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M Vaupel

Publications and source records attributed to M Vaupel.

14 recordsLinked to original sources
PubMed2005

Cyclosporine A and mycophenolate mofetil vs cyclosporine A and methotrexate for graft-versus-host disease prophylaxis after stem cell transplantation from HLA-identical siblings.

The combination of Cyclosporin A (CSA) and Methotrexate (MTX) is considered to be the standard regimen for the prevention of graft-versus-host disease (GVHD) after stem cell transplantation (SCT) from HLA-identical siblings. Mycophenolate Mofetil (MMF) has been widely used for GVHD prophylaxis after nonmyeloablative SCT, but experience following myeloablative therapy is still limited. We retrospectively compared CSA/MTX and CSA/MMF in 93 patients (median age 35 years, range 17-59 years, male subjects 48, female subjects 45) with acute myeloid leukemia (n=33), myelodysplastic syndrome (MDS) (n=3), acute lymphoblastic leukemia (ALL) (n=20) or chronic myeloid leukemia (n=37) who received CSA/MMF (n=26) or CSA/MTX (n=67) as GVHD prophylaxis following high-dose therapy and allogeneic SCT from HLA-identical siblings. No statistically significant differences were found in overall survival, relapse rate, treatment-related mortality and acute or chronic GVHD. Time to myeloid recovery was significantly shorter in patients who received CSA/MMF. We conclude that the combination of CSA/MMF appears equivalent to CSA/MTX for GVHD prophylaxis in patients receiving conventional-intensity SCT from HLA-identical siblings.

Adolescent↗
PubMed2004

Transplantation of allogeneic CD34+-selected cells followed by early T-cell add-backs: favorable results in acute and chronic myeloid leukemia.

BACKGROUND: The aim of this study was to investigate preservation of anti-leukemic activity and protection from opportunistic infections after transplantation of allogeneic + cells in patients with hematologic malignancies and bad prognosis. Methods Thirty-three patients [median age 42 years, range 23-55 years, diagnosis AML/myelodysplastic syndrome (MDS) 14, ALL nine, CML seven and multiple myeloma (MM) three] received myeloablative conditioning followed by infusion of selected CD34+ cells from matched unrelated donors (31) or HLA-identical siblings (two). Early donor lymphocyte infusions (DLI; 0.5 and 1.0 x 10(6) CD3+ cells/kg) were given while patients were on immunosuppressive therapy. RESULTS: Ninety-seven per cent of patients engrafted and 24 of 29 patients surviving more than 30 days received at least one pre-emptive DLI. Three patients (10%) developed acute (a)GvHD (two grade I-II, one grade III-IV) spontaneously, and 16 patients (67%) developed aGvHD after DLI (12 grade I-II, four grade III-IV). Eight of 24 evaluable patients developed chronic (c)GvHD (33%, six limited, two extensive). After a median follow-up of 590 days (range 138-1610 days) 18 patients were alive (55%), 16 in complete remission (CR), one in hematologic and one in molecular relapse. Seven patients died after relapse (21%) and eight died from transplantation-related causes (24%). Patients with myeloid malignancies had a significantly better survival than patients with ALL or MM (74%+/-10 vs. 30%+/-13, P<0.05). DISCUSSION: Early pre-emptive low-dose DLI following transplantation of selected CD34+ cells from unrelated donors after myeloablative conditioning is feasible and effective without undue toxicity, especially in patients with myeloid malignancies.

Adult↗
PubMed1999

The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri.

BACKGROUND: The reduction of carbon dioxide to methane in methanogenic archaea involves the tetrahydrofolate analogue tetrahydromethanopterin (H(4)MPT) as a C(1) unit carrier. In the third step of this reaction sequence, N(5)-formyl-H(4)MPT is converted to methenyl-H(4)MPT(+) by the enzyme methenyltetrahydromethanopterin cyclohydrolase. The cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri (Mch) is extremely thermostable and adapted to a high intracellular concentration of lyotropic salts. RESULTS: Mch was crystallized and its structure solved at 2.0 A resolution using a combination of the single isomorphous replacement (SIR) and multiple anomalous dispersion (MAD) techniques. The structure of the homotrimeric enzyme reveals a new alpha/beta fold that is composed of two domains forming a large sequence-conserved pocket between them. Two phosphate ions were found in and adjacent to this pocket, respectively; the latter is displaced by the phosphate moiety of the substrate formyl-H(4)MPT according to a hypothetical model of the substrate binding. CONCLUSIONS: Although the exact position of the substrate is not yet known, the residues lining the active site of Mch could be tentatively assigned. Comparison of Mch with the tetrahydrofolate-specific cyclohydrolase/dehydrogenase reveals similarities in domain arrangement and in some active-site residues, whereas the fold appears to be different. The adaptation of Mch to high salt concentrations and high temperatures is reflected by the excess of acidic residues at the trimer surface and by the higher oligomerization state of Mch compared with its mesophtic counterparts.

