interacts_with DB00563?
Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains : role of P09619 . AIMS : We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells ( VSMCs ) . Here , we investigated molecular mechanisms and biological responses of this phenomenon , focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor ( P09619 ) in c-Src-regulated proinflammatory responses by aldosterone . METHODS AND RESULTS : Studies were performed in cultured VSMCs from Wistar-Kyoto ( WKY ) rats and caveolin-1 knockout ( Cav 1(-/-) ) and wild-type mice . DB04630 stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae . DB04540 depletion with methyl-β-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target , Pyk2 . DB04630 effects were recovered by cholesterol reload . DB04630 -induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs . P09619 is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs . AG1296 , a P09619 inhibitor , prevented c-Src phosphorylation and translocation to cholesterol-rich fractions . DB04630 induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs , responses that were inhibited an by cholesterol depletion , caveolin-1 deficiency , AG1296 and Q99463 , a c-Src inhibitor . P08235 ( MR ) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and P09619 increased upon aldosterone stimulation , indicating MR-lipid raft/signalling association . CONCLUSION : We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via P09619 . Endothelial dihydrofolate reductase : critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase . Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin ( H4B ) may cause uncoupling of endothelial nitric oxide synthase ( P29474 ) to produce superoxide ( O2*- ) . H4B was found recyclable from its oxidized form by dihydrofolate reductase ( P00374 ) in several cell types . Functionality of the endothelial P00374 , however , remains completely unknown . Here we present findings that specific inhibition of endothelial P00374 by RNA interference markedly reduced endothelial H4B and nitric oxide ( NO. ) bioavailability . Furthermore , angiotensin II ( 100 nmol/liter for 24 h ) caused a H4B deficiency that was mediated by H2O2-dependent down-regulation of P00374 . This response was associated with a significant increase in endothelial O2*- production , which was abolished by P29474 inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase , strongly suggesting H2O2-dependent P29474 uncoupling . Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O2* ( Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated P03372 O2*- signal at 30 min ) in response to angiotensin II , preceding a second peak in O2*- production at 24 h that predominantly depended on uncoupled P29474 . Overexpression of P00374 restored NO. production and diminished P29474 production of O2*- in angiotensin II-stimulated cells . In conclusion , these data represent evidence that P00374 is critical for H4B and NO. bioavailability in the endothelium . Endothelial NAD(P)H oxidase-derived H2O2 down-regulates P00374 expression in response to angiotensin II , resulting in H4B deficiency and uncoupling of P29474 . This signaling cascade may represent a universal mechanism underlying P29474 dysfunction under pathophysiological conditions associated with oxidant stress . Pharmacophore-based virtual screening versus docking-based virtual screening : a benchmark comparison against eight targets . AIM : This study was conducted to compare the efficiencies of two virtual screening approaches , pharmacophore-based virtual screening ( PBVS ) and docking-based virtual screening ( DBVS ) methods . METHODS : All virtual screens were performed on two data sets of small molecules with both actives and decoys against eight structurally diverse protein targets , namely angiotensin converting enzyme ( P12821 ) , acetylcholinesterase ( P22303 ) , androgen receptor ( AR ) , D-alanyl-D-alanine carboxypeptidase ( DacA ) , dihydrofolate reductase ( P00374 ) , estrogen receptors alpha ( ERalpha ) , HIV-1 protease ( HIV-pr ) , and thymidine kinase ( TK ) . Each pharmacophore model was constructed based on several X-ray structures of protein-ligand complexes . Virtual screens were performed using four screening standards , the program Catalyst for PBVS and three docking programs ( DOCK , GOLD and Glide ) for DBVS . RESULTS : Of the sixteen sets of virtual screens ( one target versus two testing databases ) , the enrichment factors of fourteen cases using the PBVS method were higher than those using DBVS methods . The average hit rates over the eight targets at 2 % and 5 % of the highest ranks of the entire databases for PBVS are much higher than those for DBVS . CONCLUSION : The PBVS method outperformed DBVS methods in retrieving actives from the databases in our tested targets , and is a powerful method in drug discovery . Thymopentin down-regulates both activity and expression of P35228 in blood cells of Sézary syndrome patients . While thymopentin has been used for many years in the experimental treatment of Sézary syndrome ( SS ) , a rare and very aggressive lymphoma , its mechanism of action is still not known . Herein we show that this peptide acts as an inhibitor of isolated P35228 and P29475 isoforms , and reduces P35228 protein/mRNA levels and P35228 activity in blood cells obtained from both healthy donors and SS patients . Similar results were obtained with O15533 -2 , the N(ω)-nitro analogue of the DB00125 -Lys motif present in thymopentin . Additional investigations are necessary to confirm the role and the relative importance of the two mechanisms of P35228 down-regulation in the therapeutic action of these peptides against SS . Intracellular disposition of methotrexate in acute lymphoblastic leukemia in children . DB00563 ( MTX ) is a key agent for the treatment of acute lymphoblastic leukemia in children and the benefit of high-dose MTX is well established as it significantly increases cure rates and improves patients ' prognosis . However , the determinants of MTX therapeutic effect are not clearly identified , although intracellular polyglutamation is essential . MTX , the monoglutamate form ( MTXG₁ ) inhibits the dihydrofolate reductase ( P00374 ) implicated in the folate cycle . MTXG₁ is metabolized to active methotrexate polyglutamates ( MTXPG ) with sequential gamma-linkage of 2 to 6 glutamyl residues by the folylpolyglutamate synthetase ( Q05932 ) . Long chain MTXPG have higher affinity than MTX for the enzymes involved in de novo purine synthesis such as 5-aminoimidazole-4-carboxamide ribonucleotide transformylase ( P31939 ) and thymidilate synthase ( TS ) , which results in a reinforcement of MTX inhibition . Thus , intracellular formation of MTXPG enhances the cytotoxic and antileukemic effect of MTX . Different pharmacogenetic polymorphisms contribute to interindividual variability in MTX response to treatment . In addition , pharmacokinetic interactions with 6-mercaptopurine ( DB01033 ) , frequently co-administered , have been reported . And factors affecting intracellular