kinase pools mediating the DB06719 SUB response in nondesensitizing Q92820 ( 3 ) cells . A similar mechanism has been reported in other systems .

The mitochondrial pharmacogenomics of haplogroup T : P03891 * LHON 4917G and antiretroviral therapy-associated peripheral neuropathy . Peripheral neuropathy ( PN ) due to mitochondrial injury complicates HIV therapy with some nucleoside reverse transcriptase inhibitors ( NRTIs ) . Variation in the mitochondrial genome may influence susceptibility to NRTI toxicities . Two non-synonymous mitochondrial DNA polymorphisms , P03886 * LHON 4216C ( 4216C ) and P03891 * LHON 4917G ( 4917G ) were characterized in HIV-infected participants exposed to NRTIs in a randomized clinical trial . Among 250 self-identified white , non-Hispanic participants , symptomatic PN ( > or = grade 1 ) developed in 70 ( 28 % ) . Both 4216C ( odds ratio ( OR )= 1.98 ( 95 % confidence interval ( CI ) 1.05- 3.75 ) ; P= 0.04 ) and 4917G ( OR = 2.93 ( 95 % CI 1.25- 6.89 ) ; P= 0.01 ) were more frequent in PN cases . These two polymorphisms remained independently associated with PN after adjusting for age , baseline P01730 count , plasma HIV RNA level , and NRTI randomization arm ; 4216C ( OR = 2.0 ( 95 % CI 1.1- 4.0 ) P= 0.04 ) and 4917G ( OR = 5.5 ( 95 % CI 1.6- 18.7 ) P < 0.01 ) . When 4917G individuals were excluded from the analysis , the association with 4216C was no longer seen . The mitochondrial 4917G polymorphism may increase susceptibility to NRTI-associated PN .

Expression of type I P01148 receptor and in vivo and in vitro P01148 - I effects in corpora lutea of pseudopregnant rabbits . The expression of type I P01148 receptor ( P30968 - I ) and the direct role of P01148 - I on corpora lutea ( CL ) function were studied in the pseudopregnant rabbit model . Immunohistochemistry evidenced P30968 - I and P01148 - I in luteal cells at early ( day 4 pseudopregnancy ) - , mid ( day 9 ) - , and late ( day 13 ) - luteal stages . Real-time RT-PCR and western blotting revealed P30968 - I mRNA and protein at the three luteal stages . DB06719 SUB in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24 h respectively . In in vitro cultured CL , buserelin reduced progesterone secretion , increased prostaglandin F ( 2α ) ( P49763 ( 2α ) ) secretion and cyclo-oxygenase - 2 ( P35354 ) and nitric oxide synthase ( NOS ) activities at days 9 and 13 , and decreased PGE₂ at day 13 . Co-incubation with antagonists for P01148 - I ( antide ) , inositol 1,4 , 5 - trisphosphate ( IP₃ , 2 - amino-ethoxydiphenylborate ) , and diacylglycerol ( DAG , 1 - hexadecyl - 2 - acetyl glycerol ) or inhibitors for phospholipase C ( P98160 , compound 48/80 ) , and protein kinase C ( PKC , staurosporine ) counteracted the buserelin effects . DB06719 SUB co-incubated with P36551 inhibitor ( acetylsalicylic acid ) increased progesterone and decreased P49763 ( 2α ) and NOS activity at days 9 and 13 , whereas co-incubation with NOS inhibitor ( DB04223 methyl ester ) increased progesterone at the same luteal stages . These results suggest that P30968 - I is constitutively expressed in rabbit CL independently of luteal stage , whereas P01148 - I down-regulates directly CL progesterone production via P49763 ( 2α ) at mid - and late-luteal stages of pseudopregnancy , utilizing its cognate type I receptor with a post-receptorial mechanism that involves P98160 , IP₃ , DAG , PKC , P35354 , and NOS .

Dual P00533 and P42345 targeting in squamous cell carcinoma models , and development of early markers of efficacy . The epidermal growth factor receptor ( P00533 ) is a validated target in squamous cell carcinoma ( SCC ) of the head and neck . Most patients , however , do not respond or develop resistance to this agent . P42345 ( P42345 ) is involved in the pathogenesis of SCC of the head and neck ( SCCHN ) . This study aimed to determine if targeting P42345 in combination with P00533 is effective in SCC , and to develop early pharmacodynamic markers of efficacy . Two SCC cell lines , one resistant ( HEP 2 ) and one of intermediate susceptibility ( Detroit 562 ) to P00533 inhibitors , were xenografted in vivo and treated with an P42345 inhibitor ( temsirolimus ) , an P00533 inhibitor ( erlotinib ) or a combination of both . DB06287 MEN exerted superior growth arrest in both cell lines than erlotinib . The combined treatment resulted in synergistic antitumor effects in the Detroit 562 cell line . Immunohistochemical assessment of pharmacodynamic effects in fine-needle aspiration ( FNA ) biopsies early after treatment using phospho MAPK , Phospho-P 70 and Ki67 as end points demonstrated pathway abrogation in the Detroit 562 tumours treated with the combination , the only group where regressions were seen . In conclusion , an P42345 inhibitor showed antitumor activity in P00533 - resistant SCC cell lines . Marked antitumor effects were associated with dual pathway inhibition , which were detected by early FNA biopsies .

Nongenomic , glucocorticoid receptor-mediated regulation of serotonin transporter cell surface expression in embryonic stem cell derived serotonergic neurons . Depressive disorders have been linked to the combined dysregulation of the hypothalamus-pituitary-adrenal ( Q9Y251 ) - axis and the serotonergic system . The Q9Y251 - axis and serotonergic ( 5 - HT ) neurons exert reciprocal regulatory actions . It has been reported that glucocorticoid-glucocorticoid receptor ( GR ) signaling influences serotonin transporter ( 5 - HTT ) transcription but data also points to the fact that 5 - HTT expression is regulated nongenomically via redistribution of 5 - HTT from the cell surface into intracellular compartments . In order to analyze the acute effects of glucocorticoids on 5 - HTT cell surface localization we differentiated serotonergic neurons from mouse embryonic stem ( ES ) cells derived from the C57BL / 6N blastocysts . These postmitotic 5 - HT neurons express all relevant serotonergic markers following the application of a growth factor-based differentiation protocol . Increasing concentrations of the GR agonist dexamethasone ( DB00514 MEN ) resulted in enhanced , dose-dependent 5 - HTT cell surface localization in the presence of the protein synthesis inhibitor cycloheximide already 1h after incubation . Inhibition of GR function by the specific GR-antagonist mifepristone abolished the increase in 5 - HTT cell surface localization . Hence , our data account for a nongenomic upregulation of 5 - HTT cell surface expression by glucocorticoid-GR interaction which likely constitutes a rapid physiological response to increased levels of glucocorticoids as seen during stress . Taken together , we provide a cellular model to analyze and dissect glucocorticoid - P31645 interactions on a molecular level that corresponds to in vivo animal models using C57BL / 6N mice .

Salmon DB00644 and its analogues bind the human placental receptor . OBJECTIVE : The presence of DB00644 receptors in the human placenta has been recognized for a number of years . However , mammalian DB00644 , which is expressed in placental tissues , has limited affinity for the chorionic receptor . On the basis of immunological and bioactivity data , we have previously proposed that the chorionic DB00644 may differ from mammalian DB00644 . METHODS : We have studied the affinity of another isoform of DB00644 ( ie , salmon DB00644 and stable analogues of this DB00644 isoform ) , and compared their receptor affinity to that of mammalian DB00644 and its analogues . RESULTS : Using our receptor assay method with the labeled mammalian DB00644 analogue DB06719 SUB , salmon DB00644 had a twofold greater affinity for the placental P30968 than did mammalian DB00644 and for the stable salmon DB00644 analogue the affinity was increased tenfold . Using a homologous receptor assay method with a stable salmon DB00644 analogue as label , the affinity for this salmon DB00644 analogue had a K ( d ) of 101 nmol / L . CONCLUSION : The presence of these higher affinity receptors for non-mammalian DB00644 in the human placenta has led us to propose that the chorionic tissues may express more than one isoform of DB00644 and that non-mammalian DB00644 , such as salmon DB00644 , may be potent regulators of placental functions .

In vitro effects of gonadotropin-releasing hormone ( DB00644 ) analogue on cancer cell sensitivity to cis-platinum . Six endometrial cancer cell lines ( Ishikawa , EIIL , HEC 1A , 6 , 50 and 59 ) , one breast cancer cell line ( MCF - 7 ) and two ovarian cancer cell lines ( OVHS - 1 , HRA ) were treated for 24 or 168 h with a gonadotropin-releasing hormone ( DB00644 ) analogue , DB06719 SUB acetate , and the cellular growth profile was studied . All these cell lines except for the HRA line had positive P30968 mRNA expression detected by reverse transcriptase polymerase chain reaction . GnRHa suppressed cell growth after 168 h of exposure , but not after 24 h . Suppression of cell growth by the exposure to cis-platinum ( DB00515 , 10 nM for 24 h ) was significantly increased in the presence of GnRHa for 168 h . The mechanism of this growth inhibition was tested by examining both RNA components of human telomerase ( hTR ) expression and telomerase activity . The results showed that GnRHa inhibits telomerase activity without altering the RNA component of telomerase expression . The present data suggest that DB00644 analogue may modulate endometrial , breast and ovarian cancer cell growth through modifying the telomerase activity . Since GnRHa increased the cytotoxic effects of DB00515 and GnRHa is a compound of high patient compliance , the value of GnRHa as a tumor sensitizer to DB00515 should be further tested in clinical trials .

