The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T>C, c.1361C>T, c.1459G>A, c.1557-4_1557+3del7, and c.1622_1623dupTA) and three novel mutations (c.7A>T, c.1573C>T, and c.1953-6_1953-2del5) were detected. Importantly, transfection of fibroblasts of cblE patients with a wild-type MTRR minigene expression construct resulted in a significant approximately four-fold increase of methionine synthesis, indicating correction of the enzyme defect. Our study shows a link between a milder predominantly hematological presentation and homozygosity for the c.1361C>T mutation, but no other obvious genotype-phenotype correlation. The identification of mutations in the MTRR gene, together with restoration of methionine synthesis following MTRR minigene expression in cblE cells confirms that this disease is caused by defects in the MTRR gene.

• MeSH
• 5-methyltetrahydrofoláthomocystein-S-methyltransferasa nedostatek   MeSH
• běloši genetika   MeSH
• betain terapeutické užití   MeSH
• bodová mutace MeSH
• ferredoxin-NADP-reduktasa nedostatek   genetika   MeSH
• fibroblasty enzymologie   patologie   MeSH
• genetická terapie * MeSH
• haplotypy genetika   MeSH
• homocystein krev   MeSH
• homocystinurie krev   klasifikace   farmakoterapie   enzymologie   genetika   patologie   terapie   MeSH
• hydroxokobalamin terapeutické užití   MeSH
• kyselina listová terapeutické užití   MeSH
• lidé MeSH
• missense mutace MeSH
• mozek patologie   MeSH
• mutační analýza DNA MeSH
• nesmyslný kodon MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• polymorfismus genetický MeSH
• rekombinantní fúzní proteiny fyziologie   MeSH
• sekvenční delece MeSH
• substituce aminokyselin MeSH
• syntetické geny MeSH
• testy genetické komplementace MeSH
• transfekce MeSH
• transformované buněčné linie enzymologie   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5-methyltetrahydrofoláthomocystein-S-methyltransferasa MeSH
• betain MeSH
• ferredoxin-NADP-reduktasa MeSH
• homocystein MeSH
• hydroxokobalamin MeSH
• kyselina listová MeSH
• methionine synthase reductase MeSH Prohlížeč
• nesmyslný kodon MeSH
• rekombinantní fúzní proteiny MeSH

Using a recombinant vaccinia virus expressing protooncogene Bcl-2, we demonstrate opposite effects of the expressed Bcl-2 in two cell lines: apoptosis induction in BSC-40 cells and apoptosis prevention in HeLa G cells. The apparent molecular weight of the expressed Bcl-2, its amounts and its effects on the mitochondrial membrane potential are comparable in both cell lines, suggesting that the consequences of Bcl-2 expression depend on the cellular environment. To further support these findings we demonstrate the pro-apoptotic effect of the expressed Bcl-2 in several other cell lines.

• MeSH
• apoptóza genetika   MeSH
• buněčné linie cytologie   MeSH
• Cercopithecus aethiops MeSH
• chloramfenikol-O-acetyltransferasa genetika   MeSH
• druhová specificita MeSH
• epitelové buňky cytologie   MeSH
• genetické vektory genetika   MeSH
• geny bcl-2 * MeSH
• HeLa buňky cytologie   MeSH
• intracelulární membrány fyziologie   MeSH
• Jurkat buňky cytologie   MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• membránové potenciály MeSH
• mitochondrie fyziologie   MeSH
• protoonkogenní proteiny c-bcl-2 fyziologie   MeSH
• rekombinantní fúzní proteiny fyziologie   MeSH
• reportérové geny MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• chloramfenikol-O-acetyltransferasa MeSH
• kaspasy MeSH
• protoonkogenní proteiny c-bcl-2 MeSH
• rekombinantní fúzní proteiny MeSH

The 5'-enhancer-deleted genomic construct of the H2-K(b) gene, stably integrated into the genome of L(tk-) fibroblasts, retains full competence to be induced by tumor necrosis factor-alpha (TNF-alpha) treatment. The only defined regulatory region in this construct is the intragenic downstream regulatory element (H2DRE). Computational inspection uncovered two potential NF-IL6 (C/EBPbeta) binding motifs within the H2DRE. Chloramphenicol acetyltransferase (CAT) reporter gene assay revealed that NF-IL6 is able to elevate transcription from H2DRE. Moreover, transient transfection of an NF-IL6 expression vector increased both constitutive and TNF-alpha-induced mRNA levels of endogenous H2 class I genes, and transfection of an NF-IL6 dominant negative construct decreased the expression of endogenous H2 class I genes in a dose-dependent manner. Using the electrophoretic mobility shift assay (EMSA) and antibody supershift assay, we were able to qualify the two computationally identified NF-IL6 binding motifs as one high-affinity and one low-affinity binding site. We conclude that the H2-K(b) gene belongs to target genes of the NF-IL6 (C/EBPbeta) in the course of the cellular response to TNF-alpha, and we discuss some consequences of this conclusion in a general framework of inducible expression of the H2-K(b) gene.

