Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan.

In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5' and 3' rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme.

Endopolygalacturonases reveal molecular features for processivity pattern and tolerance towards acetylated pectin.

Endopolygalacturonases (EndoPGs) hydrolyse the 1-4 linkages between two alpha-d-galacturonic acids (GalA) of the smooth homogalacturonan regions of pectin. GalA may be methyl-esterified on the carboxylic group and acetyl-esterified on the hydroxylic groups. EndoPG activity most often decreases with such increasing degree of substitution. In this paper, we used bioinformatics and molecular modelling technics to explain the tolerance profile at the molecular scale and processivity scheme of three endoPGs with respect to acetylated pectin substrate; the first two enzymes originate from Aspergillus niger (AnPGI and AnPGII) and the third from Fusarium moniliforme (FmPG). Partly acetylated and methylated homogalacturonan fragments in complex with the three PGs were successively modelled in silico. The amino acid residues involved in substrate binding were identified for each enzyme. Similarly, the docking pattern of the differently decorated oligomers in the catalytic groove was individually characterized for each enzyme. This work shows full agreement with our previous extensive mass spectrometry analysis of the hydrolytic products that established distinct tolerance profiles for the three endoPGs and earlier work that ascertained processivity, specifically for AnPGI. In our previous work, AnPGI was shown to be the most powerful enzyme among the three enzymes with an enhanced tolerance towards O2- and O3-acetylated substrates. We report here amino acids of AnPGI that are unique in binding the pectin backbone and that are identified as possibly crucial for its specificity, namely S191(An)(PGI)/D240(An)(PGI). Similarly, topologically equivalent residues in AnPGII and FmPG were identified that could impede such binding; S234(An)(PGII)/S91(An)(PGII) and S245(Fm)(PG)/V89(Fm)(PG). In addition, we report here, from normal mode analysis computed on AnPG1, a shear bending motion of 15 A of amplitude that fully accredits the processive action pattern for this enzyme, with D240(An)(PGI) and R96(An)(PGI) working as crampons to favour the sliding of the substrate. Conversely, the same method clearly evidences a hinge binding motion for AnPGII and FmPG that should only authorize one hydrolytic event per enzyme/substrate encounter.

Short-chain arabinoxylans prepared from enzymatically treated wheat grain exert prebiotic effects during the broiler starter period.

Carbohydrate-degrading multi-enzyme preparations (MEP) are used to improve broiler performances. Their mode of action is complex and not fully understood. In this study, we compared the effect of water-soluble fractions isolated at the pilot scale from wheat grain incubated with (WE) and without (WC) MEP. The fractions were incorporated in a wheat-based diet (0.1% w/w) to feed Ross PM3 broilers and compared with a non-supplemented control group (NC). The body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) until d 14 were determined. At d 14, ileal and cecal contents and tissue samples were collected from euthanized animals. The intestinal contents were used to measure the short-chain fatty acids (SCFA) concentration using gas chromatography and to determine the abundance and composition of microbiota using 16S sequencing. Villi length of ileal samples was measured, while L-cell and T-cell densities were determined using immuno-histochemistry. The MEP treatment increased the amount of water-soluble arabinoxylans (AX) and reduced their molecular weight while retaining their polymer behavior. The WE fraction significantly (P < 0.05) increased FI by 13.8% and BWG by 14.7% during the first wk post hatch when compared to NC. No significant effect on FCR was recorded during the trial. The WE increased the abundance of Enterococcus durans and Candidatus arthromitus in the ileum and of bacteria within the Lachnospiraceae and Ruminococcaceae families, containing abundant butyrate-producing bacteria, in the ceca. It also increased the concentration of SCFA in the ceca, decreased the T-lymphocyte infiltration in the intestinal mucosa, and increased the glucagon-like-peptide-2 (GLP-2)-producing L-cell density in the ileal epithelium compared with WC and NC. No significant effects were observed on villi length. These results showed that AX present in the WE fraction altered the microbiota composition towards butyrate producers in the ceca. Butyrate may be responsible for the reduction of inflammation, as suggested by the decrease in T-lymphocyte infiltration, which may explain the higher feed intake leading to improved animal growth.

