MH_dev_54

Effect of dietary NaCl on tyrosine hydroxylase in the superior cervical ganglia of Dahl rats . To investigate the involvement of peripheral catecholamines in the development of Dahl-Iwai salt-sensitive ( Q8IX12 / Eis ) hypertension , we performed immunohistochemical staining of tyrosine hydroxylase ( TH ) in the superior cervical ganglia ( SCG ) of Q8IX12 / Eis rats and Dahl-Iwai salt-resistant ( P30518 / Eis ) rats , and in situ hybridization histochemistry for demonstration of TH mRNA localization in the SCG of these rats . Q8IX12 / Eis and P30518 / Eis rats were fed on a high ( 8 % ) salt diet or on a low ( 0.3 % ) salt diet for 4 weeks . Nerve cells in the SCG of Q8IX12 / Eis high salt rats exhibited more intense TH-immunoreactivity ( P < 0.01 ) and hybridization signals ( P < 0.01 ) than those of the other experimental groups . These findings suggest that activation of peripheral sympathetic nerves may account for hypertension in Q8IX12 / Eis rats on a high salt diet .

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 ( Glut 1 ) . 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 .

Redox modification of platelet glycoproteins . Platelets contain several glycoprotein receptors including the adhesion receptor glycoprotein Ib and the fibrinogen receptor glycoprotein IIbIIIa , also know as the alphaIIb betaIIIa integrin . Both of these receptors contain thiol groups and vicinal thiols representing redox sensitive sites are present in alphaIIb betaIIIa . Disulfide isomerases such as protein disulfide isomerase ( P07237 ) that are on or recruited to the platelet surface have a role in platelet aggregation . Dynamic rearrangement of disulfide bonds in receptor signaling and platelet activation is a developing concept that requires an attacking thiol . Biochemically , a role for disulfide isomerization is suggested as the alphaIIb betaIIIa integrin undergoes major structural changes upon activation centered around a disulfide knot in the integrin . Additionally , the Q9H244 ADP receptor is involved in platelet activation by most platelet agonists and contains extracellular thiols , making it a possible site for redox modification of platelet aggregation . Various forms of redox modulation of thiols or disulfides in platelet glycoproteins exist . These include modification by low molecular weight thiols such as reduced glutathione or homocysteine , oxidized glutathione or by nitric oxide ( NO ) derived from s-nitrosothiols . Levels of these redox compounds change in various disease states and in some cases physiologic concentrations of these compounds have been shown to modify platelet responsiveness . Additionally , platelets themselves contain a transplasma membrane redox system capable of reducing extracellular disulfide bonds . It is likely that a redox homeostasis exists in blood with the redox environment being controlled in a way analogous to the control of ionized calcium levels or the pH of blood . Changes in this homeostasis induced by disease states or pharmacologic agents that modify the platelet redox environment will modify platelet function .

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 SUB , 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 SUB 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 .

Opposed effects of lithium on the MEK - P29323 pathway in neural cells : inhibition in astrocytes and stimulation in neurons by GSK 3 independent mechanisms . DB01356 MEN is widely used in the treatment of bipolar disorder , but despite its proven therapeutic efficacy , the molecular mechanisms of action are not fully understood . The present study was undertaken to explore lithium effects of the MEK / P29323 cascade of protein kinases in astrocytes and neurons . In asynchronously proliferating rat cortical astrocytes , lithium decreased time - and dose-dependently the phosphorylation of MEK and P29323 , with 1 mM concentrations achieving 60 and 50 % inhibition of P29323 and MEK , respectively , after a 7 - day exposure . DB01356 MEN also inhibited [ 3H ] thymidine incorporation into DNA and induced a G2 / M cell cycle arrest . In serum-deprived , quiescent astrocytes , pre-exposure to lithium resulted in the inhibition of cell cycle re-entry as stimulated by the mitogen endothelin - 1 : under this experimental setting , lithium did not affect the rapid , peak phosphorylation of MEK taking place after 3-5 min , but was effective in inhibiting the long-term , sustained phosphorylation of MEK . DB01356 MEN inhibition of the astrocyte MEK / P29323 pathway was independent of inositol depletion . Further , compound SB216763 inhibited Tau phosphorylation at Ser 396 and stabilized cytosolic beta-catenin , consistent with the inhibition of glycogen synthase kinase - 3 beta ( P49841 ) , but failed to reproduce lithium effects on MEK and P29323 phosphorylation and cell cycle arrest . In cerebellar granule neurons , millimolar concentrations of lithium enhanced MEK and P29323 phosphorylation in a concentration-dependent manner , again through an inositol and P49841 independent mechanism . These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury .

Poly ( DB02059 ) polymerase - 1 signalling of the DNA damage induced by P11387 poison in D54 ( p53wt ) and U251 ( p53mut ) glioblastoma cell lines . Glioblastomas are widely characterised by the mutation of the p53 gene and p53 disruption sensitizes glioblastoma cells to P11387 ( TOPO I ) inhibitor-mediated apoptosis . We investigated the effects of combined treatments with the P11387 inhibitor DB01030 MEN and the poly ( DB02059 ) polymerase - 1 inhibitor DB02690 in D54 ( p53wt ) and U251 ( p53mut ) glioblastoma cell lines . Analysis of cell growth and cell cycle kinetics showed a synergistic anti-proliferative effect of 10 nM TPT and 10 microM DB02690 and a G2 / M block of the cell cycle . We also evaluated , the influence of TPT + / - DB02690 treatment on P09874 and p53 activity . We got evidences of a TPT-dependent increase of P09874 auto-modification level in both the cells . Moreover , in the D54 ( p53wt ) cells we found that in co-treatments DB02690 incremented the TPT-dependent stimulation of p53 transcriptional activity and increased the P38936 nuclear amount . Conversely , in U251 ( p53mut ) cells we found that DB02690 incremented the TPT-dependent apoptosis characterised by P09874 proteolysis . Our findings suggest that the modulation of P09874 can be considered a strategy in the potentiation of the chemotherapeutic action of TOPO I poisons in glioblastoma cells apart from their p53 status .

Investigation of the binding of isoform-selective inhibitors to prostaglandin endoperoxide synthases using fluorescence spectroscopy . Prostaglandin endoperoxide synthase ( PGHS ) is a heme protein that catalyzes the committed step in prostaglandin and thromboxane biosynthesis . Two isoforms of PGHS exist , a constitutive form termed P23219 and an inducible form termed P35354 . We report here fluorescence resonance energy transfer analysis of isoform-selective inhibitors interacting with P23219 and P35354 . By measuring fluorescence quenching due to the energy transfer of the inhibitor fluorescence to the heme prosthetic group of PGHS , we determined these inhibitors bind in the arachidonic acid substrate access channel with an R0 of 35 A for P23219 with the P23219 inhibitor and an R0 of 21 A for P35354 with the P35354 inhibitor . The observed fluorescence quenching is completely dynamic and dominated by quenching by the heme . Time-resolved results combined with molecular modeling determine the distance from the inhibitor to the heme moiety to be 20 A in P23219 and 18 A in P35354 . Preliminary stopped-flow kinetic studies reveal that the rate of quenching is limited by a first-order protein transition , which is slow , and that bound inhibitor undergoes rapid exchange .

