MH_dev_70

DB00205 SUB - 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 REA ) , and dihydropteroate synthase ( P49366 REA ) 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 REA - 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 REA 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 REA ) . CPs produce several neuroprotective peptides , which are secreted into the P04141 REA . 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 MEN ) in castrated male and female mice subjected to DB02901 MEN replacement was analyzed . We show that rat CP epithelial cells contain AR mRNA and protein . Moreover , we demonstrate that AR is downregulated by DB02901 MEN 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 mic 1 gene previously described for E . tenella transfection . DB00205 SUB resistant E . nieschulzi parasites were obtained in vitro after transfection with a plasmid encoding the Toxoplasma gondii dhfr / ts-m 2m3 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 REA - 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 REA , 3 , 4 , 5 , and 7 , as well as for P21796 REA and P13569 REA in cultured astrocytes . Moreover , we performed immunostaining with antibodies against each of these channels and showed the strongest expression of P51788 REA and P51790 REA , strong expression of P51795 REA and P21796 REA , weak expression of P51798 REA and very weak expression of P51793 REA and P13569 REA . 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 SUB acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase ( P00374 REA - TS ) . Resistance in the most important human parasite , Plasmodium falciparum , initially results from an S108N mutation in the P00374 REA 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 REA - 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 REA fromP . falciparum , the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM , but against the P00374 REA 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 REA ( 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 REA 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 HepG 2 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 HepG 2 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 CDKN 3 , LOC 10028961 2 , P00374 REA , Q99986 REA , Q99741 REA , Q96GD4 and P78334 . Genes like Q07973 REA , P38398 REA , O14965 REA , P06493 REA , P24941 REA , P11802 REA and P06213 REA 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 REA , P17936 REA , P06400 REA , P14735 REA , P01130 REA , P55157 REA , P04114 REA , MTIX , P04179 REA and P08294 REA 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 REA 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 REA ) 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 REA ) ; 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 MEN treatment increased cyclic GMP concentration in aorta of rabbits . P29474 REA 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 REA 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 REA inhibitor , retards the initiation of atherosclerosis formation through the improvement of NO bioavailability by both up-regulation of P29474 REA 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 REA exon 9 . The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator ( P13569 REA ) 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 REA 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 REA 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 REA 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 REA 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 REA ) , dihydropteroate synthase ( P49366 REA ) , and cytochrome b genes were determined by sequencing . DB00205 SUB 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 REA 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 REA 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 REA 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 ( AcH 3 ) on lysines 9 and 14 ( P35527 REA and P02533 REA ) 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 MEN ) . Chromatin immunoprecipitation ( ChIP ) on the promoters and enhancers of prostate specific antigen ( PSA ) and transmembrane protease , serine 2 ( O15393 REA ) genes was performed . qRT-PCR was used to measure the levels of PSA and O15393 REA transcripts . RESULTS : Upon stimulation with 1 nM DB02901 MEN , AR and RNA Pol II were recruited onto PSA and O15393 REA 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 MEN concentration . The ratio of AcH 3 / H3 was increased in AR-overexpressing cells . The induction of transcription of PSA and O15393 REA 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 REA ) 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 SUB - resistant parasites acquired a degree of cross resistance to cycloguanil and methotrexate , another P00374 REA 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 SUB , an antimalarial drug , was found to be able to inhibit both enzymes ( P00374 REA - TS and PTR 1 ) 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 SUB ( 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 ( Pyr 20 ) 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 . Pyr 20 still effectively inhibits both the wild-type and SP21 proteins , and the x-ray structures of these complexes show how Pyr 20 fits into both active sites without steric strain . These structural insights suggest a general approach for developing new generations of antimalarial P00374 REA 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 REA of Toxoplasma gondii by antifolates . The responses to antifolates of Toxoplasma gondii were investigated by measuring the dihydrofolate reductase ( P00374 REA ) activity , quantity of P00374 REA mRNA , and single-strand conformational polymorphism ( SSCP ) pattern . DB00205 SUB ( 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 REA 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 REA 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 REA activity .