Amino Acid Sequence↗
PubMed1998

Two F420-reducing hydrogenases in methanosarcina barkeri

F420-reducing hydrogenases are nickel iron-sulfur flavoproteins involved in CO2 reduction with H2 to methane in methanogenic archaea. Evidence is presented that Methanosarcina barkeri contains two isoenzymes for which the encoding genes have been cloned and sequenced. The genes are organized in two operons, frhADGB and freAEGB, each comprising four open reading frames. Transcription analysis revealed that both operons are transcribed during growth of Ms. barkeri on H2/CO2, on methanol, and on trimethylamine, but not during growth on acetate.

Journal Article↗
PubMed1998

Overproduction and one-step purification of the N5,N10-methenyltetrahydromethanopterin cyclohydrolase (Mch) from the hyperthermophilic Methanopyrus kandleri.

N5,N10-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an enzyme involved in methanogenesis from CO2 and H2 which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon growing at a temperature optimum of 98 degrees C. The gene mch from M. kandleri was cloned, sequenced, and expressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium sulfate. From 3.5 g cells (250 mg protein), approximately 18 mg cyclohydrolase was obtained. The purified enzyme showed essentially the same catalytic properties as the enzyme purified from M. kandleri cells. The primary structure and properties of the cyclohydrolase are compared with those of the enzyme from Methanococcus jannaschii (growth temperature optimum 85 degrees C), from Methanobacterium thermoautotrophicum (65 degrees C), and from Methanosarcina barkeri (37 degrees C). Of the four enzymes, that from M. kandleri has the lowest isoelectric point (3.8) and the lowest hydrophobicity of amino acid composition. Besides, it has the highest relative content of glutamate, leucine, and valine and the lowest relative content of isoleucine, serine, and lysine. Some of these properties are unusual for enzymes from hyperthermophilic organisms. They may reflect the observation that the cyclohydrolase from M. kandleri is not only adapted to hyperthermophilic conditions but also to the high intracellular concentrations of lyotrophic salts prevailing in this organism.

Amino Acid Sequence↗
PubMed1997

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri is not a flavoenzyme.

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane-bound enzyme composed of a 46-kDa subunit MbHdrD and a 23-kDa subunit MbHdrE. The enzyme has been shown to contain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. In addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED, revealed that both subunits also lack an FAD-binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum (MtHdr), which is a flavo iron-sulfur protein composed of the subunits MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin-binding site. Sequence comparisons revealed that the N-terminal third of MbHdrD, which contained two sequence motifs for [4Fe-4S] clusters, is similar to MtHdrC and that the C-terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrC are structurally equivalent subunits. MbHdrE shows sequence similarity to b-type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that MbHdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since MbHdrD contains only iron-sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron-transfer reactions has to be considered such as operative in ferredoxin :thioredoxin reductases from chloroplasts and cyanobacteria.

Amino Acid Sequence↗
PubMed1997

A selenium-dependent and a selenium-independent formylmethanofuran dehydrogenase and their transcriptional regulation in the hyperthermophilic Methanopyrus kandleri.

The genome of Methanopyrus kandleri was found to harbour a gene, fwuB, predicted to encode the catalytic subunit of a tungsten formylmethanofuran dehydrogenase with an active site selenocysteine, and a second gene, fwcB, encoding a tungsten formylmethanofuran dehydrogenase with an active site cysteine. Northern blot and primer-extension analysis revealed that both genes were differentially transcribed. During growth of the methanogen on medium supplemented with selenium only fwuB was transcribed, whereas transcription of both fwuB and fwcB was observed on selenium-deprived medium. Growth of M. kandleri was stimulated by tungstate and selenite but not by molybdate. The findings indicate that the hyperthermophilic archaeon contains two tungsten isoenzymes of formylmethanofuran dehydrogenase, one of which is a novel selenium enzyme. They also indicate that the hyperthermophilic methanogen probably does not contain a molybdenum formylmethanofuran dehydrogenase which appears to be present only in thermophilic and mesophilic methanogens.