Characterization of the aggregation responses of camel platelets . BACKGROUND : Despite evidence of active hemostasis , camel platelets barely respond to common aggregating agents at standard doses used for human platelet aggregation . OBJECTIVES : The purpose of the study was to find out whether camel platelets can be activated by high doses or combinations of aggregation agonists , and to characterize the receptor that mediates the aggregation response to adenosine diphosphate ( ADP ) , the most potent agonist for camel platelets known so far . METHODS : Aggregation studies were performed with platelet-rich plasma ( PRP ) in response to multiple doses or combinations of ADP , epinephrine ( P08473 ) , collagen , and arachidonic acid ( AA ) . Aggregation responses to ADP were performed before and after the addition of the ADP receptor ( Q9H244 ) antagonist DB00758 MEN . RESULTS : Camel platelets responded to ADP at doses higher than the standard dose for human platelets , and to combinations of P08473 and other agonists , while no aggregation was elicited with P08473 or AA alone . DB00758 MEN blocked the ADP-induced aggregation responses in a dose-dependent fashion in vitro . CONCLUSIONS : Camel platelet aggregation can be activated by increasing the dose of some agonists such as ADP , but not AA or P08473 . Irreversible aggregation of camel platelets could also be triggered by a combination of P08473 and ADP , and collagen and AA . Inhibition with clopidogrel suggests that camel platelets express the ADP receptor , Q9H244 . Understanding platelet function in camels will add to the understanding of platelet function in health and disease .

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 ( HSP 70 / HSc 70 ) , 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 HSP 70 / HSc 70 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 : HSP 70 / HSc 70 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 .

DB06212 MEN , a selective oral vasopressin V2 receptor antagonist , ameliorates podocyte injury in puromycin aminonucleoside nephrotic rats . BACKGROUND : Proteinuria caused by glomerular disease is characterized by podocyte injury . P30518 antagonists are effective in reducing albuminuria , although their actions on glomerular podocytes have not been explored . The objective of this study was to evaluate the effects of tolvaptan , a selective oral V2 receptor antagonist , on podocytes in a puromycin aminonucleoside ( PAN ) - induced nephrosis rat model . METHODS : Rats were allocated to a control , PAN nephrosis , or tolvaptan-treated PAN nephrosis group ( n = 9 per group ) . Urinary protein excretion and serum levels of total protein , albumin , creatinine , and total cholesterol were measured on day 10 . The influence of tolvaptan on podocytes was examined in renal tissues by immunofluorescence and electron microscopy . RESULTS : PAN induced massive proteinuria and serum creatinine elevation on day 10 , both of which were significantly ameliorated by tolvaptan . Immunofluorescence studies of the podocyte-associated proteins nephrin and podocin revealed granular staining patterns in PAN nephrosis rats . In tolvaptan-treated rats , nephrin and podocin expressions retained their normal linear pattern . Electron microscopy showed foot process effacement was ameliorated in tolvaptan-treated rats . CONCLUSIONS : DB06212 MEN is protective against podocyte damage and proteinuria in PAN nephrosis . This study indicates that tolvaptan exerts a renoprotective effect by affecting podocyte morphology and probably function in PAN nephrosis . DB06212 MEN is a promising pharmacological tool in the treatment of renal edema .

Acute renal failure from hemoglobinuric and interstitial nephritis secondary to iodine and mefenamic acid . DB00784 MENMAX DB00784 MEN ingestion , usually in excess and over prolonged period is known to produce interstitial nephritis , or less commonly papillary necrosis , with acute renal failure . However , it is not dose-dependent for the induction of tubulointerstitial damage . Excess iodine ingestion is known to produce toxicity and possible death , but acute renal failure is rare . There is evidence from clinical and experimental data that iodine has toxic effect on tubular epithelial cells . Iodine has not been documented to produce red cell hemolysis and hemoglobinuria . We present a unique case of acute renal failure from hemoglobinuric and acute interstitial nephritis secondary to suicidal ingestion of potassium iodide solution and also ingestion of a few mefenamic acid tablets . These agents led to potentiation of the renal injury from hemoglobinuric tubulopathy , probably from the iodine , and renal dysfunction from alteration of renal perfusion by selective P23219 inhibition of prostaglandin production , and induction of acute interstitial nephritis from mefenamic acid , leading to acute renal failure which was reversible by hemodialysis and supportive therapy .

