interacts_with DB00205?
DB00205 -resistant dihydrofolate reductase enzymes of Plasmodium falciparum are not enzymatically compromised in vitro . Plasmodium falciparum , the protozoan that causes the most lethal form of human malaria , has been controlled principally by two safe , affordable drugs , chloroquine and sulfadoxine-pyrimethamine ( SP ) . Studies in the laboratory and in the field have demonstrated that resistance to SP depends on non-synonymous point mutations in the dihydrofolate reductase ( P00374 ) , and dihydropteroate synthase ( P49366 ) coding regions . Parasites that carry dhfr genes with 3 or 4 point mutations ( 51I/59R/108N triple mutation or 51I/59R/108N/164L quadruple mutation ) are resistant to pyrimethamine in vitro and patients infected with these parasites respond poorly to SP treatment . The wide spread of these pyrimethamine-resistant alleles demonstrates the increased fitness over drug-sensitive alleles in the presence of the drug . However , it is not clear whether these alleles might reduce the fitness of parasites in the absence of drug pressure . As a first step , we compared the kinetic properties of the wild type , and three mutant alleles to determine whether the native P00374 -thymidylate synthase form of the mutant proteins showed compromised activity in vitro . The mutant enzymes had K(m) values for their substrate , dihydrofolate that were significantly lower than the wild type , k(cat) values in the same range as the wild type enzyme , and k(cat)/K(m) values higher than wild type . In contrast , the K(m) values for the NADPH cofactor were higher than wild type for the mutant enzymes . These observations suggest that the fitness of these parasites may not be compromised relative to those that carry the wild type allele , even without sustained SP drug pressure . P10275 is expressed in murine choroid plexus and downregulated by 5alpha-dihydrotestosterone in male and female mice . The choroid plexuses ( CPs ) of the brain form a unique interface between the peripheral blood and the cerebrospinal fluid ( P04141 ) . CPs produce several neuroprotective peptides , which are secreted into the P04141 . Despite their importance in neuroprotection , the mechanisms underlying the regulation of most of these peptides in CPs remain unknown . Androgens regulate the expression of neuroprotective peptides in several tissues where the androgen receptor ( AR ) is coexpressed , including the brain . The presence of AR in CPs has never been investigated , but recent studies in our laboratory show that the CP is an androgen-responsive tissue . In order to fulfill this gap , we investigated and characterized AR distribution and expression in male and female rat CPs and in primary cultures from rat CP epithelial cells . In addition , the response of AR to 5alpha-dihydrotestosterone ( DB02901 ) in castrated male and female mice subjected to DB02901 replacement was analyzed . We show that rat CP epithelial cells contain AR mRNA and protein . Moreover , we demonstrate that AR is downregulated by DB02901 in mice CPs . Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi ( Coccidia , Apicomplexa ) . The rat parasite Eimeria nieschulzi is a suitable model for transfection studies and was used as an additional model organism for the genus Eimeria . We describe the transfection of this apicomplexan parasites and the cultivation of transformed stages in cell culture and in vivo . The beta-galactosidase or yellow fluorescent protein was expressed in all parasitic stages up to the second merozoite generation in vitro under control of the heterologous promoter region of Eimeria tenella mic1 gene previously described for E. tenella transfection . DB00205 resistant E. nieschulzi parasites were obtained in vitro after transfection with a plasmid encoding the Toxoplasma gondii dhfr/ts-m2m3 gene . Co-transfection experiments with an YFP-plasmid resulted in pyrimethamine resistant and fluorescent parasitic stages . Infection of rats with transfected E. nieschulzi sporozoites directed to expression of beta-galactosidase or YFP in oocysts . Co-transfection with YFP/ P00374 -TS allowed selection of resistant parasites in vivo . Excreted transgenic oocysts showed arrangement of YFP expression which lead to questions about meiotic recombination frequency and mechanisms . Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes . Rat cortical astrocytes regulate their cell volume in response to hypotonic challenge . This regulation is believed to depend largely on the release of chloride or organic osmolytes through anion channels . Using whole-cell recordings , we identified weakly outwardly rectifying chloride currents that could be activated in response to hypotonic challenge . These currents exhibited the following permeability sequence upon replacement of chloride in the bathing solution with various anions : I- > NO3- > Cl- > Gluc- > or = MeS- > Ise- . Interestingly , extracellular I- , albeit showing the greatest permeability , blocked the currents with an IC50 of approximately 50 mM . Currents were almost completely inhibited by 123 microM P16860 and partially inhibited by 200 microM niflumic acid or 200 microM DIDS . Additionally , the total number of Cl- ions effluxed through the hypotonically activated channels was markedly similar to the total solute efflux during volume regulation . We therefore propose the hypotonically activated chloride channel as a major contributor to volume regulation of astrocytes . To examine potential candidate chloride channel genes expressed by astrocytes , we employed RT-PCR to demonstrate the presence of transcripts for P51788 , 3 , 4 , 5 , and 7 , as well as for P21796 and P13569 in cultured astrocytes . Moreover , we performed immunostaining with antibodies against each of these channels and showed the strongest expression of P51788 and P51790 , strong expression of P51795 and P21796 , weak expression of P51798 and very weak expression of P51793 and P13569 . Intriguingly , although we found at least seven Cl- channel proteins from three different gene families in astrocytes , none appeared to be active in resting cells . Rational drug design approach for overcoming drug resistance : application to pyrimethamine resistance in malaria . DB00205 acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase ( P00374 -TS ) . Resistance in the most important human parasite , Plasmodium falciparum , initially results from an S108N mutation in the P00374 domain , with additional mutation ( most commonly C59R or N51I or both ) imparting much greater resistance . From a homology model of the 3-D structure of P00374 -TS , rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance . Compared to pyrimethamine ( Ki 1.5 nM ) with purified recombinant P00374 fromP. falciparum , the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM , but against the P00374 bearing the double mutation ( C59R + S108N ) , the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM , respectively . The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type P00374 ( with Ki 0.30 nM ) and the doubly mutant ( C59R +S108N ) purified enzyme ( with Ki 2.40 nM ) . Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50 % inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the P04264 parasite line bearing the double mutation ( S108N + C59R ) , compared to 10.2 microM for pyrimethamine . These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice . Microarray analysis revealed different gene expression patterns in HepG2 cells treated with low and high concentrations of the extracts of Anacardium occidentale shoots . In this study , the effects of low and high concentrations of the Anacardium occidentale shoot extracts on gene expression in liver HepG2 cells were investigated . From MTT assays , the concentration of the shoot extracts that maintained 50 % cell viability ( IC(50) ) was 1.7 mg/ml . Cell viability was kept above 90 % at both 0.4 mg/ml and 0.6 mg/ml of the extracts . The three concentrations were subsequently used for the gene expression analysis using Affymetrix Human Genome 1.0 S.T arrays . The microarray data were validated using real-time qRT-PCR . A total of 246 , 696 and 4503 genes were significantly regulated ( P < 0.01 ) by at least 1.5-fold in response to 0.4 , 0.6 and 1.7 mg/ml of the extracts , respectively . Mutually regulated genes in response to the three concentrations included CDKN3 , LOC100289612 , P00374 , Q99986 , Q99741 , Q96GD4 and P78334 . Genes like Q07973 , P38398 , O14965 , P06493 , P24941 , P11802 and P06213 were significantly regulated at 0.6 mg/ml and 1.7 mg but not at 0.4 mg/ml . However , the expression of genes including O75473 , P17936 , P06400 , P14735 , P01130 , P55157 , P04114 , MTIX , P04179 and P08294 were exclusively regulated at the IC(50) concentration . In conclusion , low concentrations of the extracts were able to significantly regulate a sizable number of genes . The type of genes that were expressed was highly dependent on the concentration of the extracts used . A P04035 inhibitor possesses a potent anti-atherosclerotic effect other than serum lipid lowering effects -- the relevance of endothelial nitric oxide synthase and superoxide anion scavenging action . We have determined whether the anti-atherosclerotic effect of a 3-hydroxy-3-methyl-glutaryl- DB01992 ( HMG- DB01992 ) reductase inhibitor ( fluvastatin ) is mediated through nitric oxide ( NO ) as well as affecting plasma lipids . NO related vascular responses , endothelial nitric oxide synthase ( P29474 ) mRNA and superoxide anion ( O(2)(-) ) release were examined in vascular walls of oophorectomized female rabbits fed 0.5 % cholesterol chow for 12 weeks with or without fluvastatin ( 2 mg/kg per day ) . Serum lipid profile was not different between two groups . NO dependent responses stimulated by acetylcholine and calcium ionophore A23187 and tone related basal NO response induced by N(G)-monomethyl-L-arginine acetate ( L- Q13145 ) ; nitric oxide synthase inhibitor were all improved by fluvastatin treatment . Endothelium independent vasorelaxation induced by nitroglycerin was not different between the two groups of rabbits ' arteries . DB01095 treatment increased cyclic GMP concentration in aorta of rabbits . P29474 mRNA expression and O(2)(-) release were measured in aorta using competitive reverse transcription-polymerase chain reaction ( RT-PCR ) and with lucigenin analogue , 2-methyl-3,7-dihydroimidazol [1,2-a]pyrazine-3-one ( MCLA ) chemiluminescence methods . P29474 mRNA in the endothelial cells of aorta was significantly up-regulated and O(2)(-) production was significantly reduced in fluvastatin treated rabbit aorta . Anti-macrophage staining area , but not anti-smooth muscle cell derived actin stained area in the aorta was also reduced by fluvastatin treatment . Conclusion , fluvastatin , a P04035 inhibitor , retards the initiation of atherosclerosis formation through the improvement of NO bioavailability by both up-regulation of P29474 mRNA and decrease of O(2)(-) production in vascular endothelial cells , and this means that part of the anti-atherosclerotic effect of fluvastatin may be due to nonlipid factors . Functional analysis of cis-acting elements regulating the alternative splicing of human P13569 exon 9 . The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator ( P13569 ) mRNA is associated with monosymptomatic forms of cystic fibrosis . Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3' splice site . We have generated a minigene carrying human P13569 exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing . Transfections into human cell lines showed that T5 , but not P02786 or T7 alleles , significantly increases the alternative splicing of exon 9 . Moreover , we found that another polymorphic locus juxtaposed upstream of the T tract , and constituted by ( TG ) (n)repeats , can further modulate exon 9 skipping but only when activated by the T5 allele . Then , we extended our studies to the mouse P13569 exon 9 which does not show alternative splicing . Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site . We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart . The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate , with the UG/U 3' splice site sequences , the extent of exon 9 skipping in P13569 mRNA . In vitro susceptibility of various genotypic strains of Toxoplasma gondii to pyrimethamine , sulfadiazine , and atovaquone . Sulfadiazine , pyrimethamine , and atovaquone are widely used for the treatment of severe toxoplasmosis . Their in vitro activities have been almost exclusively demonstrated on laboratory strains belonging to genotype I . We determined the in vitro activities of these drugs against 17 strains of Toxoplasma gondii belonging to various genotypes and examined the correlations among 50 % inhibitory concentrations ( IC50s ) , growth kinetics , strain genotypes , and mutations on drug target genes . Growth kinetics were determined in THP-1 cell cultures using real-time PCR . IC50s were determined in MRC-5 cell cultures using a T. gondii-specific enzyme-linked immunosorbent assay performed on cultures . Mutations in dihydrofolate reductase ( P00374 ) , dihydropteroate synthase ( P49366 ) , and cytochrome b genes were determined by sequencing . DB00205 IC50s ranged between 0.07 and 0.39 mg/liter , with no correlation with the strain genotype but a significant correlation with growth kinetics . Several mutations found on the P00374 gene were not linked to lower susceptibility . DB01117 IC50s were in a narrow range of concentrations ( mean , 0.06 +/- 0.02 mg/liter ) ; no mutation was found on the cytochrome b gene . IC50s for sulfadiazine ranged between 3 and 18.9 mg/liter for 13 strains and were > 50 mg/liter for three strains . High IC50s were not correlated to strain genotypes or growth kinetics . A new mutation of the P49366 gene was demonstrated in one of these strains . In conclusion , we found variability in the susceptibilities of T. gondii strains to pyrimethamine and atovaquone , with no evidence of drug resistance . A higher variability was found for sulfadiazine , with a possible resistance of three strains . No relationship was found between drug susceptibility and strain genotype . P10275 overexpression alters binding dynamics of the receptor to chromatin and chromatin structure . BACKGROUND : Castration-resistant prostate cancers ( CRPCs ) overexpress often androgen receptor ( AR ) . Here , we investigated the effect of AR overexpression on the dynamics of AR loading and RNA polymerase II ( RNA Pol II ) recruitment to chromatin . Acetylation of histone 3 ( AcH3 ) on lysines 9 and 14 ( P35527 and P02533 ) was also studied . METHODS : We used an LNCaP-based AR overexpression cell line model that includes a control line and two sublines , LNCaP-ARmo and LNCaP-ARhi , which overexpress AR twofold to threefold and fourfold to fivefold , respectively . Cells were exposed to 1 or 100 nM of dihydrotestosterone ( DB02901 ) . Chromatin immunoprecipitation ( ChIP ) on the promoters and enhancers of prostate specific antigen ( PSA ) and transmembrane protease , serine 2 ( O15393 ) genes was performed . qRT-PCR was used to measure the levels of PSA and O15393 transcripts . RESULTS : Upon stimulation with 1 nM DB02901 , AR and RNA Pol II were recruited onto PSA and O15393 enhancer regions to a greater extent ( P < 0.05 ) in AR-overexpressing cells compared to control cells . The difference in AR loading between the control and AR-overexpressing cells was abolished by a higher DB02901 concentration . The ratio of AcH3/H3 was increased in AR-overexpressing cells . The induction of transcription of PSA and O15393 occurred earlier in the AR-overexpressing cells . CONCLUSIONS : Our findings suggest that the levels of AR potentiate the recruitment of the AR , as well as components of the basic transcription machinery , to chromatin and affect the acetylation of histones in the presence of low levels of androgens . These changes result in enhanced gene transcription of AR target genes . In vitro selection of Plasmodium falciparum lines resistant to dihydrofolate-reductase inhibitors and cross resistance studies . A cloned Plasmodium falciparum line was subjected to in vitro drug pressure , by employing a relapse protocol , to select progressively resistant falciparum lines to pyrimethamine and cycloguanil , the two dihydrofolate-reductase ( P00374 ) inhibitor antimalarial drugs . The falciparum lines resistant to pyrimethamine were selected much faster than those resistant to cycloguanil . In 348 days of selection/cultivation , there was 2,400-fold increase in IC50 value to pyrimethamine , whereas only about 75-fold decrease in sensitivity to cycloguanil was registered in 351 days . DB00205 -resistant parasites acquired a degree of cross resistance to cycloguanil and methotrexate , another P00374 inhibitor , but did not show any cross resistance to some other groups of antimalarial drugs . The highly pyrimethamine-resistant line was not predisposed for faster selection to cycloguanil resistance . Resistance acquired to pyrimethamine was stable . The series of resistant lines obtained form a good material to study the ' evolution ' of resistance more meaningfully at molecular level . Synthesis and in vitro evaluation of potential anti-leishmanial targeted drugs of pyrimethamine . DB00205 , an antimalarial drug , was found to be able to inhibit both enzymes ( P00374 -TS and PTR1 ) of the leishmanial folate pathway , although this effect in vivo appears only in relatively high concentrations . To reach the parasites inside macrophage cells , where they are sheltered , targeted drugs of pyrimethamine , carboxymethyldextran-thiomannopyranoside-pyrimethamine ( CMD-P ) , and succinyldextran-thiomannopyranoside-pyrimethamine ( SD-P ) , were synthesized and assayed against L.(L.) amazonensis amastigotes . CMD-P has 2.43 % and SD-P has 2.58 % of pyrimethamine attached . At a CMD-P dose of 200 microg/mL ( 4.86 microg/mL pyrimethamine ) , the results were very promising , with a destruction of approximately 50 % of the intracellular amastigotes , with no detectable toxicity to macrophage cells . SD-P in similar doses did not show good results , probably due to different patterns of drug release . These results open the possibility of treating leishmaniasis with a safe targeted drug of pyrimethamine released directly inside the macrophage cells , reducing the host systemic toxicity . Crystal structure of dihydrofolate reductase from Plasmodium vivax : pyrimethamine displacement linked with mutation-induced resistance . DB00205 ( Pyr ) targets dihydrofolate reductase of Plasmodium vivax ( PvDHFR ) as well as other malarial parasites , but its use as antimalarial is hampered by the widespread high resistance . Comparison of the crystal structures of PvDHFR from wild-type and the Pyr-resistant ( SP21 , DB00133 -58 --> DB00125 + DB00133 -117 --> DB00174 ) strain as complexes with NADPH and Pyr or its analog lacking p-Cl ( Pyr20 ) clearly shows that the steric conflict arising from the side chain of DB00174 -117 in the mutant enzyme , accompanied by the loss of binding to DB00133 -120 , is mainly responsible for the reduction in binding of Pyr . Pyr20 still effectively inhibits both the wild-type and SP21 proteins , and the x-ray structures of these complexes show how Pyr20 fits into both active sites without steric strain . These structural insights suggest a general approach for developing new generations of antimalarial P00374 inhibitors that , by only occupying substrate space of the active site , would retain binding affinity with the mutant enzymes . Changes in enzyme activity and expression of P00374 of Toxoplasma gondii by antifolates . The responses to antifolates of Toxoplasma gondii were investigated by measuring the dihydrofolate reductase ( P00374 ) activity , quantity of P00374 mRNA , and single-strand conformational polymorphism ( SSCP ) pattern . DB00205 ( Q9BRP8 ) and methotrexate ( MTX ) were tested as antifolates . When T. gondii was treated with Q9BRP8 , the viability was decreased by the increasing concentration of Q9BRP8 , P00374 activity tended to increase as the passage proceeded , and the quantity of mRNA expressed was also increased according to passages . The viability of T. gondii was decreased by the increasing concentration of MTX , but it was maintained over 40 % up to 100 microM MTX . P00374 activity was 77.4 % in the 1st passage ( 1 microM ) . 