interacts_with DB00379?
Suppression of 3-deoxyglucosone and heparin-binding epidermal growth factor-like growth factor mRNA expression by an aldose reductase inhibitor in rat vascular smooth muscle cells . Reactive carbonyl compounds and oxidative stress have been recently shown to up-regulate the expression of heparin-binding epidermal growth factor-like growth factor ( HB- P01133 ) , a potent mitogen for vascular smooth muscle cells ( SMCs ) produced by SMC themselves . Because the polyol pathway has been reported to influence the formation of carbonyl compounds and the oxidative stress in various cells , we conducted this study to investigate whether the polyol pathway affects HB- P01133 expression along with the generation of carbonyl compounds and the oxidative stress in SMCs . We found that , compared with those cultured with 5.5mM glucose , SMCs cultured with 40 mM glucose showed the accelerated thymidine incorporation , elevated levels of intracellular sorbitol , 3-deoxyglucosone ( 3-DG ) , advanced glycation end products ( AGEs ) , and thiobarbituric acid-reactive substances ( TBARS ) along with the enhanced expression of HB- P01133 mRNA . An aldose reductase inhibitor ( Q9Y4X5 ) , Q9NYY3 -860 , significantly inhibited all of these abnormalities , while aminoguanidine suppressed 3-DG levels and HB- P01133 mRNA expression independent of sorbitol levels . The results suggest that the polyol pathway may play a substantial role in SMC hyperplasia under hyperglycemic condition in part by affecting HB- P01133 mRNA expression via the production of carbonyl compounds and oxidative stress . New targeted therapies in melanoma . BACKGROUND : The previous 2 years have been an exciting time in melanoma research , due in part to the approval of vemurafenib and ipilimumab for advanced melanoma . Increased knowledge of the molecular biology leading to melanoma has led to the development of several new agents that target specific oncogenes . METHODS : The authors review the latest developments in signal transduction inhibitors and in immune modulators for the treatment of melanoma . Investigational agents currently in development are also discussed . RESULTS : DB08881 and ipilimumab have improved overall survival in patients with metastatic melanoma . Many new agents are in development , including programmed death-1 antibodies and combination signal transduction inhibitors . CONCLUSIONS : A recognition of the genetic diversity of melanoma and a better understanding of the immune system have resulted in improvements in overall survival in patients with metastatic melanoma . Refractory cases remain challenging , and combination therapies are being explored in an effort to overcome resistance mechanisms . New molecular targets need to be identified to help the subset of patients who do not harbor P15056 mutations . DB01645 potentiates the P01160 effect on a K(+)-conductance in P29320 -293 cells . P29320 -293 cells are known to reflect many features of the late distal tubule . Furthermore , they have the ability to release urodilatin , the structural analog to P01160 . RT-PCR was performed to test for the expression of natriuretic peptide receptors . While the mRNA for the human P01160 receptor ( P16066 , P16066 ) could be amplified , the P09543 -specific receptor P20594 ( P20594 ) and the receptor specific for guanylins , P25092 , could not be detected . In patch clamp experiments the effects of P01160 ( 10 nM ) on membrane voltage ( V(m) ) were monitored and P29320 -293 cells depolarized by 2.3 +/- 0.5 mV ( n=14 ) . In the presence of the P01133 receptor blocker genistein ( 10 microM ) the effect of P01160 was increased by 65 % to 3.9 +/- 0.8 mV ( n=14 ) . After removal of genistein the P01160 -mediated depolarization further increased by 147 % to 5.7 +/- 1.0 mV ( n=14 ) . P01160 given repetitively without genistein had no increasing depolarizing effect in P29320 -293 cells with time . The P01160 effect could be fully blocked by 1 mM Ba(2+) and by 1 microM of the specific PKG inhibitor KT5823 indicating that P01160 inhibits a K(+)-conductance via a cGMP-dependent protein kinase . DB01645 itself hyperpolarized the membrane voltage of P29320 -293 cells by -3.9 +/- 0.6 mV ( n=11 ) and this effect could also be fully blocked by Ba(2+) ( -0.3 +/- 0.1 mV , n=5 ) , indicating that genistein activates a K(+)-conductance which contributes significantly to the membrane potential of P29320 -293 cells . DB01169 inhibits osteosarcoma cell invasiveness via MAPK signaling pathway . DB01169 ( As(2)O(3) ) is an active ingredient in traditional Chinese medicine . Recent studies showed that it causes apoptosis in several cancer cells . However , research of