The basal subcellular distribution of beta-adrenergic receptor kinase is independent of G-protein beta gamma subunits . beta-Adrenergic receptor kinase ( beta O14965 or P25098 ) is a key regulatory protein involved in the regulation of G-protein-coupled receptors which associates with microsomal and plasma membranes . beta gamma Subunits of G-proteins have been suggested to mediate agonist-dependent membrane translocation of beta ARK , but their possible role in maintaining the complex subcellular distribution of the kinase is not known . In this study we show that lovastatin-mediated inhibition of G gamma subunits isoprenylation in P29320 - 293 cells stably transfected with beta O14965 leads to a significant release of G beta subunits to the cytosol without causing changes in total particulate beta ARK or in the association of this kinase to plasma or microsomal membrane fractions . In addition , transient overexpression of mutant forms of G gamma unable to become isoprenylated resulted in a marked sequestration of G beta to the soluble compartment , but caused no rearrangement in the distribution of cotransfected beta ARK . These results indicate that anchoring of beta ARK to cellular membranes under basal conditions is independent of the availability of heterotrimeric G-protein subunits .

The role of MAPK and Nrf 2 pathways in ketanserin-elicited attenuation of cigarette smoke-induced P10145 production in human bronchial epithelial cells . Cigarette smoking is a major risk factor in chronic obstructive pulmonary disease ( P48444 ) with chronic airway inflammation as a key feature . Blockade of serotonin receptor 2A ( 5 - HTR ( 2A ) ) with ketanserin has been found to improve lung function in P48444 patients . Furthermore , ketanserin has been shown to possess anti-inflammatory properties in vivo . In this study , we investigated the antioxidative and anti-inflammatory properties of ketanserin and its underlying mechanism of action on cigarette smoke-induced interleukin ( IL ) - 8 release in vitro . Primary normal human bronchial epithelial cells and human bronchial epithelial cell line ( BEAS - 2B ) were treated with or without ketanserin prior to exposure to cigarette smoke medium ( CSM ) . Exposure to CSM caused elevation of both mRNA and release of P10145 with increased phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 ( P27361 / 2 ) . Consistently , CSM-induced P10145 release was blocked by SB203580 , U0126 , or Q02750 small interfering RNA ( siRNA ) but not SP600125 . On the other hand , CSM caused a dose-dependent decrease in the ratio of reduced glutathione to oxidized glutathione ( rGSH / GSSG ) together with an increased translocation of Nrf 2 to the nucleus demonstrated by Western blot analysis . Knock down of Nrf 2 by siRNA completely blocked CSM-induced P10145 release . Ketanserin suppressed CSM-induced P10145 release by inhibiting p38 , P27361 / 2 MAPK , and Nrf 2 signaling pathways and partially inhibited CSM-induced reduction of rGSH / GSSG ratio . Our data demonstrated the novel antioxidative and anti-inflammatory role of ketanserin via the Nrf 2 signaling pathway in CSM-exposed human bronchial epithelial cells . This may open up new perspectives in the development of novel therapeutic targets in the treatment of cigarette smoke-related P48444 .

Induction of the Galpha ( q ) signaling cascade by the human immunodeficiency virus envelope is required for virus entry . Binding of human immunodeficiency virus type 1 ( HIV - 1 ) envelope glycoprotein ( Env ) with the primary receptor P01730 and one of two coreceptors , P61073 or P51681 , activates a signaling cascade resulting in Rac - 1 GTPase activation and stimulation of actin cytoskeletal reorganizations critical for HIV - 1 - mediated membrane fusion . The mechanism by which HIV - 1 Env induces Rac - 1 activation and subsequent actin cytoskeleton rearrangement is unknown . In this study , we show that Env-mediated Rac - 1 activation is dependent on the activation of Galpha ( q ) and its downstream targets . Fusion and Rac - 1 activation are mediated by Galpha ( q ) and phospholipase C ( P98160 ) , as shown by attenuation of fusion and Rac - 1 activation in cells either expressing small interfering RNA ( siRNA ) targeting Galpha ( q ) or treated with the P98160 inhibitor U73122 . Rac - 1 activation and fusion were also blocked by multiple protein kinase C inhibitors , by inhibitors of intracellular Ca2 + release , by Pyk 2 - targeted siRNA , and by the Ras inhibitor S-trans , trans-farnesylthiosalicylic acid ( Q9H8T0 ) . Fusion was blocked without altering cell viability or cell surface localization of P01730 and P51681 . Similar results were obtained when cell fusion was induced by Env expressed on viral and cellular membranes and when cell lines or primary cells were the target . Treatment with inhibitors and siRNA specific for Galpha ( i ) or Galpha ( s ) signaling mediators had no effect on Env-mediated Rac - 1 activation or cell fusion , indicating that the Galpha ( q ) pathway alone is responsible . These results could provide a new focus for therapeutic intervention with drugs targeting host signaling mediators rather than viral molecules , a strategy which is less likely to result in resistance .

DB08827 MEN : A novel agent for the treatment of homozygous familial hypercholesterolemia . PURPOSE : The pharmacology , pharmacokinetics , and clinical efficacy and safety of lomitapide in the management of homozygous familial hypercholesterolemia ( HoFH ) are reviewed . SUMMARY : DB08827 MEN ( Juxtapid , Aegerion Pharmaceuticals ) is an oral microsomal triglyceride transfer protein ( P55157 ) inhibitor indicated for the treatment of patients with HoFH , a rare form of hypercholesterolemia that can lead to premature atherosclerotic disease . In clinical trials , the use of lomitapide alone or in combination with other lipid-lowering modalities reduced plasma concentrations of low-density lipoprotein cholesterol ( LDL-C ) by a mean of more than 50 % . DB08827 MEN is associated with significant gastrointestinal adverse effects and increases in hepatic fat levels . DB08827 MEN undergoes hepatic metabolism via cytochrome P - 450 ( CYP ) isoenzyme 3A4 and interacts with P08684 substrates including atorvastatin and simvastatin ; dose adjustment is recommended when lomitapide is used concurrently with these agents . In patients receiving concomitant warfarin , the International Normalized Ratio ( INR ) should be closely monitored , as lomitapide use may increase INR values . The recommended initial dosage of lomitapide is 5 mg once daily , with subsequent upward dose adjustment at specified intervals according to tolerability . DB08827 MEN is contraindicated in patients with moderate-to-severe liver disease , patients with sustained abnormal liver function tests , patients taking strong or moderate P08684 inhibitors , and pregnant patients . CONCLUSION : DB08827 MEN is an oral P55157 inhibitor approved for the treatment of HoFH . This agent appears to be a realistic option for patients with HoFH who are unable to attain their LDL-C goal or can not tolerate statin therapy .

Ex vivo binding of flibanserin to serotonin P08908 and 5 - Q13049 receptors . DB04908 MEN has been reported to be an agonist at P08908 - receptors and an antagonist at 5 - Q13049 receptors , with higher affinity for P08908 receptors . Despite the fact that less receptor occupation is required by full agonists than by antagonists to exert their effects , flibanserin was shown to exert 5 - Q13049 antagonism at doses ( 4-5 mg kg - 1 ) that are lower or equal to those required to stimulate P08908 receptors . In order to understand this phenomenon , the interaction of flibanserin with P08908 and 5 - Q13049 receptors was evaluated in ex vivo binding studies . This interaction was evaluated in the prefrontal cortex , hippocampus and midbrain by using [ 3H ]8 - OH-DPAT and [ 3H ] ketanserin to label P08908 and 5 - Q13049 receptors , respectively . DB04908 MEN was given at 1 , 10 and 30 mg kg - 1 intraperitoneally . The dose of 1 mg kg - 1 displaced both radioligands preferentially in the frontal cortex . The doses of 10 and 30 mg kg - 1 reduced the binding of both radioligands in all the three brain regions non-selectively by about 50 % and 70 % , respectively . The displacement was maximal after 0.5 h and was reduced or not evident after 3 h . We conclude that 5 - HT2 antagonism brought about by low doses of flibanserin may reflect functional mechanisms more than receptor-mediated effects .

Loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia . Mutations in microsomal triglyceride transfer protein ( P55157 ) cause abetalipoproteinemia ( P00519 ) , characterized by the absence of plasma apoB-containing lipoproteins . In this study , we characterized the effects of various P55157 missense mutations found in P00519 patients with respect to their expression , subcellular location , and interaction with protein disulfide isomerase ( P07237 ) . In addition , we characterized functional properties by analyzing phospholipid and triglyceride transfer activities and studied their ability to support apoB secretion . All the mutants colocalized with calnexin and interacted with P07237 . We found that R540H and N780Y , known to be deficient in triglyceride transfer activity , also lacked phospholipid transfer activity . Novel mutants S590I and G746E did not transfer triglycerides and phospholipids and did not assist in apoB secretion . In contrast , D384A displayed both triglyceride and phospholipid transfer activities and supported apoB secretion . These studies point out that P00519 is associated with the absence of both triglyceride and phospholipid transfer activities in P55157 .

Anandamide regulates the expression of GnRH 1 , GnRH 2 , and DB00644 - Rs in frog testis . DB00644 ( either GnRH 1 or GnRH 2 ) exerts a local activity in vertebrate testis , including human testis . Relationships between endocannabinoid ( eCB ) and DB00644 systems in gonads have never been elucidated in any species so far . To reveal a cross-talk between eCBs and DB00644 at testicular level , we characterized the expression of DB00644 ( GnRH 1 and GnRH 2 ) as well as P30968 ( DB00644 - Q96GN5 , - R2 , and - R3 ) mRNA in the testis of the anuran amphibian Rana esculenta during the annual sexual cycle ; furthermore , the corresponding transcripts were localized inside the testis by in situ hybridization . The possible endogenous production of the eCB , anandamide ( AEA ) , was investigated in testis by analyzing the expression of its biosynthetic enzyme , Nape-pld . Incubations of testis pieces with AEA were carried out in the postreproductive period ( June ) and in February , when a new spermatogenetic wave takes place . In June , AEA treatment significantly decreased GnRH 1 and DB00644 - R2 mRNA , stimulated the transcription of GnRH 2 and DB00644 - Q96GN5 , and did not affect DB00644 - R3 expression . In February , AEA treatment upregulated GnRH 2 and DB00644 - R3 mRNA , downregulated DB00644 - R2 , and did not affect GnRH 1 and DB00644 - Q96GN5 expression . These effects were mediated by type 1 cannabinoid receptor ( P21554 ) since they were fully counteracted by SR141716A ( DB06155 ) , a selective P21554 antagonist . In conclusion , eCB system modulates DB00644 activity in frog testis during the annual sexual cycle in a stage-dependent fashion .