82.2 % in the 4th passage ( 10 microM ) , and 141.3 % in the 7th passage ( 100 microM ) . But no changes were detected in SSCP pattern of T. gondii exposed to Q9BRP8 and MTX , both . These results suggested that the response of T. gondii to Q9BRP8 was regulated by transcriptional level and that , in MTX , the viability of T. gondii was derived from increasing P00374 activity . 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 . Genomic comparison of Escherichia coli P04264 strains isolated from the cerebrospinal fluid of patients with meningitis . Escherichia coli is a major cause of enteric/diarrheal diseases , urinary tract infections , and sepsis . E. coli P04264 is the leading gram-negative organism causing neonatal meningitis , but the microbial basis of E. coli P04264 meningitis is incompletely understood . Here we employed comparative genomic hybridization to investigate 11 strains of E. coli P04264 isolated from the cerebrospinal fluid ( P04141 ) of patients with meningitis . These 11 strains cover the majority of common O serotypes in E. coli P04264 isolates from P04141 . Our data demonstrated that these 11 strains of E. coli P04264 can be categorized into two groups based on their profile for putative virulence factors , lipoproteins , proteases , and outer membrane proteins . Of interest , we showed that some open reading frames ( ORFs ) encoding the type III secretion system apparatus were found in group 2 strains but not in group 1 strains , while ORFs encoding the general secretory pathway are predominant in group 1 strains . These findings suggest that E. coli P04264 strains isolated from P04141 can be divided into two groups and these two groups of E. coli P04264 may utilize different mechanisms to induce meningitis . Intake of vitamin P04264 and K2 and risk of hip fractures : The Hordaland Health Study . BACKGROUND : Evidence of the effect of vitamin K on bone health is conflicting . The aim was to investigate the association between intake of vitamins P04264 and K2 and subsequent risk of hip fracture in a general population sample , as well as potential effect modification by apolipoprotein E gene ( P02649 ) status by presence of the E4 allele . METHODS : 1238 men and 1569 women 71-75 years of age were included in the community-based Hordaland Health Study 1997-1999 in Western Norway . Information on hip fracture was obtained from hospitalizations in the region from enrolment until 31 December 2009 . Information on intake of vitamins P04264 and K2 collected at baseline was used as potential predictors of hip fracture in Cox proportional hazards regression analyses . RESULTS : Participants in the lowest compared to the highest quartile of vitamin P04264 intake had increased risk of suffering a hip fracture ( hazard ratio (HR)=1.57 [ 95 % CI 1.09 , 2.26 ] ) . Vitamin K2 intake was not associated with hip fracture . Presence of APOE4-allele did not increase the risk of hip fracture , nor was there any effect modification with vitamin P04264 in relation to risk of hip fracture . CONCLUSIONS : A low intake of vitamin P04264 , but not K2 , was associated with an increased risk of hip fractures . DB00205 resistant Plasmodium falciparum : overproduction of dihydrofolate reductase by a gene duplication . The accumulation of [3H]pyrimethamine by pyrimethamine-resistant ( Pyrr ) mutants of the Plasmodium falciparum strain FCR3 was examined by measuring the accumulation of drug in infected red blood cells . [3H] DB00205 was stage specifically accumulated in trophozoites and schizont infected red blood cells . The mutant parasites accumulated drug as efficiently as FCR3 . DB00205 was associated with a high molecular weight protein that eluted from a Sephadex G200 column exactly as [3H]fluorodeoxyuridinemonophosphate ( FdUMP ) labeled parasite dihydrofolate reductase-thymidylate synthetase ( P00374 -TS ) enzyme . These results suggested that the pyrimethamine resistance was not associated with decreased drug permeability of the membrane . P00374 -TS-[3H]FdUMP enzyme complex of all the Pyrr mutants and FCR3 had a monomer of 70 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . One highly resistant mutant , FCR3-D7 , exhibited a 5-10 fold higher uptake of pyrimethamine and a proportionately higher amount of P00374 -TS protein than FCR3 but only a normal level of P00374 activity . The genomic DNA of FCR3-D7 was shown to contain at least twice as much P00374 -TS specific DNA than either FCR3- P41226 , another Pyrr mutant , or FCR3 . Preliminary results suggested some of the P00374 -TS genetic material in FCR3-D7 is associated with a gene duplication . The testis anion transporter TAT1 ( Q96RN1 ) physically and functionally interacts with the cystic fibrosis transmembrane conductance regulator channel : a potential role during sperm capacitation . The Slc26 gene family encodes several conserved anion transporters implicated in human genetic disorders , including Pendred syndrome , diastrophic dysplasia and congenital chloride diarrhea . We previously characterized the TAT1 ( testis anion transporter 1 ; Q96RN1 ) protein specifically expressed in male germ cells and mature sperm and showed that in the mouse , deletion of Tat1 caused male sterility due to a lack of sperm motility , impaired sperm capacitation and structural defects of the flagella . Ca(2+) , Cl(-) and HCO(3)(-) influxes trigger sperm capacitation events required for oocyte fertilization ; these events include the intracellular rise of cyclic adenosine monophosphate ( DB02527 ) and protein kinase A ( PKA ) -dependent protein phosphorylation . The cystic fibrosis transmembrane conductance regulator ( P13569 ) is expressed in mature sperm and has been shown to contribute to Cl(-) and HCO(3)(-) movements during capacitation . Furthermore , several members of the SLC26 family have been described to form complexes with P13569 , resulting in the reciprocal regulation of their activities . We show here that TAT1 and P13569 physically interact and that in Xenopus laevis oocytes and in CHO- P04264 cells , TAT1 expression strongly stimulates P13569 activity . Consistent with this , we show that Tat1 inactivation in mouse sperm results in deregulation of the intracellular DB02527 content , preventing the activation of PKA-dependent downstream phosphorylation cascades essential for sperm activation . These various results suggest that TAT1 and P13569 may form a molecular complex involved in the regulation of Cl(-) and HCO(3)(-) fluxes during sperm capacitation . In humans , mutations in P13569 and/or TAT1 may therefore be causes of asthenozoospermia and low fertilizing capacity of sperm . DB00205 and WR99210 exert opposing selection on dihydrofolate reductase from Plasmodium vivax . Plasmodium vivax is a major public health problem in Asia and South and Central America where it is most prevalent . Until very recently , the parasite has been effectively treated with chloroquine , but resistance to this drug has now been reported in several areas . Affordable alternative treatments for vivax malaria are urgently needed . DB00205 -sulfadoxine is an inhibitor of dihydrofolate reductase ( P00374 ) that has been widely used to treat chloroquine-resistant Plasmodium falciparum malaria . P00374 inhibitors have not been considered for treatment of vivax malaria , because initial trials showed poor efficacy against P. vivax . P. vivax can not be grown in culture ; the reason for its resistance to P00374 inhibitors is unknown . We show that , like P. falciparum , point mutations in the dhfr gene can cause resistance to pyrimethamine in P. vivax . WR99210 is a novel inhibitor of P00374 , effective even against the most pyrimethamine-resistant P. falciparum strains . We have found that it is also an extremely effective inhibitor of the P. vivax P00374 , and mutations that confer high-level resistance to pyrimethamine render the P. vivax enzyme exquisitely sensitive to WR99210 . These data suggest that pyrimethamine and WR99210 would exert opposing selective forces on the P. vivax population . If used in combination , these two drugs could greatly slow the selection of parasites resistant to both drugs . If that is the case , this novel class of P00374 inhibitors could provide effective and affordable treatment for chloroquine- and pyrimethamine-resistant vivax and falciparum malaria for many years to come . [ HPV caused pathological changes in genital system of mice ] . The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7 , Ad- P02533 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7 , Ad- P02533 -E6/E7 were used as experimental group , while pAd-CMV and pAdtrack- P02533 were used as control group . Four of them were injected through one main vein of nude mice tail respectively . These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks . Mice were anaesthesiaed with 2.5 % Avertint and the vagina , mammary gland , ovaries and uterus were dissected and fixed in 3.75 % paraformaldehyde overnight at 4 degrees C . Paraffin-embedded sections , HE staining and identification of P04637 and Bcl-2 protein via immunohistochemistry were performed . The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice . HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2 . The expression of mutant P04637 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell . Western blotting also showed that E6 protein was expressed . The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone . Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falciparum . DB00205 and cycloguanil are competitive inhibitors of the Plasmodium enzyme dihydrofolate reductase ( P00374 ) . They have been effective treatments for malaria , but rapid selection of populations of the parasite resistant to these drugs has compromised their effectiveness . Parasites resistant to either drug usually have point mutations in the dhfr gene , but the frequency of these mutations is unknown . To study drug resistance more effectively , we transferred the P00374 domain of the dhfr-thymidylate synthase gene from a drug-sensitive line of P. falciparum to a strain of the budding yeast , Saccharomyces cerevisiae , that lacks endogenous P00374 activity . Expression of the P. falciparum dhfr is controlled by the yeast dhfr 5' and 3' regulatory regions and the heterologous enzyme provided all of the functions of the yeast dhfr gene . These yeast were susceptible to pyrimethamine and cycloguanil at low concentrations that inhibit P. falciparum ( IC50 about 10(-8) and 10(-7) M , respectively ) . Yeast expressing constructs with dhfr alleles from pyrimethamine-resistant strains were resistant to both pyrimethamine and cycloguanil ( IC50 > 10(-6) M ) ; resistance of the yeast depended on the dhfr allele they expressed . The experimental drug WR99210 efficiently killed all three yeast strains ( IC50 about 10(-8) M ) but the pyrR strains showed collateral hypersensitivity to drug . The yeast transformants carrying the drug-sensitive allele can now be screened quickly and quantitatively to identify new drugs or combinations of drugs and determine which drugs select resistant parasites least efficiently . Such compounds would be excellent candidates for development of treatments with a longer life in clinical practice . DB00205 -sulfadoxine efficacy and selection for mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase in Mali . To assess pyrimethamine-sulfadoxine ( PS ) efficacy in Mali , and the role of mutations in Plasmodium falciparum dihydrofolate reductase ( P00374 ) and dihydropteroate synthase ( P49366 ) in in vivo PS resistance , 190 patients with uncomplicated P. falciparum malaria were treated with PS and monitored for 56 days . Mutation-specific polymerase chain reactions and digestion with restriction endonucleases were used to detect P00374 and P49366 mutations on filter paper blood samples from pretreatment and post-treatment infections . Only one case each of RI and RII level resistance and no cases of RIII resistance or therapeutic failure were observed . Post-PS treatment infections had significantly higher rates of P00374 mutations at codons 108 and 59 . No significant selection for P49366 mutations was seen . DB00205 -sulfadoxine is highly efficacious in Mali , and while the low level of resistance precludes assessing the utility of molecular assays for in vivo PS resistance , rapid selection of P00374 mutations supports their role in PS failure . Genetic factors influencing outcome from neurotrauma . PURPOSE OF REVIEW : Clinical outcome after neurotrauma is considerably variable and can only partly be explained by known prognostic factors . There is converging evidence from genetic research that a number of genetic variants may contribute to this variability . This review provides recent data from human studies , published in the previous year , on genetic factors influencing outcome after neurotrauma . The bibliographic databases MEDLINE , EMBASE and PsycINFO were searched to identify relevant studies . RECENT FINDINGS : Genetic susceptibility to various aspects of clinical outcome after neurotrauma was reported in recent clinical studies . Genetic loci investigated include polymorphisms in P02649 , P21397 , P23560 , NOS3 , P05231 , P12036 , P31645 , P21964 , P48454 and Q8IX03 genes . The importance of these findings and future directions are discussed . SUMMARY : Recent genetic studies have revealed emerging aspects and extended the existing knowledge regarding the pathogenesis of neurotrauma and the genetic influence on phenotypic diversity . A better understanding of the underlying biological pathways and molecular mechanisms of an individual 's response to neurotrauma may hold the promise of novel treatment strategies and improved clinical outcome . Lectin-like oxidized P01130 -1 ( P78380 ) supports adhesion of mononuclear leukocytes and a monocyte-like cell line THP-1 cells under static and flow conditions . Adhesion of mononuclear leukocytes to vascular endothelial cells appears one of the initial steps in the process of atherogenesis and inflammation . We examined if P78380 , an endothelial scavenger receptor with C-type lectin-like structure , can support adhesion of mononuclear leukocytes . Under a static condition , CHO- P04264 cells stably expressing P78380 showed more prominent adhesion of human peripheral blood mononuclear leukocytes and THP-1 cells than untransfected CHO- P04264 cells , in a temperature-independent fashion . Mononuclear leukocytes also adhered to plastic plates precoated with recombinant soluble P78380 extracellular domain . A neutralizing anti- P78380 monoclonal antibody , as well as oxidized low-density lipoprotein , significantly blocked adhesion of THP-1 cells to CHO- P04264 cells overexpressing P78380 and bovine aortic endothelial cells . Under a flow condition , increased numbers of THP-1 cells showed rolling with reduced velocities on P78380 -expressing CHO- P04264 cells , compared with those on untransfected CHO- P04264 cells . Taken together , P78380 can work as a cell surface receptor for mononuclear leukocytes under both static and flow conditions . DB00435 production stimulated by the basic fibroblast growth factor requires the synthesis of ceramide . DB00435 ( NO ) is an intracellular and intercellular mediator involved in the modulation of many physiologic and pathologic processes including the regulation of neoangiogenesis . We analyzed the effects of basic fibroblast growth factor ( P09038 ) on NO production in CHO- P04264 cells and the intracellular mechanisms involved . P09038 induces NO production through activation of the endothelial NO synthase ( P29474 ) , causing a subsequent increase in cGMP levels . In most systems , P29474 activation is a Ca(2+)-calmodulin-dependent process . In CHO- P04264 cells , NO production by P09038 is Ca(2+) and Q96HU1 kinase independent , because it was not reverted by pretreatment with intracellular Ca(2+) chelators or MEK inhibitors . Translocation of the P29474 from the plasma membrane , where it is bound to caveolin 1 , to the cytosol is the crucial step in the synthesis of NO . We demonstrate that the cytosolic translocation of P29474 is caused by increased synthesis of ceramide dependent by the P09038 activation of sphingomyelinase . Indeed , in the presence of the sphingomyelinase inhibitors D609 or desipramine , P09038 -dependent NO production is abrogated . To support this evidence we evaluated ceramide concentration using HPLC-electrospray ionization-mass spectrometry in controls and in P09038 -treated cells : after P09038 stimulation , a substantial increase in ceramide levels was observed . These data were further confirmed by the lack of NO production in response to fibroblast growth factor in fibroblasts derived from Niemann Pick patients who genetically lack the enzyme sphingomyelinase . In conclusion , ceramide in CHO- P04264 cells is responsible for a novel Ca(2+)/calmodulin-independent mechanism for P29474 activation after fibroblast growth factor stimulation . UV-induced but P04637 independent apoptotic death in CHO. P04264 cells is promoted by M phase inhibitors . Persistence of Sulfadoxine- DB00205 Resistance Despite Reduction of Drug Pressure in Malawi . BACKGROUND : In 2007 , Malawi replaced sulfadoxine-pyrimethamine ( SP ) with an artemisinin-based combination therapy as the first-line treatment for uncomplicated Plasmodium falciparum malaria in response to failing SP efficacy . Here we estimate the effect of reduced SP pressure on the prevalence of SP-resistant parasites and the characteristics of the associated selective sweeps flanking the resistance loci . METHODS : Samples obtained from individuals with clinical malaria during a period of high SP use ( 1999-2001 ) , a transitional period ( 2007-2008 ) , and a period of low SP use ( 2012 ) were genotyped for resistance markers at pfdhfr-ts codons 51 , 59 , and 108 and pfdhps codons 437 , 540 , and 581 . Expected heterozygosity was estimated to evaluate the genetic diversity flanking pfdhfr-ts and pfdhps . RESULTS : An increase in the prevalence of the resistance haplotypes P00374 51I/59R/108N and P49366 437G/540E occurred under sustained drug pressure , with no change in haplotype prevalence 5 years after reduction in SP pressure . The P49366 437G/540E/581G haplotype was observed in 2007 and increased in prevalence during a period of reduced SP pressure . Changes to the sweep characteristics flanking pfdhfr-ts and pfdhps were minimal . CONCLUSIONS : In contrast to the rapid and complete return of chloroquine-susceptible falciparum malaria after chloroquine was withdrawn from Malawi , a reemergence of SP efficacy is unlikely in the near future . Potentiator ivacaftor abrogates pharmacological correction of ΔF508 P13569 in cystic fibrosis . Cystic fibrosis ( CF ) is caused by mutations in the CF transmembrane conductance regulator ( P13569 ) . Newly developed " correctors " such as DB09280 ( VX-809 ) that improve P13569 maturation and trafficking and " potentiators " such as ivacaftor ( VX-770 ) that enhance channel activity may provide important advances in CF therapy . Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation ( G551D ) that affects only channel activity , a single compound is not sufficient to treat patients with the more common P13569 mutation , ΔF508 . Thus , patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit . However , whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro , the impact of chronic therapy has not been established . In studies of human primary airway epithelial cells , we found that both acute and chronic treatment with VX-770 improved P13569 function in cells with the G551D mutation , consistent with clinical studies . In contrast , chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated P13569 correction in ΔF508 homozygous cultures . This result reflected the destabilization of corrected ΔF508 P13569 by VX-770 , markedly increasing its turnover rate . Chronic VX-770 treatment also reduced mature wild-type P13569 levels and function . These findings demonstrate that chronic treatment with P13569 potentiators and correctors may have unexpected effects that can not be predicted from short-term studies . Combining these drugs to maximize rescue of ΔF508 P13569 may require changes in dosing and/or development of new potentiator compounds that do not interfere with P13569 stability . Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone . Chloroquine has been the mainstay of malaria chemotherapy for the past five decades , but resistance is now widespread . DB00205 or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance . Both pyrimethamine and proguanil are dihydrofolate reductase ( P00374 ) inhibitors , the proguanil acting primarily through its major metabolite cycloguanil . Resistance to these drugs arises due to specific point mutations in the dhfr gene . Cross resistance between cycloguanil and pyrimethamine is not absolute . It is , therefore , important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that P00374 inhibitor with less mutation rate is favored in drug combinations . Hence , we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil . Using erythrocytic stages of P. falciparum cultures , progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence . Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil . It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs . [ Postoperative pulmonary embolism ] . Post-operative thrombo-embolic disease remains a frequent occurrence in spite of advances in their prophylaxis . Evaluation of 60 case-reports of this disease which often includes peripheral manifestations and always pulmonary manifestations , enables to specify the role of the procedure itself ( mostly orthopaedic surgery 60 % ) , pelvic surgery 20 % , the chronology of events ( possibility of early embolism between D1 and D3 and usual occurrence of manifestations between P41226 and D18 , and the importance of the background , whether investigated or not : deficiencies in anti-thrombin III , protein C and S : 4 cases . The diagnosis is based on clinical signs ( non-specific ) and the laboratory tests , especially scintigraphy ( screening ) and angiography , absolutely necessary for the diagnosis and evaluation of the amputation coefficient ( Miller index ) . With a diagnosis of pulmonary embolism , it is always necessary to look for a proximal venous thrombosis . The treatment , calls for heparin ( quite seldom ) , thrombolytics ( DB00013 , P00747 in our experience ) , the indication of which must take into consideration the delays and the nature of the previous procedure , and finally surgery ( massive forms where thrombolytics are contraindicated ) . The thrombo-embolic manifestations with thrombogenic thrombopenia secondary to heparin are quite frequent , in a surgical environment ( 10 cases ) and difficult to treat . [ Research on hepatitis C virus entry inhibitor ] . Hepatitis c virus ( HCV ) infection has become one of the global public health problem,while there is no vaccine to prevent HCV infection , the so-called " cocktail " therapy that use a combination of drugs targeting multiple steps in the HCV infection cycle could achieve better curative effect. the process of HCV entering into host cell is the important step of drug intervention , in which HCV envelope protein El and E2 , Host cell factors including Heparan sulfate(HS) , P60033 , scavenger receptor class B type I ( Q8WTV0 ) , Q16625 ( OCLD ) , Claudin ( CLDN ) , low densitity lipoprotein receptor ( P01130 ) , dendritic cell-specific P32942 -grabbing nonintegrin ( Q9NNX6 ) , Liver/lymph node specific P32942 -grabbing integrin( Q9H2X3 ) , trans- ferrin receptor 1 ( P02786 ) and so on play a important role . The virus and the host factors can be used as targets of hcv entry inhibitors many studies have shown that as novel and promising compounds , HCV entry inhibitors combinating with other drugs can be more effective in the treatment of HCV , this paper have re- viewed targets and inhibitors of HCV enterring into host cell since 1990s . DB00205 resistant mutations in Plasmodium falciparum . Three mutations in Plasmodium falciparum yielding increased resistance to pyrimethamine were obtained following treatment with chemical mutagens and selection in presence of pyrimethamine . From parasite clone TM4/8.2 a mutant , TM4/8.2/4.1 , was produced which raised pyrimethamine resistance about 500 times and was found to involve an amino acid change in the P00374 -TS enzyme molecule from Ser108 to Asn108 . A clone of another isolate , P02786 /94 , yielded a mutant , P02786 /94/300.300 , raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile164 to Met164 . However , another mutant from P02786 /94 , P02786 /94/M1-1(b3) , although it raised the pyrimethamine resistance 100 times , did not involve any changes in the coding sequence of the P00374 -TS gene , but resulted in the production of about twice as much P00374 -TS enzyme as the original clone P02786 /94 . No amplification of the P00374 -TS gene was detected . It is concluded that changes in pyrimethamine resistance of malaria parasites may arise in at least 2 ways : ( 1 ) by structural changes in the P00374 domain of the P00374 -TS gene ( as previously found by other workers ) ; ( 2 ) by other changes , possibly affecting the expression of the P00374 -TS gene . The relative importance of these 2 mechanisms in causing resistance in wild populations of P. falciparum is discussed . [ Effect of the genetic markers Kell( P04264 ) , P1 , Km(1) , Tf ( C < B < D ) , AK and 6- P52209 on the outcome of paternity cases ] . This statistical analysis of the results of 288 paternity cases is a contribution to the discussion of those blood group systems to be selected for the basis of paternity expertise in the Federal Republic of Germany . When typing 22 blood-group systems in 288 one-man cases , we found exclusions in 101 ( 35.07 % ) of them . In only 83 ( 44.39 % ) of the 187 cases with nonexclusions did the resulting EM value correspond to the verbal predicate : " paternity practically proved. " The results of the systems of factors Kell( P04264 ) , Tf(C,B,D) , AK and 6- P52209 had the smallest rate of exclusion constellations and only inferior influence on the resulting EM values . Replacing them by isoelectric focusing of the systems P36871 , Tf , Gc , Pi and P00747 ( plasminogen ) seems to be reasonable . The factors P1 and Km(1) proved more favorable for the results of paternity cases . Prevalence of the dihydrofolate reductase DB00174 -108 mutation as the basis for pyrimethamine-resistant falciparum malaria in the Brazilian Amazon . DB00205 resistance in cultivated laboratory isolates of Plasmodium falciparum is linked to the dihydrofolate reductase mutation DB00174 -108 , a mutation that acts by interrupting drug binding within the active site of the enzyme . To determine the prevalence of this mutation in endemic regions harboring pyrimethamine-resistant malaria , we used a mutation-specific polymerase chain reaction assay to survey P. falciparum strains from a wide section of the Brazilian Amazon . Mutations were identified directly from blood samples without intervening steps of in vitro cultivation . Of 42 samples collected from four states in Brazil , 38 ( 90 % ) contained the DB00174 -108 codon AAC that confers pyrimethamine resistance , four samples contained only the wild-type DB00133 -108 codon AGC , and none contained the DB00156 -108 codon ACC found in cycloguanil-resistant pyrimethamine-sensitive strains . These findings indicate that a very high incidence of the DB00174 -108 P00374 mutation is responsible for pyrimethamine resistance in the Amazon , and they are consistent with recent failure rates reported for Fansidar ( pyrimethamine-sulfadoxine ) . We suggest that limited use of proguanil be evaluated as an alternative to pyrimethamine . [ Effects of plasminogen and streptokinase on the vital functions of nervous tissue cells in culture ] . In the protein-deficient media plasminogen stimulated the vital functions of cells and in concentrations 10(-7)-10(-10) M it protected cells of sympathetic ganglia , neocortex and continues cell lines under damaging actions of H2O2 ( 0.0001 M ) , NH4CI ( 0.01 M ) and cooling . DB00086 essentially influenced the mode of damaging effect of DB00171 ( 0.001 M ) . Even a short-term exposition ( 20 min ) of PC12 cells with both proteins ( each in the concentration 10(-9) M ) led to sharp alterations in intracellular DB00171 - or Ca(2+)-activated proteolysis . In some cases plasminogen and streptokinase provided acceleration of cultured tissue maturation , improvement of cell adhesion , high survival rate , the increase in quantity and length of processes and their arborisation . Electronic microscopy established the character of structural rearrangements of nervous tissue cells ( neurons , astrocytes , oligodendrocytes ) , reflecting the protective action of plasminogen and streptokinase . In the presence of plasminogen and especially streptokinase , the total number of cultured glioma P13671 and neuroblastoma IMR-32 cells , the intracellular contents of protein , RNA and DNA increased several-fold . Addition of plasminogen promoted formation of processes by neuroblastoma cells , this suggests initiation of differentiation of cellular elements . In cultures of sensitive and sympathetic ganglia streptokinase increased proliferation of Schwann cells . These proteins did not cause transformation of PC12 