Degradation of Differently Substituted Xylogalacturonans by Endoxylogalacturonan Hydrolse and Endopolygalacturonases

Abstract

A method was developed to make xylogalacturonans (XGAs) with different degrees of xyloslyation from gum tragacanth (XGA-25, XGA-29, XGA-35 and XGA-47), using alkali treatment at 4°C and acid treatment at 100°C. Ester linkages as well as fucose and arabinose substituents could selectively be removed by this procedure. Galactosyl- and xylosyl-linkages appeared to be more stable, while some backbone degradation of the galacturonan took place upon prolonged acid treatment. Using XGA-35, endoxylogalacturonan hydrolase (XGH) from Aspergillus tubingensis, expressed in Kluyveromyces lactis, was characterised with respect to kinetic parameters, temperature and pH effects.

XGA-25 and XGA-47 were degraded with endopolygalacturonases (PGs) from Aspergillus niger (PG1, PG2), from A. tubingensis (PF-arf), from Kluyveromyces fragilis (PG-kluyv) and XGH from A. tubingensis. The activity of the different PGs decreased with increasing degrees of xylosylation. However, for each PG a different tolerance for the presence of side chains was observed. PG-arf and PG1 were hindered most by xylosyl branching, whereas XGH appeared to have a requirement for xylosylation and was almost not active towards polygalacturonic acid. The degradability of xylogalacturonans by XGH increased with higher degrees of xylosylation. Typically, a highly substituted xylogalacturonan from pea was almost resistant to XGH treatment. XGH produces a distinctive set of oligosaccharides from XGA, which is different from the hydrolysis products of PG action.

Saponified modified hairy regions from apple (MHR-S) containing xylogalacturonan, were partially degraded by XGH. A combination of XGH and rhamnogalacturonan hydrolase was able to fully degrade the high molecular weight fraction of MHR-S. The two enzymes acted additively, no synergy being observed.     

Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.     

Saline aerosol bolus dispersion. II. The effect of conductive airway alteration.

In a companion study (Verbanck S, Schuermans D, Vincken W, and Paiva M, J Appl Physiol 90: 1754-1762, 2001), we investigated whether saline aerosol bolus tests could also be used to detect proximal, as opposed to peripheral, airway alterations. We studied 10 never-smokers before and after histamine challenge, obtaining, for various volumetric lung depths (VLD), saline bolus-derived indexes computed by discarding aerosol concentrations below either 50% of the exhaled bolus maximum (half-width, H) or below cutoffs ranging from 5 to 25% (standard deviation, sigma(5%)-sigma(25%)) and skew (sk(5)-sk(25%)). Multiple-breath N(2) washout-derived indexes of conductive (S(cond)) and acinar (S(acin)) ventilation inhomogeneity were also determined. After histamine, S(cond) significantly increased (P = 0.008) whereas S(acin) remained unaffected, indicating purely conductive airway alteration. Consistent with this observation, sk(5%) (or sk(25%)) was increased to the same extent at all VLD, and sigma(5%) was increased preferentially at low VLD. By contrast, H and sigma(25%) displayed preferential increases at high VLD, a pattern similar to that induced by peripheral alterations. The present work shows that proximal airway alteration can be reliably identified by saline bolus tests only if these include measurements at low and high VLD and if bolus dispersion is quantified as a standard deviation with a low cutoff.     

Saline aerosol bolus dispersion. I. The effect of acinar airway alteration.

We explored the possibility of using a saline aerosol for bolus dispersion measurements to detect peripheral airway alterations in smokers. Indexes of ventilation inhomogeneity in conductive (S(cond)) and acinar (S(acin)) lung zones, as derived from the multiple-breath N(2) washout (Verbanck S, Schuermans D, Van Muylem A, Noppen M, Paiva M, and Vincken W, J Appl Physiol 83: 1807-1816, 1997), were also measured. The saline bolus test consisted of inhaling 60-ml saline aerosol boluses to different volumetric lung depths (VLD) in the 1.1 liter volume above functional residual capacity. In the never-smoker group (n = 12), saline boluses showed bolus dispersion values consistent with normal values reported in the literature for 0.5- to 1-microm aerosols. In the smoker group (n = 12; 28 +/- 9 pack years, mean +/- SD), significant increases were seen on dispersion and skew of the most peripherally inhaled saline boluses (VLD = 800 ml; P < 0.05) as well as on S(acin) (P = 0.007) with respect to never-smokers. Shallow inhaled boluses (VLD = 200 ml) and S(cond) did not reveal any significant differences between smokers and never-smokers. This study shows the consistent response of two conceptually independent tests, in which both saline aerosol and gas-derived indexes point to a heterogeneous distribution of smoking-induced structural alterations in the lung periphery.     

