RESUMEN

We developed a clonal WI-38hTERT/GFP-RAF1-ER immortal cell line to study RAF-induced senescence of human fibroblasts. Activation of the GFP-RAF1-ER kinase by addition of 4-hydroxy-tamoxifen led to a robust induction of senescence within one population doubling, accompanied by the assembly of heterochromatic foci. At least two pathways contribute in parallel to this senescence leading to the accumulation of p15, p16, p21 and p27 inhibitors of cyclin-dependent kinases (CKIs). Cells that traversed S phase after RAF1 kinase activation experienced a replicative stress manifested by phosphorylation of H2AX and Chk2 and synthesis of p21. However, about half the cells in the population were blocked without passing through S phase and did not show activation of DNA-damage checkpoints. When the cells were cultivated in 5% oxygen, RAF1 activation generated minimal reactive oxygen species, but RAF-induced senescence occurred efficiently in these conditions even in the presence of anti-oxidants or inhibitors of DNA checkpoint pathways. Despite the presence of heterochromatic foci, simultaneous knockdown of p16 and p21 with inactivation of the GFP-RAF1-ER kinase led to rapid reversion of the senescent state with the majority of cells becoming competent for long-term proliferation. These results demonstrate that replicative and oxidative stresses are not required for RAF-induced senescence, and this senescence is readily reversed upon loss of CKIs.

Asunto(s)

RESUMEN

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.

Asunto(s)

RESUMEN

The Rhizobium sp. T1 strain. which induces nodule formation on alfalfa and clover roots, produces, during growth, an extracellular polysaccharide composed of D-glucose and D-glucuronic acid noted glucoglucuronan. During the bacterial growth, the pH of the medium decreases slightly. The control of pH in the growth medium slightly reduces the glucoglucuronan production. Under the conditions tested in the present work, the weight-average molecular weight of the polymers produced with or without pH control are similar: Mw approximately 2 x 10(6); the repeating unit determined by chemical and NMR analyses corresponds to the disaccharide: --> 3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1 -->.

Asunto(s)

RESUMEN

Rhamnogalacturonan II (RG-II) is a structurally complex pectic mega-oligosaccharide that is released enzymatically from the primary cell wall of higher plants. It contains roughly 30 monosaccharide units (MW approximately 5 kDa) including very unusual residues such as Kdo, Dha, aceric acid and apiose. Previous studies have demonstrated that these monomers are arranged into four structurally well-defined oligosaccharide side chains (A-D), linked to a homogalacturonan mainchain, but the specific attachment sites of these branches on the pectic backbone have not yet been elucidated. In the present work, fairly complete assignments of the 750 MHz 1H NMR spectra and partial assignments of the 13C NMR spectra of the sodium-borohydride-reduced RG-II monomer were obtained for a 5 mM sample isolated from red wine. On the whole, these data corroborate the primary structures of the sidechains previously established by methylation analysis, partial hydrolysis and FAB-MS spectrometry but some heterogeneity has been demonstrated (partial substitution at B5, B6, and A5). The preferred orientations of the majority of the sidechain glycosidic linkages in the RG-II monomer have been determined from the sequential nOe data and the solution structure is generally in good agreement with the stable conformers previously obtained by molecular modeling (MM3) of the disaccharide and sidechain oligosaccharide building blocks. All of a two-residue, a three-residue, and a four-residue segment of the backbone have been tentatively identified from long range interactions between sidechain protons as well as in the mainchain. Taking into account the length of the 9-mer galacturonan mainchain described in prior work, these building blocks constitute almost the complete structure of RG-II (Scheme 2).

Asunto(s)

RESUMEN

Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polymannuronate lyase AlxMB (only acting on two or more consecutive, nonacetylated mannuronate units) to prepare either mannuronate blocks (poly-M blocks) with dp approximately 30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp > 20. The poly-M blocks were obtained by lysis of a P. aeruginosa polymannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.