Elevated retinol binding protein 4 induces apolipoprotein B production and associates with hypertriglyceridemia . CONTEXT AND OBJECTIVE : A high level of retinol binding protein 4 ( P02753 ) is reported to be associated with insulin resistance in humans . However , evidence from large-scale populations about the relationship between serum P02753 and metabolic phenotypes is scarce . In the present study , we aimed to evaluate serum P02753 distribution and its association with metabolic phenotypes among middle-aged and elderly Chinese . DESIGN AND PARTICIPANTS : Serum concentrations of P02753 in a cross-sectional sample of 2780 Chinese population aged 50-70 years old in Guangzhou were measured by ELISA . RESULTS : The mean of serum P02753 concentration was 28.04 μg / mL for male and 37.76 μg / mL for female ( P < . 01 ) , respectively . Circulating P02753 was positively correlated with serum triglyceride and apolipoprotein B ( apoB ) concentrations . The odds ratio ( OR ) was substantially higher for hypertriglyceridemia ( OR , 3.26 ; 95 % confidence interval , 2.36- 4.51 ) in the highest P02753 quartile compared with those in the lowest quartile after multiple adjustment for confounders . Furthermore , serum P02753 was significantly associated with fasting glucose , insulin levels , and homeostasis model assessment index-insulin resistance ( HOMA-IR ) . Moreover , we showed that P02753 enhanced microsomal triglyceride transfer protein ( P55157 ) expression and activity via up-regulation of protein disulfide isomerase ( P07237 ) , suppressed low-density lipoprotein receptor ( P01130 ) expression , and impaired insulin-signaling pathway , leading to inductions in apoB secretion both in vitro and in vivo . CONCLUSIONS : Elevated circulating P02753 concentrations were associated with higher risk of hypertriglyceridemia by inducing the secretion of triglyceride-rich apoB-containing lipoproteins .

Cyclic adenosine 3 ' , 5 ' - monophosphate ( DB02527 ) and DB02527 responsive element-binding protein are involved in the transcriptional regulation of gonadotropin-releasing hormone ( DB00644 ) receptor by DB00644 and mitogen-activated protein kinase signal transduction pathway in Q92820 ( 3 ) cells . Stimulation of mouse P30968 promoter by a DB00644 agonist ( DB06719 SUB ) , or by a DB02527 analogue , significantly increased reporter ( luciferase ) activity . Overexpression of P04049 , P27361 , or P28482 partially blocked DB06719 SUB - stimulated luciferase activity . In contrast , treatment with a mitogen-activated protein kinase ( MAPK ) kinase inhibitor ( PD 98059 ) activated basal and DB06719 SUB - stimulated luciferase activity in a dose-dependent manner . Transient transfection of the deleted DB02527 response element expression vector followed by pretreatment with PD98059 prior to DB06719 SUB stimulation showed that the transcriptional response was decreased compared to wild-type promoter . A gel-mobility shift assay using a probe containing the DB02527 response element showed the presence of two specific protein-DNA complexes that contain one or more members of the DB02527 responsive element-binding ( CREB ) protein family . These results suggest that DB02527 and CREB participate in the DB00644 activation of P30968 promoter activity and that the MAPK cascade is involved in the negative regulation of basal and DB00644 - stimulated P30968 transcriptional activity .

Transient transfection of GGH 3-1 ' cells [ GH3 cells stably transfected with the gonadotropin-releasing hormone ( DB00644 ) receptor complementary deoxyribonucleic acid ] with the carboxyl-terminal of beta-adrenergic receptor kinase 1 blocks prolactin release : evidence for a role of the G protein beta gamma-subunit complex in DB00644 signal transduction . G proteins consist of heterotrimeric alpha - , beta - , and gamma-subunits . To assess the role of the beta gamma-subunit complex in P30968 - mediated signal transduction , GGH 3-1 ' cells were transfected with plasmids PRK 5 - beta O14965 ( 495-689 ) containing complementary DNA ( cDNA ) of the carboxyl-terminal ( Gly 495 - Leu 689 ) of beta-adrenergic receptor kinase 1 ( beta O14965 ) . GGH 3-1 ' cells are GH3 cells that have been stably transfected with rat P30968 cDNA . The carboxyl region of beta O14965 ( Gly 495 - Leu 689 ) binds to the beta gamma complex and thereby inhibits its action . Twenty-four hours after stimulation , PRL release , DB02527 release , and inositol phosphate ( IP ) production were measured in these cells and in control cells transfected with vector PRK 5 cDNA alone . In cells expressing the beta O14965 - ( 495-689 ) sequence there was inhibition of basal PRL release ( 69.3 % ) , DB02527 release ( 61.2 % ) , and IP production ( 75.5 % ) compared to cells containing vector only . When cells expressing the beta O14965 fragment were stimulated with a DB00644 analog ( DB06719 SUB ; 10 ( - 7 ) M ) , maximal responses were inhibited ( 66.1 % for PRL release , 52.3 % for DB02527 release , and 79.1 % for IP production ) . Scatchard analysis of DB00644 analog binding was also performed in the two groups of transfected cells . No significant differences in Kd or receptor numbers were found between beta O14965 - ( 495-689 ) - transfected cells and control cells containing the vector alone . These data suggest a role for the beta gamma complex in mediation of DB02527 release , IP production , and hormone release in response to agonist occupancy of the P30968 .

Prostate cancer cell proliferation is strongly reduced by the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in vitro on human cell lines and primary cultures . PURPOSE : To investigate the effects of the epidermal growth factor receptor tyrosine kinase inhibitor ( P00533 - TKI ) ZD1839 ( ' DB00317 ' ) on the cellular proliferation of androgen-sensitive and androgen-independent human prostatic cancer cell lines and primary cultures in vitro . EXPERIMENTAL DESIGN : In this study , we investigated the effects of the quinazoline ZD1839 , a potent , selective P00533 - TKI , on the P00533 autophosphorylation and cellular proliferation of androgen-sensitive ( P03886 , LNCaP , and ALVA - 31 ) and androgen-independent ( PC3 , DU145 , and TSU-Pr 1 ) human prostatic cancer cell lines and 20 primary cultures derived from human prostatic cancer tissue . RESULTS : P00533 was present and phosphorylated in all cell lines tested . ZD1839 reduced P00533 autophosphorylation in intact cell lines with IC ( 50 ) s of 0.46- 0.97 microM , and inhibited cellular proliferation with IC ( 50 ) s of 0.37- 1.03 microM . Constitutive P00533 autophosphorylation was low in primary cell cultures , but addition of P01133 ( 50 ng / ml ) caused marked P00533 autophosphorylation ; cellular proliferation in the presence of P01133 was inhibited by ZD1839 with a mean IC ( 50 ) of 0.45 microM . At doses > 1 microM , ZD1839 induced apoptosis in both androgen-dependent and androgen-independent PCa cell lines . CONCLUSION . Our experiments suggest that P00533 - TKIs such as ZD1839 may have potential in blocking the growth and progression of human prostatic cancers even in early phases of the disease .

Antiinflammatory steroid action in human ovarian surface epithelial cells . The human ovarian surface epithelium ( OSE ) is subject to serial injury and repair during ovulation , which is a natural inflammatory event . We asked whether there is a compensatory antiinflammatory component to this process , involving steroid hormones produced locally at the time of ovulation . Quantitative RT-PCR analysis of total RNA from cultured human OSE cell monolayers showed that exposure to proinflammatory IL1alpha ( 500 pg / ml ) increased mRNA levels of cyclooxygenase - 2 ( P35354 ) ( P < 0.01 ) at 48 h . The P35354 mRNA response to IL1alpha was associated with an approximate 18 - fold ( P < 0.01 ) increase in mRNA levels of 11beta - hydroxysteroid dehydrogenase type 1 ( 11betaHSD1 ) , encoding the steroid dehydrogenase that reversibly reduces cortisone to antiinflammatory cortisol . Addition of cortisol to OSE cell culture medium dose-dependently suppressed the P35354 mRNA response to IL1alpha ( P < 0.01 ) but reciprocally enhanced the 11betaHSD1 mRNA response ( P < 0.05 ) , with both effects strongest at 1 microm cortisol . Presence of glucocorticoid receptor-alpha mRNA and protein was established in OSE cell monolayers and treatment with IL1alpha shown to significantly up-regulate the glucocorticoid receptor-alpha mRNA level ( P < 0.05 ) . P04150 antagonist ( DB00834 MEN , 10 microm ) fully reversed the inhibitory effect of 1 microm cortisol on IL1alpha - stimulated P35354 mRNA expression . Progesterone also suppressed IL1alpha - induced P35354 mRNA expression but had no significant effect on IL1alpha - stimulated 11betaHSD1 expression . These data provide direct evidence for antiinflammatory actions of cortisol and progesterone in human OSE cells .