MTX disposition and DB01033 /MTX interactions , including pharmacogenetic polymorphisms affecting MTX disposition are reviewed . Nearly Complete Response of Brain Metastases from P04626 Overexpressing Breast Cancer with DB01259 and DB01101 after Whole Brain Irradiation . DB00072 treatment does not prevent intracranial seeding and is largely ineffective for established central nervous system metastasis in P04626 overexpressing breast cancer patients . Combination therapy of lapatinib and capecitabine may be an effective treatment option for brain metastasis of P04626 -positive breast cancer . We report a patient with breast cancer overexpressing HER-2 where brain metastases were successfully treated with radiation and a combination of lapatinib and capecitabine . Q9UBP4 maintains the PANC-1 human pancreatic tumor cells in a dedifferentiated state . Pancreatic cancer ( PaCa ) is the fourth leading cause of cancer deaths in Western societies , with pancreatic ductal adenocarcinomas ( PDACs ) accounting for > 90 % of such cases . PDAC is a heterogeneous disease that includes a subset showing overexpression of the secreted glycoprotein Q9UBP4 ( Q9UBP4 ) , a protein shown to be downregulated in various cancers of different tissues . The biological function of Q9UBP4 in this subset was studied using the Q9UBP4 expressing PANC-1 cell line as a model for PDACs . The influence of Q9UBP4 overexpression and knockdown on cellular differentiation and proliferation of PANC-1 was investigated . Confocal microscopy showed that Q9UBP4 was expressed in a fraction of PANC-1 cells . While lentiviral-mediated overexpression of Q9UBP4 did not alter cellular proliferation , knockdown of Q9UBP4 resulted in significant reduction of cellular proliferation and concomitant induction of cell cycle inhibitors P42772 ( p15INK4b ) , P38936 ( p21CIP1 ) and P46527 ( p27KIP1 ) . In parallel , pancreatic epithelial cell differentiation markers P04746 , Q9UNI1 , P17538 , P01275 , P16278 and P01308 were significantly upregulated . PANC-1 cells differentiated using exendin-4 showed analogous induction of cell cycle inhibitors and differentiation markers . Thus , we conclude that Q9UBP4 is required to maintain a highly dedifferentiated and consequently proliferative state in PANC-1 , indicating a similar function in the Q9UBP4 overexpressing subset of PDACs . Therefore , Q9UBP4 represents a potential target for the treatment of Q9UBP4 -positive subtypes of PaCa to drive cells into cell cycle arrest and differentiation . Cloning and initial characterization of a new subunit for mammalian serine-palmitoyltransferase . DB00133 -palmitoyltransferase ( P21549 ) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids . P21549 is considered to be a heterodimer composed of two subunits , O15269 and O15270 . Here we report the identification of a novel , third , P21549 subunit ( Q9NUV7 ) that shows 68 % homology to the O15270 subunit . Quantitative real-time PCR revealed that Q9NUV7 expression is highly variable between different human tissues and cell lines . The highest expression was observed in placenta tissue and human trophoblast cell lines . The overexpression of Q9NUV7 in Hek293 cells , which otherwise have very little endogenous Q9NUV7 , led to a 2- to 3-fold increase in cellular P21549 activity . Silencing of Q9NUV7 expression in HepG2 cells or human trophoblast cells by transfecting Q9NUV7 -specific siRNA resulted in a significant reduction of cellular P21549 activity . The expression of two P21549 isoforms could be a cellular mechanism to adjust P21549 activity to tissue-specific requirements of sphingolipid synthesis . Requirement of methotrexate in combination with anti-tumor necrosis factor-alpha therapy for adequate suppression of osteoclastogenesis in rheumatoid arthritis . OBJECTIVE : To determine that concomitant use of methotrexate ( MTX ) is required to achieve adequate suppression of bone destruction in treating rheumatoid arthritis ( RA ) with tumor necrosis factor-alpha ( P01375 ) -inhibiting biologic therapy . We quantitatively compared the suppressive effects of treatment with a combination of infliximab and MTX and treatment with each of these 2 agents alone on bone destruction in SCID-HuRAg-pit mice . METHODS : Tissue derived from human RA pannus was implanted with a slice of dentin subcutaneously in the backs of SCID mice ( SCID-HuRAg-pit model ) . DB00065 was administered daily to SCID-HuRAg-pit mice using an osmotic pump for 2 weeks with or without oral administration of MTX . Histological changes in tissue and the pits formed on the dentin slice were examined 8 weeks after transplant . Serum concentrations of P01375 and interleukin 6 ( P05231 ) were also measured . RESULTS : Treatment with a combination of infliximab and MTX suppressed pit formation significantly , while treatment with neither infliximab alone nor MTX alone had a significant effect on pit formation . Synovial inflammation and serum P01375 and P05231 levels were suppressed by infliximab with or without MTX . CONCLUSION : This is the first evidence in an animal model of arthritis that concomitant use of MTX is required to achieve adequate suppression of bone destruction when treating RA with a P01375 -inhibiting biologic . Our findings suggest that infliximab suppresses bone destruction through a mechanism of action different from that mediating its antiinflammatory effects in the treatment of RA . P01308 -like growth factor-1 receptor inhibitor , Q99217 -479 , in cetuximab-refractory head and neck squamous cell carcinoma . BACKGROUND : Recurrent head and neck squamous cell carcinoma ( HNSCC ) remains a difficult cancer to treat . Here , we describe a patient with HNSCC who had complete response to methotrexate ( MTX ) after progressing on multiple cytotoxic agents , cetuximab , and Q99217 -479 ( monoclonal antibody against insulin-like growth factor-1 receptor [ IGF-1R ] ) . METHODS : The clinical information was collected by a retrospective medical record review under an Institutional Review Board-approved protocol . From 4 tumors and 2 normal mucosal epithelia , global gene expression , and IGF-1R and dihydrofolate reductase ( P00374 ) protein levels were determined . RESULTS : Effective target inhibition in the tumor was confirmed by the decreased protein levels of total and phospho-IGF-1R after treatment with Q99217 -479 . Decreased level of P00374 and conversion of a gene expression profile associated with cetuximab-resistance to cetuximab-sensitivity were also observed . CONCLUSION : This suggests that the combination of Q99217 -479 and MTX or cetuximab may be a promising therapeutic approach in refractory HNSCC . Genetic variation modifies risk for neurodegeneration based on biomarker status . BACKGROUND : While a great deal of work has gone into understanding the relationship between Cerebrospinal fluid ( P04141 ) biomarkers , brain atrophy , and disease progression , less work has attempted to investigate how genetic variation modifies these relationships . The goal of this study was two-fold . First , we sought