Arabinose is metabolized via a phosphoketolase pathway in Clostridium acetobutylicum ATCC 824.

In this report, a novel zymogram assay and coupled phosphoketolase assay were employed to demonstrate that Clostridium acetobutylicum gene CAC1343 encodes a bi-functional xylulose-5-P/fructose-6-P phosphoketolase (XFP). The specific activity of purified recombinant XFP was 6.9 U/mg on xylulose-5-P and 21 U/mg on fructose-6-P, while the specific activity of XFP in concentrated C. acetobutylicum whole-cell extract was 0.094 and 0.52 U/mg, respectively. Analysis of crude cell extracts indicated that XFP activity was present in cells grown on arabinose but not glucose and quantitative PCR was used to show that CAC1343 mRNA expression was induced 185-fold during growth on arabinose when compared to growth on glucose. HPLC analysis of metabolites revealed that during growth on xylose and glucose more butyrate than acetate was formed with final acetate:butyrate ratios of 0.72 and 0.83, respectively. Growth on arabinose caused a metabolic shift to more oxidized products with a final acetate:butyrate ratio of 1.95. The shift towards more oxidized products is consistent with the presence of an XFP, suggesting that arabinose is metabolized via a phosphoketolase pathway while xylose is probably metabolized via the pentose phosphate pathway.

Syntheses of imido-substituted glycosans and their photocyclisation towards highly functionalised heterotricycles.

Reaction of O-protected amino-1,6-anhydro-beta-D-hexopyranoses with succinic or glutaric anhydride and subsequent intramolecular acylation afforded the succinimido- and glutarimido-substituted glycosans. Irradiation with UV light of 254 nm wavelength led to gamma-hydrogen abstraction at the pyranose ring by the excited carbonyl function. The stereoselective recombination of the resulting 1,4-diradicals gave annelated azetidinols, which fragmented by a retrotransannular ring opening reaction to give the glycosan-annelated azepanedione and azocanedione systems, respectively.

Solution structure of lariat RNA by 500 MHz NMR spectroscopy and molecular dynamics studies in water.

A 500 MHz NMR study of the lariat RNA tetramer 1 and pentamer 2 mimicking the naturally occurring lariat RNA is reported. The conformational properties of 1 and 2 were compared with those of a linear branched RNA tetramer 3, which show that the conformational features of the two lariat RNAs, 1 and 2, are quite constrained and significantly different from those observed for the linear branched RNA tetramer 3. The conformation of all sugar residues forming the lariat ring in 1 and 2 are locked in a rigid South-type conformation. All residues in both lariat RNAs have a high population of gamma+ (67-85%) and beta t (95-100%) rotamers except guanosine where the gamma+ population is low. The conformation around the glycosidic bond is anti for all residues except for guanosine where NOE data indicates an equilibrium of syn<-->anti. In both lariat RNAs, 1 and 2, the temperature dependent 1H and 31P chemical shifts as well as the oligomerization shifts, with respect to adenosine 2',3',5'-triethyl-phosphate (Sund et al., 1992, Tetrahedron 48, 695) suggests that the 3'-->5' linked U4 or C4 residue is stacked on guanosine. Subsequently, 1H-1H, 1H-31P and 13C-31P coupling constants derived torsional constraints were used for molecular dynamics study in water with counter sodium ions for a total of 226 ps. The MD simulations were first carried out with harmonic torsional constraints which were derived from J couplings (0-86 ps) and then completely without constraints (96-226 ps). The lack of any major changes in the conformation of the two lariat-RNA structures upon releasing the NMR constraints indicate that the conformers generated in the MD simulation in water agree well with the structural features suggested by experimental observables. This means that the ensemble of conformers generated during the MD trajectory of 226 ps are not artificially held in these conformations due to the NMR constraints, suggesting that these conformers can be considered to be good representatives of the actual NMR observed solution structures.

A comparative conformational study of thymidylyl(3'----5')-thymidine, thymidylyl(3'----5')-5'-thio-5'- deoxythymidine and thymidinylacetamido-[3'(O)----5'(C)]-5'-deoxythymidine.

A comparative 270 MHz NMR spectroscopic study on the solution structure of the dimer d(TpT) 1, and its two analogues, namely, d(TpST) 2, and NH2d(TcmT) 4 has been reported. Analysis of chemical shifts and coupling constants indicate that: (i) The sugar moieties of the constituent nucleotides are not affected by modification of the internucleotide linkages and adopt preferentially an S-type conformation. (ii) The C4'-C5' bond in the pT part of the modified dimers 2 and 4 shows a large conformational freedom (gamma+ = 32% and 35%, respectively) compared to 1 (gamma+ = 75%). (iii) The population of the trans conformer about C5'-O5' is less important in d(TpST) 2 compared to d(TpT) 1. (iv) The C3'-O3' bond in 2 adopts a trans conformation as in 1. (v) The glycosidic bonds in the modified dimers 2 and 4 showed preferential syn conformation. UV and CD data show that the modified dimers 2 and 4 have poor tendency to stack intramolecularly, they also base pair less efficiently with d(ApA) as compared to d(TpT) 1.

Sensitive detection of genes by sandwich hybridization and time-resolved fluorometry.

Europium has been used as a non-radioactive marker in immunoassays as this metal can be detected with high sensitivity by time-resolved fluorometry. In this work streptavidin labeled with europium was used to detect biotinylated probes in a sandwich nucleic-acid hybridization assay with microtitration strips as the solid phase. pBR 322 plasmids were detected with a sensitivity of 4 x 10(5) molecules. As the sample is added in solution in sandwich hybridization, fast and simple sample pre-treatment can be used without encountering background problems. The method was applied to test bacterial samples of uropathogenic Escherichia coli strains for the presence of the beta-lactamase gene.