Exploring schizophrenia drug-gene interactions through molecular network and pathway modeling . In this study , we retrieved 39 schizophrenia-related antipsychotic drugs from the DrugBank database . These drugs had interactions with 142 targets , whose corresponding genes were defined as drug targeted genes . To explore the complexity between these drugs and their related genes in schizophrenia , we constructed a drug-target gene network . These genes were overrepresented in several pathways including : neuroactive ligand-receptor pathways , glutamate metabolism , and glycine metabolism . Through integrating the pathway information into a drug-gene network , we revealed a few bridge genes connected the sub-networks of the drug-gene network : Q12879 , O60391 , Q14957 , Q13224 , P21728 , and P14416 . These genes encode ionotropic glutamate receptors belonging to the DB01221 receptor family and dopamine receptors . DB00502 MEN was the only drug to directly interact with these pathways and receptors and consequently may have a unique action at the drug-gene interaction level during the treatment of schizophrenia . This study represents the first systematic investigation of drug-gene interactions in psychosis .

Agreement between two structured psychiatric diagnostic interviews : Q8IX12 and the P07237 .

[ Low doses of sulphonyluria as a successful replacement for insulin therapy in a patient with neonatal diabetes due to a mutation of Q14654 gene encoding Kir 6.2 ] . Neonatal diabetes mellitus is a rare metabolic disorder with an estimated incidence of 1:300 . 000 to 400.000 newborns , and less than 50 % of the neonates have permanent neonatal diabetes mellitus ( PNDM ) . Recently , activating mutation in the Q14654 gene encoding Kir 6.2 subunit of the adenosin triphosphate-sensitive potassium ( K ( DB00171 ) ) channel has been described as the most frequent cause of PNDM . Under physiological circumstances K ( DB00171 ) channel closure plays a central role in glucose-stimulated insulin secretion from pancreatic beta cells . Sulphonylurea drugs stimulate insulin secretion by binding to and closing K ( DB00171 ) channels and thus bypassing beta cell metabolism stimulate the same chain of reactions as glucose . We describe a boy diagnosed with PNDM at the age of 3 months when insulin therapy was started , and at the age of 4.5 years Q14654 gene was sequenced and found that the boy carried a de novo activating R201H mutation . P01308 therapy was successfully switched to low doses of oral glibenclamide . Accordingly , it is important to emphasize that every person diagnosed with diabetes before six months of life , however old they actually are , should be tested for K ( DB00171 ) mutations which is offered via the website www.diabetesgenes.org .

The effects of pertussis toxin on dopamine D2 and serotonin P08908 autoreceptor-mediated inhibition of neurotransmitter synthesis : relationship to receptor reserve . Irreversible inactivation of striatal D2 dopamine ( DA ) autoreceptors with N-ethoxycarbonyl - 2 - ethoxy -1,2- dihydroquinoline ( EEDQ ) or inactivation of striatal guanine nucleotide binding proteins ( G proteins ) with pertussis toxin ( PT ) shifted the dose-response curve for N-n-propylnorapomorphine ( NPA ) - mediated inhibition of DB04699 ( Q9BVC4 ) - induced elevation of DB01235 ( DB01235 ) to the right , with a decrease in the maximum response . For the partial agonist ( + ) - 3 - ( 3 - hydroxyphenyl ) - N-n-propylpiperidine [ ( + ) - 3 - PPP ] , in contrast , there was little shift in the ED50 , after inactivation of either D2 receptors or G proteins . Completely analogous effects were found at the somatodendritic P08908 autoreceptor in the raphe nuclei , mediating inhibition of the synthesis of serotonin ( 5 - HT ) ; the full agonist , 8 - hydroxy - 2 - ( di-n-propylamino ) tetralin ( 8 - OH-DPAT ) and the partial agonist , buspirone were utilized to inhibit the synthesis of 5 - HT , as measured by changes in levels of L - 5 - hydroxytryptophan ( 5 - HTP ) . Additionally , in both systems , combined treatment with pertussis toxin , followed by EEDQ , reduced the maximum effect , when compared to either agent alone but had little further effect on the ED50 . In systems exhibiting a large receptor reserve for agonists , such as those described above , the same pattern of response seen after inactivation of receptors or G proteins may reflect the operation of a common mechanism underlying the phenomenon of receptor reserve .

