Richard Bourbouze

Poly(3-hydroxyoctanoate) containing pendant carboxylic groups for the preparation of nanoparticles aimed at drug transport and release

Copolymers of poly(3-hydroxyoctanoate) (PHO) with carboxylic groups in lateral chains have been prepared by chemical modification of unsaturated bacterial polyesters. The oxidation of the pendant alkenes is complete and important loss in molecular weight of polymer was observed. The presence of repeating units containing pendant carboxy groups in the proportion of 25% has enhanced hydrophilicity of these new polymeric structures. Nanoparticles have been prepared from PHO and two functionalized derivatives, characterized by electronic microscopy and compared in view of bioactive molecules transport and release.

Inhibition of A and B N-acetyl-beta-D-glucosaminidase urinary isoenzymes by urea.

A urinary fraction which inhibits the activity of N-acetyl-beta-D-glucosaminidase (NAG) has been isolated and identified as being urea. Usually present in high concentration, urea appears to be the only urinary component responsible for the frequently observed urinary NAG inhibition. The inhibition of the two urinary NAG isoenzymes A and B is competitive with respective Ki values of about 70 mmol/l and 60 mmol/l. With routine assay conditions, it seems that a dilution of urine prior to enzyme assay is sufficient to abolish the inhibition of the two isoenzymes A and B by endogenous urea.

Characteristics and regulation of 11β-hydroxysteroid dehydrogenase of proximal and distal nephron

Enzymatic properties of the enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD), which confers mineralocorticoid selectivity, have been explored in the aldosterone-sensitive collecting duct (CCD) and the aldosterone-insensitive Pars Recta (PR) of the rat kidney. After incubation of freshly isolated tubular segments with [3H]corticosterone (3H-B) or [3H]dehydrocorticosterone (3H-A), the rate of transformation of 3H-B into 3H-A (dehydrogenase activity), or the reverse reaction (reductase activity) were measured by HPLC, Vmax for dehydrogenase activity was found to be 8- to 10-fold higher in CCD than PR. The enzyme functions over a very wide range (0.1-5000 nM) of corticosterone concentration. In CCD, enzyme kinetics suggest either the presence of two 11-HSD forms, differing by their affinity for corticosterone, or complex kinetics. Addition of NAD or NADP to permeabilized tubules revealed that dehydrogenase activity is NAD-dependent in CCD and NADP-dependent in PR. Cofactor addition was ineffective in intact tubules. CCD exhibited an exclusive dehydrogenase activity, whereas in PR dehydrogenase and reductase activity were found. No regulation of dehydrogenase activity could be evidenced in adrenalectomized rats receiving or not aldosterone, corticosterone or dexamethasone, for 2 h, 3 days or 4 days. We conclude that 11-HSD in the CCD and PR differs by its Vmax and cofactor dependence. Corticosteroid hormones do not influence 11-HSD activity.

Early monitoring of human renal transplantations by N-acetyl-β-d-glucosaminidase isoenzyme activities in urines

Monitoring of variations in N-acetyl-beta-D-glucosaminidase (NAG) urinary activity, following renal transplantation, has been proposed for the early diagnosis of rejection episodes. In this study, the measurement of urinary NAG-B activity was conducted as a complement to total NAG (A + B) measurement, which is normally used alone. Selective measurement of NAG-B activity is carried out after fixation of NAG-A on ion exchanger in test tubes. Results of NAG (A + B) activity confirm that the assay of urinary NAG is a useful indicator of rejection, but a positive correlation between NAG-B and NAG (A + B) activities was observed during the various complications which can occur after transplantation. The specific measurement of this isoenzyme does not, therefore, seem to provide additional information in the early monitoring of human renal transplantations. Apart from rejection episodes, other factors are likely to produce marked NAG-B excretion, e.g. gentamicin therapy.

N-acetyl-β-D-glucosaminidase (NAG) isoenzymes release from human monocyte-derived macrophages in response to zymosan and human recombinant interferon-γ

Abstract Secretion of N -acetyl- β -D-glucosaminidase (NAG) isoenzymes by human blood monocyte-derived macrophages in response to zymosan and human recombinant interferon-γ was studied. Macrophages were found to release NAG in response to zymosan, but interferon-γ has no effect on secretion. Isoenzyme separation by isoelectric focusing demonstrates that non stimulated and zymosan or interferon-γ treated macrophages release predominantly NAG B and, to a lesser extent, NAG A isoenzymes. In all these conditions, the intracellular intermediate form NAG I could not be detected in the media. Thus, activated macrophages may not be the source of NAG intermediate forms I and P in pathological or maternal serum. In contrast, macrophages could contribute to a significant elevation of urinary activity and NAG B excretion in response to inflammatory conditions.

