interacts_with DB03615?
Genetic markers in the EET metabolic pathway are associated with outcomes in patients with aneurysmal subarachnoid hemorrhage . Preclinical studies show that epoxyeicosatrienoic acids ( EETs ) regulate cerebrovascular tone and protect against cerebral ischemia . We investigated the relationship between polymorphic genes involved in EET biosynthesis/metabolism , cytochrome P450 ( CYP ) eicosanoid levels , and outcomes in 363 patients with aneurysmal subarachnoid hemorrhage ( aSAH ) . Epoxyeicosatrienoic acids and dihydroxyeicosatetraenoic acid ( DHET ) cerebrospinal fluid ( P04141 ) levels , as well as acute outcomes defined by delayed cerebral ischemia ( P42126 ) or clinical neurologic deterioration ( CND ) , were assessed over 14 days . Long-term outcomes were defined by Modified Rankin Scale ( P59665 ) at 3 and 12 months . P10632 *4 allele carriers had 44 % and 36 % lower mean EET and DHET P04141 levels ( P=0.003 and P=0.007 ) and were 2.2- and 2.5-fold more likely to develop P42126 and CND ( P=0.039 and P=0.041 ) , respectively . P34913 55Arg , P51589 *7 , P10632 *1B , and P10632 g.36785A allele carriers had lower EET and DHET P04141 levels . P10632 g.25369T and P10632 g.36755A allele carriers had higher EET levels . Patients with P10632 *2C and P34913 404del variants had worse long-term outcomes while those with P34913 287Gln , P51589 *7 , and P11712 g.816G variants had favorable outcomes . Epoxyeicosatrienoic acid levels were associated with Fisher grade and unfavorable 3-month outcomes . Dihydroxyeicosatetraenoic acids were not associated with outcomes . No associations passed Bonferroni multiple testing correction . These are the first clinical data demonstrating the association between the EET biosynthesis/metabolic pathway and the pathophysiology of aSAH . Leishmania major protein disulfide isomerase as a drug target : enzymatic and functional characterization . Leishmaniasis is a major health problem worldwide and tools available for their control are limited . Effective vaccines are still lacking , drugs are toxic and expensive , and parasites develop resistance to chemotherapy . In this context , new antimicrobials are urgently needed to control the disease in both human and animal . Here , we report the enzymatic and functional characterization of a Leishmania virulence factor , Leishmania major Protein disulfide isomerase ( LmPDI ) that could constitute a potential drug target . LmPDI possesses domain structure organization similar to other P07237 family members ( a , a ' , b , b ' and c domains ) , and it displays the three enzymatic and functional activities specific of P07237 family members : isomerase , reductase and chaperone . These results suggest that LmPDI plays a key role in assisting Leishmania protein folding via its capacity to catalyze formation , breakage , and rearrangement of disulfide bonds in nascent polypeptides . Moreover , DB00626 , a reductase activity inhibitor , and DB03615 , a chaperone activity inhibitor , were tested in LmPDI enzymatic assays and versus Leishmania promastigote in vitro cultures and Leishmania amastigote multiplication inside infected THP-1-derived macrophages . DB00626 inhibited both isomerase and reductase activities , while DB03615 had no effect on the chaperone activity . Interestingly , DB00626 blocked in vitro promastigote growth as well as amastigote multiplication inside macrophages with EC(50) values of 39 μM . These results suggest that LmPDI may constitute an interesting target for the development of new anti-Leishmania drugs . P10275 is expressed in murine choroid plexus and downregulated by 5alpha-dihydrotestosterone in male and female mice . The choroid plexuses ( CPs ) of the brain form a unique interface between the peripheral blood and the cerebrospinal fluid ( P04141 ) . CPs produce several neuroprotective peptides , which are secreted into the P04141 . Despite their importance in neuroprotection , the mechanisms underlying the regulation of most of these peptides in CPs remain unknown . Androgens regulate the expression of neuroprotective peptides in several tissues where the androgen receptor ( AR ) is coexpressed , including the brain . The presence of AR in CPs has never been investigated , but recent studies in our laboratory show that the