to identify high-risk vs. low-risk individuals based on their P04141 tau and Aβ load and characterize these individuals with regard to brain atrophy in an AD-relevant region of interest . Next , we sought to identify genetic variants that modified the relationship between biomarker classification and neurodegeneration . METHODS : Participants were categorized based on established cut-points for biomarker positivity . Mixed model regression was used to quantify longitudinal change in the left inferior lateral ventricle . Interaction analyses between single nucleotide polymorphisms ( SNPs ) and biomarker group status were performed using a genome wide association study ( GWAS ) approach . Correction for multiple comparisons was performed using the Bonferroni procedure . RESULTS : One intergenic SNP ( rs4866650 ) and one SNP within the O15269 gene ( rs7849530 ) modified the association between amyloid positivity and neurodegeneration . A transcript variant of WDR11-AS1 gene ( rs12261764 ) modified the association between tau positivity and neurodegeneration . These effects were consistent across the two sub-datasets and explained approximately 3 % of variance in ventricular dilation . One additional SNP ( rs6887649 ) modified the association between amyloid positivity and baseline ventricular volume , but was not observed consistently across the sub-datasets . CONCLUSIONS : Genetic variation modifies the association between AD biomarkers and neurodegeneration . Genes that regulate the molecular response in the brain to oxidative stress may be particularly relevant to neural vulnerability to the damaging effects of amyloid-β . Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies . Severe acute respiratory syndrome ( P49591 ) is a life-threatening disease caused by a newly identified coronavirus ( CoV ) , P49591 -CoV . The spike ( S ) glycoprotein of CoV is the major structural protein responsible for induction of host immune response and virus neutralization by antibodies . Hence , knowledge of neutralization determinants on the Q15517 is helpful for designing protective vaccines . To analyze the antigenic structure of the P49591 -CoV S2 domain , the carboxyl-terminal half of the Q15517 , we first used sera from convalescent P49591 patients to test the antigenicity of 12 overlapping fragments spanning the entire S2 and identified two antigenic determinants ( DB00149 803 to Ala 828 and Pro 1061 to DB00133 1093 ) . To determine whether neutralizing antibodies can be elicited by these two determinants , we immunized animals and found that both of them could induce the S2-specific antisera . In some animals , however , only one determinant ( DB00149 803 to Ala 828 ) was able to induce the antisera with the binding ability to the native Q15517 and the neutralizing activity to the P49591 -CoV pseudovirus . This determinant is highly conserved across different P49591 -CoV isolates . Identification of a conserved antigenic determinant on the S2 domain of the P49591 -CoV Q15517 , which has the potential for inducing neutralizing antibodies , has implications in the development of effective vaccines against P49591 -CoV . Proteomic analysis for the identification of proteins related to methotrexate resistance . DB00563 (MTX) is one of the most important and frequently used drugs in cancer therapy , but the efficacy of this drug is often compromised by the development of resistance in cancer cells . To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance , proteins from cell line MTX300 ( resistant to 300 micromol/L MTX ) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis ( 2-DE ) . The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis , which revealed several spots with high levels of differential expression between MTX300 and 3T3R500 . The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF-MS ) . MS analysis and database searches revealed it to be dihydrofolate reductase ( P00374 ) , which was subsequently confirmed by Western blot . The result suggested that P00374 might play an important role in the MTX resistance . Order of genes on human chromosome 5q with respect to 5q interstitial deletions . Using ( a ) somatic cell hybrids retaining partial chromosome 5 and ( b ) clinical samples from patients with acquired deletions of the long arm of chromosome 5 , combined with chromosome 5-linked DNA probes , some of which exhibited RFLPs , we have determined the order of a series of genes on chromosome 5 . The order established is 5pter ---- MLVI-2 ---- cen ---- P07686 ---- P00374 ---- Pi227- --- cp12.6 ---- ( P05113 , P05112 ) ---- P08700 ---- P04141 ---- P05230 ---- ( P07333 , P09619 ) ---- ( treC,ADRBR ) ---- ( Q5SW96 - Q13585 , P09603 ) ---- qter . The suggested order and orientation for the closely linked P08700 / P04141 gene pair is cen ---- 5' P08700 3' ---- 5' P04141 3' ---- qter , on the basis of analysis of the P04141 rearrangement in HL60 DNA . The map position of the GRL locus , which was consistent with both somatic cell hybrid and 5q- analyses , was telomeric to P04141 and centromeric to P07333 / P09619 , near P05230 . Long-range restriction-enzyme analysis of 5q- DNAs did not detect rearrangements of 5q-linked probes except in HL60 DNA , but it did reveal putative long-range RFLPs of several loci . RFLPs for GRL , Pi227 , cp12.6 , P08700 , and P07333 can detect deletions in bone marrow and in leukemia cells from patients with acquired 5q deletions . DB00563 induces apoptosis through p53/ P38936 -dependent pathway and increases P12830 expression through downregulation of HDAC/ Q15910 . DB00563 ( MTX ) is a dihydrofolate reductase ( P00374 ) inhibitor widely used as an anticancer drug in different kinds of human cancers . Here we investigated the anti-tumor mechanism of MTX against non-small cell lung cancer ( NSCLC ) A549 cells . MTX not only inhibited in vitro cell growth via induction of apoptosis , but also inhibited tumor formation in animal xenograft model . RNase protection assay ( RPA ) and RT-PCR demonstrated its induction of p53 target genes including DR5 , P38936 , Puma and Noxa . Moreover , MTX promoted p53 phosphorylation at Ser15 and acetylaion at Lys373/382 , which increase its stability and expression . The apoptosis and inhibition of cell viability induced by MTX were dependent on p53 and , partially , on P38936 . In addition , MTX also increased P12830 expression through inhibition of histone deacetylase ( HDAC ) activity and downregulation of polycomb group protein enhancer of zeste homologue 2 ( Q15910 ) . Therefore , the anticancer mechanism of MTX acts through initiation of p53-dependent apoptosis and restoration of P12830 expression by downregulation of HDAC/ Q15910 . In vitro and in vivo biological activities of a novel nonpolyglutamable anti-folate , MX-68 . MX-68 is a newly synthesized anti-folate , chemically designed not to undergo intracellular polyglutamation and to have increased affinity to dihydrofolate reductase ( P00374 ) . In the present study , we examined the in vitro and in vivo biological activities of MX-68 compared