Rapid & sensitive polymerase chain reaction based detection & typing of foot-and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related viruses

&lt;p&gt;Reverse transcription followed by the polymerase chain reaction method (PCR) allowed the detection of foot-and-mouth disease virus (FMDV), regardless of the serotype. A primer set corresponding to highly conserved regions of the 2B sequence was selected. By combining in a single reaction tube specific primer pairs for FMDV, swine vesicular disease virus, (SVDV), encephalomyocarditis virus (EMCV) and bovine viral diarrhea virus (BVDV), all four viruses could be identified and differentiated in one amplification reaction, based on the different lengths of the respective amplified segments. In a similar way, FMDV types O, A, C, SAT 2 and Asia 1 could be identified and differentiated, using primers selected from the ID (VP1) genome region. All results were confirmed by direct sequencing of the PCR product. The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within three hours after samples (cell culture isolates as well as clinical material) arrived, which is of great importance in case of an FMDV suspicion. Furthermore, a very high sensitivity was achieved (less than one PFU). Therefore, our powerful detection assay by means of PCR for FMDV as well as for SVDV, EMCV and BVDV, has advantages compared to the presently used procedures.&lt;/p&gt;</p

Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule

Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases.

D-Xylose isomerases are metal-ion (Mn2+, Co2+, Mg2+)-requiring tetrameric enzymes. Both the stoichiometry and the binding constants have been determined by titrating the metal-ion-free enzymes from five organisms (Actinomycetaceae and more divergent bacteria) with the respective metal ions using the enzyme activity as indicator of active complex-formation. The following characteristics have been observed for each specific isomerase: (i) two essential metal ion sites (one structural and one catalytic) exist per subunit; (ii) the metal ion binding at one site does not affect the binding at the other site; (iii) of the four possible configurations E, aE, Eb and aEb, only the double-occupied enzyme is active; (iv) the metal ion activation is a time-dependent process; (v) the dissociation constants for both the structural and catalytic sites may be identical or may differ by one or higher orders of magnitude; (vi) metal ion binding is stronger in the order Mn2+ greater than Co2+ much greater than Mg2+; (vii) pronounced increases in Km values concomitant with decreasing equivalents of metal ion added are only observed in the presence of Mg2+ ions