• MeSH
• 5' přiléhající oblast DNA genetika   MeSH
• aktivace transkripce * účinky léků   MeSH
• buněčné linie MeSH
• buňky 3T3 MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• geny MHC třídy I * MeSH
• H-2 antigeny genetika   MeSH
• myši MeSH
• NF-kappa B metabolismus   MeSH
• protein beta vázající zesilovač transkripce CCAAT genetika   fyziologie   MeSH
• regulace genové exprese účinky léků   MeSH
• regulační geny * MeSH
• rekombinantní fúzní proteiny fyziologie   MeSH
• rekombinantní proteiny farmakologie   MeSH
• reportérové geny MeSH
• retardační test MeSH
• TNF-alfa farmakologie   MeSH
• transfekce MeSH
• vazebná místa MeSH
• zánět MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• H-2 antigeny MeSH
• H-2Kb protein, mouse MeSH Prohlížeč
• NF-kappa B MeSH
• protein beta vázající zesilovač transkripce CCAAT MeSH
• rekombinantní fúzní proteiny MeSH
• rekombinantní proteiny MeSH
• TNF-alfa MeSH

Experiments were designed to characterize cytolytic effector cells from mice with SMRTD treated with IL-2 gene therapy. Mice were inoculated with syngeneic murine MK16 carcinoma cells. When the tumours reached 8-12 mm in diameter, they were excised and the operated mice were randomized into two groups. The first group without any further treatment was designated as operated-only; the second group, vaccinated 3 days after the operation with IL-2-producing tumour vaccine, is referred to as operated-vaccinated. Tumour recurrence rate in the operated-only mice was 90 percent; in the operated-vaccinated group the recurrence rate was 38.5 percent (progressors). The remaining 61.5 percent of mice were permanently protected (regressors). On day 53, the tumour progressors, regressors and healthy controls were sacrificed, and their spleen cells were used for 51Cr microcytotoxicity assay. Splenocytes from any group of mice were not cytolytic when allowed to react with MK16, YAC-1 (NK sensitive) and C1498 (NK resistant) targets. However, when grown for 3 days in IL-2-containing medium, the splenocytes from all groups of mice could develop cytolytic activity. The cytolytic activity of splenocytes from tumour progressors and regressors was substantially lower then that of splenocytes from healthy controls. In addition, significantly lower cytolytic activity was observed with IL-2-activated splenocytes from tumour progressors as compared to that of tumour regressors. Depletion of NK1.1+ cells or CD4+ plus CD8+ cells prevented the induction of significant IL-2-stimulated cytotoxicity directed against MK16 and C1498 targets in spleen cell cultures from tumour progressors, regressors, and healthy control mice, indicating that both, NK1.1+ and CD4+ plus CD8+, cells participate in the antitumour effect of IL-2 gene therapy. This was further supported by the finding that after depletion of CD4+ plus CD8+ cells, a residual cytolytic activity directed exclusively against NK-sensitive YAC-1 cells was observed.

• MeSH
• aktivní imunoterapie * MeSH
• buňky NK imunologie   MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• cytotoxické T-lymfocyty imunologie   MeSH
• cytotoxické testy imunologické MeSH
• experimentální sarkom metabolismus   patologie   MeSH
• genetická terapie * MeSH
• interleukin-2 genetika   MeSH
• karcinom imunologie   patologie   chirurgie   terapie   MeSH
• kombinovaná terapie MeSH
• komplementární DNA genetika   MeSH
• lokální recidiva nádoru prevence a kontrola   terapie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buňky kultivované imunologie   účinky záření   transplantace   MeSH
• progrese nemoci MeSH
• protinádorové vakcíny terapeutické užití   MeSH
• rekombinantní fúzní proteiny fyziologie   MeSH
• reziduální nádor MeSH
• slezina imunologie   MeSH
• transfekce MeSH
• transplantace nádorů MeSH
• vakcinace * MeSH
• zkřížené reakce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interleukin-2 MeSH
• komplementární DNA MeSH
• protinádorové vakcíny MeSH
• rekombinantní fúzní proteiny MeSH