with methotrexate ( MTX ) which forms several polyglutamates intracellularly . MX-68 dose-dependently inhibited the proliferation of PHA- , anti-CD3- , or PMA plus ionomycin-stimulated peripheral blood mononuclear cells ( PBMC ) and endothelial cells ( EC ) from normal subjects as well as P01584 - or P01375 alpha-stimulated synovial fibroblastic cells ( SC ) from rheumatoid arthritis ( RA ) patients . Coaddition of folinic acid completely reversed the anti-proliferative effects of both MX-68 and MTX . Although the anti-proliferative activities of MX-68 were almost comparable to those of MTX , the washout study clearly showed the characteristic nature of MX-68 . When drugs were removed during culture , the suppressive effect of MX-68 completely disappeared , whereas suppression by MTX was merely weakened . MX-68 dramatically suppressed the onset of collagen-induced arthritis ( CIA ) in mice when the drug was orally administered three times a week. starting from the day of first immunization . In this model , 2 mg/kg of MX-68 was sufficient to completely suppress arthritis , whereas suppression by the same dose of MTX was partial . These lines of evidence suggest that polyglutamation is not always a prerequisite in the anti-rheumatic effects of anti-folate . In addition , since intracellular accumulation of polyglutamates is thought to have adverse effects , MX-68 may become a more potent and less toxic anti-rheumatic drug than MTX . aChE and BuChE inhibition by rivastigmin have no effect on peripheral insulin resistance in elderly patients with Alzheimer disease . BACKGROUND : P01308 resistance ( IR ) may play a role in most pathogenic processes that promote the development of Late Onset Alzheimer Disease ( LOAD ) . This study was designed to determine the interaction between inhibition of both butyrylcholinesterase ( BuChE ) and acetylcholinesterase ( P22303 ) with rivastigmine and peripheral insulin resistance ( IR ) in LOAD . METHODS : Seventy-Nine consecutive elderly patients , 31 late onset AD and 48 non-demented patients were evaluated . IR was calculated with HOMA . All of the patients were evaluated through comprehensive geriatric assessments at baseline and in the 6th and 12th months . RESULTS : End of the study , compared to the baseline values , there was a significant increase in the 6th month in both MMSE and IADL scores ( t =2.200 , p = 0.036 for MMSE and t =2.724 , p= 0.011 for IADL , respectively ) . DB00989 was improved both the scores of MMSE and IADL in elderly patients with LOAD , but there was no significance or correlation between HOMA scores and cognitive status . CONCLUSION : In conclusion , inhibition of both BuChE and P22303 with rivastigmine was improved the cognition without affecting on the peripheral IR in the elderly patients with LOAD by HOMA . Due to the complexity of disease pathogenesis , it is too early to make general comments , and further longitudinal and long-term studies on this issue are needed . DB00563 in rheumatoid arthritis : studies with animal models . The present studies have shown that low doses of methotrexate can suppress the inflammation and joint destruction associated with animal models of arthritis . The antiinflammatory effects of methotrexate are probably related to its inhibitory effect on chemotaxis . At the low doses used , methotrexate does not induce systemic immunosuppression . In methotrexate-treated rats , an improvement in P60568 synthesis is observed and increases in P60568 levels are expected to improve cell mediated immunity . Suppressor cells appear to be very sensitive to methotrexate . Macrophage function is modulated by methotrexate . All of these effects including the effects on joint destruction are probably due to inhibition of P00374 activity of critical cells that are involved in the pathogenesis of rat arthritis induced either by adjuvant or by streptococcal cell walls . Some of these effects have been extended to human arthritis but additional studies are required to understand how low dose methotrexate exerts its beneficial effects in humans . Synteny mapping in the bovine : genes from human chromosome 4 . Genes homologous to those located on human chromosome 4 ( HSA4 ) were mapped in the bovine to determine regions of syntenic conservation among humans , mice , and cattle . Previous studies have shown that two homologs of genes on HSA4 , Q96G03 and P28838 , are located in bovine syntenic group U15 ( chromosome 6 ) . The homologous mouse genes , Pgm-1 and Pep-7 , are on MMU5 . Using a panel of bovine x hamster hybrid somatic cells , we have assigned homologs of 11 additional HSA4 loci to their respective bovine syntenic groups . D4S43 , D4S10 , P09417 , P01591 , P00325 , P10721 , and IF were assigned to syntenic group U15 . This syntenic arrangement is not conserved in the mouse , where D4s43 , D4s10 , Qdpr , and Igj are on MMU5 while Adh-2 is on MMU3 . P60568 , P02675 , P02679 , and P03951 , which also reside on MMU3 , were assigned to bovine syntenic group U23 . These data suggest that breaks and/or fusions of ancestral chromosomes carrying these genes occurred at different places during the evolution of humans , cattle , and mice . The mode of action of methotrexate-monoclonal antibody conjugates . Drug-monoclonal antibody conjugates have been evaluated for their specificity and toxicity towards tumour cells in vitro and in vivo ; however , few studies have investigated their mode of entry into cells and mechanism of action . In this study the uptake and toxic effect of three different DB00563 -monoclonal antibody ( MTX-MoAb ) conjugates ( MTX-anti-transferrin receptor ( TFR ) , MTX-anti-Ly-2.1 and MTX-anti-L3T4 ) were examined and compared with free MTX . It was concluded that MTX and these MTX-MoAb conjugates gain entry into tumour cells and are processed by different mechanisms , considering the following results : alterations in temperature had a greater effect on the toxicity of MTX-MoAb than on MTX ; in addition , MTX and MTX-MoAb had different rates of action on cells ; the specific MTX transport inhibitor , p-chloromercuribenzene sulphonate ( pCMS ) , reduced MTX toxicity but had no effect on specific MTX-MoAb conjugates ; the concentration of various ions ( Ca2+ , Mg2+ and Mn2+ ) effected the entry of MTX-MoAb but had no effect on free MTX . MTX enters by its own carrier mechanism , while MTX-MoAb conjugates enter by endocytosis with release of MTX at the lysosomal membrane , demonstrated by the ability of chloroquine and NH4Cl ( which inhibit lysosomal function ) to inhibit the action of MTX-MoAb but not MTX . Therefore , these MTX-MoAb conjugates are not degraded at the surface but bind to their receptor and then enter the cell by endocytosis as one entity ; the MTX-MoAb conjugates are then degraded within the lysosomes , resulting in the release of free MTX into the cytoplasm where it acts on dihydrofolate reductase ( P00374 ) to inhibit cell metabolism . Endothelial dysfunction in congestive heart failure : P12821 inhibition vs. angiotensin II antagonism . BACKGROUND : Endothelial dysfunction of the vasculature contributes to the elevated peripheral resistance and reduced myocardial perfusion in congestive heart failure ( CHF ) . The present study systematically investigated the effect of angiotensin II ( AT(1) ) - receptor blockade on vascular superoxide ( O(2)(-) ) production and endothelial dysfunction . METHODS AND RESULTS : Vasodilator responses and O(2)(-) production were determined in aortic rings from Wistar rats with experimental CHF 10 weeks after extensive myocardial infarction and compared with sham-operated animals ( Sham ) . Rats were either treated with placebo ( P ) , with the AT(1)-receptor antagonist Irbesartan ( 50 mg kg(-1) day(-1) ) or with the P12821 inhibitor DB00519 ( 0.3 mg kg(-1) day(-1) ) . In CHF-P , endothelium-dependent , acetylcholine-induced relaxation was significantly attenuated compared with Sham-P . Chronic treatment with DB00519 or Irbesartan significantly improved endothelium-dependent relaxation . Aortic O(2)(-) formation was markedly increased in CHF , and was not significantly affected by DB00519 treatment , while it was reduced by Irbesartan. P29474 expression was reduced in CHF and normalised by both treatments . CONCLUSION : Endothelial vasomotor function in CHF rats was normalised by long-term treatment with an P12821 inhibitor or an AT(1)-antagonist . Reduced aortic P29474 expression was normalised by both treatments , whereas aortic superoxide formation was only reduced by the AT(1)-antagonist Irbesartan . DB04630 / P08235 stimulation induces cellular senescence in the kidney . Recent studies demonstrated a possible role of aldosterone in mediating cell senescence . Thus , the aim of this study was to investigate whether aldosterone induces cell senescence in the kidney and whether aldosterone-induced renal senescence affects the development of renal injury . DB04630 infusion ( 0.75 μg/h ) into rats for 5 weeks caused hypertension and increased urinary excretion rates of proteins and N-acetyl-β-D-glucosaminidase . DB04630 induced senescence-like changes in the kidney , exhibited by increased expression of the senescence-associated β-galactosidase , overexpression of p53 and cyclin-dependent kinase inhibitor ( P38936 ) , and decreased expression of Q96EB6 . These changes were abolished by eplerenone ( 100 mg/kg/d ) , a mineralocorticoid receptor ( MR ) antagonist , but unaffected by hydralazine ( 80 mg/liter in drinking water ) . Furthermore , aldosterone induced similar changes in senescence-associated β-galactosidase , P38936 , and Q96EB6 expression in cultured human proximal tubular cells , which were normalized by an antioxidant , N-acetyl L-cysteine , or gene silencing of MR . DB04630 significantly delayed wound healing and reduced the number of proliferating human proximal tubular cells , while gene silencing of P38936 diminished the effects , suggesting impaired recovery from tubular damage . These findings indicate that aldosterone induces renal senescence in proximal tubular cells via the MR and P38936 -dependent pathway , which may be involved in aldosterone-induced renal injury . Genetic polymorphism and activities of human lung alcohol and aldehyde dehydrogenases : implications for ethanol metabolism and cytotoxicity . DB00898 dehydrogenase ( DB00067 ) and aldehyde dehydrogenase ( ALDH ) exhibit genetic polymorphism and tissue specificity . DB00067 and ALDH isozyme phenotypes from 39 surgical Chinese lung specimens were identified by agarose isoelectric focusing . The identity of the lung beta-ADHs was further demonstrated by their characteristic pH-activity profiles for ethanol oxidation , Km values for NAD and ethanol , and inhibition by DB01213 or 1,10-phenanthroline . The beta 2 allele , coding for beta 2 polypeptide , was found to be predominant in the lung specimens studied . The DB00067 activities in the lungs with the homozygous phenotype P00325 2-2 ( exhibiting beta 2 beta 2 ) and P00325 1-1 ( exhibiting beta 1 beta 1 ) and the heterozygous phenotype P00325 P35326 ( exhibiting beta 2 beta 2 , beta 2 beta 1 , and beta 1 beta 1 ) were determined to be 999 +/- 77 , 48 +/- 17 , and 494 +/- 61 nmol/min/g tissue , respectively . Fifty-one percent of the specimens studied lacked the P05091 activity band on the isoelectric focusing gels . The activities in the lung tissues with the P05091 -active phenotype and the inactive phenotype were determined to be 30 +/- 3 and 17 +/- 1 nmol/min/g tissue , respectively . These findings indicate that human pulmonary ethanol-metabolizing activities differ significantly with respect to genetic polymorphism at both the P00325 and the P05091 loci . The results suggest that individuals with high Vmax beta 2- DB00067 and deficient in low-Km mitochondrial P05091 , accounting for approximately 45 % of the Chinese population , may end up with acetaldehyde accumulation during alcohol consumption , rendering them vulnerable to tissue injury caused by this highly reactive and toxic metabolite . Role of the chaperonin cofactor P61604 in protein folding and sorting in yeast mitochondria . Protein folding in mitochondria is mediated by the chaperonin P10809 , the homologue of E. coli GroEL . Mitochondria also contain a homologue of the cochaperonin GroES , called P61604 , which is a functional regulator of the chaperonin . To define the in vivo role of the co-chaperonin , we have used the genetic and biochemical potential of the yeast S. cerevisiae . The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated . Our results identify P61604 as an essential component of the mitochondrial protein folding apparatus , participating in various aspects of P10809 function . P61604 is required for the folding and assembly of proteins imported into the matrix compartment , and is involved in the sorting of certain proteins , such as the Rieske DB01592 / Q15517 , passing through the matrix en route to the intermembrane space . The folding of the precursor of cytosolic dihydrofolate reductase ( P00374 ) , imported into mitochondria as a fusion protein , is apparently independent of P61604 function consistent with observations made for the chaperonin-mediated folding of P00374 in vitro . The temperature-sensitive mutations in P61604 map to a domain ( residues 25-40 ) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature . Oxidation of alcohols and reduction of aldehydes derived from methyl- and dimethylpyrenes by cDNA-expressed human alcohol dehydrogenases . Some methylated pyrenes can form DNA adducts in rat tissues after benzylic hydroxylation and sulpho conjugation . However , oxidation of the intermediate alcohols to carboxylic acids is an important competing pathway leading to detoxification . We previously showed that co-administration of ethanol or DB01213 strongly enhances DNA adduct formation by 1-hydroxymethylpyrene , indicating an involvement of alcohol dehydrogenases ( ADHs ) in the detoxification . This mechanism may be involved in the observed synergism of smoking and alcohol consumption in certain human cancers . In a preceding study , cDNA-expressed