Effects of retroviral-mediated P08183 REA 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 ( HaMDR 1 ) , a variant of human dihydrofolate reductase ( HaDHFR ) , or both P08183 REA and P00374 REA 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 REA , 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 HaMDR 1 - 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 HaMDR 1 - 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 REA - 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 REA 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 REA is the leading gram-negative organism causing neonatal meningitis , but the microbial basis of E . coli P04264 REA meningitis is incompletely understood . Here we employed comparative genomic hybridization to investigate 11 strains of E . coli P04264 REA isolated from the cerebrospinal fluid ( P04141 REA ) of patients with meningitis . These 11 strains cover the majority of common O serotypes in E . coli P04264 REA isolates from P04141 REA . Our data demonstrated that these 11 strains of E . coli P04264 REA 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 REA strains isolated from P04141 REA can be divided into two groups and these two groups of E . coli P04264 REA may utilize different mechanisms to induce meningitis .

Intake of vitamin P04264 REA 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 REA and K2 and subsequent risk of hip fracture in a general population sample , as well as potential effect modification by apolipoprotein E gene ( P02649 REA ) 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 REA 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 REA 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 APOE 4 - allele did not increase the risk of hip fracture , nor was there any effect modification with vitamin P04264 REA in relation to risk of hip fracture . CONCLUSIONS : A low intake of vitamin P04264 REA , but not K2 , was associated with an increased risk of hip fractures .

DB00205 SUB 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 FCR 3 was examined by measuring the accumulation of drug in infected red blood cells . [ 3H ] DB00205 SUB was stage specifically accumulated in trophozoites and schizont infected red blood cells . The mutant parasites accumulated drug as efficiently as FCR 3 . DB00205 SUB 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 REA - TS ) enzyme . These results suggested that the pyrimethamine resistance was not associated with decreased drug permeability of the membrane . P00374 REA - TS - [ 3H ] FdUMP enzyme complex of all the Pyrr mutants and FCR 3 had a monomer of 70 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . One highly resistant mutant , FCR 3 - D7 , exhibited a 5-10 fold higher uptake of pyrimethamine and a proportionately higher amount of P00374 REA - TS protein than FCR 3 but only a normal level of P00374 REA activity . The genomic DNA of FCR 3 - D7 was shown to contain at least twice as much P00374 REA - TS specific DNA than either FCR 3 - P41226 REA , another Pyrr mutant , or FCR 3 . Preliminary results suggested some of the P00374 REA - TS genetic material in FCR 3 - D7 is associated with a gene duplication .

The testis anion transporter TAT 1 ( Q96RN1 ) physically and functionally interacts with the cystic fibrosis transmembrane conductance regulator channel : a potential role during sperm capacitation . The Slc 26 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 TAT 1 ( testis anion transporter 1 ; Q96RN1 ) protein specifically expressed in male germ cells and mature sperm and showed that in the mouse , deletion of Tat 1 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 REA ) is expressed in mature sperm and has been shown to contribute to Cl ( - ) and HCO ( 3 ) ( - ) movements during capacitation . Furthermore , several members of the SLC 26 family have been described to form complexes with P13569 REA , resulting in the reciprocal regulation of their activities . We show here that TAT 1 and P13569 REA physically interact and that in Xenopus laevis oocytes and in CHO - P04264 REA cells , TAT 1 expression strongly stimulates P13569 REA activity . Consistent with this , we show that Tat 1 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 TAT 1 and P13569 REA may form a molecular complex involved in the regulation of Cl ( - ) and HCO ( 3 ) ( - ) fluxes during sperm capacitation . In humans , mutations in P13569 REA and / or TAT 1 may therefore be causes of asthenozoospermia and low fertilizing capacity of sperm .