Novel piperazine-based compounds inhibit microtubule dynamics and sensitize colon cancer cells to tumor necrosis factor-induced apoptosis . We recently identified a series of mitotically acting piperazine-based compounds that potently increase the sensitivity of colon cancer cells to apoptotic ligands . Here we describe a structure-activity relationship study on this compound class and identify a highly active derivative ( ( 4 - ( 3 - chlorophenyl ) piperazin - 1 - yl ) ( 2 - ethoxyphenyl ) methanone ) , referred to as AK301 , the activity of which is governed by the positioning of functional groups on the phenyl and benzoyl rings . AK301 induced mitotic arrest in HT29 human colon cancer cells with an ED50 of ≈ 115 nm . Although AK301 inhibited growth of normal lung fibroblast cells , mitotic arrest was more pronounced in the colon cancer cells ( 50 % versus 10 % ) . Cells arrested by AK301 showed the formation of multiple microtubule organizing centers with O14965 and γ-tubulin . Employing in vitro and in vivo assays , tubulin polymerization was found to be slowed ( but not abolished ) by AK301 . In silico molecular docking suggests that AK301 binds to the colchicine-binding domain on β-tubulin , but in a novel orientation . Cells arrested by AK301 expressed elevated levels of P19438 on their surface and more readily activated caspases - 8 , - 9 , and - 3 in the presence of P01375 . Relative to other microtubule destabilizers , AK301 was the most active P01375 - sensitizing agent and also stimulated Fas - and P50591 - induced apoptosis . In summary , we report a new class of mitosis-targeting agents that effectively sensitizes cancer cells to apoptotic ligands . These compounds should help illuminate the role of microtubules in regulating apoptotic ligand sensitivity and may ultimately be useful for developing agents that augment the anti-cancer activities of the immune response .

Higher levels of activation markers and chemokine receptors on T lymphocytes in the cervix than peripheral blood of normal healthy women . Heterosexual transmission of human immunodeficiency virus ( HIV - 1 ) is the predominant mode of infection world-wide . To better understand sexual transmission of HIV - 1 in women we have analysed virus co-receptor and cellular activation marker expression on T lymphocyte subsets from the cervical epithelium and have made comparisons with peripheral blood T cells . Intraepithelial cervical T lymphocytes were obtained with a cytobrush , immunolabelled and analysed by flow cytometry . Activation markers ( Q07108 , CD25 and HLA-DR ) were found to be more highly expressed on cervical than on blood T lymphocytes . These higher levels of activation on cervical T lymphocyte subsets could facilitate HIV - 1 infection . P61073 was expressed at marginally higher levels than P51681 on T cells from the cervical epithelium and peripheral blood . Thus , the preferential transmission of macrophage tropic strains of HIV - 1 following sexual contact can not be explained solely on the expression of chemokine co-receptors by T lymphocyte subsets .

Oleocanthal inhibits proliferation and MIP - 1α expression in human multiple myeloma cells . Multiple myeloma ( MM ) is a plasma cell malignancy that causes devastating bone destruction by activating osteoclasts in the bone marrow milieu . MM is the second of all hematological malignancies . Thus , the search for new pharmacological weapons is under intensive investigation being MM a critically important public health goal . Recently , it has been demonstrated that macrophage inflammatory protein 1 - alpha ( MIP - 1 α ) is crucially involved in the development of osteolytic bone lesions in MM . Phenolic components of extra virgin olive oil are reported to have anti tumor activity . However , the underlying mechanisms and specific targets of extra virgin olive oil remain to be elucidated . In the present study , we investigated the effects of a recently isolated novel extra virgin olive oil polyphenol , oleocanthal , on the human multiple myeloma cell line Q5SW96 - 77 . Here we report that this natural compound has a remarkable in vitro activity by inhibiting MIP - 1 α expression and secretion in MM cells . In addition , we also demonstrated that oleocanthal inhibits MM cells proliferation by inducing the activation of apoptosis mechanisms and by down-regulating P27361 / 2 and AKT signal transduction pathways . This in vitro study suggests a therapeutic potential of oleocanthal in treating multiple myeloma .

DB00227 MEN , a 3 - hydroxy - 3 - methylglutaryl coenzyme A reductase inhibitor , induces apoptosis and differentiation in human anaplastic thyroid carcinoma cells . Although only 1 % of differentiated thyroid cancers transform into anaplastic thyroid cancer , this disease is always fatal . Differentiation therapy may provide a new therapeutic approach to increasing the survival rate in such patients . 3 - Hydroxy - 3 - methylglutaryl coenzyme A ( HMG - DB01992 ) reductase inhibitors are reported to promote cellular apoptosis and differentiation in many cancer cells ; these effects are unrelated to lipid reduction . Recently , we found that TNFalpha induces cytomorphological differentiation in anaplastic thyroid cancer cells and increases thyroglobulin expression ; however , P01375 is cytotoxic for normal human tissue . The aim of this study was to determine whether lovastatin , an P04035 inhibitor , could induce apoptosis and differentiation in anaplastic thyroid cancer cells . Anaplastic thyroid cancer cells were treated with lovastatin , then examined for cellular apoptosis and cytomorphological differentiation by DNA fragmentation , phosphatidylserine externalization / flow cytometry , and electron microscopy . Thyroglobulin levels in the culture medium were also measured . Our results showed that at a higher dose ( 50 micro M ) , lovastatin induced apoptosis of anaplastic thyroid cancer cells , whereas at a lower dose ( 25 micro M ) , it promoted 3 - dimensional cytomorphological differentiation . It also induced increased secretion of thyroglobulin by anaplastic cancer cells . Our results show that lovastatin not only induces apoptosis , but also promotes redifferentiation in anaplastic thyroid cancer cells , and suggest that it and other P04035 inhibitors merit further investigation as differentiation therapy for the treatment of anaplastic thyroid cancer .

Oral keratinocytes support non-replicative infection and transfer of harbored HIV - 1 to permissive cells . BACKGROUND : Oral keratinocytes on the mucosal surface are frequently exposed to HIV - 1 through contact with infected sexual partners or nursing mothers . To determine the plausibility that oral keratinocytes are primary targets of HIV - 1 , we tested the hypothesis that HIV - 1 infects oral keratinocytes in a restricted manner . RESULTS : To study the fate of HIV - 1 , immortalized oral keratinocytes ( OKF 6 / O14746 - 2 ; O14746 - 2 cells ) were characterized for the fate of HIV-specific RNA and DNA . At 6 h post inoculation with X4 or R5 - tropic HIV - 1 , HIV - 1gag RNA was detected maximally within O14746 - 2 cells . Reverse transcriptase activity in O14746 - 2 cells was confirmed by VSV-G-mediated infection with HIV-NL 4-3 Deltaenv-EGFP . DB00495 MEN inhibited EGFP expression in a dose-dependent manner , suggesting that viral replication can be supported if receptors are bypassed . Within 3 h post inoculation , integrated HIV - 1 DNA was detected in O14746 - 2 cell nuclei and persisted after subculture . Multiply spliced and unspliced HIV - 1 mRNAs were not detectable up to 72 h post inoculation , suggesting that HIV replication may abort and that infection is non-productive . Within 48 h post inoculation , however , virus harbored by P01730 negative O14746 - 2 cells trans infected co-cultured peripheral blood mononuclear cells ( PBMCs ) or MOLT 4 cells ( P01730 + P51681 + ) by direct cell-to-cell transfer or by releasing low levels of infectious virions . Primary tonsil epithelial cells also trans infected HIV - 1 to permissive cells in a donor-specific manner . CONCLUSION : Oral keratinocytes appear , therefore , to support stable non-replicative integration , while harboring and transmitting infectious X4 - or R5 - tropic HIV - 1 to permissive cells for up to 48 h .

Effects of gonadotropin-releasing hormone agonist on steroidogenesis in the rat ovary . To assess the regulatory roles of gonadotropin-releasing hormone ( DB00644 ) in ovarian function , the kinetics of the ovarian P30968 and the effects of the DB00644 superagonist buserelin on steroidogenesis in ovarian cell culture were examined . Scatchard analysis of buserelin-binding to crude ovarian cell membrane revealed a specific high-affinity P30968 . DB06719 SUB together with follicle-stimulating hormone stimulated estradiol ( E2 ) production in immature follicles in hypophysectomized and DES-treated rats . On the other hand , applied to developing follicles of rats treated with pregnant-mare-serum gonadotropin buserelin suppressed E2 production to terminate follicle maturation and simultaneously stimulated progesterone ( P4 ) production to induce luteinization . With ovarian cells luteinized by human chorionic gonadotropin in vitro , buserelin suppressed production of both P4 and E2 , leading to luteolysis . DB06719 SUB affected steroid production by modulating activities of key enzymes in steroid synthesis . These findings indicate that buserelin action depended on the gonadotropin priming of ovarian cells , and suggest the possible involvement of DB00644 in the regulation of steroidogenesis throughout the ovulatory cycle .

Tunicamycin and neuraminidase effects on luteinizing hormone ( LH ) - releasing hormone binding and LH release from rat pituitary cells in culture . We have studied the effects of tunicamycin ( TM ) and neuraminidase on the binding of 125I - labeled DB06719 SUB , a DB00644 agonist , and on DB00644 - stimulated LH release in cultured rat pituitary cells . Treatment with TM , an antibiotic which inhibits protein glycosylation , abolished the development of elongated cell processes without any effect on cell viability . Concomitantly , TM caused a time - and dose-dependent inhibition of specific binding of DB06719 SUB and of DB00644 - stimulated LH release . The inhibition of binding was due to a decrease in the number of DB00644 receptors without any significant effect on binding affinity . Protein synthesis was not affected under these experimental conditions , suggesting that the aglycosylated DB00644 receptors are probably intracellularly accumulated and are not expressed on the cell surface . Treatment with neuraminidase inhibited only 50 % of DB00644 agonist binding and did not affect DB00644 - stimulated LH release . These results indicate that the oligosaccharide portion is essential for the functional properties of the P30968 .