CP is an androgen-responsive tissue . In order to fulfill this gap , we investigated and characterized AR distribution and expression in male and female rat CPs and in primary cultures from rat CP epithelial cells . In addition , the response of AR to 5alpha-dihydrotestosterone ( DB02901 ) in castrated male and female mice subjected to DB02901 replacement was analyzed . We show that rat CP epithelial cells contain AR mRNA and protein . Moreover , we demonstrate that AR is downregulated by DB02901 in mice CPs . Systemic sclerosis - a systematic overview : part 2 - immunosuppression , treatment of SSc-associated vasculopathy , and treatment of pulmonary arterial hypertension . Here we give an overview over treatment recommendations propagated by the European League Against Rheumatism ( EULAR ) , EULAR Scleroderma Trials and Research Group , the German Network for Systemic Sclerosis , the European Respiratory Society , and the International Society of Heart and Lung Transplantation . As response to immunosuppressant ( IS ) therapy is usually weaker in systematic sclerosis ( SSc ) compared to other connective tissue disorders IS should be considered with caution . To prevent scleroderma renal crisis steroid doses should not exceed 15 mg/d . The definitive role of a number of new immunosuppressant drugs and the effects of autologous stem cell transplantation in systemic clerosis ( SSc ) have to be elucidated . Prostanoids , especially iloprost , are widely used as intravenous formulas for the treatment of severe Raynaud 's phenomenon ( RP ) and digital ulcers ( DU ) . DB01373 antagonists are of limited therapeutic value . DB00559 , an oral endothelin receptor antagonists ( P25101 ) , was shown to prevent new DU , but failed to heal existing DU , while the oral phopshodiesterase inhibitor ( P07237 ) DB00203 reduces the occurrence of RP and might be effective in ulcer healing . Combination therapies of P07237 with P25101 are currently evaluated . Therapy of pulmonary arterial hypertension ( PAH ) is usually started as oral monotherapy , frequently using an P25101 . When this first-line therapy is not tolerated P25101 is substituted by P07237 . If treatment goals are not reached with monotherapy combinationtherapy is started , for example by adding a P07237 to an existing P25101 . In general , treatment of PAH in patients with connective tissue disease follows the same algorithms as in idiopathic PAH . The antiangiogenic effects of a vascular endothelial growth factor decoy receptor can be monitored in vivo using contrast-enhanced ultrasound imaging . The development of antiangiogenic therapies has stimulated interest in noninvasive imaging methods to monitor response . We investigated whether the effects of a vascular endothelial growth factor decoy receptor ( DB08885 , Regeneron Pharmaceuticals , Tarrytown , NY ) could be monitored in vivo using contrast-enhanced ultrasonography ( CEUS ) . Twenty nude mice ( in two groups ) were implanted with a human melanoma cell line ( DB-1 ) . The active group received DB08885 ( 4 × 25 mg/kg over 2 weeks ) , whereas the control group received an inactive protein . An ultrasound contrast agent was injected followed by power Doppler imaging ( P07237 ) and pulse inversion harmonic imaging ( PIHI ; regular and intermittent ) . Specimens were sectioned in the same planes as the images and stained for endothelial cells ( CD31 ) , cyclooxygenase-2 ( P35354 ) , P15692 , and hypoxia ( Glut1 ) . Measures of tumor vascularity obtained with the different imaging modes were compared to immunohistochemical markers of angiogenesis . Mean tumor volume was smaller in the active group than in the control group ( 656 ± 225 vs 1,160 ± 605 mm3 ) . Overall , P07237 and P15692 correlated ( r = .34 ; p = .037 ) . Vascularity decreased from control to treated mice with intermittent PIHI , as did the expression of CD31 and P35354 ( p ≤ .02 ) , whereas P15692 increased ( p = .05 ) . CEUS appears to allow in vivo monitoring of the antiangiogenic effects of DB08885 in the DB-1 human melanoma xenograft model . [ Quantitative analysis of P11387 activity in human and rat glioma : characterization and mechanism of resistance to antitopoisomerase chemical , camptothecin-11 ] . DB00762 ( CPT-11 ) is a new derivation of camptothecin , a plant alkaloid