As(2)O(3) in osteosarcoma is sparse . In our present study , an inhibitory effect of As(2)O(3) on osteosarcoma cell adhesion and metastasis was observed with a cell adhesion , migration and invasion test . The impact of As(2)O(3) on the activities of P14780 and MAPK pathway-related downstream factors was analyzed by western blotting . Our results showed that As(2)O(3) significantly inhibited motility , migration and invasion in Q9UKB1 and MNNG cells in a concentration-dependent manner at concentrations ranging from 0.5-2 μM , and led to cytoskeletal rearrangements . As(2)O(3) exerted an inhibitory effect on the phosphorylation of P27361 /2 and MEK , which are the members of the MAPK family . Additionally , treatment with As(2)O(3) in combination with inhibitors specific for MEK ( U0126 ) in Q9UKB1 and MNNG cells resulted in a marked inhibition of cell invasion and As(2)O(3) could significantly reduce PMA-induced invasion . In conclusion , we demonstrate the inhibitory effects of As(2)O(3) on the invasiveness of Q9UKB1 and MNNG cells , which may be due at least partly to inactivation of the MAPK signaling pathway . Adipocyte pseudohypoxia suppresses lipolysis and facilitates benign adipose tissue expansion . Prolyl hydroxylase enzymes ( PHDs ) sense cellular oxygen upstream of hypoxia-inducible factor ( HIF ) signaling , leading to HIF degradation in normoxic conditions . In this study , we demonstrate that adipose Q9GZT9 inhibition plays a key role in the suppression of adipocyte lipolysis . Adipose Phd2 gene ablation in mice enhanced adiposity , with a parallel increase in adipose vascularization associated with reduced circulating nonesterified fatty acid levels and normal glucose homeostasis . Phd2 gene-depleted adipocytes exhibited lower basal lipolysis in normoxia and reduced β-adrenergic-stimulated lipolysis in both normoxia and hypoxia . A selective P20941 inhibitor suppressed lipolysis in murine and human adipocytes in vitro and in vivo in mice . Q9GZT9 genetic ablation and pharmacological inhibition attenuated protein levels of the key lipolytic effectors hormone-sensitive lipase and adipose triglyceride lipase ( Q96AD5 ) , suggesting a link between adipocyte oxygen sensing and fatty acid release . Q9GZT9 mRNA levels correlated positively with mRNA levels of AB-hydrolase domain containing-5 , an activator of Q96AD5 , and negatively with mRNA levels of lipid droplet proteins , perilipin , and O60664 in human subcutaneous adipose tissue . Therapeutic pseudohypoxia caused by Q9GZT9 inhibition in adipocytes blunts lipolysis and promotes benign adipose tissue expansion and may have therapeutic applications in obesity or lipodystrophy . A mechanism for the synergistic antimalarial action of atovaquone and proguanil . A combination of atovaquone and proguanil has been found to be quite effective in treating malaria , with little evidence of the emergence of resistance when atovaquone was used as a single agent . We have examined possible mechanisms for the synergy between these two drugs . While proguanil by itself had no effect on electron transport or mitochondrial membrane potential ( DeltaPsim ) , it significantly enhanced the ability of atovaquone to collapse DeltaPsim when used in combination . This enhancement was observed at pharmacologically achievable doses . DB01131 acted as a biguanide rather than as its metabolite cycloguanil ( a parasite dihydrofolate reductase [ P00374 ] inhibitor ) to enhance the atovaquone effect ; another P00374 inhibitor , pyrimethamine , also had no enhancing effect . DB01131 -mediated enhancement was specific for atovaquone , since the effects of other mitochondrial electron transport inhibitors , such as myxothiazole and antimycin , were not altered by inclusion of proguanil . Surprisingly , proguanil did not enhance the ability of atovaquone to inhibit mitochondrial electron transport in malaria parasites . These results suggest that proguanil in its prodrug form acts in synergy with atovaquone by lowering the effective concentration at which atovaquone collapses DeltaPsim in malaria parasites . This could explain the paradoxical success of the atovaquone-proguanil combination even in regions where proguanil alone is ineffective due to resistance . The results also suggest that the atovaquone-proguanil combination may act as a site-specific uncoupler of parasite mitochondria in a selective manner . Long-term outcomes in a family with nephrogenic syndrome of inappropriate antidiuresis . We report a familial case of the nephrogenic syndrome of inappropriate antidiuresis ( NSIAD ) , including 30-year followup data on two patients . The proband and one maternal uncle presented in their infancy