human P00325 efficiently oxidised 1- , 2- and 4-hydroxymethylpyrene ; these reactions were inhibited in the presence of ethanol or DB01213 . Here we report that P00326 , P00326 and P08319 also show substantial activity towards these substrates and two further congeners , 1-hydroxymethyl-6-methylpyrene and 1-hydroxymethyl-8-methylpyrene . All four DB00067 forms also catalysed the reverse reaction , implying that the aldehydes have to be sequestered by other enzymes , such as aldehyde dehydrogenases , for final detoxification . P00326 and P08319 activities towards hydroxymethylpyrenes were more strongly inhibited in the presence of ethanol and DB01213 than those of P00325 . P00326 was only inhibited at very high concentrations of the modulators . In conclusions , several human ADHs are capable of detoxifying benzylic alcohols of alkylated polycyclic aromatic hydrocarbons . However , some competing substrates and inhibitors may affect all these redundant detoxification systems , although to various extents . Role of arginine in superficial wound healing in man . DB00125 supplementation has been identified as advantageous in experimental wound healing . However , the mechanisms underlying this beneficial effect in tissue repair remain unresolved . Animal studies suggest that the beneficial role of arginine supplementation is mediated , at least in part through NO . The latter component mediates processes involved in tissue repair , including angiogenesis , epithelialization and collagen formation . This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man . Expression of enzymes , known to be involved in arginine metabolism , was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation . Plasma and wound fluid levels of arginine metabolites ( ornithine , citrulline , nitrate and nitrite = NOx ) were measured using High Performance Liquid Chromatography . Expression of P35228 , P29474 , arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue . P05089 concentration was measured in plasma and wound fluid using ELISA . Arginase-2 was determined using Western blot analysis . We observed increased levels of citrulline , ornithine , NOx and arginase-1 in wound fluid when compared with plasma . Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma . P35228 was expressed by neutrophils , macrophages , fibroblasts , keratinocytes and endothelial cells upon wounding , whereas P29474 reactivity was observed in endothelial cells and fibroblasts . P05089 was expressed in neutrophils post-wounding , while arginase-2 staining was observed in endothelial cells , keratinocytes , fibroblasts , macrophages and neutrophils . For the first time , human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin . P01375 -α-mediated cardiorenal injury after rhabdomyolysis in rats . The P01375 -α serum level increases after rhabdomyolysis and is involved in the subsequent cardiorenal injury . In the present study , we investigated the P01375 -α-dependent cell signaling pathways implicated in cellular injury in these organs . Rhabdomyolysis was induced by intramuscular glycerol injection in rats . Renal function , cardiac and renal pathology , and activation of caspases were evaluated during the first 24 h after glycerol injection . P01375 -α blockade with infliximab reduced tubular necrosis and cardiorenal apoptosis . Cellular Fas-associated protein with death domain-like IL-1β-converting enzyme inhibitory protein ( cFLIP ) , an inhibitor of caspase-8 , was overexpressed in the kidney but not in the heart . The inhibitory effect of cFLIP blunted caspase-8 activation in the kidney . In this condition , the cellular response to the P01375 -α stimulus was driven to receptor-interacting protein-1 ( Q13546 ) -mediated necroptosis . Treatment with Q13546 inhibitor ( necrostatin-1 ) isolated or in combination with infliximab showed a similar reduction in tubular necrosis , underscoring the importance of P01375 -α-mediated tubular necroptosis in this model . P01375 -α played a positive regulatory role in the transcription of proapoptotic Bax and p53-upregulated modulator of apoptosis ( PUMA ) proteins . DB00065 treatment reduced caspase-9-mediated apoptosis in both organs . Treatment with a caspase-8 inhibitor showed that caspase-8 participated in the process of apoptosis only in the heart , upstream of caspase-9 activation . P01375 -α-mediated necroptosis is the predominant form of tubular injury observed in the glycerol model . P01375 -α up regulates Bax and PUMA proapoptotic proteins , resulting in activation of the intrinsic pathway of apoptosis in the kidney and heart . Flow cytometric analysis of mammalian glial cultures treated with methotrexate . DB00563 ( MTX ) is an antineoplastic drug that acts by competitive inhibition of the enzyme dihydrofolate reductase ( P00374 ) . MTX treatment of cultured cell lines leads to the emergence of resistant cell populations . Studies using stepwise selection procedures have demonstrated that MTX resistance conferred by overproduction of P00374 can be caused by P00374 gene amplification . We examined the effect of MTX on cells whose origin more closely approximates the in vivo condition by developing a culture system using dissociated brain tissue from 17-19 day old mouse embryos . At the first passage , cultures were divided into control and MTX groups . Cells were treated with the same or successively higher concentrations of MTX at each passage over a 3-4 month period . The first passage eliminated neurons and left a glial culture comprised of approximately 90 % astrocytes . We used the Fluorescence Activated Cell Sorter in conjunction with fluorescent dyes to measure P00374 content , DNA content , size , and viability of glial cells following MTX treatment . MTX-treated cells divided but grew more slowly and were larger than untreated cells . Stepwise selection in 30/60/90 nM or 60/120 nM MTX resulted in significant two- to threefold increases in fluorescence , and hence P00374 levels . Slot hybridizations assays demonstrated a threefold increase in P00374 gene copy number in the DNA from the 30/60/90 cultures . Thus , our findings were consistent with the results obtained from somatic cell lines , and lend support to the hypothesis that gene amplification may be a common mechanism for the acquisition of resistance in many types of cells . They also indicate that glial cells may be a specific target for cytotoxic effects of MTX on the central nervous system . Circulating cytokines and risk of B-cell non-Hodgkin lymphoma : a prospective study . Cytokines play important roles in B-cell activation , proliferation , and apoptosis , thus may be etiologically related to risk of B-cell non-Hodgkin lymphoma ( B- Q9NZ71 ) . However , the association between circulating levels of cytokines and B- Q9NZ71 risk has not been prospectively studied in non-HIV populations . The objective of this study was to assess this association by conducting a case-control study nested within a prospective cohort of non-HIV-infected , healthy women . Fifteen cytokines were measured in samples collected a median of 8.2 years prior to diagnosis in 92 cases and two matched controls per case . Only cytokines that showed adequate temporal reproducibility over a two-year period were included . The odds ratio ( OR ) for the highest tertile relative to the lowest was elevated for soluble P60568 receptor ( sIL-2R ) ( OR = 2.5 , 95 % CI = 1.4-4.7 , p ( trend ) < 0.01 ) and decreased for P35225 ( OR = 0.5 , 95 % CI = 0. P35326 .0 , p ( trend ) = 0.05 ) . Three other cytokines were marginally associated with risk of B- Q9NZ71 : P01375 ( OR = 1.7 , 95 % CI = 0.9-3.3 , p ( trend ) = 0.11 ) , sTNF-R2 ( OR = 1.9 , 95 % CI = 0.9-3.5 , p ( trend ) = 0.06 ) , and P05113 ( OR = 0.5 , 95 % CI = 0.3-1.0 , p ( trend ) = 0.06 ) . No association was observed between B- Q9NZ71 risk and levels of the other cytokines measured ( IL-1beta , IL-1RA , P60568 , P05112 , P05231 , P22301 , IL-12 , IL-12p70 , CRP and sTNF- Q96GN5 ) . This study suggests that dysregulated cytokines may be involved in B- Q9NZ71 development . Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced P28906 + cell-derived human dendritic cells . The efficient genetic modification of P28906 + cell-derived dendritic cells ( DC ) will provide a significant advancement towards the development of immunotherapy protocols for cancer , autoimmune disorders and infectious diseases . Recent reports have described the transduction of P28906 + cells via retrovirus- and lentivirus-based gene transfer vectors and subsequent differentiation into functional DC . Since there is significant apprehension regarding the clinical uses of HIV-based vectors , in this report , we compare a murine leukemia virus ( MLV ) - and a human immunodeficiency virus ( HIV ) -based bicistronic vector for gene transfer into human P28906 + cells and subsequent differentiation into mature DC . Each vector expressed both EGFP and the dominant selectable marker P00374 (L22Y) allowing for the enrichment of marked cells in the presence of the antifolate drug trimetrexate ( TMTX ) . Both MLV-based and HIV-based vectors efficiently transduced cytokine mobilized human peripheral blood P28906 + cells . However , in vitro expansion and differentiation in the presence of GM- P04141 , P01375 , Flt-3L , P21583 and P05112 resulted in a reduction in the percentage of DC expressing the transgene . Selection with TMTX during differentiation increased the percentage of marked DC , resulting in up to 79 % ( MLV vector ) and up to 94 % ( lentivirus-vector ) transduced cells expressing EGFP without loss of DC phenotype . Thus , MLV-based vectors and in vitro selection of transduced human DC show great promise for immunotherapy protocols . Temporal profiling of lapatinib-suppressed phosphorylation signals in P00533 / P04626 pathways . DB01259 is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor ( P00533 ) and EGFR2 ( also known as P04626 ) . However , there is only limited information about the in vivo effects of lapatinib on P00533 / P04626 and downstream signaling targets . Here , we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics . Among 4953 identified phosphopeptides from 1548 proteins , a small proportion ( 5-7 % ) was regulated at least twofold by 1-10 μm lapatinib . We obtained a comprehensive phosphorylation map of 21 sites on P00533 / P04626 , including nine novel sites on P04626 . Among them , serine/threonine phosphosites located in a small region of P04626 ( amino acid residues 1049-1083 ) were up-regulated by the drug , whereas all other sites were down-regulated . We show that DB02527 -dependent protein kinase is involved in phosphorylation of this particular region of P04626 and regulates P04626 tyrosine kinase activity . Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation , such as Q96LT9 complexes ( i.e. hnRNP , snRNP , telomerase , ribosome ) , are linked to P00533 / P04626 signaling networks . To our knowledge , this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor . The results provide new insights into P00533 / P04626 regulation through region-specific phosphorylation , as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib . Modulation of a Mr 175,000 c-neu receptor isoform in Q9UBA6 / P00374 cells by serum starvation . The neu proto-oncogene product has been found to exist in two interconvertible forms in Q9UBA6 / P00374 mouse fibroblasts . The 185-kilodalton form ( p185 ) present in growing cells is replaced by a 175-kilodalton form ( p175 ) under conditions of serum starvation . This low molecular weight form accounts almost exclusively for the phosphotyrosine content of the receptor and is associated with increased tyrosine kinase activity . Addition of serum , platelet-derived growth factor or tumor promoter induces conversion of p175 to p185 within minutes , and this increase in molecular weight is associated with phosphorylation of serine and threonine ; removal of serum growth factors is followed by replacement of p185 with p175 over several hours . Unlike Q9UBA6 / P00374 cells , the human breast cancer cell line SK-Br-3 expresses a high molecular weight neu/ P04626 receptor with unchanged phosphotyrosine content in both serum-starved and serum-stimulated cultures . These findings indicate that activation of the neu proto-oncogene product in Q9UBA6 / P00374 cells may be regulated in part by protein kinase C-mediated receptor transmodulation rather than by ligand availability alone . Expression Enhancement in DB00072 Therapeutic Monoclonal Antibody Production using Genomic Amplification with DB00563 . BACKGROUND : DB00072 ( Herceptin ) is a humanized monoclonal antibody ( mAb ) which is used for specific treatment of metastatic breast cancer in patients with overexpression of P04626 /neu receptor . In this study , we have attempted to develop a biosimilar version of trastuzumab mAb . METHODS : According to in silico studies , the heavy and light chains of trastuzumab mAb were designed and constructed . The recombinant constructs were co-transfected in CHO DG44 cell line . Stable transformants were selected on a semi solid medium . Genomic amplification with methotrexate was achieved for heavy chain gene amplification . Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method . RESULTS : Three folds of amplification were obtained after seven rounds of methotrexate treatments . The results indicated the equal expression level of heavy and light chains . The yield of purified mAb was between 50 to 60 mg/l /day . According to the results , the produced mAb had similar affinity to P04626 (+) tumor cells to that of Herceptin . CONCLUSION : High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker , such as Dihydrofolate Reductase ( P00374 ) . It is usually accepted that P00374 gene can be amplified in P00374 (－) CHO cells , which consequently leads to amplification of the co-linked target gene , and finally amplification of recombinant protein . In this research , with the aim of producing a biosimilar version of herceptin , the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR . Impact of fish oil enriched total parenteral nutrition on DNA synthesis , cytokine release and receptor expression by lymphocytes in the postoperative period . A prospective randomized study on sixty patients was conducted to investigate the effects of a fish oil containing total parenteral nutrition ( O15533 ) regimen in the postoperative period on lymphocyte subset distribution , proliferation , cytokine production and interleukin-2 receptor ( IL-2R ) expression . Patients who underwent large bowel surgery were divided into three groups . Nineteen patients received O15533 with fish oil ( 0.2 g/kg body weight per day ) plus soybean oil ( 1.0 g/kg per day ) , twenty patients received soybean oil ( 1.2 g/kg per day ) , and twenty-one patients who were on a fat-free regimen served as the control group . Natural killer ( NK ) cells , total , B- , T- , DB00451 - , T8-lymphocytes , proliferation of lymphocytes , in vitro production of P60568 , P01579 , P01375 , and IL-2R expression were measured . Fish oil administration did not affect subset distribution and proliferation of lymphocytes . Production of interleukin-2 ( P60568 ) , interferon gamma ( P01579 ) and tumor necrosis factor alpha ( P01375 ) was augmented , and IL-2R expression less enhanced compared with the controls . It is concluded that administration of 0.2 g/kg per day fish oil after a moderate surgical stress is not immunosuppressive , but enhances the production of P01579 , P01375 and possibly P60568 . P00797 status does not predict the anti-hypertensive response to angiotensin-converting enzyme inhibition in African-Americans . DB00519 Multicenter Study Group . The angiotensin-converting enzyme ( P12821 ) inhibitor trandolapril , a non-sulfhydryl prodrug which is hydrolysed into trandolaprilat , was studied in 322 hypertensives of African-American descent using a double-blind , randomised , placebo-controlled , parallel study design . Following 6 weeks of double-blind treatment with placebo or 0.25 to 16 mg/day trandolapril , an analysis of drug effect on trough blood pressure ( BP ) stratified by age , gender , weight , pre-treatment plasma renin activity , and trandolaprilat concentration was performed . Two mg was the lowest effective trandolapril dose , whereas doses above 4 mg did not significantly reduce trough BP . Reduction in BP did not correlate with trough plasma trandolaprilat concentration . Pre-treatment plasma renin activity was not a reliable indicator of anti-hypertensive response , as similar reductions in BP occurred even in patients with the lowest renin levels . There were no observable differences based on age , gender or measurements of the renin-angiotensin-aldosterone axis . In conclusion , neither age , gender or plasma renin activity influenced anti-hypertensive response to angiotensin-converting enzyme inhibition in African-Americans . Effects of retroviral-mediated P08183 expression on hematopoietic stem cell self-renewal and differentiation in culture . Ex vivo expansion of hematopoietic stem cells would be useful for bone marrow transplantation and gene therapy applications . Toward this goal , we have investigated whether retrovirally-transduced murine stem cells could be expanded in culture with hematopoietic cytokines . Bone marrow cells were transduced with retroviral vectors expressing either the human multidrug resistance 1 gene ( HaMDR1 ) , a variant of human dihydrofolate reductase ( HaDHFR ) , or both P08183 and P00374 in an internal ribosomal entry site ( IRES ) -containing bicistronic vector ( HaMID ) . Cells were then expanded for 15 days in cultures stimulated with interleukin ( IL ) -3 , P05231 , and stem cell factor . When very low marrow volumes were injected into lethally irradiated recipient mice , long-term reconstitution with 100 % donor cells was seen in all mice injected with HaMDR1- or HaMID-transduced cells . By contrast , engraftment with HaDHFR- or mock-transduced cells ranged from partial to undetectable despite injection of significantly larger marrow volumes . In addition , mice transplanted with expanded HaMDR1- or HaMID-transduced stem cells developed a myeloproliferative disorder that was characterized by an increase in abnormal peripheral blood leukocytes . These results show that P08183 -transduced stem cells can be expanded in vitro with hematopoietic cytokines , but indicate that an increased stem cell division frequency can lead to stem cell damage . Pharmacological properties of thalidomide and its analogues . Thalidomide and its immunomodulatory imide drugs ( IMiDs ) analogues DB00480 ( DB00480 , DB00480 ) and CC-4047 ( Actimid , DB08910 ) have been used as anti-inflammatory and anticancerous drugs in the recent years . Thalidomide and IMiDs inhibit the cytokines tumour necrosis factor-alpha ( P01375 ) , interleukins ( IL ) 1-beta , 6 , 12 , and granulocyte macrophage-colony stimulating factor ( GM- P04141 ) . They also costimulate primary human T , NKT and NK lymphocytes inducing their proliferation , cytokine production , and cytotoxic activity . On the other hand , the compounds are anti-angiogenic , anti-proliferative , and pro-apoptotic . Thalidomide analogues have been used as inhibitors of alpha glucosidase and could be potential drugs for diabetes treatment . In this review , we explore the current trend of the different structures , the new patents , and the possible new applications in different pathologies . DB00563 selectable retroviral vectors for Gaucher disease . To develop a gene therapy protocol suitable for the treatment of a benign disease such as Gaucher disease , we have developed two bicistronic vectors that allow transduced cells to be selected for with methotrexate ( MTX ) . The two vectors differ in the presence or absence of a mutant polyoma enhancer ( delta Mo + PyF101 ) replacing the wild-type retroviral enhancer in the LTR . Infection of human TF-1 and K562 cells , Gaucher type II fibroblasts and murine hemopoietic bone marrow cells conferred MTX resistance and glucocerebrosidase ( GC ) expression . Upon increasing MTX concentrations , the number of proviral copies and GC activity increased , demonstrating in vitro selection of retrovirus-transduced cells . At high MTX selection pressure , up to 140 microM for infected Gaucher type II fibroblasts , no endogenous wild-type P00374 amplification could be detected , indicating that both retroviral constructs provide sufficient P00374 protein levels . Upon transduction , murine bone marrow cells were protected against otherwise lethal MTX concentrations ( range 1-5 microM MTX ) . Flow cytometry specific for human GC ( hGC ) demonstrated that in vitro selection resulted in increased percentages of hGC-positive murine cells . In conclusion , the generated bicistronic vectors are ideally suited to investigate whether an in vivo selection approach for retrovirus-transduced cells is feasible . Such a strategy might abolish the need for a high initial transduction efficiency and might result in a gene therapy protocol devoid of the undesirable need for marrow ablative treatment of the recipient .