antitumor agent . Previous studies indicated that antitumor activity of CPT-11 was mediated through interaction of the drugs with its target enzyme , P11387 ( topo I ) . In this study , we studied the relation between sensitivity to CPT-11 and topo I activity of glioma cells . Furthermore , we established CPT-11 resistant cell lines in order to elucidate potential mechanisms of drug resistance . A clear correlation between the sensitivities to CPT-11 and topo I activities in surgical glioma specimens was demonstrated . Activities of topo I in CPT-11 sensitive group ( IC50 values for CPT-11 ; < 50 micrograms/ml ) tended to be higher than those in CPT-11 resistant group ( IC50 values ; > or = 50 ) . Topo I activity may serve as a novel marker to predict the sensitivity of gliomas to topo inhibitors . CPT-11 resistance cell lines ( T98G/CPT-11 and P13671 ) respectively exhibit a 5.4- and 7.3-fold increase in resistance to CPT-11 . No differences in topo I activity and intracellular accumulation of CPT-11 were observed between parent and CPT-11 resistant lines . On the other hand , topo I from T98G/CPT-11 and P13671 /CPT-11 cells were at least 4- and 2-fold resistant to the inhibitory effect of the CPT-11 on the relaxation activity of topo I in comparison with their parent lines . This enzymological difference may be responsible for the resistance to CPT-11 . DB01098 , a new P04035 inhibitor , reduces the colonic inflammatory response in dextran sulfate sodium-induced colitis in mice . The aim of the present study was to elucidate the beneficial effects of rosuvastatin , a new P04035 inhibitor , on colonic mucosal damage and on the inflammatory response in a dextran sulfate sodium ( DSS ) colitis model . Acute colitis was induced using 8 % DSS in female BALB/c mice . Colonic mucosal inflammation was evaluated clinically , biochemically , and histologically . Mucosal protein contents and mRNA levels of tumor necrosis factor ( P01375 ) -alpha were determined by immunoassay and real time-PCR . The mRNA levels of endothelial nitric oxide synthase ( P29474 ) were determined by real-time PCR . Disease activity scores in DSS-induced colitis model mice , as determined by weight loss , stool consistency , and blood in stool , were significantly lower in the rosuvastatin-treated mice than in control mice . Shortening of the colon was significantly reversed by rosuvastatin . Increases in tissue-associated myeloperoxidase activity and thiobarbituric acid-reactive substances after DSS administration were both significantly inhibited by treatment with rosuvastatin . DB01098 also inhibited increases in intestinal P01375 protein and mRNA expression after DSS administration , respectively . The mucosal mRNA levels of P29474 were decreased after DSS administration , but preserved in mice treated with rosuvastatin . These results suggest that rosuvastatin prevents the development of DSS-induced colitis in mice via the inhibition of mucosal inflammatory responses associated with the preservation of P29474 transcription . Surface proteins of P13671 /36 cells involved in dengue virus 4 binding and entry . Dengue virus ( DENV ) is the causative agent of the most important mosquito-borne viral disease , which is endemic to over 100 countries in tropical and subtropical areas of the world . It is transmitted to humans by Aedes mosquitoes . The first step in the viral infection of host cells is virion attachment to the plasma membrane , which is mediated by specific surface molecules . There are several molecules that participate in DENV infection of mosquitoes , but only a few have been identified . In this work , we co-purified 4 proteins from P13671 /36 cells using a recombinant DENV 4 E protein and identified them as 70 kDa Heat Shock and 70 kDa Heat Shock cognate proteins ( HSP70/HSc70 ) , Binding immunoglobulin protein ( P11021 ) , P10599 /protein disulphide isomerase ( P07237 ) , and 44 kDa Endoplasmic reticulum resident protein ( Q9BS26 ) via matrix-assisted laser desorption/ionisation time of flight ( Maldi-ToF ) analysis . Using immunofluorescence and flow cytometry assays , we observed re-localisation of HSP70/HSc70 and , to a lesser extent , P11021 to the plasma membrane under stress conditions , such as during DENV infection . By performing binding and