with severe recurrent hyponatremia , and clinical pictures consistent with the syndrome of inappropriate antidiuretic hormone ( SIADH ) in the absence of an elevated DB00067 level . They were both confirmed to be hemizygous for the R137C mutation on the P30518 gene ( P30518 ) , the same locus of the gain of function mutation demonstrated in the original reports of this condition . The proband 's mother was identified as an asymptomatic carrier of this X-linked condition . Our case describes a favourable long-term outcome for NSIAD , in particular , successful treatment with oral urea during the infancy period , and with self-regulated precautions on fluid intake into adult life . Development of stable cell lines expressing different subtypes of GABAA receptors . The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors . For this , we developed an original two step selection strategy , in which the first step consisted of transfecting P29320 293 cells with rat GABAA receptor alpha and beta subunits . G 418 resistant colonies isolated at this step were screened for [ 3H ] muscimol binding to select for those that coexpressed alpha- and beta-subunits . The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant P00374 gene . After a second round of selection , this time in presence of methotrexate , those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [ 3H ] flumazenil as a probe . This strategy was applied to the isolation of 3 GABAA receptor clones , alpha 1 beta 2 gamma 2s , alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s , that expressed relatively high levels of these proteins . These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations . These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes . Mutational profiling of kinases in glioblastoma . BACKGROUND : Glioblastoma is a highly malignant brain tumor for which no cure is available . To identify new therapeutic targets , we performed a mutation analysis of kinase genes in glioblastoma . METHODS : Database mining and a literature search identified 76 kinases that have been found to be mutated at least twice in multiple cancer types before . Among those we selected 34 kinase genes for mutation analysis . We also included O75874 , P48735 , P60484 , P04637 and P01111 , genes that are known to be mutated at considerable frequencies in glioblastoma . In total , 174 exons of 39 genes in 113 glioblastoma samples from 109 patients and 16 high-grade glioma ( HGG ) cell lines were sequenced . RESULTS : Our mutation analysis led to the identification of 148 non-synonymous somatic mutations , of which 25 have not been reported before in glioblastoma . Somatic mutations were found in P04637 , P60484 , O75874 , P42336 , P00533 , P15056 , P29320 , P01111 , P37173 , P36888 and Q96S38 . Mapping the mutated genes into known signaling pathways revealed that the large majority of them plays a central role in the PI3K-AKT pathway . CONCLUSIONS : The knowledge that at least 50 % of glioblastoma tumors display mutational activation of the PI3K-AKT pathway should offer new opportunities for the rational development of therapeutic approaches for glioblastomas . However , due to the development of resistance mechanisms , kinase inhibition studies targeting the PI3K-AKT pathway for relapsing glioblastoma have mostly failed thus far . Other therapies should be investigated , targeting early events in gliomagenesis that involve both kinases and non-kinases . Gating properties of Q14524 mutations and the response to mexiletine in long-QT syndrome type 3 patients . BACKGROUND : DB00379 ( Mex ) has been proposed as a gene-specific therapy for patients with long-QT syndrome type 3 ( LQT3 ) caused by mutations in the cardiac sodium channel gene ( Q14524 ) . The degree of QT shortening and the protection from arrhythmias vary among patients harboring different mutations . We tested whether the clinical response to Mex in LQT3 could be predicted by the biophysical properties of the different mutations . METHODS AND RESULTS : We identified 4 Q14524 mutations in 5 symptomatic LQT3 patients with different responses to Mex ( 6 to 8 mg . kg(-1) . d(-1) ) . We classified the mutations as sensitive to Mex ( P1332L , R1626P ; >/= 10 % of QTc shortening and QTc < 500 ms or no arrhythmias ) or insensitive to Mex ( S941N , M1652R ; negligible or no QTc shortening and sudden death ) . We measured Na(+) current from P29320 293 cells transfected with wild-type ( WT ) or mutant Nav1.5 . All mutations showed impaired inactivation of Na(+) current , but the mutations identified in patient responders to Mex ( P1332L , R1626P ) showed a hyperpolarizing shift of V(1/2) of steady-state inactivation . Furthermore , Mex produced use-dependent block with the order R1626P=P1332L > S941N=WT > M1652R , suggesting that Mex-sensitive mutants present prolonged recovery from Mex block . CONCLUSIONS : We propose that voltage dependence of channel availability and shifts of V(1/2) of steady-state inactivation correlate with the clinical response observed in LQT3 patients . This supports the view that the response to Mex is mutation specific and that in vitro testing may help to predict the response to therapy in LQT3 . Phosphorylation and recycling kinetics of G protein-coupled receptors . The rate of ligand-induced phosphorylation of the V2 and V1a vasopressin receptors was characterized in P29320 293 cells . Both receptors were phosphorylated predominantly by GRKs , and the V1a receptor was also phosphorylated by protein kinase C regardless of the presence or absence of ligand . Phosphorylation of the P37288 catalyzed by GRKs reached maximal values at the shortest measured time : 15 seconds , and decayed rapidly with a t1/2 of 6 min in the continuous presence of AVP . In agreement with the hypothesis that dephosphorylation must precede receptor recycling to the cell surface , the P37288 returned rapidly to the cell surface after removal of the hormone from the medium . Phosphate incorporation into the P30518 proceeded at a slower pace , and the internalized phosphorylated receptor failed to recycle to the cell surface and retained its phosphate for a long time in the presence or absence of ligand . A single mutation in the carboxy terminus of the P30518 accelerated de-phosphorylation of the protein and conferred recycling properties to the P30518 . These experiments provided molecular evidence for the hypothesis that internalization is required for de-phosphorylation and recycling of reactivated G protein coupled receptors to the cell surface . Establishment of a cell-based drug screening model for identifying agonists of human peroxisome proliferator-activated receptor gamma ( PPARγ ) . OBJECTIVES : P37231 ( PPARγ ) plays a critical role in regulation of diverse biological processes , including lipid metabolism and adipogenesis , cell division and apoptosis , and is involved in variety of disease conditions , such as obesity , atherosclerosis , inflammation and tumour . Developing a cell-based reporter gene model targeting PPARγ would be useful to screen human PPARγ agonists that could be beneficial to patients with these diseases . METHODS : We stably co-transfected human embryonic kidney ( P29320 ) cell line 293T cells with phPPARγ-IRES2-EGFP vector to express human PPARγ ( hPPARγ ) , a reporter vector pPPRE×3-TK-LUC , and control vector pRL-CMV . The efficiency of the co-transfection was evaluated with flow cytometry of hPPARγ expressing cells . Specificity of hPPARγ activity was determined by dual luciferase reporter assay of co-transfected cells exposed to PPARγ agonist rosiglitazone , PPARα agonist WY14643 and retinoic acid receptor alpha ( RARα ) agonist all-trans-retinoic acid ( DB00755 ) . KEY FINDINGS : The phPPARγ-IRES2-EGFP co-transfected HEK293T cells showed concentration- and time-dependent luciferase induction upon exposure to the rosiglitazone , while WY14643 and DB00755 were unable to activate the co-transfected HEK293T cells . CONCLUSIONS : These data indicated that the HEK293T cells could be stably transfected with hPPARγ . This cell-based drug screening platform could be used targeting specific nuclear receptor of hPPARγ with effectiveness and specificity for hPPARγ agonists discovery . Additive role of tiotropium in severe asthmatics and Arg16Gly in P07550 as a potential marker to predict response . BACKGROUND : Recent findings have raised new interests about the use of anticholinergics , especially tiotropium , for the treatment of asthma . This study was performed to determine whether an additional improvement in lung function is obtained when tiotropium is administrated in addition to conventional therapies in severe asthmatics , and to identify factors capable of predicting the response to tiotropium , using a pharmacogenetic approach . METHODS : A total of 138 severe asthmatics on conventional medications and with decreased lung function were randomly recruited . DB01409 18 microg was added once a day and lung functions were measured every 4 weeks . Responders were defined as those with an improvement of > or = 15 % ( or 200 ml ) in the forced expiratory volume in 1 s ( FEV1 ) that was maintained for at least 8 successive weeks . Eleven single nucleotide polymorphisms ( SNPs ) in P11229 -3 ( coding muscarinic receptors one to three ) which were identified by re-sequencing , and Arg16Gly and Gln27Glu in P07550 ( coding beta(2) adrenoreceptor ) were scored in 80 of the 138 asthmatics . RESULTS : Forty-six of the 138 asthmatics ( 33.3 % ) responded to tiotropium treatment . Logistic regression analyses ( controlled for age , gender , and smoking status ) showed that Arg16Gly in P07550 [ P = 0.003 , OR ( 95 % CI ) = 0.21 ( 0.07-0.59 ) in a minor allele-dominant model ] was significantly associated with response to tiotropium . CONCLUSIONS : As many as 30 % of severe asthmatics on conventional medications with reduced lung function were found to respond to adjuvant tiotropium . The presence of Arg16Gly in P07550 may predict response to tiotropium . Transcriptional regulation of Q96NT5 by O43474 , HNF4α , Q99626 and C/EBPα : implication in its site-specific expression in the small intestine . Q96NT5 ( Q96NT5 ) , which is responsible for the intestinal uptake of folates and analogs , is expressed only in the proximal region in the small intestine . The present study was to examine its transcriptional regulation , which may be involved in such a unique expression profile and potentially in its alteration , using dual-luciferase reporter assays in human embryonic kidney ( P29320 ) 293 cells . The luciferase activity derived from the reporter construct containing the 5'-flanking sequence of -1695/+96 of the human Q96NT5 gene was enhanced most extensively by the introduction of Krüppel-like factor 4 ( O43474 ) . The O43474 -induced luciferase activity was further enhanced by hepatocyte nuclear factor 4α ( HNF4α ) synergistically . To the contrary , caudal-type homeobox transcription factor 2 ( Q99626 ) and CCAAT/enhancer-binding protein α ( C/EBPα ) extensively suppressed the luciferase activity induced by O43474 alone and also that induced by O43474 and HNF4α . Western blot analysis using the rat small intestine indicated uniform expression of O43474 along the intestinal tract , proximal-oriented expression of HNF4α , distal-oriented expression of Q99626 and C/EBPα . These results suggest that the activity of Q96NT5 promoter is basically induced by O43474 and the gradiented expression profile of Q96NT5 may be at least in part accounted for by those of HNF4α , Q99626 and C/EBPα . DB00126 is dispensable for oxygen sensing in vivo . Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors ( HIFs ) . In vitro function of HIF prolyl-4-hydroxylase domain ( P20941 ) enzymes requires oxygen and 2-oxoglutarate as cosubstrates with iron(II) and vitamin C serving as cofactors . Although vitamin C deficiency is known to cause the collagen-disassembly disease scurvy , it is unclear whether cellular oxygen sensing is similarly affected . Here , we report that vitamin C-deprived Gulo(-/-) knockout mice show normal HIF-dependent gene expression . The systemic response of Gulo(-/-) animals to inspiratory hypoxia , as measured by plasma erythropoietin levels , was similar to that of animals supplemented with vitamin C . Hypoxic HIF induction was also essentially normal under serum- and vitamin C-free cell-culture conditions , suggesting that vitamin C is not required for oxygen sensing in vivo . Glutathione was found to fully substitute for vitamin C requirement of all 3 P20941 isoforms in vitro . Consistently , glutathione also reduced HIF-1α protein levels , transactivation activity , and endogenous target gene expression in cells exposed to CoCl(2) . A Cys201Ser mutation in Q9GZT9 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro , suggesting that this surface-accessible Q9GZT9 cysteine residue is a target of antioxidative protection by vitamin C and glutathione . DB00169 and its nuclear receptor increase the expression and activity of the human proton-coupled folate transporter . Folates are essential for nucleic acid synthesis and are particularly required in rapidly proliferating tissues , such as intestinal epithelium and hemopoietic cells . Availability of dietary folates is determined by their absorption across the intestinal epithelium , mediated by the proton-coupled folate transporter ( Q96NT5 ) at the apical enterocyte membranes . Whereas transport properties of Q96NT5 are well characterized , regulation of Q96NT5 gene expression remains less elucidated . We have studied the mechanisms that regulate Q96NT5 promoter activity and expression in intestine-derived cells . Q96NT5 mRNA levels are increased in Caco-2 cells treated with 1,25-dihydroxyvitamin D(3) ( vitamin D(3) ) in a dose-dependent fashion , and the duodenal rat Pcft mRNA expression is induced by vitamin D(3) ex vivo . The Q96NT5 promoter region is transactivated by the vitamin D receptor ( P11473 ) and its heterodimeric partner retinoid X receptor-alpha ( RXRalpha ) in the presence of