Action pattern of Fusarium moniliforme endopolygalacturonase towards pectin fragments: Comprehension and prediction.

The structures of complexes of Fusarium moniliforme endopolygalacturonase (endoPG) with non-methylated or partly methylated homogalacturonan fragments were modeled to identify the residues involved in substrate binding and to correlate the cleavage pattern with the experimental productive modes. The conformational space of the complex was extensively explored and malto- to hexo-oligogalacturonates were modeled in the active cleft. To select the most highly probable productive complex for each oligomer between DP2 and 6, four energetic criteria were defined. Noteworthingly, the results were in accordance with the experimental results showing the mode of action of this enzyme towards un-methyl-esterified oligogalacturonates. Furthermore, the amino-acid residues involved in the binding were confirmed by similar studies performed on other endoPGs. Then, the oligomers were gradually methyl-esterified at one or more positions and similar docking experiments were carried out. Markedly, the docking energies were not significantly modified by the methyl-esterification of the substrate and it is likely that the methyl-esterification of the substrate does not alter the mode of action of the enzyme. Finally, 1D sequence and 3D structure of the endopolygalacturonase of Aspergillus niger II, known to be strictly non-tolerant to methylesters, were compared with the sequence and structure of the tolerant F. moniliforme endopolygalacturonase to get to a structural comprehension of the tolerant-or not-behaviour of endoPGs with methyl-esterified pectins.

A new approach for studying interaction of the polygalacturonase-inhibiting proteins with pectins.

A method for determination of the interaction between pectins and proteins was developed using cross-linked polygalacturonic acid (CLPG) as the pectic substrate and polygalacturonase-inhibiting proteins (PGIPs). Defined water-insoluble pectins were prepared by chemical substitutions with acetyl or methoxyl groups on CLPG. In the presence of 0.1 M NaCl, PGIPs fully bound to CLPG but not to cross-linked alginic acid (CLAL), which had a similar pK(a) to CLPG, suggesting that the inhibitor was not simply bound to the substrate by nonspecific electrostatic interaction. Optimum binding of PGIPs to CLPG occurred at pH 2.4 to 4.7. The binding ability of the inhibitor to CLPGs with degree of methylation (DM) of 66% or degree of acetylation (DAc) of 133% was not significantly changed. In contrast, the DM of 82% or 95% decreased the binding. These results indicated that the carboxylic groups of galacturonic acid residues were involved in the recognition of the substrate by PGIPs.

Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan.

Two exo-polygalacturonases (EC 3.2.1.67) were purified from a commercial Aspergillus niger enzyme preparation by ammonium sulfate precipitation, preparative electrofocusing, anion-exchange and size-exclusion chromatographies. The enzymes had molar masses of 82 kDa (exo-PG1) and 56 kDa (exo-PG2). Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Addition of 0.01 mM HgCl(2) increased the exo-PG2 activity 3.4 times but did not affect exo-PG1. Analysis of the reaction products of (reduced) pentagalacturonate by high-performance anion-exchange chromatography revealed that both enzymes split the substrate from the non-reducing end in a multi-chain attack mode. Exo-PG1 had a broad specificity towards oligogalacturonates with different degrees of polymerisation, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid and Xyl-GalA disaccharide, as identified by electrospray ionization-ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing galacturonic acid and acetylated galacturonic acid, as shown by ESI-ITMS.

Hydrolysis of pectins with different degrees and patterns of methylation by the endopolygalacturonase of Fusarium moniliforme.

