Methylmalonyl-CoA decarboxylase from Propionigenium modestum--cloning and sequencing of the structural genes and purification of the enzyme complex.

Methylmalonyl-CoA decarboxylase catalyses the only energy-conserving step during succinate fermentation by Propionigenium modestum: the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA is coupled to the vectorial transport of Na+ across the cytoplasmic membrane, thereby creating a sodium ion motive force that is used for ATP synthesis. By taking advantage of the sequence similarity between the beta-subunits of other Na+-transport decarboxylases, a portion of the P. modestum beta-subunit gene was amplified by PCR with degenerated primers. The cloned PCR product then served as homologous probe for cloning suitable fragments from genomic DNA. Sequence analysis of a 3.7-kb region identified four genes which probably form a transcriptional unit, mmdADCB. Remarkably, a mmdE gene which is present in the homologous mmdADECB cluster from Veillonella parvula and encodes the 6-kDa epsilon-subunit, is missing in P. modestum. By sequence comparisons, the following functions could be assigned to the P. modestum proteins: MmdA (56.1 kDa; alpha-subunit), carboxyltransferase; MmdB (41.2 kDa; beta-subunit), carboxybiotin-carrier-protein decarboxylase; MmdC (13.1 kDa; gamma-subunit), biotin carrier protein. MmdD (14.2 kDa; delta-subunit) presumably is essential for the assembly of the complex, as shown for the corresponding V. parvula protein. Methylmalonyl-CoA decarboxylase was solubilized from membranes of P. modestum with n-dodecylmaltoside and enriched 15-fold by affinity chromatography on monomeric avidin resin. The purified protein was composed of four subunits, three of which were identified by N-terminal sequence analysis as MmdA, MmdD, and MmdC. The purified enzyme exhibited a specific activity of up to 25 U/mg protein and an apparent Km value for (S)-methylmalonyl-CoA of approximately 12 microM. Compared to the five-subunit complex of V. parvula, the four-subunit enzyme of P. modestum appeared to be more labile, presumably a consequence of the lack of the epsilon-subunit.

Randomized double-blind crossover ondansetron-dexamethasone versus ondansetron-placebo study for the treatment of chemotherapy-induced nausea and vomiting in pediatric patients with malignancies.

PURPOSE: To determine whether the addition of dexamethasone to ondansetron (OND + DEX) is a more effective antiemetic regimen than ondansetron (OND) alone in children receiving chemotherapy. PATIENTS AND METHODS: Children who had solid tumors and who were receiving highly emetogenic chemotherapy, including cisplatin, carboplatin, cyclophosphamide, and ifosfamide, were randomized (1:1) in a double-blind fashion to receive either OND 0.15 mg/kg intravenously (i.v.) 30 min before and 4 and 8 h after chemotherapy and placebo, or OND + DEX (same OND doses plus DEX 8 mg/m2 i.v. 30 min before chemotherapy, followed by 16 mg/m2 in divided doses) as antiemetics. The patients were crossed over to the other antiemetic regimen when receiving an identical course of chemotherapy. Patients were monitored for emetic episodes, nausea, appetite, sense of well-being, and antiemetic adverse events. RESULTS: A total of 33 patients were evaluated. Sixty-one percent of the patients receiving OND + DEX regimens had a complete response for emetic episodes as compared with 23% with OND alone. Combined complete and major responses (two or less emetic episodes) were 86% for OND + DEX and 67% for OND. Failure for emetic episodes (more than five vomitings/day) were seen only in 7-10% of the total population. Minimal or no nausea was experienced in 74% of OND + DEX courses and in 52% of the OND courses. Appetite was better in OND + DEX courses (p = 0.006). Both antiemetic arms had similar adverse events. Mild to moderate sedation occurred in about half of the courses, followed by restlessness (29%), headache (17%), diarrhea (17%), and hiccups (2%). CONCLUSION: The combination of ondansetron and dexamethasone is superior to ondansetron alone to control emetic episodes in children receiving highly emetogenic chemotherapy (p = 0.04).