vitamin D(3) . In silico analyses predicted a P11473 response element ( VDRE ) in the Q96NT5 promoter region -1694/-1680 . DNA binding assays showed direct and specific binding of the P11473 :RXRalpha heterodimer to the Q96NT5 (-1694/-1680) , and chromatin immunoprecipitations verified that this interaction occurs within living cells . Mutational promoter analyses confirmed that the Q96NT5 (-1694/-1680) motif mediates a transcriptional response to vitamin D(3) . In functional support of this regulatory mechanism , treatment with vitamin D(3) significantly increased the uptake of [(3)H]folic acid into Caco-2 cells at pH 5.5 . In conclusion , vitamin D(3) and P11473 increase intestinal Q96NT5 expression , resulting in enhanced cellular folate uptake . Pharmacological treatment of patients with vitamin D(3) may have the added therapeutic benefit of enhancing the intestinal absorption of folates . Stereospecific interaction of a novel spirosuccinimide type aldose reductase inhibitor , DB05327 , with aldose reductase . P15121 ( AR ) is an NADPH-dependent enzyme implicated in diabetic complications . DB05327 [ ( R ) -(-)-2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-4-spiro-3'-pyrrolidine-1,2',3,5'-tetrone ] is a structurally novel and potent Q9Y4X5 with an inhibitor constant ( K(i) = 10(-)(10) M ) 2000-fold lower than that of its optical antipode ( S-isomer ) . To elucidate the inhibition modes and the stereochemical differences in their inhibitory potencies , we examined the interaction of these R- and S-isomers with AR under physiological conditions . Enzyme kinetic analysis , which was performed by using physiological substrates at 37 degrees C , showed that both isomers selectively act on the E-NADP(+) complex in both the forward and reverse reactions of AR . However , fluorometric titration analysis demonstrated that the affinities of the isomers for the E-NADP(+) complex are about the same as those for the E-NADPH complex and the apoenzyme . These results suggested that the selective binding to the E-NADP(+) complex arises from the predominance of this enzyme form during steady-state turnover rather than from binding specificity . Both the competition with a known active site-directed Q9Y4X5 and the protective effect on AR inactivation by N-bromosuccinimide showed that the isomers bind to the active site of the enzyme , but the thermodynamic parameters for the binding to AR indicated that additional hydrogen bonds and/or van der Waals interactions contribute to the energetic stabilization in the E-R-isomer complex . Molecular modeling , together with the deductions from spectroscopic studies , suggested that the succinimide ring and the 4-bromo-2-fluorobenzyl group of the R-isomer are optimally located for formation of a hydrogen-bonding network with AR , and that the latter benzyl group is also effective for the differentiation between AR and aldehyde reductase ( a closely related enzyme ) . Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover . Although neutral lipid storage droplets are ubiquitous in eukaryotic cells , very little is known about how their synthesis and turnover are controlled . Adipocyte differentiation-related protein ( Q99541 ; also known as adipophilin ) is found on the surface of lipid droplets in most mammalian cell types . To learn how Q99541 affects lipid storage , we stably expressed the protein in human embryonic kidney 293 ( P29320 293 ) cells , which express little endogenous Q99541 . As expected , Q99541 was targeted to the surface of lipid droplets and caused an increase in triacylglycerol ( TAG ) mass under both basal and oleate-supplemented conditions . At least part of the increased mass resulted from a 50 % decrease in the rate of TAG hydrolysis in Q99541 -expressing cells . Furthermore , Q99541 expression increased the fraction of total cellular TAG that was stored in lipid droplets . Q99541 expression induced a striking decrease in the association of adipose triglyceride lipase ( Q96AD5 ) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins . Transient expression of Q99541 in two other cell lines also reduced the lipid droplet association of catalytically inactive Q96AD5 . We conclude that the reduced lipid droplet association of Q96AD5 and/or other lipases may explain the decrease in TAG turnover observed in Q99541 -expressing P29320 293 cells . Q9H4A3 activates large-conductance Ca2+-activated K+ channels through modulation of P27361 /2 signaling . With no lysine ( WNK ) kinases are members of the serine/threonine kinase family . We previously showed that Q96J92 inhibits renal large-conductance Ca(2+)-activated K(+) ( BK ) channel activity by enhancing its degradation through