infection assays independently , we found that all 4 proteins participate in both processes , but to differing extents : HSP70/HSc70 is the most critical component , while Q9BS26 is less important . Viral infection was not inhibited when the cells were incubated with antibodies against all of the surface proteins after virus binding , which suggests that DENV entry to P13671 /36 cells is mediated by these proteins at the same step and not sequentially . TGBp3 triggers the unfolded protein response and P63208 -dependent programmed cell death . The Potato virus X ( PVX ) triple gene block protein 3 ( TGBp3 ) , an 8-kDa membrane binding protein , aids virus movement and induces the unfolded protein response ( UPR ) during PVX infection . TGBp3 was expressed from the Tobacco mosaic virus ( TMV ) genome ( TMV-p3 ) , and we noted the up-regulation of P63208 and several endoplasmic reticulum ( ER ) -resident chaperones , including the ER luminal binding protein ( P11021 ) , protein disulphide isomerase ( P07237 ) , calreticulin ( CRT ) and calmodulin ( P62158 ) . Local lesions were seen on leaves inoculated with TMV-p3 , but not TMV or PVX . Such lesions were the result of TGBp3-elicited programmed cell death ( P61457 ) , as shown by an increase in reactive oxygen species , DNA fragmentation and induction of P63208 expression . UPR-related gene expression occurred within 8 h of TMV-p3 inoculation and declined before the onset of P61457 . TGBp3-mediated cell death was suppressed in plants that overexpressed P11021 , indicating that UPR induction by TGBp3 is a pro-survival mechanism . Anti-apoptotic genes Bcl-xl , DB01333 -9 and Op-IAP were expressed in transgenic plants and suppressed N gene-mediated resistance to TMV , but failed to alleviate TGBp3-induced P61457 . However , TGBp3-mediated cell death was reduced in P63208 -silenced Nicotiana benthamiana plants . The combined data suggest that TGBp3 triggers the UPR and elicits P61457 in plants . A case study of acenocoumarol sensitivity and genotype-phenotype discordancy explained by combinations of polymorphisms in Q9BQB6 and P11712 . To determine the cause of a genotype-phenotype discordancy for acenocoumarol sensitivity . Methods A patient , highly sensitive to acenocoumarol , and previously determined to carry only a single P11712 *3 allele , was genotyped for additional functionally defective alleles in the P11712 and Q9BQB6 genes . Family members were also analyzed to trace the pedigree . Results The acenocoumarol-sensitive patient was found to possess , in addition to P11712 *3 allele , a P11712 *11 allele and the Q9BQB6 AA diplotype which were all traced back through the parental lines . Conclusions DB01418 sensitivity in this subject is the consequence of inheritance of multiple functionally defective alleles in both the P11712 and Q9BQB6 genes . The study provides additional data in support of diminished P11712 activity due to the presence of the rare *11 allele . DB03615 inhibits the chaperone activity of protein disulfide isomerase . In the process of screening of proteins binding to ribostamycin in bovine liver using the affinity column chromatography , we found that ribostamycin inhibited the chaperone activity of protein disulfide isomerase ( P07237 ) , but it did not inhibit the isomerase activity . P07237 was identified by SDS-PAGE , Western blotting , and N-terminal amino acid sequence analysis . A 100:1 molar ratio of ribostamycin to P07237 was almost sufficient to completely inhibit the chaperone activity of P07237 . The binding affinity of ribostamycin to purified bovine P07237 was determined by the Biacore system , which gave a K(D) value of 3.19 x 10(-4) M . This suggests that ribostamycin binds to region distinct from the CGHC motif of P07237 . This is the first report to describe the inhibitor of the chaperone activity of P07237 . Cardiac channelopathies associated with infantile fatal ventricular arrhythmias : from the cradle to the bench . BACKGROUND : Fatal ventricular arrhythmias in the early period of life have been associated with cardiac channelopathies for decades , and postmortem analyses in P22304 victims have provided evidence of this association . However , the prevalence and functional properties of cardiac ion channel mutations in infantile fatal arrhythmia cases are