Nedd 9 restrains renal cystogenesis in Pkd 1 - / - mice . Mutations inactivating the cilia-localized Pkd 1 protein result in autosomal dominant polycystic kidney disease ( ADPKD ) , a serious inherited syndrome affecting ∼ 1 in 500 people , in which accumulation of renal cysts eventually destroys kidney function . Severity of ADPKD varies throughout the population , for reasons thought to involve differences both in intragenic Pkd 1 mutations and in modifier alleles . The scaffolding protein Q14511 , commonly dysregulated during cancer progression , interacts with Aurora-A ( O14965 ) kinase to control ciliary resorption , and with Src and other partners to influence proliferative signaling pathways often activated in ADPKD . We here demonstrate Nedd 9 expression is deregulated in human ADPKD and a mouse ADPKD model . Although genetic ablation of Nedd 9 does not independently influence cystogenesis , constitutive absence of Nedd 9 strongly promotes cyst formation in the tamoxifen-inducible Pkd 1fl / fl ; Cre / Esr 1 ( + ) mouse model of ADPKD . This cystogenic effect is associated with striking morphological defects in the cilia of Pkd 1 ( - / - ); Nedd 9 ( - / - ) mice , associated with specific loss of ciliary localization of adenylase cyclase III in the doubly mutant genotype . Ciliary phenotypes imply a failure of Aurora-A activation : Compatible with this idea , Pkd 1 ( - / - ); Nedd 9 ( - / - ) mice had ciliary resorption defects , and treatment of Pkd 1 ( - / - ) mice with a clinical Aurora-A kinase inhibitor exacerbated cystogenesis . In addition , activation of the ADPKD-associated signaling effectors Src , Erk , and the P42345 effector S6 was enhanced , and Ca ( 2 + ) response to external stimuli was reduced , in Pkd 1 ( - / - ); Nedd 9 ( - / - ) versus Pkd 1 ( - / - ) mice . Together , these results indicated an important modifier action of Nedd 9 on ADPKD pathogenesis involving failure to activate Aurora-A .

Cross-talk between PKA-Cβ and p65 mediates synergistic induction of Q07343 by roflumilast and NTHi . Phosphodiesterase 4B ( Q07343 ) plays a key role in regulating inflammation . DB01656 MEN , a phosphodiesterase ( PDE ) 4 - selective inhibitor , has recently been approved for treating severe chronic obstructive pulmonary disease ( P48444 ) patients with exacerbation . However , there is also clinical evidence suggesting the development of tachyphylaxis or tolerance on repeated dosing of roflumilast and the possible contribution of Q07343 up-regulation , which could be counterproductive for suppressing inflammation . Thus , understanding how Q07343 is up-regulated in the context of the complex pathogenesis and medications of P48444 may help improve the efficacy and possibly ameliorate the tolerance of roflumilast . Here we show that roflumilast synergizes with nontypeable Haemophilus influenzae ( NTHi ) , a major bacterial cause of P48444 exacerbation , to up-regulate PDE 4B2 expression in human airway epithelial cells in vitro and in vivo . Up-regulated PDE 4B2 contributes to the induction of certain important chemokines in both enzymatic activity-dependent and activity-independent manners . We also found that protein kinase A catalytic subunit β ( PKA-Cβ ) and nuclear factor-κB ( NF-κB ) p65 subunit were required for the synergistic induction of PDE 4B2 . PKA-Cβ phosphorylates p65 in a DB02527 - dependent manner . Moreover , Ser 276 of p65 is critical for mediating the PKA-Cβ-induced p65 phosphorylation and the synergistic induction of PDE 4B2 . Collectively , our data unveil a previously unidentified mechanism underlying synergistic up-regulation of PDE 4B2 via a cross-talk between PKA-Cβ and p65 and may help develop new therapeutic strategies to improve the efficacy of DB05876 inhibitor .

Polymorphisms associated with egg number at 300 days of age in chickens . We looked for variations that could be associated with chicken egg number at 300 days of age ( EN300 ) in seven genes of the hypothalamic-pituitary-gonadal axis , including gonadotrophin-releasing hormone-I ( DB00644 ) , P30968 ( GnRHR ) , neuropeptide Y ( P01303 ) , dopamine D2 receptor ( P14416 ) , vasoactive intestinal polypeptide ( P01282 ) , P01282 receptor - 1 ( VIPR - 1 ) , prolactin ( PRL ) , and the QTL region between 87 and 105 cM of the Z chromosome . Ten mutations in the seven genes were chosen to do marker-trait association analyses in a population comprising 1310 chickens , which were obtained from a company located in Guangdong Province of China . The C1704887T of VIPR - 1 was found to have a highly significant association with EN300 . The T5841629C of P14416 and the C1715301T of VIPR - 1 were significantly associated with EN300 . A highly significant association was also found between the C1704887T - C1715301T haplotypes of VIPR - 1 and EN300 . H1H3 had the highest EN300 . Four PCR-RFLP variations in the candidate QTL region were selected to investigate their genetic effects on EN300 . The haplotypes of T32742468C - G32742603A in this region showed a highly significant association with EN300 . Bioinformatics analyses showed that both T32742468C and G32742603A were located in intron 1 of the SH3 - domain P62993 - like 2 ( Q99962 ) gene . We conclude that five SNPs , including C1704887T and C1715301T of VIPR - 1 , T5841629C of P14416 , and T32742468C and G32742603A of Q99962 , would be useful as markers for breeding to increase chicken EN300 .

Transcriptional regulation of the gonadotropin-releasing hormone receptor gene is mediated in part by a putative repressor element and by the cyclic adenosine 3 ' , 5 ' - monophosphate response element . The levels of the P30968 ( GnRHR ) and its messenger RNA depend on the pattern of administration of DB00644 . In this study , internal deletion mutants in a luciferase reporter gene vector ( GnRHR-pXP 2 ) containing a 1226 - bp promoter fragment of mouse GnRHR gene were used to examine the regulation of GnRHR gene transcription in GGH 3 cells . Our results indicate that the mouse GnRHR promoter contains one putative repressor element located at position - 343 / - 335 . When this sequence was deleted , the GnRHR promoter activity was significantly increased in both basal and DB00644 agonist ( DB06719 SUB ) - , phorbol ester - , and forskolin-stimulated cells . Gel mobility shift assay showed that the sequence - 343 / - 335 is capable of binding GGH 3 nuclear proteins . With deletion of the DB02527 response element ( - 107 / - 100 ) , basal and DB06719 SUB - stimulated transcription was decreased . The same response was observed after stimulation with forskolin . Stimulation with ( Bu ) 2cAMP did not alter transcription above basal levels . The stimulation with phorbol ester resulted in an attenuated increase in transcriptional activity , suggesting that this sequence of the GnRHR promoter is a DB02527 response element . These results suggest that the transcriptional activity of the GnRHR gene is mediated in part by a putative repressor element and by the DB02527 response element .

Genetic structure of candidate genes for litter size in Italian Large White pigs . The aim of this work was to verify whether polymorphisms in candidate genes for litter size segregate in Italian Large White ( ITLW ) pigs . We genotyped 120 sows that belonged to six different farms for 10 single nucleotide polymorphisms ( SNPs ) of 10 different genes . Polymorphisms in the chosen genes had already been associated with litter-size traits in other pig populations and were candidates for function and / or chromosomal location . The results indicated that the O14967 , pDAZL , and RFN 4 SNPs were not segregating in the genotyped samples . The remaining seven markers were polymorphic with minor allele frequencies ranging from 0.10 ( AFP ) to 0.48 ( P02753 ) . Because of the observed genetic variabilities in the investigated loci , the polymorphisms in the AFP , O00238 , P02778 , Q92731 , P30968 , Q9Y2E5 , and P02753 genes can be considered suitable markers for association studies with litter-size traits in ITLW pigs .

Phosphodiesterase - 4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 ( P25098 ) in P29320 - 293beta2 cells and cardiac myocytes . Membrane-recruitment of P25098 ( G-protein receptor kinase 2 ) provides a fundamental step in the desensitization process controlling GPCRs ( G-protein-coupled receptors ) , such as the beta 2AR ( beta 2 - adrenergic receptor ) . In the present paper , we show that challenge of P29320 - 293beta2 [ human embryonic kidney cells stably overexpressing the FLAG-tagged beta 2AR - GFP ( green fluorescent protein ) ] cells with the beta-adrenoceptor agonist , isoprenaline , causes P25098 to become phosphorylated by PKA ( DB02527 - dependent protein kinase ) . This action is facilitated when DB02527 - specific DB05876 ( phosphodiesterase - 4 ) activity is selectively inactivated , either chemically with rolipram or by siRNA ( small interfering RNA ) - mediated knockdown of Q07343 and Q08499 . DB05876 - selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of P25098 , phosphorylation of the beta 2AR by P25098 , membrane translocation of beta-arrestin and internalization of beta 2ARs . DB05876 - selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of P25098 in cardiac myocytes . In the absence of isoprenaline , rolipram-induced inhibition of DB05876 activity in P29320 - 293beta2 cells acts to stimulate PKA phosphorylation of P25098 , with consequential effects on P25098 membrane recruitment and P25098 - mediated phosphorylation of the beta 2AR . We propose that a key role for DB05876 enzymes is : ( i ) to gate the action of PKA on P25098 , influencing the rate of P25098 phosphorylation of the beta 2AR and consequential recruitment of beta-arrestin subsequent to beta-adrenoceptor agonist challenge , and ( ii ) to protect P25098 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of DB02527 .

Differential involvement of Galpha 16 in CC chemokine-induced stimulation of phospholipase Cbeta , P29323 , and chemotaxis . Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes . Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G ( 16 ) and its close relative , G ( 14 ) . Yet , many chemokine receptors utilize pertussis toxin-sensitive G ( i ) proteins for signaling . Given that both G ( 16 ) and G ( 14 ) are capable of linking G ( i ) - coupled receptors to the stimulation of phospholipase Cbeta , we examined the capacity of six CC chemokine receptors ( P32246 , CCR 2a , CCR 2b , P51677 , P51681 and P32248 ) to interact with G ( 14 ) and G ( 16 ) in a heterologous expression system . Among the CC chemokine receptors tested , P32246 , CCR 2b , and P51677 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G ( 14 ) or G ( 16 ) . The G ( 14 ) / G ( 16 ) - mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin ( PTX ) treatment . In contrast , CCR 2a , P51681 and P32248 were unable to interact with G ( 14 ) and G ( 16 ) . Under identical experimental conditions , all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G ( i ) as well as stimulating phospholipase Cbeta via 16z44 , a G ( 16 / z ) chimera that possesses increased promiscuity toward G ( i ) - coupled receptors . Moreover , P32246 - mediated P27361 / 2 phosphorylation was largely PTX-insensitive in THP - 1 monocytic cells that endogenously express Galpha ( 16 ) . In addition , P32246 agonist was less efficacious in mediating chemotaxis of THP - 1 cells following the knockdown of Galpha ( 16 ) by overexpressing siRNA , indicating the participation of Galpha ( 16 ) in P32246 - induced cell migration . These results show that different CC chemokine receptors can discriminate against G ( 14 ) and G ( 16 ) for signal transduction .