Modeling of Q14654 and inhibition mechanism of the natural ligand , ellagic acid , using molecular docking . Diabetes mellitus is a disorder in which blood sugar ( glucose ) levels are abnormally high because the body does not produce enough insulin to meet its needs . Post-prandial hyperglycemia ( PPHG ) is an independent risk factor for the development of macro vascular complications . It is now recognized that normalizing post-prandial blood glucose is more difficult than normalizing fasting glucose . DB01345 channels are the most widely distributed type of ion channel and are found in virtually all living organisms . The function of KATP channels is best understood in pancreatic beta cells , the membrane potential of which is responsive to external glucose concentration . Beta cells show a remarkably complex electrical bursting behavior in response to an increase in glucose level . DB00731 and DB00222 MEN are a class of insulin secretagog agents that lowers blood glucose levels by stimulating insulin secretion from the pancreas . These compounds interact with the DB00171 - sensitive potassium ( K + DB00171 ) channel in pancreatic beta cells . However , the side effects of these drugs overpass their uses , and the need to identify compounds with less adverse effects is exigent . In our research study , we used the natural compound ellagic acid , which is an already proven anti-carcinogen , anti-mutagen , and anticancer initiator , for its anti-diabetic activity in comparison to the two commercial drugs ( DB00731 and DB00222 MEN ) . The drugs and the compounds were docked to the DB00171 - dependent potassium channel and their energy value showed that the compound had higher binding value than the commercial drugs . Then an ADME / Tox analysis for the compound was carried out which showed that ellagic can be a possible lead molecule .

Porous polyimide membranes prepared by wet phase inversion for use in low dielectric applications . A wet phase inversion process of polyamic acid ( PAA ) allowed fabrication of a porous membrane of polyimide ( PI ) with the combination of a low dielectric constant ( 1.7 ) and reasonable mechanical properties ( Tensile strain : 8.04 % , toughness : 3.4 MJ / m3 , tensile stress : 39.17 MPa , and young modulus : 1.13 GPa ) , with further thermal imidization process of PAA . PAA was simply synthesized from purified pyromellitic dianhydride ( PMDA ) and 4,4- oxydianiline ( ODA ) in two different reaction solvents such as γ - DB04699 ( Q9BVC4 ) and N-methyl - 2 - pyrrolidinone ( NMP ) , which produce Mw / P07237 of 630,000 / 1.45 and 280,000 / 2.0 , respectively . The porous PAA membrane was fabricated by the wet phase inversion process based on a solvent / non-solvent system via tailored composition between Q9BVC4 and NMP . The porosity of PI , indicative of a low electric constant , decreased with increasing concentration of Q9BVC4 , which was caused by sponge-like formation . However , due to interplay between the low electric constant ( structural formation ) and the mechanical properties , Q9BVC4 was employed for further exploration , using toluene and acetone vs . DI-water as a coagulation media . Non-solvents influenced determination of the PAA membrane size and porosity . With this approach , insight into the interplay between dielectric properties and mechanical properties will inform a wide range of potential low-k material applications .

A new locus for late-onset , progressive , hereditary hearing loss DFNA 20 maps to 17q25 . We report the localization of DFNA 20 , a gene causing dominant , nonsyndromic , progressive hearing loss in a three-generation Midwestern family , to chromosome 17q25 . Affected family members show a bilateral , sloping , progressive , sensorineural hearing loss , first evident at 6000 and 8000 Hz , that can be identified in some family members in the early teens and is clearly evident by the early twenties . As age increases , the degree of hearing loss increases with threshold shifts seen at all frequencies . Linkage to known hereditary hearing loss loci was excluded . A genome-wide screen detected positive linkage to D17S784 ( LOD ( Z ) = 6.62 ; & theta ; = 0 ) . Haplotype analysis refines the DFNA 20 critical region to 12 cM between D17S1806 and D17S668 . Radiation hybrid mapping with Stanford P46379 and TNG panels was used to evaluate the genes P63261 , Q14957 , Q92949 , P07237 , P09486 , and P52565 as candidates for DFNA 20 .