not clear . METHODS AND RESULTS : Seven infants with potentially lethal arrhythmias at age < 1 year ( 5 males , age of onset 44.1 ± 72.1 days ) were genetically analyzed for P51787 , Q12809 , P15382 -5 , P63252 , Q14524 , P36382 , and P62158 by using denaturing high-performance liquid chromatography and direct sequencing . Whole-cell currents of wildtype and mutant channels were recorded and analyzed in Chinese hamster ovary cells transfected with Q14524 and Q12809 cDNA . In 5 of 7 patients , we identified 4 mutations ( p.N1774D , p.T290fsX53 , p.F1486del and p.N406K ) in Q14524 , and 1 mutation ( p.G628D ) in Q12809 . N1774D , F1486del , and N406K in Q14524 displayed tetrodotoxin-sensitive persistent late Na(+) currents . By contrast , Q14524 -T290fsX53 was nonfunctional . Q12809 -G628D exhibited loss of channel function . CONCLUSION : Genetic screening of 7 patients was used to demonstrate the high prevalence of cardiac channelopathies . Functional assays revealed both gain and loss of channel function in Q14524 mutations , as well as loss of function associated with the Q12809 mutation . P07237 -mediated ER retention and proteasomal degradation of procollagen I in corneal endothelial cells . Procollagen I in corneal endothelial cells ( CECs ) is intracellularly degraded immediately after its synthesis . In this study , we investigated the mechanism of intracellular degradation of procollagen I by determining the role of protein disulfide isomerase ( P07237 ) in endoplasmic reticulum ( ER ) retention and further determined the degradation pathway of procollagen I in CECs . When association of P07237 to monomeric proalpha chains or the trimeric procollagen I carboxyl propeptides ( PICPs ) was analyzed , immune complex precipitated with anti-PICP antibody contained more P07237 than that precipitated with antibodies to monomeric chains . PICPs were completely colocalized with P07237 . When CECs were transfected with P07237 vector , procollagen I and the recombinant P07237 were colocalized in the ER , whereas CECs transfected with P07237 minus KDEL ( the ER retrieval sequence ) vector demonstrated that the two proteins were localized in the Golgi and were subsequently secreted into the medium . DB03615 ( an inhibitor of the chaperone activity of P07237 ) blocked colocalization of P07237 and procollagen I . Cells treated with chloroquine ( lysosome inhibitor ) did not alter the subcellular localization of procollagen I , because the inhibitor failed to induce the accumulation of procollagen I at Golgi . On the other hand , procollagen I was colocalized with ubiquitin in the cytoplasm , and proteasomal inhibitors further facilitated the colocalization of the two proteins and accumulation of ubiquitinated procollagen I ladders . These results suggest that association of P07237 with procollagen I , whether monomeric or trimeric , leads to ER retention of procollagen I before intracellular degradation via the ubiquitin-proteasome pathway . Development and evaluation of high throughput functional assay methods for Q12809 potassium channel . Three functional hERG channel assay methods have been developed and evaluated . The methods were tested against five known hERG channel inhibitors : dofetilide , terfenadine ( Seldane ) , sertindole ( DB06144 ) , astemizole ( Hismanal ) , and cisapride ( Propulsid ) . The DiBAC4(3)-based assays were found to be the most economical but had high false-hit rates as a result of the interaction of dye with the test compounds . The membrane potential dye assay had fewer color-quenching problems but was expensive and still gave false hits . The nonradioactive Rb+ efflux assay was the most sensitive of all the assays evaluated and had the lowest false-hit rate . [ DB09053 : A new drug of B-cell malignancies ] . DB09053 ( Imbruvica® ) is a first-in-class , orally administered once-daily , that inhibits B-cell antigen receptor signaling downstream of Bruton 's tyrosine kinase ( Q06187 ) . DB09053 has been approved in USA in February 2014 and in France in October 2014 for the treatment of patients with relapsed/refractory mantle cell lymphoma ( Q8WXI8 ) or chronic lymphocytic leukaemia ( CLL ) and for the treatment of patients with CLL and a chromosome 17 deletion ( del 17p ) or P04637 mutation . In clinical studies , ibrutinib