An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117-131), Loop 2 (residues 192-201) and the substrate recognition peptide (residues 94-102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.

Impact of gemcitabine and cisplatin with radiotherapy in locally advanced or metastatic transitional cell carcinoma of urinary bladder

The primary object of this study is to evaluate the efficacy and safety of Gemcitabine and Cisplatin along with radiotherapy in transitional cell carcinoma of urinary bladder. Patients and Twenty patients with locally advanced or metastatic TCC of urinary bladder were enrolled during the 22-months period from January, 1999 to October, 2000 and followed up till March 2002.Three patients received 4 cycles, five patients received 5 cycles and twelve patients received 6 cycles of Gemcitabine 1250mg / m2 on day 1 and day 8 and Cisplatin 80mg / m2 on day 1; administered every 3 weeks. No patient received prior chemotherapy, radiotherapy or surgery. However, four patients received prior intravesical chemotherapy. All patients received radiotherapy after completion of chemotherapy regimen. Nineteen patients achieved complete response at the end of the treatment. The complete response rate was 95%. The confidence interval was at 95%, level of confidence ranged from 85% to 100%. Median duration of clinical benefit was 21 months. Six patients [30%] were documented neutropenia, three patients [15%] documented thrombocytopenia. No life threatening toxicity was observed. Gemcitabine and Cisplatin along with radiotherapy in locally or metastatic Transitional cell carcinoma of urinary bladder, exhibited pronounced response rate among all the patients. The toxicity profile remained extremely low and disease free survival enhanced. The above investigation may further be continued at a larger scale encompassing a wide band of subjects

Pyrimidine nucleoside catabolism in penicillium chrysoyenum

P. Chrysogenum utilized cytidine recognized as the sole source of nitrogen. This pyrimidine nucleoside supported the good growth of P. chrysogenum compared to that obtained with sodium nitrate. Dailyzed cell-free extracts of this organism hydrolyzed cytidine and uridine to the correspondings base and ribose by the pyrimidine nucleoside hydrolase. Pyrimidine nucleoside hydrolase was found to be constitutive. It was proved experimentally that this enzyme had hydrological activity and no phosphorylation occurred. Thymidine can not be cleaved under these experimental conditions. The pH optimum for cytidine and uridine hydrolysis was the same 6.2, and the optimum temperature was found to be 40C. It has been proved that pyrimidine nucleoside hydrolase differs from the purine nucleoside hydrolase in the same organism. The Km value of the enzyme was calculated and fond to be 13.5 x 10-3 M for cytidine and 16.6 x 10-3 M for uridine