Aldehyde Oxidoreductases↗
PubMed1996

Primary structure of cyclohydrolase (Mch) from Methanobacterium thermoautotrophicum (strain Marburg) and functional expression of the mch gene in Escherichia coli.

The gene mch encoding N5,N10-methenyltetrahydromethanopterin cyclohydrolase (Mch) in Methano-bacterium thermoautotrophicum (strain Marburg) was cloned and sequenced. The gene, 963 bp, was found to be located at the 3' end of a 3.5-kbp BamHI fragment. Upstream of the mch gene two open reading frames were recognized, one encoding for a 25-kDa protein with sequence similarity to deoxyuridylate hydroxymethylase and the other encoding for a 34.6-kDa protein with sequence similarity to cobalamin-independent methionine synthase (MetE). The N-terminal amino acid sequence deduced for the deoxyuridylate hydroxymethylase was identical to that previously published for thymidylate synthase (TysY) from M. thermoautotrophicum. The 3' end of the tysY gene overlapped by 8 bp with the 5' end of the mch gene. Despite this fact, the mch gene appeared to be transcribed monocistronically as evidenced by Northern blot analysis and primer-extension experiments. The mch gene was overexpressed in Escherichia coli yielding an active enzyme of 37 kDa with a specific activity of 30 U/mg cell extract protein.

Amino Acid Sequence↗
PubMed1996

A polyferredoxin with eight [4Fe-4S] clusters as a subunit of molybdenum formylmethanofuran dehydrogenase from Methanosarcina barkeri.

Formylmethanofuran dehydrogenase (Fmd) from Methanosarcina barkeri is a molybdenum iron-sulfur protein involved in methanogenesis. The enzyme contains approximately 30 mol non-heme iron/mol and 30 mol acid-labile sulfur/mol. We report here the cloning and sequencing of the encoding genes, and that these genes form a transcription unit fmdEFACDB. Evidence is provided that the subunit FmdB harbours the molybdenum-containing active site and may bind one [4Fe-4S] cluster. fmdF encodes a protein with four tandemly repeated bacterial-ferredoxin-like domains and is predicted to be a polyferredoxin that could contain as many as 32 iron atoms in eight [4Fe-4S] clusters. The other genes code for proteins without sequence motifs characteristic for iron-sulfur proteins. These findings suggest that most of the iron-sulfur clusters present in the purified formylmethanofuran dehydrogenase are associated with the subunit FmdF. The finding that FmdF forms a tight complex with the other subunits of formylmethanofuran dehydrogenase indicates a function of the polyferredoxin in the reaction catalyzed by the enzyme. fmdE encodes a protein not present in the purified enzyme. All six genes of the fmd operon were expressed in Escherichia coli and yielded proteins of expected molecular masses. A malE-fmdF gene fusion was constructed and expressed in E. coli, making the apoprotein of the polyferredoxin available in preparative amounts.

Aldehyde Oxidoreductases↗
PubMed1995

Coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductase (Mer) from Methanobacterium thermoautotrophicum strain Marburg. Cloning, sequencing, transcriptional analysis, and functional expression in Escherichia coli of the mer gene.

The gene encoding the F420-dependent N5,N10-methylenetetrahydromethanopterin reductase (Mer), which catalyzes an intermediate step in methanogensis, was cloned and sequenced from the thermophilic Methanobacterium thermoautotrophicum strain Marburg. The gene was identified on a 3.8-kbp BamHI fragment of M. thermoautotrophicum genomic DNA using a homologous probe. The mer gene encoded an acidic protein of 321 amino acids, corresponding to a calculated molecular mass of 33,492 Da. Sequence analysis revealed the presence of a ribosome binding site, a putative promoter, and a possible terminator structure. The size of the mer mRNA was estimated as 1 kb indicating monocistronic transcription. The mer gene was expressed in Escherichia coli yielding an active enzyme of 36 kDa consistent with the apparent molecular mass described for the enzyme from M. thermoautotrophicum. Sequence comparisons revealed similarities between the F420-dependent N5,N10-methylenetetrahydromethanopterin reductase and a F420-dependent reductase involved in lincomycin biosynthesis in Streptomyces lincolnensis.

Amino Acid Sequence↗