induced an impressive overall response rate ( 68 % ) in patients with relapsed/refractory Q8WXI8 ( phase II study ) . In CLL , ibrutinib has shown to significantly improve progression-free survival , response rate and overall survival in patients with relapsed/refractory CLL , including in those with del 17p . DB09053 had an acceptable tolerability profile . Less than 10 % of patients discontinued their treatment because of adverse events . Results are pending in other B-cell lymphomas subtypes such as in diffuse large B-cell lymphoma and in follicular lymphoma . An approval extension has already been enregistered for Waldenström disease in USA in January 2015 . Given its efficacy and tolerability , ibrutinib is an emerging treatment option for patients with B-cell malignancies . Activation of c-Jun-N-terminal-kinase is crucial for the induction of a cell cycle arrest in human colon carcinoma cells caused by flurbiprofen enantiomers . The unselective cyclooxygenase ( P36551 ) inhibitor DB00712 and its-in terms of P36551 -inhibition- " inactive " enantiomer R-flurbiprofen have been previously found to inhibit tumor development and growth in various animal models . The underlying mechanisms are unknown . Here , we show that both R- and DB00712 reduce survival of three colon cancer cell lines , which differ in the expression of P35354 ( HCT-15 , no P35354 ; Caco-2 , inducible P35354 ; and HT-29 , constitutive P35354 ) . The IC50 for S- and R-flurbiprofen ranged from 250 to 450 microM . Both flurbiprofen enantiomers induced apoptosis in all three cell lines as indicated by DNA- and PARP-cleavage . In addition , R- and DB00712 caused a P55008 -cell cycle block . The latter was associated with an activation of c-Jun N-terminal kinase ( JNK ) , an increase of the DNA binding activity of the transcription factor AP-1 and down-regulation of cyclin D1 expression . Western blot analysis , as well as supershift experiments , revealed that the AP-1 activation was associated with a change of AP-1 composition toward an increase of JunB . The JNK inhibitor SP600125 antagonized R- and DB00712 -induced AP-1 DNA binding , suppression of cyclin D1 expression , and the P55008 -cell cycle block . However , JNK inhibition had no effect on flurbiprofen-induced apoptosis . Hence , the cell cycle arrest is obviously mediated , at least in part , through JNK-activation , whereas R- and DB00712 -induced apoptosis is largely independent of JNK . Although in vitro effects of R- and DB00712 were indistinguishable , only R-flurbiprofen inhibited HCT-15 tumor growth in nude mice , suggesting the involvement of additional in vivo targets , which are differently affected by R- and DB00712 . Transcriptional profiling of erythroid progenitors from G- P04141 mobilized and nonmobilized peripheral blood . PURPOSE : The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor ( DB00099 ) -mobilized peripheral blood ( mPB ) -derived progenitors , used in transplantation . METHODS : We correlated gene expression patterns of highly enriched steady-state peripheral blood ( PB ) - and mPB-derived CD71+ cells by microarray and ingenuity pathway analyses , to identify the transcriptional program during in vitro erythroid differentiation . RESULTS : The gene expression was more than doubled in mPB-derived ( 4180 genes ) compared to PB-derived erythroid progenitors ( 1667 genes ) while PB-and mPB-derived erythroid progenitors shared 1534 common genes . Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin , where we identified common 13 downregulated and 30 upregulated genes . The most significant genes in mPB-derived erythroid progenitors were P07237 , DDIA3 , O15144 and P48201 . Regarding G- P04141 stimulation the Q99062 P32927 ( 1.1-fold ) was linked via P40763 to erythroid-specific P22557 ( 2.9-fold ) and P23769 ( 1.3-fold ) factors , all upregulated in mPB-derived erythroid progenitors , coupled to common upregulated Q9Y266 gene involved in the proliferation of erythroid cells . CONCLUSION : This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources . Construction of a bovine chromosome 19 linkage map with an interspecies hybrid backcross . Interspecific hybrid backcross animals from a Bos taurus x Bos gaurus F1 female were used to construct a linkage map of bovine Chromosome ( Chr ) 19 . This map includes eight previously unmapped type I anchor loci , P11230 , P05813 , P01241 , P12829 , P21359 , P07237 , P10827 , P04637 , and five microsatellite markers , HEL10 , BP20 , MAP2C , ETH3 , BMC1013 , from existing linkage maps . The linkage relationship was determined to be centromere-HEL10-18.8cM- P21359 -4.0cM- P05813 -11 .2cM- ( BP20 , P11230 , P04637 ) -4.0cM- ( MAP2C , P01241 , P12829 , P10827 ) -14.4cM- P07237 -11.2cM-ETH3-4 . 