Effects of Asn 318 and Asp 87Asn318 mutations on signal transduction by the gonadotropin-releasing hormone receptor and receptor regulation . P30968 ( P30968 ) contains Asn 87 and Asp 318 instead of the more frequently observed Asp 87 and Asn 318 found in other G protein-coupled receptors . In the present study , site-directed mutagenesis was used to introduce Asn 318 and Asp 87Asn318 into P30968 . The effect on coupling and regulation of P30968 was studied by stable expression of wild and mutant mouse P30968 in the lactotropic GH3 cells ; these normally release PRL in response to TRH stimulation . The responses to DB06719 SUB ( a metabolically stable DB00644 analog ) in three different cell lines , M1 , N8 , and P03886 ( expressing wild-type , Asn 318 mutant , and Asp 87Asn318 mutant mouse P30968 , respectively ) were compared with that observed in the previously characterized GGH 3-1 ' cells , which stably express rat P30968 . The Asn 318 and Asp 87Asn318 mutations had no measurable effect on ligand binding , but abolished the initial down-regulation of receptor that was observed in M1 and GGH 3-1 ' cells , suggesting that the normal location of Asn 87 and Asp 318 in P30968 is involved in the regulation of P30968 . In N8 and P03886 cells , DB06719 SUB - stimulated inositol phosphate ( IP ) production was attenuated , but the release of both DB02527 and PRL was stimulated in a dose - and time-dependent manner . These mutations apparently impaired the coupling between P30968 and G proteins involved in IP production , but not those involved in DB02527 release . In M1 cells , DB06719 SUB stimulation produced a significant increase in IP production , but neither DB02527 nor PRL release was significantly stimulated . These findings are consistent with the previous suggestion that DB00644 - stimulated PRL release is mediated by a DB02527 second messenger system in transfected GGH 3 cells .

Microsomal transfer protein ( P55157 ) inhibition-a novel approach to the treatment of homozygous hypercholesterolemia . Homozygous familial hypercholesterolemia ( HoFH ) represents the most severe lipoprotein disorder , generally attributable to mutation ( s ) of the low-density lipoprotein receptor ( LDL-R ) , i . e . autosomal dominant hypercholesterolemia type 1 ( P07327 ) . Much lower percentages are due to alterations of apolipoprotein B ( P00325 ) , or gain-of-function mutations of proprotein convertase subtilisin / kexin type 9 ( Q8NBP7 ) ( P00326 ) . In certain geographical areas a significant number of patients may be affected by an autosomal recessive hypercholesterolemia ( Q5SW96 ) . Mutations may be also combined ( two mutations of the same gene , compound heterozygosity ) , or two in different genes ( double heterozygosity ) . Among the most innovative therapeutic approaches made available recently , inhibitors of the microsomal transfer protein ( P55157 ) system have shown a high clinical potential . P55157 plays a critical role in the assembly / secretion of very-low-density lipoproteins ( VLDL ) , and its absence leads to apo B deficiency . P55157 antagonists dramatically lower LDL-cholesterol ( LDL-C ) in animals , although a reported increase of liver fat delayed their clinical development . DB08827 MEN , the best-studied P55157 inhibitor , reduces LDL-C by 50 % or more in HoFH patients , with modest , reversible , liver steatosis . Recent US approval has confirmed an acceptable tolerability , provided patients adhere to a strictly low-fat regimen . There are no clinical data on atherosclerosis reduction / regression , but animal models provide encouraging results .

DB00644 analogues reduce the proliferation of endometrial stromal cells but not endometriotic cells . AIMS : We investigated the potential of gonadotropin-releasing hormone ( DB00644 ) agonists and DB00644 antagonists to inhibit cell proliferation in endometriotic and endometrial stromal cells . METHODS : Twenty patients with ovarian endometriomas and 18 patients with uterine fibromas were recruited . Endometriotic and endometrial stromal cells were obtained from the ovarian chocolate cyst linings and the eutopic endometria of premenopausal women with uterine fibromas , respectively . RESULTS : DB00644 agonist or antagonist treatment attenuated tumor necrosis factor ( P01375 ) - α-induced cell proliferation in the endometrial stromal cells , whereas endometriotic stromal cells did not respond to treatment . The endometriotic stromal cells exhibited a decreased expression of the type I P30968 compared with the endometrial stromal cells . DB00644 agonists or antagonists did not repress P01375 - α-induced P10145 production in endometriotic stromal cells . CONCLUSION : DB00644 agonists and antagonists have similar effects in slowing the growth of endometrial stromal cells . Endometriotic stromal cells resist the antiproliferative effect of DB00644 agonists and antagonists .

Interferon-gamma-induced dephosphorylation of P40763 and apoptosis are dependent on the P42345 pathway . Interferon-gamma ( P01579 ) exhibits diverse biological activities , including control of cell growth and tumor suppression . Here , we report that the treatment of M12 cells , a human metastatic prostate cancer cell line , with P01579 , resulted in marked inhibition of cell proliferation and induced apoptosis . These effects were not seen with either IFN-alpha or IFN-beta . M12 cells , like many other human cancer cells , contain constitutively activated signal transducer and activator of transcription 3 ( P40763 ) . The basal levels of both Akt and P27361 / 2 phosphorylation are also markedly elevated in M12 cells . Strikingly , P01579 - induced apoptosis and growth inhibition of M12 cells were associated with persistent suppression of the constitutive tyrosine-phosphorylated P40763 ( pY - P40763 ) . The P01579 - induced dephosphorylation of pY - P40763 , however , was inhibited when the P42345 pathway was specifically blocked by rapamycin . Inhibition of PI - 3K with low-dose LY294002 , or MAPK with PD98059 also suppressed the P42345 / P08133 S6k pathway , and correlated with the blockage of P01579 - induced dephosphorylation of pY - P40763 . Simultaneously , treatment with LY294002 , PD98059 , or rapamycin abolished P01579 - induced apoptosis in M12 cells . The inhibition of the P42345 pathway , however , did not affect P01579 - induced activation of P42224 pathway , and suppression of P42224 expression by siRNA had no effect on P01579 - induced dephosphorylation of pY - P40763 . Taken together , these results demonstrate that an intact P42345 pathway is critical for P01579 - induced suppression of pY - P40763 and apoptosis . Our study thus provides novel insights into the contributions of signaling pathways other than the classical JAK / P42224 pathway in the anti-proliferative , proapoptotic actions of P01579 .

Cyclooxygenase isozymes are expressed in human myeloma cells but not involved in anti-proliferative effect of cyclooxygenase inhibitors . Considering possible tumorigenic activity of cyclooxygenase ( P36551 ) isozymes in myeloma , we examined expression levels of P23219 and - 2 in seven human myeloma cell lines ( Q5SW96 - 77 , IM - 9 , RPMI - 8226 , HPC , HS-Sultan , TSPC - 1 , and U - 266 ) . As analyzed by reverse transcriptase-polymerase chain reaction ( RT-PCR ) , all the cell lines constitutively expressed P23219 , while P35354 levels markedly varied among different cell lines . Induction of P35354 by phorbol ester was observed in RPMI - 8226 and HPC cells . In contrast , P35354 was constitutively expressed in Q5SW96 - 77 and IM - 9 cells . Moreover , the high expression level of P35354 protein in Q5SW96 - 77 cells was verified by Western blotting . Intact cells of Q5SW96 - 77 converted 14C - labeled arachidonic acid to prostaglandin E2 , F2alpha , and D2 , and this activity was dose-dependently inhibited by selective P35354 inhibitors ( SC - 58125 and NS - 398 ) , a non-selective P36551 inhibitor ( indomethacin ) , and relatively high concentrations of a selective P23219 inhibitor ( SC - 560 ) . These P36551 inhibitors also suppressed the proliferation of Q5SW96 - 77 cells , but significant suppression was seen only at 100 microM , a much higher concentration than those sufficient for the P36551 inhibition . Moreover , proliferation of the myeloma cells lacking P35354 was also suppressed by 100 microM of SC - 58125 . These results suggested that the anti-proliferative effect of the P36551 inhibitors is independent of the inhibition of P35354 .

Evolution of subterranean diving beetles ( Coleoptera : Dytiscidae : Hydroporini , Bidessini ) in the arid zone of Australia . Calcrete aquifers in arid inland Australia have recently been found to contain the world ' s most diverse assemblage of subterranean diving beetles ( Coleoptera : Dytiscidae ) . In this study we test whether the adaptive shift hypothesis ( ASH ) or the climatic relict hypothesis ( P06850 ) is the most likely mode of evolution for the Australian subterranean diving beetles by using a phylogeny based on two sequenced fragments of mitochondrial genes ( CO1 and 16S - tRNA - P03886 ) and linearized using a relaxed molecular clock method . Most individual calcrete aquifers contain an assemblage of diving beetle species of distantly related lineages and / or a single pair of sister species that significantly differ in size and morphology . Evolutionary transitions from surface to subterranean life took place in a relatively small time frame between nine and four million years ago . Most of the variation in divergence times of the sympatric sister species is explained by the variation in latitude of the localities , which correlates with the onset of aridity from the north to the south and with an aridity maximum in the Early Pliocene ( five mya ) . We conclude that individual calcrete aquifers were colonized by several distantly related diving beetle lineages . Several lines of evidence from molecular clock analyses support the P06850 , indicating that all evolutionary transitions took place during the Late Miocene and Early Pliocene as a result of aridification .

