Evidence for a radical mechanism in biocatalytic degradation of synthetic dyes by fungal laccases mediated by violuric acid

The bleaching activity of the Pleurotus ostreatus POXC laccase isoenzyme has been tested against selected single textile acid dyes (two anthraquinonic and two azo dyes), as well as towards a solution mimicking a real acid dye waste-water for wool. The catalytic reaction of POXC has been investigated both in the presence and in the absence of the synthetic mediator violuric acid (VIO) (–NOH type of mediator). In all the cases tested, the presence of the mediator enhanced the reaction rate and the percentage of decolorization, apart from one of the dyes (Acid Blue 62), which is itself a good substrate for the laccase-catalyzed oxidation. Electron paramagnetic resonance (EPR) experiments, after the addition of an excess of VIO to the solution of laccase, showed the presence of a strong and stable radical signal that was assigned to a neutral radical form of VIO

Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin.

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogenbonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization

Characterization of radical intermediates in the laccase-mediator systems. A multifrequency EPR, ENDOR, and DFT/PCM investigation

Suitable low molecular-weight compounds, called mediators, can be used in combination with the phenol-oxidase enzyme laccase to indirectly oxidize large organic substrates, such as environmental pollutants, which are not laccase natural substrates. The oxidation of two different synthetic redox mediators, violuric acid (VIO) and 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) has been studied under catalysis of two laccases from white-rot fungi (Trametes versicolor and Pleurotus ostreatus). VIO was selected as a prototype of the –NOH type of mediators and compared to ABTS, a well-known two-step redox system. To characterize the radical intermediates formed from both mediators after the enzymatic oxidation, a multifrequency EPR approach has been adopted. The radical species have been investigated employing 9.4 GHz (X-band), 34 GHz (Q-band) and 244 GHz (high field) EPR and pulse electron nuclear double resonance (ENDOR) techniques. Theoretical calculations based on density functional theory (DFT/PCM) have been performed to support and further interpret the experimental EPR and ENDOR data. This integrated approach allowed us to obtain a complete characterization of both radicals and to elucidate the type of the radical state (neutral or cationic)

Structure of the type IX Group B Streptococcus capsular polysaccharide and its evolutionary relationship with types V and VII

The Group B Streptococcus capsular polysaccharide type IX was isolated and purified, and the structure of its repeating unit was determined. Type IX capsule \u21924)[NeupNAc-\u3b1-(2\u21923)- Galp-\u3b2-(1\u21924)-GlcpNAc- \u3b2-(1\u21926)]-\u3b2-GlcpNAc-(1\u21924)-\u3b2-Galp- (1\u21924)-\u3b2- Glcp-(1\u2192 appears most similar to types VII and V, although it contains two GlcpNAc residues. Genetic analysis identified differences in cpsM, cpsO, and cpsI gene sequences as responsible for the differentiation between the three capsular polysaccharide types, leading us to hypothesize that type V emerged from a recombination event in a type IX background

Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen : Comparing Lysine and Tyrosine-directed Conjugation

Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates. (Figure Presented)

ECTOPIC EXPRESSION OF FSH RECEPTOR ISOFORMS IN NEOPLASTIC BUT NOT IN ENDOTHELIAL CELLS FROM PANCREATIC NEUROENDOCRINE TUMOURS

FSH receptor (FSHR) expression is restricted to gonads, where it drives FSH-dependent cell differentiation; in addition, FSHR plays an important role in the regulation of ovarian angiogenesis. Recently, FHSR expression has been shown in blood vessels of various tumours. However, pancreatic neuroendocrine tumours (p-NET), which have high degree blood supply, were not included in that study. The aim of this study was to evaluate FSHR expression in p-NET. FSHR expression was evaluated in tumour samples from 30 patients with p-NET by immunohistochemistry and Western blot (WB); fluorescence microscopy was used to localize FSHR in specific cells from tissue samples. von Willebrand factor (vWF) and chromograninA (chrA) was used as blood vessel and NET cells marker, respectively, to co-localize FSHR. FSHR expression was detected in all p-NET by immunohistochemistry. Western blot confirmed FSHR expression on p-NETs although different FSHR isoforms, ranging from 240 kD to 55 kD were found in the samples studied. Surprisingly, FSHR colocalized with chromogranin A but not with vWF, suggesting that neoplastic cells of neuroendocrine origin rather than blood vessels expressed FSHR. No relationship was found between degree of FSHR expression and histology of p-NET. FSHR may be aberrantly expressed in neoplastic cells from p-NET and not in tumour blood vessels; however, its biological significance as well as its clinical relevance remains to be elucidated

ECTOPIC EXPRESSION OF FSH RECEPTOR ISOFORMS IN NEOPLASTIC BUT NOT IN ENDOTHELIAL CELLS FROM PANCREATIC NEUROENDOCRINE TUMOURS

FSH receptor (FSHR) expression is restricted to gonads, where it drives FSH-dependent cell differentiation; in addition, FSHR plays an important role in the regulation of ovarian angiogenesis. Recently, FHSR expression has been shown in blood vessels of various tumours. However, pancreatic neuroendocrine tumours (p-NET), which have high degree blood supply, were not included in that study. The aim of this study was to evaluate FSHR expression in p-NET. FSHR expression was evaluated in tumour samples from 30 patients with p-NET by immunohistochemistry and Western blot (WB); fluorescence microscopy was used to localize FSHR in specific cells from tissue samples. von Willebrand factor (vWF) and chromograninA (chrA) was used as blood vessel and NET cells marker, respectively, to co-localize FSHR. FSHR expression was detected in all p-NET by immunohistochemistry. Western blot confirmed FSHR expression on p-NETs although different FSHR isoforms, ranging from 240 kD to 55 kD were found in the samples studied. Surprisingly, FSHR colocalized with chromogranin A but not with vWF, suggesting that neoplastic cells of neuroendocrine origin rather than blood vessels expressed FSHR. No relationship was found between degree of FSHR expression and histology of p-NET. FSHR may be aberrantly expressed in neoplastic cells from p-NET and not in tumour blood vessels; however, its biological significance as well as its clinical relevance remains to be elucidated

Phosphorylation of the Synthetic Hexasaccharide Repeating Unit Is Essential for the Induction of Antibodies to Clostridium difficile PSII Cell Wall Polysaccharide

Clostridium difficile is emerging worldwide as a major cause of nosocomial infections. The negatively charged PSII polysaccharide has been found in different strains of C. dif f icile and, thereby, represents an important target molecule for a possible carbohydrate-based vaccine. In order to identify a synthetic fragment that after conjugation to a protein carrier could be able to induce anti-PSII antibodies, we exploited a combination of chemical synthesis with immunochemistry, confocal immunofluorescence microscopy, and solid state NMR. We demonstrate that the phosphate group is crucial in synthetic glycans to mimic the native PSII polysaccharide; both native PSII and a phosphorylated synthetic hexasaccharide repeating unit conjugated to CRM197 elicit comparable immunogenic responses in mice. This finding can aid design and selection of carbohydrate antigens to be explored as vaccine candidates