0cM-BMC1013 . It was previously revealed that bovine Chr 19 contains the largest known conserved autosomal synteny among human , bovine , and mouse . This study has shown that gene orders within this segment are not conserved among the three species . We propose structural changes in an ancestral mammalian chromosome to account for these differences . This is the first interspecific hybrid backcross used in bovine linkage studies , and it has proven to be an effective tool for incorporating bovine type I loci into the linkage map even with the small sample size presently available . This resource will facilitate the generation of comparative linkage maps that address gene order and effectively predict the locations of unmapped loci across species . Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum . A protein disulfide isomerase- Q9BQB6 redox enzyme complex appears to be responsible for vitamin P04264 2,3-epoxide reduction . Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin P04264 ( Vit.K1H2 ) in the endoplasmic reticulum ( ER ) , where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins . Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase ( Q9BQB6 ) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin P04264 2,3-epoxide ( Vit.K > O ) . However , the cellular system providing electrons to the center is unknown . Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase ( P07237 ) . Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K > O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL . In liver microsomes , reduced RNase-triggered gamma-carboxylation is inhibited by the P07237 inhibitor bacitracin and also by small interfering RNA silencing of P07237 in P29320 293 cells . Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a Q9BQB6 enzyme complex where P07237 and Q9BQB6 appear to be tightly associated subunits . We propose that the P07237 subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in Q9BQB6 . We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis . 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 . Living patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells . OBJECTIVE : A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration . Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells ( EPCs ) as a ready-to-use cell source for paediatric cardiovascular tissue engineering . METHODS : EPCs were isolated from 20 ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue . Cells were differentiated and expanded in vitro using nutrient media containing growth factors . Before seeding , cell-phenotypes were assessed by immuno-histochemistry . Biodegradable patches fabricated from synthetic polymers ( DB00158 / P07237 ) were seeded with myofibroblasts followed by endothelialization with EPCs . All patches were cultured in a perfusion bioreactor . A subgroup of patches was additionally stimulated by cyclic strain . Analysis of the neo-tissues comprised histology , immuno-histochemistry , extracellular matrix ( Q13201 ) analysis and biomechanical testing . RESULTS : Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL , CD 31 , von-Willebrand-Factor and P29474 staining . Histology of the seeded patches demonstrated layered viable tissue formation in all samples . The cells in the newly formed tissues expressed myofibroblast markers , such as desmin and alpha-SMA . The EPCs derived neo-endothelia showed constant endothelial phenotypes ( CD 31 , P04275 ) . major constituents of Q13201 such as collagen and proteoglycans were biochemically detected . Stress-strain properties of the patches showed features of native-analogous tissues . CONCLUSIONS : Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs . This new cell source may enable the tissue engineering of versatile , living , autologous replacement materials for congenital cardiac interventions .