Tonic , but not phasic corticosterone , constrains stress activatedextracellular-regulated-kinase 1 / 2 immunoreactivity within the hypothalamic paraventricular nucleus . The negative-feedback actions of corticosterone ( O00230 ) depend on both phasic and tonic O00230 secretion patterns to regulate hypothalamic-pituitary-adrenal ( Q9Y251 ) axis activity . How these two different O00230 secretion pattens influence specific intracellular signal transduction pathway activity within the cellular elements of the Q9Y251 axis has not been determined . For example , it is unknown whether O00230 has suppressive actions over signal transduction events within medial parvocellular paraventricular nucleus ( PVN ) corticotrophin-releasing hormone ( P06850 ) neurones , nor whether these suppressive actions are responsible for alterations in PVN transcriptional processes and neurohormone secretion associated with stress . The extracellular-regulated kinase ( P29323 ) is a stress activated intracellular signalling molecule that is potentially subject to glucocorticoid negative-feedback regulation . We tested the ability of O00230 to modulate levels of the active ( phosphorylated ) form of P29323 ( pERK 1/2 ) in the PVN of rats . Acute psychological stress ( restraint ) produced a rapid increase in the number of PVN pERK 1/2 immunopositive cells within P06850 neurones . Absence of tonic O00230 via adrenalectomy ( P10109 ) produced no change in basal pERK 1/2 cell counts but augmented the increased pERK 1/2 cell counts elicited by acute restraint . Treatment of P10109 rats with O00230 in the drinking water normalised this enhanced pERK 1/2 response to stress . By contrast , treatment of P10109 rats with a phasic increase in O00230 1 h before restraint had no effect on pERK 1/2 cell counts , despite substantially suppressing stress-induced PVN crh gene expression and adrenonocorticotrophic hormone secretion . This tonic O00230 inhibition of stress-induced activation of P27361 / 2 may involve both alteration of the activity of stress-dependent neural inputs to PVN P06850 neurones and alteration within those neurones of stress-dependent intracellular signalling mechanisms associated with P29323 activation .

[ The effects of DB00644 agonist on steroidogenesis in the rat ovary ] . The effects of DB00644 agonist ( buserelin ) on in vitro ovarian steroidogenesis were studied using DES-treated immature rats and PMS-treated immature rats . The estradiol and progesterone secreted by the cultured ovarian cells and the activities of various enzymes of steroid-metabolism were examined with or without gonadotropins ( DB00094 or hCG ) , and the effects of 10 ( - 6 ) - 10 ( - 12 ) M of buserelin on those indices were observed for 3-72 hours . In addition , the kinetic study of ovarian P30968 was performed using 125I - labelled buserelin and crude ovarian cell membrane fraction of PMS-treated rats . The Scatchard analysis revealed the specific high affinity and low capacity ovarian P30968 ( Kd = 0.92 nM and Bmax = 0.57 fmol / mg tissue ) . The DB00094 - stimulated cholesterol side chain cleavage enzyme ( CSCC ) activity of the DES-treated rats was suppressed in a dose-dependent manner by buserelin . Estradiol release and aromatase activity were increased by 10 (-8 ) M buserelin within 48 hours from the beginning of the incubation of the PMS-treated rat ovarian cells , but were suppressed after 48 hours . DB06719 SUB increased basal progesterone secretion and both activities of CSCC and of 3 beta-hydroxysteroid dehydrogenase of PMS-treated rat ovarian cells incubated without hCG , which were suppressed by buserelin co-incubated with 100 IU / ml of hCG . These results suggested that DB00644 plays a physiological role in ovarian steroidogenesis binding the specific receptor and that DB00644 promotes the development of the follicle through increased estrogen synthesis in the early stage of the folliculogenesis and the luteinization in the late stage of the follicular development through increased progesterone and decreased estradiol production and the luteolysis in the luteinized cells by hCG through decreased progesterone secretion .

P04150 antagonism disrupts the reconsolidation of social reward-related memories in rats . Reconsolidation is the process whereby consolidated memories are destabilized upon retrieval and restabilized to persist for later use . Although the neurobiology of the reconsolidation of both appetitive and aversive memories has been intensively investigated , reconsolidation of memories of physiologically relevant social rewards has received little attention . Social play , the most characteristic social behaviour displayed by young mammals , is highly rewarding , illustrated by the fact that it can induce conditioned place preference ( CPP ) . Here , we investigated the role of signalling mechanisms implicated in memory processes , including reconsolidation , namely glucocorticoid , mineralocorticoid , DB01221 glutamatergic and P21554 cannabinoid receptors , in the reconsolidation of social play-induced CPP in rats . Systemic treatment with the glucocorticoid receptor antagonist mifepristone before , but not immediately after , retrieval disrupted the reconsolidation of social play-induced CPP . DB00834 MEN did not affect social play-induced CPP in the absence of memory retrieval . Treatment with the DB01221 receptor antagonist MK - 801 modestly affected the reconsolidation of social play-induced CPP . However , the reconsolidation of social play-induced CPP was not affected by treatment with the mineralocorticoid and P21554 cannabinoid receptor antagonists spironolactone and rimonabant , respectively . We conclude that glucocorticoid neurotransmission mediates the reconsolidation of social reward-related memories in rats . These data indicate that the neural mechanisms of the reconsolidation of social reward-related memories only partially overlap with those underlying the reconsolidation of other reward-related memories .

Signaling and antiproliferative effects of type I and II gonadotropin-releasing hormone receptors in breast cancer cells . DB00644 receptors ( DB00644 - Rs ) mediate direct antiproliferative effects on hormone-dependent cancer cells . DB00644 - Rs can be grouped according to ligand specificity ( for DB00644 and - II ) , and there is evidence that type II DB00644 ligands and / or receptors can inhibit proliferation . Type I DB00644 - Rs ( e . g . human and sheep ) lack the C-terminal tails found in other G protein-coupled receptors including type II DB00644 - Rs ( e . g . Xenopus ; XGnRH-R ) . This underlies the remarkable resistance of type I DB00644 - Rs to desensitization and may be important for chronic effects on proliferation . To test this , we have compared the antiproliferative effects of DB00644 - Rs expressed in MCF 7 breast cancer cells using recombinant adenovirus ( Ad ) . Endogenous DB00644 - Rs were not detected , but infection with Ad-expressing sheep DB00644 - Rs ( sGnRH-R ) facilitated proliferation inhibition by DB06719 SUB , and maximum inhibition required only 10,000- 20,000 sGnRH-Rs . XGnRH-Rs were much less efficient at inhibiting proliferation and were internalized faster than sGnRH-Rs . Thus , the type II P30968 is less efficient at inhibiting proliferation , presumably because it is rapidly desensitized and / or internalized . Moreover , comparisons of human P30968 , sGnRH-R , and XGnRH-R , as well as chimeric receptors ( type I DB00644 - Rs with C-terminal tails from XGnRH-Rs ) , revealed that C-terminal tail addition increases receptor expression and thereby increases the efficiency with which the vector facilitates the antiproliferative effect .

DB00133 residues 338 and 339 in the carboxyl-terminal tail of the type II gonadotropin-releasing hormone receptor are critical for beta-arrestin-independent internalization . Cloned mammalian type II DB00644 receptors have a carboxyl-terminal tail in contrast to the mammalian type I DB00644 receptors , which uniquely lack a carboxyl-terminal tail . Because this domain mediates internalization of many serpentine receptors , the internalization pathway of the marmoset monkey type II P30968 and the functional role of the carboxyl-terminal tail in internalization was studied . The internalization pathway of the type II P30968 was investigated in COS - 1 cells by coexpressing G protein-coupled receptor kinases ( GRKs ) , dynamin - 1 , and beta-arrestins . Internalization of the receptor requires GRKs and dynamin but does not require beta-arrestin . The type II P30968 can also internalize via beta-arrestin in the presence of exogenous beta-arrestins , suggesting that the receptor can use two distinct internalization pathways . Receptor internalization appears to occur via clathrin-coated pits and caveolae because disruption of either structure inhibits internalization . Progressive truncations of the carboxyl-terminal tail identified a region containing serine residues 338 and 339 as critical for receptor internalization . Substitution of these serine residues with alanine residues inhibited internalization , whereas substitutions with glutamic acid residues rescued internalization . Furthermore , a dominant-negative P25098 did not inhibit internalization of receptors having these serine substitutions , although it inhibited internalization of the wild-type receptor . These results together identify serine residues 338 and 339 in the carboxyl-terminal tail as critical for internalization of the type II P30968 and suggest that these residues undergo phosphorylation by GRKs . However , neither of these residues , nor the carboxyl-terminal tail , is required for beta-arrestin-dependent internalization .

Array-comparative genomic hybridization to detect genomewide changes in microdissected primary and metastatic oral squamous cell carcinomas . Oral squamous cell carcinoma ( OSCC ) is a common worldwide malignancy . However , it is unclear what , if any , genomic alterations occur as the disease progresses to invasive and metastatic OSCC . This study used genomewide array-CGH in microdissected specimens to map genetic alterations found in primary OSCC and neck lymph node metastases . We used array-based comparative genomic hybridization ( array-CGH ) to screen genomewide alterations in eight pairs of microdissected tissue samples from primary and metastatic OSCC . In addition , 25 primary and metastatic OSCC tissue pairs were examined with immunohistochemistry for protein expression of the most frequently altered genes . The highest frequencies of gains were detected in P12524 , Q04864 , TERC , P42336 , P10242 , P08183 , P01112 , GARP , P30279 , P07332 , P04626 , P01127 , and Q05066 . The highest frequencies of losses were detected in p44S10 , O15164 , P06858 , Q13126 , P35226 , P11161 , and Q13163 . Genomic alterations in TGFbeta 2 , cellular retinoid-binding protein 1 gene ( P09455 ) , P42336 , P28222 , P01112 , P21860 , and O14965 differed significantly between primary OSCC and their metastatic counterparts . Genomic alterations in Q05513 , P00519 , and P08620 were significantly different in patients who died compared with those who survived . Immunohistochemistry confirmed high P42336 immunoreactivity in primary and metastatic OSCC . Higher P08620 immunoreactivity in primary OSCC is associated with a worse prognosis . Loss of P09455 immunoreactivity is evident in primary and metastatic OSCC . Our study suggests that precise genomic profiling can be useful in determining gene number changes in OSCC . As our understanding of these changes grow , this profiling may become a practical tool for clinical evaluation .

