Beta 1,4 galactosyl- and alpha 2,6 sialyltransferase (gal-T EC 2.4.1.22 and sialyl-T EC 2.4.99.1) sequentially elongate and terminate complex N-glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi-disturbing agents. Polyclonal, peptide-specific antibodies to human sialyl-T expressed as a beta-galactosidase-sialyl-T fusion protein in E. coli were developed and applied together with mABs to human milk gal-T. In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl-T and gal-T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal-T followed by sialyl-T; in the reassembled Golgi apparatus sialyl-T and gal-T were co-localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal-T positive Golgi elements while sialyl-T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal-T and sialyl-T remained co-localized. Treatment with chloroquine affected Golgi structures less than monensin and led to condensation of gal-T positive and to slight enlargement of sialyl-T positive structures. Sequential recovery from BFA of gal-T and sialyl-T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.
[4‐13C]Nicotinate was synthesised and used to support the growth of a nicotinate auxotrophic mutant of Pseudomonas putida. 13C‐NMR spectroscopy of the isolated urocanase confirmed the efficient incorporation of 13C into C4 of the nicotinamide ring of the tightly bound NAD+ cofactor. β‐([2′‐13C]Imidazol‐4‐yl)propionate was synthesised according to known procedures and used for inhibition of the 13C‐labelled urocanase. An increase in the absorbance at 330 nm indicated adduct formation between enzyme‐bound NAD+ and inhibitor. The adduct was stabilised by oxidation with phenazine methosulfate and isolated using a slight modification of the procedure of Matherly et al. [Matherly, L. H., DeBrosse, C. W. & Phillips, A. T. (1982) Biochemistry 21, 2789–2794]. The 13C‐NMR spectrum of the doubly labelled adduct, [4‐13C]NAD‐[2′‐13C]imidazolylpropionate, showed no one‐bond 13C‐13C coupling between labelled sites. The 1H‐NMR spectrum of this adduct in 2H2O showed only one imidazole signal, which appeared as a doublet (1JC‐H= 212 Hz), confirming the presence of a proton at the labelled C2′. The lack of a C5′ signal and further NMR data provide evidence for a C‐C bond between C4 of the nicotinamide and C5′ of the imidazole ring. The revised structure for the enzymatically formed addition complex suggests a novel mechanism for the urocanase reaction which is not only chemically plausible but also explains the previously observed urocanase‐catalysed exchange of the C5 proton of urocanate and of β‐(imidazol‐4‐yl)propionate.
To determine the spread of Staphylococcus aureus within and between nursing home (NH) residents in the Euregion Meuse-Rhine, a cross-border region of the Netherlands and Germany, we investigated the prevalence of antibiotic resistance, genetic background and population structure of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates. A total of 245 S. aureus isolates were collected from NH residents. Susceptibility testing was performed with microbroth dilution. The genetic background was determined using spa typing, SCCmec typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Differences in the prevalence of resistance between the German and Dutch MSSA isolates were observed for the macrolides (15 % vs. 2 %, p = 0.003), clindamycin (15 % vs. 0 %, p = 0.003) and ciprofloxacin (34 % vs. 25 %). The macrolide and ciprofloxacin resistance varied between the NHs, while trimethoprim-sulfamethoxazole resistance was low in all residents. The MRSA prevalence was 3.5 % and <1 % among the German and Dutch NH residents, respectively (p = 0.005). The German MRSAs, isolated in 7 out of 10 NHs, belonged to ST22-MRSA-IV or ST225-MRSA-II. spa clonal complexes (spa-CCs) 015 and 002 were prevalent among the German MSSA isolates and spa-CCs 024 and 1716 were prevalent among the Dutch MSSA isolates. The antibiotic resistance of MSSA and the MRSA prevalence were significantly higher among the German NH residents. The spread of two MRSA clones was observed within and between the German NHs, but not between the Dutch and German NHs. Differences in the prevalence of resistance and the prevalence of MRSA between NHs on both sides of the border warrant the continuation of surveillance at a local level.
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