No abstract

We describe a near-infrared spectroscopy (NIRS) method for fast-screening Viagra tablets, counterfeit Viagra tablets, and imitations of Viagra. The method can (1) check the homogeneity of a batch; (2) distinguish counterfeits and imitations from authentic Viagra; (3) screen for the presence of sildenafil citrate, the pharmacologically active substance in Viagra, irrespectively of the excipients present; (4) and detect whether similar samples have been previously analysed. We applied the method to 103 samples with a diversity of appearance, chemical composition, and origin. Other analytical methods confirmed the positive screening results for sildenafil citrate and the presence of other pharmacological active substances. The NIRS screening indicated the absence of sildenafil citrate in the presence of another pharmacological substance for only 2 samples, where the reference methods showed the presence of sildenafil citrate in addition to that of clomifene citrate. Otherwise, the method gave no false positive or negative results. The NIRS screening method is very fast and reliable for detecting counterfeits and imitations, and it correctly predicts the presence or absence of sildenafil citrate in 98% of the samples.

The structure of unknown compounds present in herbal products was elucidated using liquid chromatography-electrospray ionization-mass spectrometry, direct-infusion electrospray ionization-mass spectrometry, and nuclear magnetic resonance. Compounds 1-3 were identified as sildenafil analogues, 1 bearing an N-ethylpiperazine moiety instead of an Nmethylpiperazine, and an acetyl group instead of the sulfonyl group, named acetildenafil, 2 bearing an N-ethylpiperazine moiety instead of an N-methylpiperazine (homosildenafil), and 3 bearing an N-hydroxylethylpiperazine moiety instead of an N-methylpiperazine, named hydroxyhomosildenafil. When analysing products marketed for penile erectile dysfunction or marketed as aphrodisiacs, attention should be given to the possible presence of these components.

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2-O-methylfucose residue at the reducting-terminal N-acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum). Here we show that transfer of nodZ to Rhizobium leguminosarum blovar (bv) viciae, which produces LCOs that are not modified at the reducing-terminal N-acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N-acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum, Glycine soja, Vigna unguiculate and Leucaena leucocephala. Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.

The nodZ gene, which is present in various soil bacteria such as Bradyrhizobium japonicum, Azorhizobium caulinodans, and Rhizobium loti, is involved in the addition of a fucosyl residue to the reducing N-acetylglucosamine residue of lipochitin oligosaccharide (LCO) signal molecules. Using an Escherichia coli strain that produces large quantities of the NodZ protein of B. japonicum, we have purified the NodZ protein to homogeneity. The purified NodZ protein appears to be active in an in vitro transfucosylation assay in which GDP-␤-fucose and LCOs or chitin oligosaccharides are used as substrates. The products of the in vitro reaction using chitin oligosaccharides as substrate were studied by using mass spectrometry, linkage analysis, and composition analysis. The data show that one fucose residue is added to C6 of the reducing-terminal N-acetylglucosamine residue. The substrate specificity of NodZ protein was analyzed in further detail, using radiolabeled GDP-␤-fucose as the donor. The results show that chitin oligosaccharides are much better substrates than LCOs, suggesting that in Rhizobium NodZ fucosylates chitin oligosaccharides prior to their acylation. The free glycan core pentasaccharides of N-linked glycoproteins are also substrates for NodZ. Therefore, the NodZ enzyme seems to have an activity equivalent to that of the enzyme involved in the addition of the C6-linked fucosyl substituent in the glycan core of N-linked glycoproteins in eukaryotes. Oligosaccharides that contain only one N-acetylglucosamine at the reducing terminus are also substrates for NodZ, although in this case very high concentrations of such oligosaccharides are needed. An example is the leukocyte antigen Lewis-X, which can be converted by NodZ to a novel fucosylated derivative that could be used for binding studies with E-selectin.The symbiotic relationship between legumes and rhizobia (i.e., Rhizobium, Bradyrhizobium, or Azorhizobium) can result in the formation of a nitrogen-fixing root organ, the nodule. The development of legume nodules is largely controlled by reciprocal signal exchange between the symbiotic partners. Legume roots secrete specific flavonoids or isoflavonoids that induce the transcription of many bacterial genes governing the early steps of this interaction (nod, nol, and noe genes). Many of these genes are involved in the synthesis and secretion of signal molecules, which are lipochitin oligosaccharides (LCOs). The chitin oligosaccharide backbone of all LCOs (also known as Nod factors) is N-acylated on the non-reducing-terminal residue. The basic structure of LCOs is synthesized by the cooperative action of NodA, NodB, and NodC (1-6). Additional gene products provide chemical decorations which, in some cases, have been shown to determine host specificity (for reviews see refs. 7-11). For instance, in a recent study (12) it was shown that transfer of the Bradyrhizobium japonicum nodZ gene to Rhizobium leguminosarum biovar viciae leads to the biosynthesis of LCOs that are fucosylated on C6 of the reducing-...