The mode of action of the endopolygalacturonase from Fusarium moniliforme was studied towards a series of pectins with different amounts and distribution patterns of methyl-ester groups. The enzyme hydrolysed the linkages between two galacturonic acid residues according to a multi-chain attack mechanism, at least at the early stage of the reaction. The final percentage of hydrolysis decreased with increasing the degree of methylation. The distribution pattern of the methyl groups affected the rate of hydrolysis as well as the final percentage of hydrolysis, a blockwise distribution being more favourable than a random one. The final products, as analysed by mass spectrometry, included methyl-esterified oligogalacturonates. The detailed analysis of the structure of the oligomers showed that the enzyme was able to accommodate methylated galacturonic acid in its active site, but that methyl-esterification negatively affected the affinity of the enzyme.

Study of the mode of action of endopolygalacturonase from Fusarium moniliforme.

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.

Fifteen new mutations (-195C>T, L-12X, 298-2A>G, T117N, A159T, R229S, 997+2T>A, E274X, A331T, H364R, D389G, 1256delC, R433H, N461I, C472S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with hypophosphatasia.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and liver/bone/kidney-type alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 12 families affected by severe or mild hypophosphatasia. Twenty distinct mutations were found, 5 of which were previously reported. Nine of the 15 new mutations were missense mutations (T117N, A159T, R229S, A331T, H364R, D389G, R433H, N461I, and C472S). The others were 2 nonsense mutations (L-12X and E274X), one single nucleotide deletion (1256delC), 2 mutations affecting splicing (298-2A>G, 997+2T>A), and a mutation in the major transcription start site (-195C>T). Hum Mutat 15:293, 2000.

Intraindividual variability in orthostatic blood pressure changes among older adults: the influence of meals.

OBJECTIVES: To examine the influence of time of day and of meals on postural blood pressure (BP) changes in older adults. DESIGN: Prevalence study of BP changes in response to orthostasis. SETTING: A geriatric short-stay department PARTICIPANTS: A total of 126 inpatients (91 women and 35 men; mean age: 81.4+/-7.9, range 61-95 years) were included in the study. MEASUREMENTS: Two sets of BP and heart rate measurements were obtained for each subject by one examiner using a standard mercury manometer: (1) in mid-morning (between 10:00 and 10:30 a.m.) and (2) within 30 to 60 minutes after lunch (between 1:00 and 1:30 p.m.). Orthostatic hypotension (OH) was defined as a systolic blood pressure (SBP) decline > or = 20 mm Hg within 3 minutes after standing. RESULTS: Sixty-one participants (48%) experienced significant orthostatic BP decline on at least one reading. Among them, 46 (37%) had OH in the mid-morning, and 32 (25%) had OH after lunch (P = .05). Only 17 (13%) had OH on both readings (persistent OH). Forty-four patients (35%) had variable OH. Patients with persistent OH were more likely to exhibit symptoms of dizziness and had a lower body mass index and a higher mean basal supine SBP. There was a positive correlation between basal supine SBP and postural SBP decline. CONCLUSIONS: Because of the variability of postural BP changes, the diagnosis of OH should not be based on a single orthostatic BP measurement but requires repeated testing, at best under circumstances similar to those in which the symptoms occurred. The postprandial period is not particularly favorable to OH, suggesting that the ingestion of a meal does not worsen orthostatic BP changes in most aged patients.

Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp, (Ulvales, Chlorophyta).

A marine bacterium degrading the water-soluble cell wall polysaccharides from Ulva sp. (ulvan) has been isolated. The good correlation between ulvan degradation monitored by reducing-power, UV absorbance and viscosimetry, indicated that the crude enzymatic extract contains essentially an endo-ulvan lyase activity. This activity was rapidly inhibited by the reaction products which consisted of a series of ulvanobiouronic acid A 3-sulfate [-->4)-beta-D-GlcpA-(1-->4)-alpha-L-Rhap 3-sulfate-(1-->]n with 4-deoxy-L-threo-hex-4-enopyranosiduronic acid at the non-reducing end. Other deviant repeating structures with beta-D-Xylp or alpha-IdopA replacing beta-D-GlcpA in the repeating ulvanobiouronic acid disaccharide and the presence of two consecutive (1-->4) linked beta-D-Glc pA demonstrated the great variability and complexity of ulvan chemical structure.