a lysosomal pathway . In this study , we investigated the effect of Q9H4A3 on Q12791 activity . In HEK293 cells stably expressing the α subunit of BK ( P29320 -BKα cells ) , siRNA-mediated knockdown of Q9H4A3 expression significantly inhibited both BKα channel activity and open probability . Knockdown of Q9H4A3 expression also significantly inhibited BKα protein expression and increased P27361 /2 phosphorylation , whereas overexpression of Q9H4A3 significantly enhanced BKα expression and decreased P27361 /2 phosphorylation in a dose-dependent manner in HEK293 cells . Knockdown of P27361 /2 prevented Q9H4A3 siRNA-mediated inhibition of BKα expression . Similarly , pretreatment of P29320 -BKα cells with the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of Q9H4A3 siRNA on BKα expression in a dose-dependent manner . Knockdown of Q9H4A3 expression also increased the ubiquitination of BKα channels . Notably , mice fed a high-K(+) diet for 10 days had significantly higher renal protein expression levels of BKα and Q9H4A3 and lower levels of P27361 /2 phosphorylation compared with mice fed a normal-K(+) diet . These data suggest that Q9H4A3 enhances Q12791 function by reducing P27361 /2 signaling-mediated lysosomal degradation of the channel . The role of peroxisome proliferator-activated receptor-gamma and estrogen receptors in genistein-induced regulation of vascular tone in female rat aortas . BACKGROUND : DB01645 ( Q86UG4 ) is a phytoestrogen that binds estrogen receptors ( ER ) to produce a protective cardiovascular effect . It also has been shown binding peroxisome proliferator-activated receptor-gamma ( P37231 ) . METHODS : In the present study , we assessed the role of P37231 and ER in Q86UG4 -mediated regulation of vascular tone in female rat aortas by in vitro tension measurement , immunohistochemistry , immunofluorescence , immunoprecipitation and Western blot analysis . RESULTS : In aortas pretreated with GW9662 ( inhibitor of P37231 ) , ICI182780 ( inhibitor of ER ) and a combination of GW9662 and ICI182780 , the magnitudes of Q86UG4 -induced dilatation were attenuated . N(G)-nitro-L-arginine methyl ester had a similar effect . Q92731 and P37231 colocalized in all 3 layers of the aortas , while P03372 and P37231 colocalized only in the vascular endothelium and adventitia . In Q86UG4 -treated whole-cell protein samples , we demonstrated coimmunoprecipitation of Q92731 ( not P03372 ) and P37231 . The expression of Q92731 and P37231 in nuclear protein from Q86UG4 -treated samples increased , which was partially reversed by either GW9662 or ICI182780 and more efficiently reversed using a combination of GW9662 and ICI182780 . CONCLUSION : Our findings suggest that Q86UG4 can relax phenylephrine-induced vascular contraction in female rat aortas , which is mediated by P37231 and Q92731 . Mass spectrometric analysis of agonist effects on posttranslational modifications of the beta-2 adrenoceptor in mammalian cells . Posttranslational modifications ( PTMs ) of the beta-2 adrenoceptor ( P07550 ) play a fundamental role in receptor regulation by agonists . We have examined the effects of several agonists on net levels of P07550 palmitoylation and phosphorylation using epitope tagging in stably transfected human embryonal kidney ( P29320 ) 293 cells , immunoaffinity purification , and mass spectrometry combined with the method of stable isotope labeling by amino acids in cell culture ( SILAC ) . Palmitoylation of Cys341 was confirmed and did not change detectably after 30 min exposure of cells to saturating concentrations of dopamine , epinephrine , or isoproterenol . However , all of these agonists produced a marked increase in net phosphorylation . Phosphorylation of the third cytoplasmic loop was increased to a similar degree by all three agonists , whereas differences between agonists were observed in net phosphorylation of the carboxyl-terminal cytoplasmic domain ( isoproterenol approximately epinephrine >> dopamine ) . Interestingly , agonist-induced phosphorylation of the carboxyl-terminal cytoplasmic domain was observed exclusively in a proximal portion ( between residues 339-369 ) . None of the agonists produced detectable phosphorylation in a distal portion of the cytoplasmic tail , which contains all sites of agonist-induced phosphorylation identified previously by in vitro reconstitution . These results provide insight to agonist-dependent regulation of the P07550 in intact cells , suggest the existence of significant differences in regulatory phosphorylation events occurring between in vitro and in vivo conditions , and outline a general analytical approach to investigate regulated PTM of receptors in mammalian cells .