DB00205 SUB 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 SUB - sulfadoxine is an inhibitor of dihydrofolate reductase ( P00374 REA ) that has been widely used to treat chloroquine-resistant Plasmodium falciparum malaria . P00374 REA 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 REA 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 REA , 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 REA , 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 REA 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-E 6 / E7 , Ad - P02533 REA - E6 / E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E 6 / E7 , Ad - P02533 REA - E6 / E7 were used as experimental group , while pAd-CMV and pAdtrack - P02533 REA 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 REA 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 REA 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 SUB and cycloguanil are competitive inhibitors of the Plasmodium enzyme dihydrofolate reductase ( P00374 REA ) . 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 REA 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 REA 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 SUB - 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 REA ) and dihydropteroate synthase ( P49366 REA ) 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 REA and P49366 REA 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 REA mutations at codons 108 and 59 . No significant selection for P49366 REA mutations was seen . DB00205 SUB - 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 REA 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 REA , P21397 REA , P23560 REA , NOS 3 , P05231 REA , P12036 , P31645 REA , P21964 REA , P48454 REA 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 REA - 1 ( P78380 REA ) 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 REA , an endothelial scavenger receptor with C-type lectin-like structure , can support adhesion of mononuclear leukocytes . Under a static condition , CHO - P04264 REA cells stably expressing P78380 REA showed more prominent adhesion of human peripheral blood mononuclear leukocytes and THP - 1 cells than untransfected CHO - P04264 REA cells , in a temperature-independent fashion . Mononuclear leukocytes also adhered to plastic plates precoated with recombinant soluble P78380 REA extracellular domain . A neutralizing anti - P78380 REA monoclonal antibody , as well as oxidized low-density lipoprotein , significantly blocked adhesion of THP - 1 cells to CHO - P04264 REA cells overexpressing P78380 REA and bovine aortic endothelial cells . Under a flow condition , increased numbers of THP - 1 cells showed rolling with reduced velocities on P78380 REA - expressing CHO - P04264 REA cells , compared with those on untransfected CHO - P04264 REA cells . Taken together , P78380 REA 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 REA ) on NO production in CHO - P04264 REA cells and the intracellular mechanisms involved . P09038 REA induces NO production through activation of the endothelial NO synthase ( P29474 REA ) , causing a subsequent increase in cGMP levels . In most systems , P29474 REA activation is a Ca ( 2 + ) - calmodulin-dependent process . In CHO - P04264 REA cells , NO production by P09038 REA 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 REA 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 REA is caused by increased synthesis of ceramide dependent by the P09038 REA activation of sphingomyelinase . Indeed , in the presence of the sphingomyelinase inhibitors D609 or desipramine , P09038 REA - dependent NO production is abrogated . To support this evidence we evaluated ceramide concentration using HPLC-electrospray ionization-mass spectrometry in controls and in P09038 REA - treated cells : after P09038 REA 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 REA cells is responsible for a novel Ca ( 2 + ) / calmodulin-independent mechanism for P29474 REA activation after fibroblast growth factor stimulation .

UV-induced but P04637 REA independent apoptotic death in CHO . P04264 REA cells is promoted by M phase inhibitors .