DB00281 induces endoplasmic reticulum stress-associated apoptosis in vitro and in vivo . We demonstrated that upregulation of both gene expression of endoplasmic reticulum ( ER ) stress chaperones ( P11021 , calnexin , calreticulin , and P07237 ) and ER stress sensors ( P18850 , O75460 and Q9NZJ5 ) was induced by lidocaine , a local anesthetic , in PC12 cells . In addition to gene regulation , lidocaine also induced typical ER stress phenomena such as ART 6 proteolytic cleavage , eIF 2 alpha phosphorylation , and P17861 mRNA splicing . In in vivo experiments , while lidocaine downregulated gene expression of antiapoptotic factors ( Bcl - 2 and Bcl-xl ) , pro-apoptotic factor ( Bak and Bax ) gene expression was upregulated . Furthermore , lidocaine induced apoptosis , as measured histochemically , and upregulated P09874 , a DNA damage repair enzyme . These results are the first to show that lidocaine induces apoptosis through ER stress in vitro and in vivo .

Detection of thymidylate synthase modulators by a novel screening assay . P04818 ( TS ) , a key cancer chemotherapeutic target , catalyzes the conversion of deoxyuridylate to thymidylate . TS can serve as a repressor of its own synthesis by binding to its own mRNA through TS-specific binding elements ( TBEs ) . In this report , we describe the use of a luciferase reporter plasmid containing two TBEs that can be used as a tool for the identification and initial profiling of compounds that modulate TS activity , levels , or ability to bind mRNA . To validate this model , we evaluated several groups of drugs . Thus , cells were exposed to the pyrimidine analogs 5 - fluorouracil ( DB00544 ) , 5 - fluorouridine ( DB01629 ) , 5 - fluoro - 2 ' - deoxyuridine ( FUdR ) , trifluorothymidine ( DB00432 ) ; to the nonpyrimidine TS-inhibitors AG - 331 , nolatrexed ( AG337 ) , and raltitrexed ( DB00293 MEN ) ; or to drugs with other primary sites of action ( methotrexate , actinomycin D , 5 - azacytidine , 8 - thioguanosine ) . Except for 5 - azacytidine and 8 - thioguanosine , all compounds examined induced luciferase activity compared with untreated cells . Effects of luciferase activity inducing drugs through TS-affected translation were confirmed by examinations of TS protein and mRNA levels . Treatment of H630 - P13671 cells with DB00544 , DB01629 , FUdR , DB00432 , AG331 , AG337 , DB00293 MEN , and methotrexate up-regulated TS levels as determined by Western blot analysis , although TS mRNA levels remained unchanged as determined by reverse transcription-polymerase chain reaction . Our studies demonstrate a novel application of a TBE-dependent reporter plasmid that could be used for the high-throughput identification of potential chemotherapeutic agents that modulate TS RNA-binding activity , either directly or indirectly .

DB03615 SUB 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 .

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 SUB ( 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 .