Biosynthetic stereocopolymer of 3-methylmalic acid as hydrolyzable and biocompatible polyester for temporary therapeutic applications

Abstract A mixture of (2 S ,3 S ) and (2 S ,3 R )-3-methylaspartic acid, obtained by bioconversion of mesaconic acid in the presence of 3-methylaspartase as enzymatic catalyst, was transformed into the corresponding benzyl 3-methylmalolactonate stereoisomers using a multiple-step synthesis. A mixture of (3 R ,4 R ) and (3 S ,4 R ) (80/20 (mol/mol) ratio) benzyl-3-methylmalolactonate was transformed by anionic ring-opening polymerization to an optically active and stereoregular stereocopolymer constituted by 80xa0mol% of benzyl (3 R ,4 S ) 3-methylmalate repeating units and 20xa0mol% of benzyl (3 S ,4 S ) 3-methylmalate units, as determined by 1 H NMR. The corresponding optically active poly( β -3-methylmalic acid) was obtained by catalytic hydrogenolysis of the protecting benzyl ester groups, in N -methylpyrrolidone as solvent. This functionalized hydrosoluble polyester was degraded by simple hydrolysis in a phosphate buffer at pH 7, as shown by SEC measurements. It is worth noting that the kinetic profile was equivalent to the one of poly( β -malic acid). Moreover, at 37°C, the hydrolysis was complete within six weeks, yielding to the corresponding optically active 3-methylmalic acid. In order to use this polymeric material for temporary therapeutic applications, polymers containing both diastereoisomers as repeating units as well as their ultimate products of degradation were evaluated based on harmlessness towards their environment. No toxicity was detected against a human cell line, HepG2.

Partial characterization of intracellular and secreted glycosidases from rabbit articular chondrocytes in culture.

N-Acetyl-beta-hexosaminidase, beta-galactosidase and beta-glucuronidase activities were shown to be present in cultured rabbit articular chondrocytes. Secretion of enzyme activity seems to preferentially result in the accumulation of N-acetyl-beta-hexosaminidase. Three days after seeding, the amount of N-acetyl-beta-hexosaminidase activity found in the medium accounts for about 140% of the total N-acetyl-beta-hexosaminidase activity after complete disruption of the cell pellet. Optimal conditions of incubation time, cell numbers, substrate concentration, and pH for glycosidase activities were determined in 0.1% Triton X-100. Intracellular and secreted glycosidases have shown similar elution profiles by chromatofocusing. N-acetyl-beta-hexosaminidase exhibits two major forms which may play a role in the catabolism of glycosaminoglycans.

N-Acetyl-β-d-glucosaminidase (NAG) isoenzymes in primary cultures of rabbit kidney proximal tubule cells: a cellular model for studies on nephrotoxicity?

N-Acetyl-beta-D-glucosaminidase (NAG) isoenzyme profile in primary cultures of rabbit kidney proximal tubule cells was studied. Confluent cells had high levels of NAG activity, but ion exchange chromatography showed that the NAG isoenzyme profile in cultured cells was different from that of rabbit renal cortex homogenates and freshly isolated cells. Confluent cultured cells contained an atypical acidic isoform, absent in homogenates and freshly isolated cells in which the predominant isoform is NAG-A (a heterodimer alpha beta). The fact that this atypical isoform was able to hydrolyse the synthetic substrate 4-methylumbelliferyl-beta-N-acetylglucosaminide-6-sulphate indicated that it probably was an alpha-subunit homodimer. These results suggest subunit rearrangement within NAG polypeptide chains linked to down-regulation of beta-subunit production in cultured rabbit proximal cells. The change in isoenzyme profile in cultured cells may make it difficult to use primary cultures of rabbit proximal tubule cells to establish correlations between in vitro and in vivo studies using NAG isoenzymes as a nephrotoxicity index, as illustrated by the effects of gentamicin.

Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor.

beta-methylaspartate ammonia-lyase, EC 4.3.1.2, (beta-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b, respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)-3a yielded the alpha, beta-substituted beta-lactone (3R,4R) 6 with a very high diastereoisomeric excess (> 95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl beta-(2R,3S)-3-methylmalate] 8, prepared by an anionic ring opening polymerization, was highly isotactic as determined by 13C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7, to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of beta-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions.

Macromolecular aggregation of F1 and P1 fractions of RU 41740 an immunomodulating compound isolated from Klebsiella pneumoniae, as revealed by size exclusion high-performance liquid chromatography and light scattering detection

RU 41740 isolated from Klebsiella pneumoniae possesses high immunomodulating activity. This immunomodulating agent has been described as composed of two glyeoprotein F1 and P1 fractions. Some characteristics of native RU 41740 and F1 and P1 fractions were investigated using size exclusion high-performance liquid chromatography and light scattering detection. Particle size distribution and apparent molecular mass data suggest that RU 41740 is a macromolecular aggregate of numerous F1 and P1 subunits. Findings suggest an effect of orally administered RU 41740 on the function of Peyers patch cells.