Persistence of Sulfadoxine - DB00205 SUB 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 REA 51I / 59R / 108N and P49366 REA 437G / 540E occurred under sustained drug pressure , with no change in haplotype prevalence 5 years after reduction in SP pressure . The P49366 REA 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 REA in cystic fibrosis . Cystic fibrosis ( CF ) is caused by mutations in the CF transmembrane conductance regulator ( P13569 REA ) . Newly developed " correctors " such as DB09280 MEN ( VX - 809 ) that improve P13569 REA 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 REA 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 REA 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 REA correction in ΔF508 homozygous cultures . This result reflected the destabilization of corrected ΔF508 P13569 REA by VX - 770 , markedly increasing its turnover rate . Chronic VX - 770 treatment also reduced mature wild-type P13569 REA levels and function . These findings demonstrate that chronic treatment with P13569 REA 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 REA may require changes in dosing and / or development of new potentiator compounds that do not interfere with P13569 REA 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 SUB 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 REA ) 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 REA 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 REA 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 REA 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 REA , scavenger receptor class B type I ( Q8WTV0 ) , Q16625 REA ( OCLD ) , Claudin ( CLDN ) , low densitity lipoprotein receptor ( P01130 REA ) , dendritic cell-specific P32942 REA - grabbing nonintegrin ( Q9NNX6 ) , Liver / lymph node specific P32942 REA - grabbing integrin ( Q9H2X3 ) , trans - ferrin receptor 1 ( P02786 REA ) 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 SUB 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 REA - TS enzyme molecule from Ser 108 to Asn 108 . A clone of another isolate , P02786 REA / 94 , yielded a mutant , P02786 REA / 94/300 . 300 , raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile 164 to Met 164 . However , another mutant from P02786 REA / 94 , P02786 REA / 94 / M1 - 1 ( b3 ) , although it raised the pyrimethamine resistance 100 times , did not involve any changes in the coding sequence of the P00374 REA - TS gene , but resulted in the production of about twice as much P00374 REA - TS enzyme as the original clone P02786 REA / 94 . No amplification of the P00374 REA - 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 REA domain of the P00374 REA - TS gene ( as previously found by other workers ) ; ( 2 ) by other changes , possibly affecting the expression of the P00374 REA - 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 REA ) , P1 , Km ( 1 ) , Tf ( C < B < D ) , AK and 6 - P52209 REA 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 REA ) , Tf ( C , B , D ) , AK and 6 - P52209 REA 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 REA , Tf , Gc , Pi and P00747 REA ( 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 SUB 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 REA 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 MEN 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 REA 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 REA 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 REA and P60709 REA as the best reference genes to quantify DB00013 P00747 REA 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 REA Activator was used as a gene of interest . FINDINGS : The expression of six HKs ( P02786 REA , P08236 REA , P04406 REA , P60709 REA , P00492 REA and P05388 REA ) was investigated using geNorm and NormFinder softwares in forty breast tumor , four normal and eight adjacent tissues . P05388 REA and P04406 REA revealed maximum M value , while P02786 REA demonstrated lowest M value . CONCLUSIONS : In the present study the most and the least stable genes were P02786 REA and P05388 REA respectively . P02786 REA and P60709 REA 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 REA 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 REA 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 REA ) , possibly reflecting differences in cellular accumulation . The significantly lower activity of both compounds under folate-independent conditions indicated P00374 REA as the primary target . DB00205 SUB ( 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 REA 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 REA 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 REA 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 REA ) 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 REA . Because this enzyme is a plasmid-encoded P00374 REA 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 REA . 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 REA , 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 REA 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 Ser 108Asn ( S108N ) mutants of parasite dihydrofolate reductase ( P00374 REA ) , 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 REA 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 SUB 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 REA . 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 REA ) 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 REA inhibitors were shown to combine effectively with dapsone and sulphamethoxazole . No synergy could be demonstrated between atovaquone and P00374 REA inhibitors or macrolides . A mild synergic effect was noted when macrolides were combined with dapsone and sulphamethoxazole . DB00205 SUB ( 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 MENMAX DB00623 MEN 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 REA 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 REA ) , and endothelial BH4 bioavailability is a critical factor in regulating the balance between NO and superoxide production ( P29474 REA 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 REA uncoupling . Conversely , dihydrofolate reductase ( P00374 REA ) can regenerate BH4 from BH2 , but whether P00374 REA is functionally important in maintaining P29474 REA coupling remains unclear . To investigate the mechanism by which P00374 REA might regulate P29474 REA coupling in vivo , we treated wild-type , BH4 - deficient ( hph - 1 ) , and GTPCH-overexpressing ( P30793 REA - Tg ) mice with methotrexate ( MTX ) , to inhibit BH4 recycling by P00374 REA . 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 REA - Tg mice . Attenuated P29474 REA activity was observed in MTX-treated hph - 1 but not wild-type or P30793 REA - Tg mouse lung , suggesting that inhibition of P00374 REA in BH4 - deficient states leads to P29474 REA uncoupling . Taken together , these data reveal a key role for P00374 REA in regulating the BH4 vs BH2 ratio and P29474 REA coupling under conditions of low total biopterin availability in vivo .

Suppression of tumor growth and metastasis by a P17948 REA antagonizing peptide identified from a phage display library . Although the P15692 REA - Flk - 1 - pathway has been known as the major driving force of angiogenesis , new evidence has shown that P17948 REA / 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 REA receptor Flt - 1 , of which peptide F56 ( WHSDMEWWYLLG ) almost abolished P15692 REA binding to receptor Flt - 1 in vitro . In vivo , F56 fused with P00374 REA ( P00374 REA - F56 ) inhibited angiogenesis in a P62158 assay . Moreover , P00374 REA - 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 REA - F56 . In the severe combined immunodeficiency disease ( SCID ) mouse model for studying metastasis of the human breast cancer cell line BICR-H 1 , 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 REA and receptor Flt - 1 . Thus , short peptide F56 may have clinical potential in tumor therapy .