Asunto(s)

RESUMEN

Structural studies were performed in two atypical polysaccharides, PS-1 and PS-2 isolated from the broth of a Tn5 mutant strain of Xanthomonas campestris. Sugar composition, methylation and nuclear magnetic resonance analyses were determined. PS-1 is composed by repeating trisaccharide units containing D-glucose, D-mannose and having the structure. carbohydrate sequence [see text]. Preliminary studies on the PS-2 show a polymer composed in a large extent of rhamnose. Unexpectedly, this polysaccharide is soluble in alcoholic solutions.

Asunto(s)

RESUMEN

The extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83 was found to be composed of D-glucose and D-galactose in a molar ratio of 2:3. The primary structure of the polysaccharide was shown by sugar analysis, methylation analysis, FABMS, partial acid hydrolysis and nuclear magnetic resonance (NMR) spectroscopy to consist of a pentasaccharide repeating unit having the following structure: -->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6 )-alpha-D -Glcp-(1-->3)-beta-D-Galf-(1-->

Asunto(s)

RESUMEN

The title compound is a cyclic oligosaccharide having six glucopyranose residues linked alternatively by alpha-(1-->4) and beta-(1-->6) glycosidic linkages. Like cyclodextrin analogues it is expected to exhibit an internal cavity and to form inclusion complexes with other species. In order to investigate its conformational preferences, an extensive conformational search was carried out using a combination of Metropolis Monte-Carlo (MMC) procedure in the glycosidic torsion angle space and molecular mechanics procedures. To this end a specific program (METROCYCLIX) was developed. To reduce the MMC search, conformational maps of parent disaccharides were considered as starting entries. Fully minimized conformations were gathered into families using a clustering technique based on RMS fitting over the glycosidic torsion angle values. A wide range of local energy minima were identified in spite of ring closure conditions that constrained the structure of the oligosaccharide. Low energy conformers were stabilized by intramolecular interactions between distant residues. From the Bolzmann population of the best structures derived from the clustering results, various average properties were calculated and compared with experimental data obtained by high resolution NMR. Interpretation of these experimental values (heteronuclear coupling constants, rotating frame nuclear Overhauser effects, relaxation times) relies on the use of Karplus like equations (coupling constants) and analysis of the full relaxation rate matrix treatment (ROE). The quality of the molecular modelling strategy used is assessed by the agreement obtained between calculated and measured observables.

Asunto(s)

RESUMEN

The 1H and 13C NMR chemicals shifts of the various saturated and unsaturated timers obtained by acid or enzymatic depolymerisation of homopolymeric blocks of alginates are reported. In addition, 13C NMR chemical shifts are assigned for several saturated oligomers of higher polymerisation degrees. Breakdown of alginate and of homopolymeric alginate blocks by Haliotis tuberculata alginate lyase was monitored with 1H NMR spectroscopy and the signals relevant to the identification of the lyase products are pointed out. The enzymes performs beta-elimination on the mannuronic acid residues, independently of their immediately surrounding neighbours. Application of this approach to the analysis of the substrate specificity of alginate lyases is discussed.

Asunto(s)

RESUMEN

Beta-Xylosidases are grouped in families 39 and 43 of a general classification of glycosyl hydrolases based on amino acid sequence similarities. The Beta-xylosidase from Butyrivibrio fibrisolvens, which belongs to family 43, has been shown to operate by a molecular mechanism which results in the inversion of the anomeric configuration. Thermoanaerobacterium saccharolyticum B6A-RI Beta-xylosidase which belongs to family 39 was purified as a recombinant enzyme from Escherichia coli. The stereochemistry of the hydrolysis of p-nitrophenyl Beta-D-xylopyranoside was followed by 1H NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm (J approximately 10 Hz) assignable to H1 of free Beta-xylose with a small amount of alpha-xylose (5.05 ppm, J approximately 3 Hz) attributable to mutarotation. This result indicates that T. saccharolyticum Beta-xylosidase operates with overall retention of the anomeric configuration. This result, with the lack of sequence similarity between the two families of Beta-xylosidases, suggests that these two families have major differences in their active-site geometries. Consistent with its retaining mechanism, Beta-xylosidase of T. saccharolyticum B6A-RI also displayed transglycosylating activity:reverse-phase HPLC showed approximately 30% conversion of p-nitrophenyl Beta-D-xylopyranoside into a number of higher nitrophenyl oligosaccharides after 5 min incubation with the enzyme. The structure of the most abundant oligosaccharides could be determined by total correlation spectroscopy NMR and showed that the enzyme can build Beta-1,4, Beta-1,3- and Beta-1,2-linked xylo-oligosaccharides.