enterochromaffine cells to neurons , though plasminogen facilitated it . P00747 addition to cell cultures did not increase fibrinolytic activity of the culture medium in the culture medium , and streptokinase did not lose its plasminogen-activating capacity . P02786 and P60709 as the best reference genes to quantify DB00013 P00747 Activator in breast cancer . BACKGROUND : Biomedical researchers have long looked for ways to diagnose and treat cancer patients at the early stages through biomarkers . Although conventional techniques are routinely applied in the detection of biomarkers , attitudes towards using Real-Time PCR techniques in detection of many biomarkers are increasing . Normalization of quantitative Real-Time PCR is necessary to validate non-biological alteration occurring during the steps of RNA quantification . Selection of variably expressed housekeeping genes ( HKs ) will affect the validity of the data . The aim of the present study was to identify uniformly expressed housekeeping genes in order to use in the breast cancer gene expression studies . DB00013 P00747 Activator was used as a gene of interest . FINDINGS : The expression of six HKs ( P02786 , P08236 , P04406 , P60709 , P00492 and P05388 ) was investigated using geNorm and NormFinder softwares in forty breast tumor , four normal and eight adjacent tissues . P05388 and P04406 revealed maximum M value , while P02786 demonstrated lowest M value . CONCLUSIONS : In the present study the most and the least stable genes were P02786 and P05388 respectively . P02786 and P60709 were verified as the best combination of two genes for breast cancer quantification . The result of this study shows that in each gene expression analysis HKs selection should be done based on experiment conditions . Uptake , cytotoxicity and metabolism of m-azidopyrimethamine and related lipophilic antifolates in SV- P02533 human keratinocytes in vivo . The growth-inhibitory properties of a series of lipophilic diaminopyrimidine antifolates were evaluated in comparison with methotrexate ( MTX ) against SV- P02533 human keratinocytes in vitro under folate-dependent and folate-independent conditions . Under folate-dependent conditions metoprine ( DDMP ) proved more cytotoxic than MTX , despite the greater inhibitory activity of the latter compound against mammalian dihydrofolate reductase ( P00374 ) , possibly reflecting differences in cellular accumulation . The significantly lower activity of both compounds under folate-independent conditions indicated P00374 as the primary target . DB00205 ( Q9BRP8 ) , m-azidopyrimethamine ( MZP ) and m-aminopyrimethamine ( Q96HU1 ) , a metabolite of MZP , were approximately equiactive but less cytotoxic than MTX or DDMP . The unexpected activity of Q96HU1 , an inferior P00374 inhibitor , suggests differences in the mechanism of action or cellular transport of the drug , although the reduction of cytotoxicity observed under folate-independent conditions indicate folate metabolism as the cytotoxic locus . In contrast , the cytotoxicity of Q9BRP8 or MZP was not reduced under folate-independent conditions implying an alternative mechanism of action . The uptake of 2-[14C]pyrimethamine by SV- P02533 keratinocytes was rapid with steady-state intracellular concentrations being observed after approximately 100 min , partition of drug into the plasma membrane preceding redistribution and extensive accumulation within the particulate cell components . The previously reported NADPH-dependent metabolism of MZP to Q96HU1 by murine liver microsome preparations was not observed with SV- P02533 keratinocytes nor with murine skin homogenates in the present study . Vibrational structure of dihydrofolate bound to R67 dihydrofolate reductase . R67 is a Type II dihydrofolate reductase ( P00374 ) that catalyzes the reduction of dihydrofolate ( DHF ) to DB00116 by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to P13671 . Because this enzyme is a plasmid-encoded P00374 from trimethoprim-resistant bacteria , extensive studies on R67 with various methods have been performed to elucidate its reaction mechanism . Here , Raman difference measurements , conducted on the ternary complex of R67.NADP(+).DHF believed to be an accurate mimic of the productive P00374 .NADPH.DHF complex , show that the pK(a) of N5 in the complex is less than 4 . This is in clear contrast to the behavior observed in Escherichia coli P00374 , a substantially more efficient enzyme , where the pK(a) of bound DHF at N5 is increased to 6.5 compared with its solution value of 2.6 . A comparison of the ternary complexes in R67 and E. coli DHFRs suggests that enzymic raising of the pK(a) at N5 can significantly increase the catalytic efficiency of the hydride transfer step . However , R67 shows that even without such a strategy an effective P00374 can still be designed . Development of 2,4-diaminopyrimidines as antimalarials based on inhibition of the S108N and C59R+S108N mutants of dihydrofolate reductase from pyrimethamine-resistant Plasmodium falciparum . The reduced binding of pyrimethamine to Ser108Asn ( S108N ) mutants of parasite dihydrofolate reductase ( P00374 ) , which forms the basis of resistance of Plasmodium falciparum to pyrimethamine , is largely due to steric constraint imposed by the bulky side chain of N108 on Cl of the 5-p-Cl-phenyl group . This and other S108 mutants with bulky side chains all showed reduced binding to pyrimethamine and cycloguanil . Less effect on binding to some bulky mutants was observed for trimethoprim , with greater flexibility for the 5-substituent . S108N P00374 also binds poorly with other pyrimethamine derivatives with bulky groups in place of the p-Cl , and the binding was generally progressively poorer for the double ( C59R+S108N ) mutant . Removal of the p-Cl or replacement with m-Cl led to better binding with the mutant DHFRs . DB00205 analogues with unbranched hydrophobic 6-substituents showed generally good binding with the mutant DHFRs . A number of compounds were identified with high affinities for both wild-type and mutant DHFRs , with very low to no affinity to human P00374 . Some of these compounds show good antimalarial activities against pyrimethamine-resistant P. falciparum containing the mutant DHFRs with low cytotoxicity to three mammalian cell lines . In-vitro activity of atovaquone , sulphamethoxazole and dapsone alone and combined with inhibitors of dihydrofolate reductase and macrolides against Pneumocystis carinii . The anti-Pneumocystis carinii activity of atovaquone , dapsone and sulphamethoxazole alone and combined with dihydrofolate reductase ( P00374 ) inhibitors and macrolides was investigated against five clinical isolates of P. carinii . The susceptibility tests were performed by inoculation of the organisms on to cell monolayer and parasite count after 72 h incubation at 37 degrees C . Culture plates were added to Dulbecco 's modified Eagle 's medium containing serial dilutions of atovaquone , dapsone and sulphamethoxazole alone or in combination with diaveridine , pyrimethamine , trimethoprim , azithromycin , clarithromycin and roxithromycin . DB01117 , dapsone and sulphamethoxazole were found to be effective at levels well below the concentrations that could be achieved clinically , while P00374 inhibitors were shown to combine effectively with dapsone and sulphamethoxazole . No synergy could be demonstrated between atovaquone and P00374 inhibitors or macrolides . A mild synergic effect was noted when macrolides were combined with dapsone and sulphamethoxazole . DB00205 ( 0.5 mg/L ) combined with dapsone and trimethoprim ( 0.5 mg/L ) combined with sulphamethoxazole exerted the strongest inhibitory effect . P62158 and troponin C : affinity chromatographic study of divalent cation requirements for troponin I inhibitory peptide ( residues 104-115 ) , mastoparan and fluphenazine binding . The different conformations induced by the binding of Mg2+ or Ca2+ to troponin C ( TnC ) and calmodulin ( P62158 ) results in the exposure of various interfaces with potential to bind target compounds . The interaction of TnC or P62158 with three affinity columns with ligands of either the synthetic peptide of troponin I ( TnI ) inhibitory region ( residues 104-115 ) , mastoparan ( a wasp venom peptide ) , or fluphenazine ( a phenothiazine drug ) were investigated in the presence of Mg2+ or Ca2+ . TnC and P62158 in the presence of either Ca2+ or Mg2+ bound to the TnI peptide 104-115 . The cation specificity for this interaction firmly establishes that the TnI inhibitory region binds to the high affinity sites of TnC ( most likely the N-terminal helix of site III ) and presumably the homologous region of P62158 . Mastoparan interacted strongly with both proteins in the presence of Ca2+ but , in the presence of Mg2+ , did not bind to TnC and only bound weakly to P62158 . DB00623 bound to TnC and P62158 only in the presence of Ca2+ . When the ligands interacted with either proteins there was an increase in cation affinity , such that TnC and P62158 were eluted from the TnI peptide or mastoparan affinity column with 0.1 M DB00974 compared with the 0.01 M DB00974 required to elute the proteins from the fluphenazine column . The interaction of these ligands with their receptor sites on TnC and P62158 require a specific and spatially correct alignment of hydrophobic and negatively charged residues on these proteins. ( ABSTRACT TRUNCATED AT 250 WORDS ) P00374 protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency . DB00360 ( BH4 ) is a required cofactor for the synthesis of NO by endothelial nitric oxide synthase ( P29474 ) , and endothelial BH4 bioavailability is a critical factor in regulating the balance between NO and superoxide production ( P29474 coupling ) . Biosynthesis of BH4 is determined by the activity of GTP-cyclohydrolase I ( GTPCH ) . However , BH4 levels may also be influenced by oxidation , forming 7,8-dihydrobiopterin ( BH2 ) , which promotes P29474 uncoupling . Conversely , dihydrofolate reductase ( P00374 ) can regenerate BH4 from BH2 , but whether P00374 is functionally important in maintaining P29474 coupling remains unclear . To investigate the mechanism by which P00374 might regulate P29474 coupling in vivo , we treated wild-type , BH4-deficient ( hph-1 ) , and GTPCH-overexpressing ( P30793 -Tg ) mice with methotrexate ( MTX ) , to inhibit BH4 recycling by P00374 . MTX treatment resulted in a striking elevation in BH2 and a decreased BH4:BH2 ratio in the aortas of wild-type mice . These effects were magnified in hph-1 but diminished in P30793 -Tg mice . Attenuated P29474 activity was observed in MTX-treated hph-1 but not wild-type or P30793 -Tg mouse lung , suggesting that inhibition of P00374 in BH4-deficient states leads to P29474 uncoupling . Taken together , these data reveal a key role for P00374 in regulating the BH4 vs BH2 ratio and P29474 coupling under conditions of low total biopterin availability in vivo . Suppression of tumor growth and metastasis by a P17948 antagonizing peptide identified from a phage display library . Although the P15692 -Flk-1-pathway has been known as the major driving force of angiogenesis , new evidence has shown that P17948 /Flt-1 plays important roles during the neovascularization under pathological conditions including tumor , atherosclerosis and arthritis . In search of Flt-1 receptor antagonizing peptides , we screened a phage display 12-mer-peptide library with recombinant Flt-1 protein . Seven candidate peptides were identified that specifically bound to P15692 receptor Flt-1 , of which peptide F56 ( WHSDMEWWYLLG ) almost abolished P15692 binding to receptor Flt-1 in vitro . In vivo , F56 fused with P00374 ( P00374 -F56 ) inhibited angiogenesis in a P62158 assay . Moreover , P00374 -F56 significantly inhibited the growth of nodules of human gastric cancer cell line MGC-803 in BALB/c nude mice . Histological analyses showed that necrosis of the implanted tumor was markedly enhanced following treatment with P00374 -F56 . In the severe combined immunodeficiency disease ( SCID ) mouse model for studying metastasis of the human breast cancer cell line BICR-H1 , synthetic peptide F56 significantly inhibited tumor growth and lung metastases . Taken together , our results have demonstrated that peptide F56 , as a Flt-1 receptor antagonist , fulfilled the antiangiogenic and antimetastatic effects by specifically interfering with the interaction between P15692 and receptor Flt-1 . Thus , short peptide F56 may have clinical potential in tumor therapy . 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 . Plasmodium falciparum dihydrofolate reductase alleles and pyrimethamine use in pregnant Ghanaian women . Drug resistance in Plasmodium falciparum affects prevention of malaria in pregnancy . In a cross-sectional study of 530 pregnant Ghanaian women , P. falciparum dihydrofolate reductase ( P00374 ) gene mutations linked with pyrimethamine resistance were assessed and associations with pyrimethamine intake were analyzed . P. falciparum infected 69 % of women without pyrimethamine use , 59 % of those who had a history of pyrimethamine consumption but a negative urine test , and 53 % of individuals with a positive urine test . Eighty-one percent , 43 % , and 74 % of the isolates contained the mutations DB00174 -108 , DB00167 -51 , and DB00125 -59 , respectively . DB00156 -108 occurred in 8 % . DB00205 use was associated with increased frequencies of DB00174 -108 and DB00125 -59 but not of DB00167 -51 or DB00156 -108 . In women with prophylaxis , wild-type parasites were absent and anemia tended to be more common with an increasing number of P00374 gene mutations . DB00205 appears to be not adequately effective in this part of Ghana , most likely due to the predominance of resistant parasites . Selection for resistance following insufficient prophylaxis could possibly affect the efficacy of future intermittent sulfadoxine-pyrimethamine treatment . In silico design : extended molecular dynamic simulations of a new series of dually acting inhibitors against P00533 and P04626 . Based on the hit structures that have been identified in our previous studies against P00533 and P04626 , new potential inhibitors that share the same scaffold of the hit structures are designed and screened in silico . Insights into understanding the potential inhibitory effect of the new inhibitors against both P00533 and P04626 receptors is obtained using extended molecular dynamics ( MD ) simulations and different scoring techniques . The binding mechanisms and dynamics are detailed with respect to two approved inhibitors against P00533 ( lapatinib ) and P04626 ( SYR127063 ) . The best scoring inhibitor ( P02786 ) is chosen for additional in silico investigation against both the wild-type and T790M mutant strain of P00533 and the wild-type P04626 . The results reveal that certain substitution patterns increase the stability and assure stronger binding and higher H-bond occupancy of the conserved water molecule that is commonly observed with kinase crystal structures . Furthermore , the new inhibitor ( P02786 ) forms stable interactions with the mutant strain as a direct consequence of the enhanced ability to form additional hydrogen bonding interactions with binding site residues . Human mitochondrial import receptor , Tom20p . Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins . The cytosolic domain of the human mitochondrial protein import receptor , hTom20 , has been expressed as a fusion protein with glutathione S-transferase ( Q86UG4 ) in bacteria and the purified protein immobilized on Sepharose beads . To discriminate between specific binding of precursor proteins with the receptor and non-specific binding , precursors were recovered as a complex with Q86UG4 -hTom20 following competitive elution from the beads with reduced glutathione . Here , we describe the specificity of this assay and demonstrate that the cytosolic domain of hTom20 interacts directly with the transcription-translation product of precursor proteins that bear a diverse array of targeting signals . Such proteins include a matrix protein ( pODHFR ) , a polytopic integral protein of the inner membrane ( uncoupling protein ) , a beta-barrel protein of the outer membrane ( P21796 /porin ) as well as bitopic integral proteins which are inserted into the outer membrane by either an NH2-terminal or COOH-terminal signal anchor sequence ( yTom70(1-29) P00374 and Bcl-2 , respectively ) . [ DB00707 sodium ( Photofrin-II ) ] . DB00707 sodium ( DB00707 ) is a photosensitizer which distributes selectively to tumor tissues , and causes tumor cell death by combination with light irradiation . Photodynamic therapy ( PDT ) by combination of porfimer sodium and laser was developed as a new cancer therapy . Tumor selectivity of porfimer sodium are based on the following reasons ; 1 ) high affinity for lipoprotein , especially , low density lipoprotein ( LDL ) , 2 ) elevation of P01130 activity in cancer tissue , and 3 ) lack or imcompleteness of lymphatic system in cancer tissue . DB00707 sodium is activated by laser irradiation at 630 nm , which can reacts with tissue oxygen to produce highly reactive excited siglet oxygen ( 1O2 ) . This highly reactive molecule is subsequently capable of killing tumor cells through oxidation of cellular component like mitochondrial enzymes . In addition , this highly reactive intermediate causes destruction of the tumor capillaries , which accelerates tumor cell death . The growth suppression or lethal damage to tumor cells by PDT of porfimer sodium and excimer dye laser were observed in experimental tumor models . In human clinical trials , the rates of complete response ( CR ) for roentgenographically occult lung cancer , stage I lung cancer , superficial esophageal cancer , superficial gastric cancer and carcinoma in situ or dysplasia of the cervix were 84.8 % , 50.0 % , 90.0 % , 87.5 % and 94.4 % , respectively . The major side effects were cutaneous symptoms e.g. photosensitivity , pigmentation , increasing GOT , GPT but these symptoms were not severe . PDT using porfimer sodium and excimer dye laser must be clinically useful for the treatment of inoperable early cancer or conservation of organ functions . 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 . MiR-24 tumor suppressor activity is regulated independent of p53 and through a target site polymorphism . MicroRNAs ( miRNAs ) are predicted to regulate approximately 30 % of all human genes ; however , only a few miRNAs have been assigned their targets and specific functions . Here we demonstrate that miR-24 , a ubiquitously expressed miRNA , has an anti-proliferative effect independent of p53 function . Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation , cell cycle control , gene regulation and cellular transformation . Overexpression of miR-24 in six different cell lines , independent of p53 function , inhibited cell proliferation and resulted in G2/S cell cycle arrest . MiR-24 over expression in cells with wt-p53 upregulated P04637 and P38936 protein ; however , in p53-null cells miR-24 still induced cell cycle arrest without the involvement of P38936 . We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme , dihydrofolate reductase ( P00374 ) a target of the chemotherapeutic drug methotrexate ( MTX ) . Of interest , we found that a miR-24 target site polymorphism in P00374 3' UTR that results in loss of miR-24-function and high P00374 levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation . Of clinical significance , we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24 . A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed . Fitness effects of P00374 -TS mutations associated with pyrimethamine resistance in apicomplexan parasites . DB00205 resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase ( P00374 ) domain of the bifunctional dihydrofolate reductase-thymidylate synthase ( P00374 -TS ) gene . We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii P00374 -TS , which can readily be expressed as recombinant protein for enzymatic studies , or as allelic replacements in transgenic parasites . In order to directly assess the costs of pyrimethamine-resistance in vivo , we have carried out competition studies between mixtures of T. gondii tachyzoites harbouring wild-type or mutant P00374 -TS alleles , both in tissue culture and in mice . Arg59+Asn108 mutants ( using the P. falciparum numbering system ) exhibit no significant fitness defects in vitro , but a fitness defect of 1.8 % per generation in mice . Arg59+Ser223 mutants exhibit fitness defects of > 2.8 % per generation both in vitro and in vivo , which may explain why this highly pyrimethamine-resistant allele has not been observed in the field . It is important to note that long-term propagation of parasites in vitro or in vivo can produce adaptations affecting fitness by > 3.7 % per generation , necessitating careful attention to background in head-to-head competition studies . A sensitive PCR-based assay permits different growth rates to be assessed even in the absence of a drug resistance marker that can be scored by plaque assay . Genetic and biochemical markers in patients with Alzheimer 's disease support a concerted systemic iron homeostasis dysregulation . Alzheimer 's disease ( AD ) is the most common form of dementia in the elderly individuals , resulting from a complex interaction between environmental and genetic factors . Impaired brain iron homeostasis has been recognized as an important mechanism underlying the pathogenesis of this disease . Nevertheless , the knowledge gathered so far at the systemic level is clearly insufficient . Herein , we used an integrative approach to study iron metabolism in the periphery , at both genotypic and phenotypic levels , in a sample of 116 patients with AD and 89 healthy control subjects . To assess the potential impact of iron metabolism on the risk of developing AD , genetic analyses were performed along with the evaluation of the iron status profile in peripheral blood by biochemical and gene expression studies . The results obtained showed a significant decrease of serum iron , ferritin , and transferrin concentrations in patients compared with the control subjects . Also , a significant decrease of ferroportin ( Q9NP59 ) and both transferrin receptors P02786 and Q9UP52 transcripts was found in peripheral blood mononuclear cells from patients . At the genetic level , significant associations with AD were found for single nucleotide polymorphisms in TF , Q9UP52 , P21399 , and Q9NP59 genes . P02649 gene , a well-known risk factor for AD , was also found significantly associated with the disease in this study . Taken together , we hypothesize that the alterations on systemic iron status observed in patients could reflect an iron homeostasis dysregulation , particularly in cellular iron efflux . The intracellular iron accumulation would lead to a rise in oxidative damage , contributing to AD pathophysiology . DB00205 and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase : polymerase chain reaction methods for surveillance in Africa . As chloroquine resistance spreads across Africa , the dihydrofolate reductase ( P00374 ) inhibitors pyrimethamine and proguanil are being used as alternative first-line drugs for the treatment and prevention of Plasmodium falciparum malaria . Resistance to these drugs is conferred by point mutations in parasite P00374 . These point mutations can be detected by polymerase chain reaction ( PCR ) assays , but better methods for sample collection , DNA extraction , and a diagnostic PCR are needed to make these assays useful in malaria-endemic areas . Here we report methods for collecting fingerstick blood onto filter paper strips that are air-dried , then stored and transported at room temperature . Cell lysis and DNA extraction are accomplished by boiling in Chelex-100 . We also report a nested PCR technique that has improved sensitivity and specificity . These procedures readily detect mixed infections of parasites with both sensitive and resistant genotypes ( confirmed by direct sequencing ) and are reliable at parasite densities less than 250/mm3 in field surveys . Is phentermine an inhibitor of monoamine oxidase ? A critical appraisal . DB00191 produces a spectrum of concentration-dependent biochemical effects . It interacts with NE transporters at 0.1 microM , DA transporters at about 1 microM , 5-HT transporters at 15 microM and P21397 at about 100 microM . When administered at typical anorectic doses , phentermine primarily interacts with DA and NE transporters and does not produce biochemical or neurochemical effects which would occur if it were inhibiting P21397 . Some other explanation other than MAO inhibition must be sought to explain how oral phentermine increases platelet 5-HT , since platelet P27338 does not metabolize platelet 5-HT , and since amphetamine-type drugs are even weaker inhibitors of P27338 than P21397 . Clinical studies in humans have shown that amphetamine , which is a more potent inhibitor of P21397 than phentermine , does not inhibit P21397 at therapeutic doses . Neither phentermine alone , fluoxetine alone or their combined use have been associated with cardiac valvulopathy , and clinical experience has shown their combined use to be free of significant adverse effects . Viewed collectively , there appears to be no data to support the hypothesis that phentermine inhibits MAO at typical therapeutic doses . P00374 hysteresis and its effect of inhibitor binding analyses . Escherichia coli dihydrofolate reductase was shown to follow slow transient kinetics ( hysteresis ) . Nonlinear reaction velocities were detected during the enzyme assay and required 10-15 min to reach a steady-state rate . The degree of hysteresis was influenced by the enzyme concentration and the order of substrate addition . Incubation of the enzyme with NADPH before addition of dihydrofolate resulted in slow initial velocities that increased up to 2-fold during the course of the assay . Increasing the enzyme concentration from 0.2 to 1 nM resulted in diminished hysteresis . NADPH-initiated reactions were linear at all enzyme concentrations tested . Certain drugs had profound effects on hysteresis . DB00205 practically eliminated the hysteresis of dihydrofolate-started reactions , whereas trimethoprime augmented the non-linearities in the sense that hysteresis was detected in both enzyme- and NADPH-started reactions . The shape of these reaction tracings makes trimethoprim is not a slow-binding inhibitor when assayed under conditions that eliminate hysteresis . Contrary to this , sulfamethoxazole did not affect hysteresis or augment inhibition of the enzyme by trimethoprim . Sulfamethoxazole alone ( at 6 mM ) did not inhibit the hysteresis and allow reliable determinations of Ki values of both weak and tight binding inhibitors . For example , Ki values for pyrimethamine , trimethoprim , and methotrexate were found to be 214 nM , 1.3 nM , and 0.021 nM , respectively . Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome . Chinese hamster ovary ( CHO ) cells are widely used for the stable production of recombinant proteins . Gene amplification techniques are frequently used to improve of protein production , and the dihydrofolate reductase ( P00374 ) gene amplification system is most widely used in the CHO cell line . We previously constructed a CHO genomic bacterial artificial chromosome ( BAC ) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and one BAC clone ( Cg0031N14 ) containing the CHO genomic DNA sequence adjacent to Dhfr was selected . To identify the specific chromosomal region adjacent to the exogenous Dhfr-amplified region in the CHO cell genome , we performed further screening of BAC clones to obtain other Dhfr-amplified regions in the CHO genome . From the screening by high-density replica filter hybridization using a digoxigenin-labeled pSV2-dhfr/hGM- P04141 probe , we obtained 8 new BAC clones containing a Dhfr-amplified region . To define the structures of the 8 BAC clones , Southern blot analysis , BAC end sequencing and fluorescence in situ hybridization ( Q5TCZ1 ) were performed . These results revealed that all the selected BAC clones contained a large palindrome structure with a small inverted repeat in the junction region . This suggests that the obtained amplicon structure in the Dhfr-amplified region in the CHO genome plays an important role in exogenous gene amplification . [ DB09053 : A new drug of B-cell malignancies ] . DB09053 ( Imbruvica® ) is a first-in-class , orally administered once-daily , that inhibits B-cell antigen receptor signaling downstream of Bruton 's tyrosine kinase ( Q06187 ) . DB09053 has been approved in USA in February 2014 and in France in October 2014 for the treatment of patients with relapsed/refractory mantle cell lymphoma ( Q8WXI8 ) or chronic lymphocytic leukaemia ( CLL ) and for the treatment of patients with CLL and a chromosome 17 deletion ( del 17p ) or P04637 mutation . In clinical studies , ibrutinib induced an impressive overall response rate ( 68 % ) in patients with relapsed/refractory Q8WXI8 ( phase II study ) . In CLL , ibrutinib has shown to significantly improve progression-free survival , response rate and overall survival in patients with relapsed/refractory CLL , including in those with del 17p . DB09053 had an acceptable tolerability profile . Less than 10 % of patients discontinued their treatment because of adverse events . Results are pending in other B-cell lymphomas subtypes such as in diffuse large B-cell lymphoma and in follicular lymphoma . An approval extension has already been enregistered for Waldenström disease in USA in January 2015 . Given its efficacy and tolerability , ibrutinib is an emerging treatment option for patients with B-cell malignancies . Bayesian analysis and the GUSTO trial . Global Utilization of DB00086 and Tissue P00747 Activator in Occluded Arteries .