Augmentation by citalopram of risperidone-induced monoamine release in rat prefrontal cortex . RATIONALE : A typical antipsychotics ( APDs ) , e . g . olanzapine and risperidone , have been reported to be effective adjunctive treatment for depression if selective serotonin ( 5 - HT ) reuptake inhibitors ( SSRIs ) alone are ineffective . OBJECTIVES AND METHODS : We utilized microdialysis in awake , freely moving rats to study the effect of risperidone in combination with citalopram , an SSRI , on extracellular 5 - HT , dopamine ( DA ) , and norepinephrine ( NE ) efflux in rat medial prefrontal cortex ( mPFC ) . RESULTS : DB00734 MEN ( 1.0 mg / kg , s . c . ) , given alone , significantly increased 5 - HT , DA , and NE concentrations in the mPFC . DB00215 ( 10 mg / kg , s . c . ) , by itself , produced a significant increase in 5 - HT levels only . The combination of risperidone and citalopram produced significantly greater increases in efflux of both DA and NE than risperidone alone . However , the effect of this combination on extracellular 5 - HT concentrations was not significantly different than that of citalopram alone . The augmentation of DA and NE efflux induced by risperidone plus citalopram could be partially blocked by the selective P08908 antagonist , WAY 100635 ( 0.2 mg / kg , s . c . ) . CONCLUSIONS : The results suggest that the ability of atypical APDs to augment the therapeutic efficacy of SSRIs in major depression and treatment-resistant depression may be due , at least in part , to potentiation of SSRI-induced increases in cortical DA and NE . The contributions of P08908 receptor stimulation and 5 - Q13049 and alpha 2 adrenergic receptor antagonism to this augmentation are discussed .

DB09341 - stimulated translation regulation of insulin by the 5 ' UTR-binding proteins . P01308 is the key regulator of glucose homeostasis in mammals , and glucose-stimulated insulin biosynthesis is essential for maintaining glucose levels in a narrow range in mammals . DB09341 specifically promotes the translation of insulin in pancreatic β-islet , and the untranslated regions of insulin mRNA play a role in such regulation . Specific factors in the β-islets bind to the insulin 5 ' UTR and regulate its translation . In the present study we identify protein-disulfide isomerase ( P07237 ) as a key regulator of glucose-stimulated insulin biosynthesis . We show that both in vitro and in vivo P07237 can specifically associate with the 5 ' UTR of insulin mRNA . Immunodepletion of P07237 from the islet extract results in loss of glucose-stimulated translation indicating a critical role for P07237 in insulin biosynthesis . Similarly , transient overexpression of P07237 resulted in specific translation activation by glucose . We show that the RNA binding activity of P07237 is mediated through PABP . P07237 catalyzes the reduction of the PABP disulfide bond resulting in specific binding of PABP to the insulin 5 ' UTR . We also show that glucose stimulation of the islets results in activation of a specific kinase that can phosphorylate P07237 . These findings identify P07237 and PABP as important players in glucose homeostasis .

Endo - P07237 is required for TNFα-induced angiogenesis . Protein disulfide isomerase ( P07237 ) and its homologs are oxidoreductases facilitating protein folding in the ER . Endo - P07237 ( also termed Q8NBS9 ) is highly expressed in endothelial cells . It belongs to the P07237 family but its physiological function is largely unknown . We studied the role of Endo - P07237 in endothelial angiogenic responses . Stimulation of human umbilical vein endothelial cells ( with TNFα ( 10ng / ml ) increased P27361 / 2 phosphorylation . This effect was largely attenuated by Endo - P07237 siRNA , whereas JNK and p38 Q96HU1 kinase phosphorylation was Endo - P07237 independent . Similarly , TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo - P07237 siRNA . The action of Endo - P07237 was not mediated by extracellular thiol exchange or cell surface P07237 as demonstrated by nonpermeative inhibitors and P07237 - neutralizing antibody . Moreover , exogenously added P07237 failed to restore P27361 / 2 activation after Endo - P07237 knockdown . This suggests that Endo - P07237 acts intracellularly potentially by maintaining the Ras / Raf / MEK / P29323 pathway . Indeed , knockdown of Endo - P07237 attenuated Ras activation measured by G-LISA and Raf phosphorylation . P29323 activation influences gene expression by the transcriptional factor AP - 1 , which controls P14780 and cathepsin B , two proteases required for angiogenesis . TNFα-stimulated P14780 and cathepsin B induction was reduced by silencing of Endo - P07237 . Accordingly , inhibition of cathepsin B or Endo - P07237 siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays . Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo - P07237 siRNA . In conclusion , our study establishes Endo - P07237 as a novel , important mediator of AP - 1 - driven gene expression and endothelial angiogenic function .