Asunto(s)

RESUMEN

The cgkA gene of Alteromonas carrageenovora encodes a kappa-carrageenase with a predicted mass of 44212 Da, much larger than the 35 kDa estimated from SDS/PAGE of the protein purified from culture supernatants. Immunoblotting experiments showed the presence of a protein of 44 +/- 2 kDa in both native and recombinant bacterial intracellular extracts, suggesting that the kappa-carrageenase is produced as a preproprotein which undergoes proteolytic processing twice during secretion. To determine the exact site of C-terminal cleavage, the precise mass of the purified extracellular kappa-carrageenase was measured by electrospray-ionization/mass spectrometry and found to be 31,741 +/- 3 Da. The mature kappa-carrageenase of A. carrageenovora thus appears to be composed of 275 amino acids, from residue Ala26 to residue Asn301 of the cgkA gene product. To assess the molecular mechanism of this member of family 16 of glycosyl hydrolases, hydrolysis of neocarrahexaitol by the kappa-carrageenase was monitored by gel filtration chromatography and 13C-NMR. Results show that neocarrabiitol and beta-neocarratetraose are initially formed, demonstrating that the enzyme operates with a molecular mechanism retaining the anomeric configuration. Consistent with this result, the enzyme was also shown to be able to catalyze transglycosylation.

Asunto(s)

Asunto(s)

RESUMEN

Chitinases A1 and D were purified from the periplasmic proteins produced by Escherichia coli HB101 harbouring recombinant plasmids carrying respectively the chiA and chiD genes of Bacillus circulans WL-12. HPLC analysis indicated that during the hydrolysis of chitotriose, both chitinases initially produce N-acetylglucosamine and only one anomer of chitobiose. 1H NMR spectroscopy of the hydrolysis of chitotetraitol showed that this anomer corresponds to beta-chitobiose, demonstrating that chitinases A1 and D act by a molecular mechanism that retains the anomeric configuration. This mechanism is similar to that of lysozymes although both chitinases belong to a family of proteins sharing no demonstrable amino acid sequence similarity with lysozymes.

Asunto(s)

RESUMEN

The use of the n.m.r. method in the investigation of chitosan carboxymethylation was evaluated. It seems to be the most effective technique to determine concurrently the degree and the position of substitution of the carboxymethylated chitosan derivatives. The 13C-n.m.r., by the DEPT method, 1H-1H and 1H-13C-n.m.r. correlations give much valuable information from the chemical shifts of the complex carboxymethylchitosan spectra. The relative reactivity of the functional groups of chitosan towards carboxymethylation was also determined assuming a higher reactivity of the C-6 position.

Asunto(s)

RESUMEN

Endoglucanase Z from the phytopathogenic bacterium Erwinia chrysanthemi (strain 3937) was purified by affinity chromatography on microcrystalline cellulose Avicel PH101. A kinetic characterization using p-nitrophenyl beta-D-cellobioside and p-nitrophenyl beta-D-lactosde as substrates was conducted: endoglucanase Z exhibited Km values of 3 mM and 7.5 mM and Vm values of 129 and 40 nmol.min-1.mg-1 towards p-nitrophenyl beta-D-cellobioside (kcat = 0.1 s-1) and p-nitrophenyl beta-D-lactoside (kcat = 0.03 s-1), respectively). The hydrolysis of cellotetraitol by endoglucanase Z was followed by HPLC and 1H NMR. Results show that cellobiitol and beta-cellobiose are initially formed, demonstrating that the enzyme is acting by a molecular mechanism retaining the anomeric configuration. This suggests the involvement of a glycosyl-enzyme intermediate.

Asunto(s)

RESUMEN

This paper concerns the characterization of the chemical structure of a DP3 glucosamine oligomer. The assignments of nearly all protons are reported. Variations of 1H chemical shifts with pD and temperature are correlated to the pKa of amino groups, and not with substantial conformational changes.

Asunto(s)