Preliminary characterization of a new exo-beta-(1,4)-galactanase with transferase activity.

As a prerequisite to the study of the fine chemical structure of the branched region of pectin, an exo-beta-(1,4)-galactanase was purified from a commercial preparation (Pectinex AR). Purification was carried out by precipitation with 70% saturated ammonium sulfate, preparative electrofocusing, anion-exchange chromatography and affinity chromatography on cross-linked alginate. Exogalactanase specific activity was 992 nkat mg-1 and the enzyme was devoid of beta-(1,3)- or beta-(1,6)-galactanase, arabinanase, beta-D-galactosidase and alpha-L-arabinofuranosidade activities. Residual exopolygalacturonase activity represented 2.9% of the galactanase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing showed two close bands with molecular weights of 120,000 and 90,000 and pHi of 3.8 and 4.1, respectively. The enzyme acted in an exo manner and its activity was optimum at pH 3.5 and 60 degrees C. When incubated with galacto-oligosaccharides, new oligosaccharides with a higher degree of polymerization appeared, indicating the ability of the enzyme to transfer galactose residues.

Development of a quality assurance program for determination of ultratrace background levels of lead and cadmium in raw agricultural crops by differential pulse anodic stripping voltammetry.

Data on the background levels of lead and cadmium in the food supply are essential in order to establish a baseline from which to evaluate the extent of contamination in transport, processing, industrial atmospheric particulate fallout, and soil treatment (e.g., fertilizers, sewage sludge, etc.). This requires the establishment of site selection and sampling criteria as well as the development of a rigorous analytical method capable of performing routine analyses of Pb and Cd at ultratrace levels. The method used in this study, which was published previously, was designed to provide high sample throughput with minimal contamination. This involved control and measurement of blank levels and the establishment of quality control procedures to maintain confidence in the accuracy and precision of the method.

Chromatographic study of highly methoxylated lime pectins deesterified by different pectin methyl-esterases.

The inter-molecular distribution of free carboxyl groups of two highly methoxylated pectins enzymatically deesterified by plant and fungus pectin methyl-esterases were investigated by size-exclusion (SEC) and ion-exchange chromatography (IEC). "Homogeneous" populations with respect to molar mass or charge density were thereby obtained and their chemical composition and physico-chemical properties (transport parameter for monovalent cations and calcium, calcium activity coefficient) were studied. Chemical analysis showed that the composition varies from one SEC fraction to another, the highest molar mass fraction being richer in rhamnose and galactose and exhibiting a slightly higher degree of methylation. Separation of pectins by IEC revealed a quite homogeneous charge density distribution for F58 contrary to P60 which exhibited a large distribution of methoxyl groups. The free carboxyl groups distributions and calcium binding behaviours of SEC and IEC fractions were shown to differ widely for highly methoxylated pectins deesterified by plant and fungus pectin methyl-esterases.

Determination of background levels of lead and cadmium in raw agricultural crops by using differential pulse anodic stripping voltammetry.

A method is described for the simultaneous determination of ultratrace levels of lead and cadmium in selected agricultural crop samples by differential pulse anodic stripping voltammetry. Samples are dry ashed at high temperature with H2SO4 as an ashing aid. Techniques are described to control the lead and cadmium blank levels of 2 ng and 0.4 ng, respectively. Typical relative standard deviations for the crop analyses are 13% at 100 ng/g and 25% at 10 ng/g for lead, and 5% at 100 ng/g and 10% at 10 ng/g for cadmium. The lowest quantifiable level, based on 3 g dry sample, is 2 ng/g for lead and 1 ng/g for cadmium. Recovery studies, precision studies, and analyses of NBS Standard Reference Materials demonstrate the accuracy and reproducibility of this technique. A summary of results for over 1700 crop samples is reported.

Therapeutic preparations of normal polyspecific IgG (IVIg) induce apoptosis in human lymphocytes and monocytes: a novel mechanism of action of IVIg involving the Fas apoptotic pathway.