P30968 concentration differentially regulates intracellular signaling pathways in GGH 3 cells . Pituitary cell lines ( GGH 3 ) expressing the P30968 ( GnRHR ) were used to investigate the effect of GnRHR concentration on the ability of a DB00644 agonist to activate second messenger systems . Four different strategies were utilized to generate cells expressing functionally different concentrations of receptors : ( 1 ) transient transfection with different concentrations of wild type GnRHR into GH3 cells , ( 2 ) utilization of two cell lines derived from a common stably transfected line expressing high ( 4,209 + / - 535 receptors / cell ) or low ( 1,031 + / - 36 receptors / cell ) concentrations of GnRHR , ( 3 ) co-incubation of GGH 3-1 ' cells with a DB00644 agonist ( DB06719 SUB ) and a DB00644 antagonist to compete for binding sites , and ( 4 ) photo-affinity binding to GnRHR with a DB00644 antagonist to change effective receptor concentration . A range of receptor concentrations ( 1,000- 8,000 receptors / cell ) were generated by these techniques . Inositol phosphate ( IP ) and DB02527 accumulation were quantified to assess the effect of receptor concentration on receptor-effector coupling . Under all four paradigms , the efficacy and potency of DB06719 SUB stimulated IP production was dependent on receptor concentration . In contrast , DB06719 SUB stimulated DB02527 release was relatively unchanged at varying concentrations of GnRHR . This suggests that the cellular concentration of GnRHR affects the induction of cell signaling pathways . These results demonstrate that a single ligand-receptor-complex can differentially activate second messenger systems and present a mechanism by which multiple physiological endpoints can be differentially regulated by a single hormone / receptor interaction .

17 DB00783 overcomes a P55008 block induced by P04035 inhibitors and fosters cell cycle progression without inducing P27361 and - 2 Q96HU1 kinases activation . P04035 inhibitors , such as DB00227 MEN and Simvastatin , cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media . Cells are induced to pause in P55008 and can readily resume growth upon removal of the enzymatic block . DB00286 , acting via their nuclear receptor , are mitogens for different normal and transformed cell types , where they foster cell cycle progression and cell division . In estrogen-responsive MCF - 7 human breast cancer cells , but not in non responsive cells , 17 beta-estradiol ( E2 ) induces cells arrested with DB00227 MEN or Simvastatin to proliferate in the presence of inhibitor , without restoring P04035 activity or affecting the protein prenylation pattern . Mitogenic stimulation of P55008 - arrested MCF - 7 cells with E2 includes primary transcriptional activation of c-fos , accompanied by transient binding in vivo of the estrogen receptor and / or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene , as detected by dimethylsulphate genomic footprinting analysis . Mitogenic stimulation of growth-arrested MCF - 7 cells by E2 occurs , under these conditions , without evident activation of P27361 and - 2 kinases , and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes .

DB00644 II ( DB00644 II ) mediates the anorexigenic actions of α-melanocyte-stimulating hormone ( α-MSH ) and corticotropin-releasing hormone ( P06850 ) in goldfish . Intracerebroventricular ( ICV ) administration of gonadotropin-releasing hormone II ( DB00644 II ) , which plays a crucial role in the regulation of reproduction in vertebrates , markedly reduces food intake in goldfish . However , the neurochemical pathways involved in the anorexigenic action of DB00644 II and its interaction with other neuropeptides have not yet been identified . Alpha-melanocyte-stimulating hormone ( α-MSH ) , corticotropin-releasing hormone ( P06850 ) and P06850 - related peptides play a major role in feeding control as potent anorexigenic neuropeptides in goldfish . However , our previous study has indicated that the DB00644 II-induced anorexigenic action is not blocked by treatment with melanocortin 4 receptor ( P32245 ) and P06850 receptor antagonists . Therefore , in the present study , we further examined whether the anorexigenic effects of α-MSH and P06850 in goldfish could be mediated through the P30968 neuronal pathway . ICV injection of the P32245 agonist , melanotan II ( 80 pmol / g body weight ; BW ) , significantly reduced food intake , and its anorexigenic effect was suppressed by ICV pre-administration of the DB00644 type I receptor antagonist , antide ( 100 pmol / gBW ) . The P06850 - induced ( 50 pmol / gBW ) anorexigenic action was also blocked by treatment with antide . ICV injection of P06850 ( 50 pmol / gBW ) induced a significant increase of the DB00644 II mRNA level in the hypothalamus , while ICV injection of melanotan II ( 80 pmol / gBW ) had no effect on the level of DB00644 II mRNA . These results indicate that , in goldfish , the anorexigenic actions of α-MSH and P06850 are mediated through the DB00644 type I receptor-signaling pathway , and that the DB00644 II system regulates feeding behavior .

P06401 activation of extranuclear signaling pathways in regulating p53 expression in vascular endothelial cells . We previously showed that progesterone ( P4 ) inhibited the proliferation of human umbilical vein endothelial cells ( HUVECs ) through a p53 - dependent pathway . Now we investigated further the molecular mechanism underlying the hormone activity . In cultured HUVECs , P4 increased the protein levels of phosphorylated Src ( p-Src ) , P04049 , and P29323 . The levels of p-Src and p-Src-progesterone receptor complex in HUVECs were increased by P4 treatment . These effects were blocked by pretreatment with a progesterone receptor antagonist , DB00834 MENMAX DB00834 MEN . The P4 - induced increase in p53 transactivity was abolished by pretreatment with Src kinase inhibitors . Moreover , administration with cSrc antisense oligonucleotide prevented the P4 - induced increases of the levels of p53 mRNA and protein . These data suggest that P4 - induced up-regulation of p53 might be mediated through activation of cSrc . Pretreatment with Src kinase inhibitors also prevented P4 - induced membrane translocation of Kras and increases of the protein levels of phosphorylated Raf and phosphorylated P29323 . Transfection with dominant-negative P28482 prevented the P4 - induced increases of protein level and promoter activity of p53 and a decrease of thymidine incorporation . P4 also increased nuclear factor-κB ( NF-κB ) nuclear translocation and NF-κB binding onto the p53 promoter . These effects were abolished by pretreatment with P29323 inhibitors . The P4 - induced up-regulation of the p53 promoter activity was prevented by preadministration with dominant-negative P28482 or NF-κB inhibitors . Taken together , our data suggest that the cSrc / Kras / P04049 / P28482 / NF-κB signaling pathway contributes to the P4 - induced up-regulation of p53 in HUVECs . These findings highlight progesterone receptor activation of extranuclear signaling pathways in regulating p53 and cell cycle progression in HUVECs .

[ DB00391 MEN in the management of functional dyspepsia and delayed gastric emptying ] . DB00391 MEN is a sulpiride isomer that exerts its prokinetic action through a dual mechanism : 1 ) as a P14416 antagonist and 2 ) as a serotonin 5HT ( 4 ) receptor agonist , conferring this drug with a cholinergic effect . At a dosage of 25mg three times daily , levosulpiride accelerates gastric and gallbladder emptying . Clinical trials have shown that this agent is more effective than placebo in reducing the symptoms of dyspepsia , while comparative studies have demonstrated that its effect is similar or superior to that of other dopamine antagonists . The safety profile of levosulpiride is good and the frequency of adverse events is similar to that of other D ( 2 ) dopamine antagonists . Therefore , this drug is a useful therapeutic option in the management of patients with functional dyspepsia , as well as in those with delayed gastric emptying .

DB00227 MEN induces the expression of bradykinin type 2 receptors in cultured human coronary artery endothelial cells . Cardioprotective bradykinin type - 2 receptors ( BK - 2Rs ) are downregulated in the myocardial endothelium of both human and rat failing hearts . Statins are cardioprotective drugs that reduce the level of plasma cholesterol but also exert cholesterol-independent pleiotropic effects . Here we examined the effect of lovastatin on BK - 2R expression in cultured human coronary artery endothelial cells . The effect of lovastatin on the expression of BK receptors in human coronary artery endothelial cells ( HCAECs ) was examined by real-time PCR , Western blot analysis and immunocytochemistry . DB00227 MEN induced a time - and concentration-dependent increase in both BK - 2R and BK - 1R mRNA expression in the cultured HCAECs . Also , the number of functional BK - 2Rs capable of inducing BK-mediated NO production and cGMP signaling was increased in the lovastatin-treated HCAECs . Mevalonate , the direct metabolite of P04035 , reversed the effect of lovastatin . Furthermore , lovastatin inhibited Rho activation and a selective inhibitor of Rho-associated kinases , Y - 27632 , induced a similar increase in BK - 2R expression as lovastatin . In contrast , a specific inhibitor of P35354 , NS398 , significantly inhibited the lovastatin-induced expression of BK - 2Rs . Here we show for the first time that lovastatin induces the expression of BK - 2Rs in cultured human coronary artery endothelial cells through a novel cholesterol-independent pleiotropic mechanism that involves RhoA kinase inhibition and P35354 activation . Thus , reported beneficial effects of statins in cardiovascular diseases may be partly mediated by an increased expression of cardioprotective BK - 2Rs in the endothelial cells of the coronary tree . Moreover , the use of P35354 inhibitors may affect the level of endothelial BK - 2Rs in a negative fashion .