Functional analysis of chimeras derived from the Sinorhizobium meliloti and Mesorhizobium loti nodC genes identifies regions controlling chitin oligosaccharide chain length

The rhizobial nodulation gene nodC encodes an N-acetylglucosaminyltransferase that is responsible for the synthesis of chitin oligosaccharides. These oligosaccharides are precursors for the synthesis of the lipo-chitin oligosaccharides that induce cell division and differentiation during the development of nitrogen-fixing root nodules in leguminous plants. The NodC proteins of Mesorhizobium loti and Sinorhizobium meliloti yield chitinpentaose and chitintetraose as their main products, respectively. In order to localize regions in these enzymes that are responsible for this difference in product chain length, a set of six chimeric enzymes, comprising different combinations of regions of the NodC proteins from these two bacteria, was expressed in Escherichia coli. The oligosaccharides produced were analyzed using thin-layer chromatography. The major conclusion from this work is that a central 91-amino acid segment does not play any obvious role in determining the difference in the chain length of the major product. Furthermore, the characteristically predominant synthesis of chitintetraose by S. meliloti NodC is mainly dependent on a C-terminal region of maximally 164 amino acids, exchange of only this C-terminal region is sufficient to completely convert the M. loti chitinpentaose synthase into an S. meliloti-like chitintetraose synthase. The N-terminal region of 170 amino acids also plays a role in restricting the length of the major product to a tetramer. However, the role of the C-terminal region is clearly dominant, since exchanging the N-terminal region has no effect on the relative amounts of chitintetraose and -pentaose produced when the C-terminal region of S. meliloti NodC is present. The length of a predicted beta-strand around residue 300 in the C-terminal region of various NodC proteins is the only structural element that seems to be related to the length of the chitin oligosaccharides produced by these enzymes; the higher the amount of chitintetraose relative to chitinpentaose, the shorter the predicted beta-strand. This element may therefore be important for the effect of the C-terminal 164 amino acids on chitin oligosaccharide chain length.Microbial Biotechnolog

Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells.

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue

Microbial Biotechnolog

Chemical synthesis of N-acetylglucosamine derivatives and their use as glycosyl-acceptors by the Mesorhizobium loti chitin oligosaccharides synthase NodC

Bio-organic SynthesisMicrobial Biotechnolog

A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functional analysis of nodE genes was constructed. By testing chimeric nodE genes constructed by the exchange of poiymerase chain reaction (PCR)-generated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues). Mass spectrometric analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans-2. trans-4. trans-6. cis-11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R. leguminosarum bv. viciae. The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C1 8:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively. Therefore, the main difference between the nodE-determined LCOs of biovar viviae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly developed spot application assay, we show that the 18:2- and C18:3-containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results, we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the poly-unsaturated fatty acyl moieties of their LCOs, which is mediated by the host-specific central domain of the NodE protein.Animal science

Saccharomyces cerevisiae chitin biosynthesis activation by N-acetylchitooses depends on size and structure of chito-oligosaccharides

<p>Abstract</p> <p>Background</p> <p>To explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on <it>in vitro </it>chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a <it>Saccharomyces cerevisiae </it>membrane fraction.</p> <p>Findings</p> <p><it>N</it>-acetylchito-tetra, -penta and -octaoses resulted in 11 to 25% [¹⁴C]-GlcNAc incorporation into [¹⁴C]-chitin, corresponding to an increase in the initial velocity. The activation appeared specific to <it>N</it>-acetylchitooses as it was not observed with oligosaccharides in other series, such as beta-(1,4), beta-(1,3) or alpha-(1,6) glucooligosaccharides.</p> <p>Conclusions</p> <p>The effect induced by the <it>N</it>-acetylchitooses was a saturable phenomenon and did not interfere with free GlcNAc and trypsin which are two known activators of yeast chitin synthase activity <it>in vitro</it>. The magnitude of the activation was dependent on both oligosaccharide concentration and oligosaccharide size.</p

Functional domains in the chitin oligosaccharide synthase NodC and related ß-polysaccharide synthases

Microbial Biotechnolog

Simultaneous liquid-chromatographic determination of vitamin K1 and vitamin E in serum.

Abstract We describe a high-performance liquid chromatographic procedure for the simultaneous measurement of vitamins K1 and E in human serum. Delipidated human serum (free of vitamins K1 and E) was used to make standard solutions of these vitamins, and cetyl naphthoate and alpha-tocopheryl acetate were the internal standards for vitamin K1 and vitamin E, respectively. A simple, novel separation method utilizing liquid-liquid partition chromatography was used as a preparative "clean-up" procedure. Cetyl naphthoate and vitamin K1 (after post-column reduction) were detected by fluorescence, alpha-tocopheryl acetate and vitamin E by ultraviolet absorption. Sensitivity (detection limit) of the assay was 30 pg for vitamin K1 and 5 ng for vitamin E per injection. The method is specific, precise, and more rapid than previously described procedures. Within- and between-assay CVs were 8.1% and 12.9%, respectively, for vitamin K1; 3.5% and 6.0%, respectively, for vitamin E. Analytical recoveries of vitamins K1 and E were 80% and 93%, respectively, from serum and from delipidated serum (standards). The average neonatal serum concentration of vitamin K1 was 83 ng/L, 2.5 mg/L for vitamin E; for normolipidemic adults, the values were 343 ng/L and 7.9 mg/L, respectively, and for hyperlipidemic adults, 541 ng/L and 11.1 mg/L, respectively.</jats:p

Ziekenthuis: Verplaatsing van zorg van het Ziekenhuis naar Thuis

Dit artikel richt zich op de gewenste verplaatsing van complexe zorg van het ziekenhuis naar thuis waarbij complexe medische technologie gebruikt wordt