Therapeutic preparations of normal human IgG for i.v. use (i.v.Ig) exhibit a broad spectrum of immunoregulatory activities in vitro and in vivo. I.v.Ig has been shown to inhibit the proliferation of activated B and T lymphocytes and of several autonomously growing cell lines. In this study, we demonstrate that i.v.Ig induces apoptosis in leukemic cells of lymphocyte and monocyte lineage and in CD40-activated normal tonsillar B cells, involving, at least in part, Fas (CD95/APO-1) and activation of caspases. I.v.Ig-induced apoptosis was higher in Fas-sensitive HuT78 cells than in Fas-resistant HuT78.B1 mutant cells, and soluble Fas inhibited IVIg-induced apoptosis. I.v.Ig immunoprecipitated Fas from Fas-expressing transfectants and recognized purified Fas/glutathione-S-transferase fusion proteins upon immunoblotting. Affinity-purified anti-Fas Abs from i.v.Ig induced apoptosis of CEM T cells at a 120-fold lower concentration than unfractionated i.v.Ig. Inhibitors of cysteine proteases of the caspase family, caspase 1 (IL-1beta-converting enzyme) and caspase 3 (Yama/CPP32b), partially inhibited i.v.Ig-induced apoptosis of CEM cells. Furthermore, cleavage of poly(A)DP-ribose polymerase into an 85-kDa signature death fragment was observed in CEM cells following i.v.Ig treatment. Thus, normal IgG induces apoptosis in lymphocytes and monocytes. Our results provide evidence for a role of Fas, bring new insights into the mechanisms of action of i.v.Ig in autoimmune diseases, and suggest a role of normal Ig in controlling cell death and proliferation.

Normal human immunoglobulin suppresses experimental myasthenia gravis in SCID mice.

Serum IgM has been shown to participate in the control of IgG autoreactivity in healthy subjects. We have recently shown that an immunoglobulin preparation of pooled normal human IgM (IVIgM) contains anti-idiotypic antibodies against disease-associated IgG autoantibodies in autoimmune patients and protects rats from experimental autoimmunity. The aim of the present study was to asses the in vitro and in vivo immunomodulatory effects of IVIgM in comparison with IgG, in SCID mice reconstituted with thymic cells from a myasthenia gravis patient. Non-leaky SCID mice were injected i.p. with 60 x 10(6) thymic cells from a patient with myasthenia gravis and 1 day later boosted with 10(6) irradiated acetylcholine receptor (AchR)-expressing TE671 cells. On days 14, 21 and 28, mice were treated with IVIgM or with equimolar amounts of human serum albumin. The level of anti-AchR antibodies in the sera of three out of four IgM-treated animals was less than 1 nM. Further, there was a significant decrease in the loss of endplate AchR on the diaphragms of IgM-treated SCID mice. These findings indicate that pooled normal IgM exerts an immunoregulatory role in experimental myasthenia gravis, and suggests that IgM may be considered as an alternative approach in the therapy of autommune diseases.

Normal human serum contains natural antibodies reactive with autologous ABO blood group antigens.

It is widely accepted that the serum of healthy individuals contains natural antibodies only against those blood group A or B antigens that are not expressed on the individual's red blood cells. The mechanisms involved in tolerance to autologous blood group antigens remain unclear. In the present study, we show that IgM and IgG antibodies reactive with autologous blood group antigens are present in the immunoglobulin fraction of normal human serum. Natural IgG anti-A antibodies purified by affinity chromatography from IgG of individuals of blood group A exhibited an affinity for A trisaccharide antigen in the micromolar range and agglutinated A red cells at sixfold higher concentrations than those required for agglutination with affinity-purified anti-A IgG of individuals of blood group B. Whereas autoantibodies reactive with self A and B antigens are readily detected in purified IgG and IgM fractions, their expression is restricted in whole serum as a result of complementary interactions between variable regions of antibodies. These observations suggest that tolerance to autologous ABO blood group antigens is dependent on peripheral control of antibody autoreactivity.