Nearly Complete Response of Brain Metastases from P04626 REA Overexpressing Breast Cancer with DB01259 MEN 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 REA overexpressing breast cancer patients . Combination therapy of lapatinib and capecitabine may be an effective treatment option for brain metastasis of P04626 REA - 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 REA ) 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 SUB 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 REA gene mutations . DB00205 SUB 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 REA and P04626 REA . Based on the hit structures that have been identified in our previous studies against P00533 REA and P04626 REA , 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 REA and P04626 REA 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 REA ( lapatinib ) and P04626 REA ( SYR 127063 ) . The best scoring inhibitor ( P02786 REA ) is chosen for additional in silico investigation against both the wild-type and T790M mutant strain of P00533 REA and the wild-type P04626 REA . 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 REA ) 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 , Tom 20p . Use of glutathione to reveal specific interactions between Tom 20 - glutathione S-transferase and mitochondrial precursor proteins . The cytosolic domain of the human mitochondrial protein import receptor , hTom 20 , 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 - hTom 20 following competitive elution from the beads with reduced glutathione . Here , we describe the specificity of this assay and demonstrate that the cytosolic domain of hTom 20 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 REA / 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 ( yTom 70 ( 1-29 ) P00374 REA and Bcl - 2 , respectively ) .

[ DB00707 MEN sodium ( Photofrin-II ) ] . DB00707 MEN sodium ( DB00707 MEN ) 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 REA activity in cancer tissue , and 3 ) lack or imcompleteness of lymphatic system in cancer tissue . DB00707 MEN 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 REA cells by serum starvation . The neu proto-oncogene product has been found to exist in two interconvertible forms in Q9UBA6 / P00374 REA 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 REA cells , the human breast cancer cell line SK-Br - 3 expresses a high molecular weight neu / P04626 REA 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 REA 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-p 53 upregulated P04637 REA and P38936 REA protein ; however , in p53 - null cells miR - 24 still induced cell cycle arrest without the involvement of P38936 REA . We show that miR - 24 regulates p53 - independent cellular proliferation by regulating an S-phase enzyme , dihydrofolate reductase ( P00374 REA ) a target of the chemotherapeutic drug methotrexate ( MTX ) . Of interest , we found that a miR - 24 target site polymorphism in P00374 REA 3 ' UTR that results in loss of miR - 24 - function and high P00374 REA 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 REA - TS mutations associated with pyrimethamine resistance in apicomplexan parasites . DB00205 SUB resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase ( P00374 REA ) domain of the bifunctional dihydrofolate reductase-thymidylate synthase ( P00374 REA - TS ) gene . We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii P00374 REA - 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 REA - TS alleles , both in tissue culture and in mice . Arg 59 + Asn 108 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 . Arg 59 + Ser 223 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 REA 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 REA , and Q9NP59 genes . P02649 REA 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 SUB 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 REA ) 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 REA . 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 MEN 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 REA 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 REA . Some other explanation other than MAO inhibition must be sought to explain how oral phentermine increases platelet 5 - HT , since platelet P27338 REA does not metabolize platelet 5 - HT , and since amphetamine-type drugs are even weaker inhibitors of P27338 REA than P21397 REA . Clinical studies in humans have shown that amphetamine , which is a more potent inhibitor of P21397 REA than phentermine , does not inhibit P21397 REA 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 REA 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 SUB 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 REA ) 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 pSV 2 - dhfr / hGM - P04141 REA 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 MEN : A new drug of B-cell malignancies ] . DB09053 MEN ( Imbruvica ® ) is a first-in-class , orally administered once-daily , that inhibits B-cell antigen receptor signaling downstream of Bruton ' s tyrosine kinase ( Q06187 REA ) . DB09053 MEN 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 REA 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 MEN 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 MEN and Tissue P00747 REA Activator in Occluded Arteries .