Nonreversible conductive airway ventilation heterogeneity in mild asthma.

A multiple-breath washout technique was used to assess residual ventilation heterogeneity in the conductive and acinar lung zones of asthmatic patients after maximal beta(2)-agonist reversibility. Reversibility was assessed in 13 patients on two separate visits corresponding to a different baseline condition in terms of forced expiratory volume in 1 s [FEV(1); average FEV(1) over 2 visits: 92 +/- 21% of predicted (SE)]. On the visit corresponding to each patient's best baseline, 400 micro g salbutamol led to normal acinar ventilation heterogeneity, normal FEV(1), and normal peak expiratory flow; i.e., none was significantly different from that obtained in 13 matched controls. By contrast, conductive ventilation heterogeneity and forced expiratory flow after exhalation of 75% forced vital capacity remained significantly different from controls (P < or = 0.005 on both indexes). In addition, the degree of postdilation conductive ventilation heterogeneity was similar to what was previously obtained in asthmatic individuals with a 19% lower baseline FEV(1) and twofold larger acinar ventilation heterogeneity (Verbanck S, Schuermans D, Noppen M, Van Muylem A, Paiva M, and Vincken W. Am J Respir Crit Care Med 159: 1545-1550, 1999). We conclude that, even in the mildest forms of asthma, the most consistent pattern of non-beta(2)-agonist-reversible ventilatory heterogeneity is in the conductive lung zone, most probably in the small conductive airways.     

Cumulative Risk of Bovine Mastitis Treatments in Denmark,Finland, Norway and Sweden

Data from the national dairy cow recording systems during 1997 were used to calculate lactation-specific cumulative risk of mastitis treatments and cumulative risk of removal from the herds in Denmark, Finland Norway and Sweden. Sweden had the lowest risk of recorded mastitis treatments during 305 days of lactation and Norway had the highest risk. The incidence risk of recorded mastitis treatments during 305 days of lactation in Denmark, Finland, Norway and Sweden was 0.177, 0.139, 0.215 and 0.127 for first parity cows and 0.228, 0.215, 0.358 and 0.204 for parities higher than three, respectively. The risk of a first parity cow being treated for mastitis was almost 3 times higher at calving in Norway than in Sweden. The period with the highest risk for mastitis treatments was from 2 days before calving until 14 days after calving and the highest risk for removal was from calving to 10 days after calving in all countries.The study clearly demonstrated differences in bovine mastitis treatment patterns among the Nordic countries. The most important findings were the differences in treatment risks during different lactations within each country, as well as differences in strategies with respect to the time during lactation mastitis was treated.     

Hydroxycinnamic acids are ester-linked directly to glucosyl moieties within the lignan macromolecule from flaxseed hulls

In flaxseed hulls, lignans are present in an oligomeric structure. Secoisolariciresinol diglucoside (SDG), ester-linked to hydroxy-methyl-glutaric acid (HMGA), forms the backbone of this lignan macromolecule. The hydroxycinnamic acids p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) are also part of the lignan macromolecule. However, their position and type of linkage are still unknown. The aim of this study was to investigate how CouAG and FeAG are linked within the lignan macromolecule from flaxseed hulls. Fragments of the lignan macromolecule were obtained by partial saponification. After isolation of the fragments by preparative RP-HPLC, several key structures were identified by MS and NMR. Within the lignan macromolecule, CouAG is attached to the C-6 position of a glucosyl moiety of SDG. FeA is linked to the C-2 position of a glucosyl moiety of SDG. FeAG is ester-linked within the lignan macromolecule with its carboxyl group, but it remains unclear whether FeAG links to the C-2 or C-6 position of SDG. Attachment of HMGA to the glucosyl moiety of CouAG or FeAG was not observed. The results clearly show that within the lignan macromolecule, the hydroxycinnamic acids are linked directly via an ester bond to the glucosyl moiety of SDG.     

The Ischemic Stroke Genetics Study (ISGS) Protocol

Background  

The molecular basis for the genetic risk of ischemic stroke is likely to be multigenic and influenced by environmental factors. Several small case-control studies have suggested associations between ischemic stroke and polymorphisms of genes that code for coagulation cascade proteins and platelet receptors. Our aim is to investigate potential associations between hemostatic gene polymorphisms and ischemic stroke, with particular emphasis on detailed characterization of the phenotype.     

A comparison of the illness beliefs of people with angina and their peers: a questionnaire study

Background

Systolic compression of a coronary artery by overlying myocardial tissue is termed myocardial bridging. Myocardial bridging usually has a benign prognosis, but some cases resulting in myocardial ischemia, infarction and sudden cardiac death have been reported. We are reporting a case of myocardial bridging which was complicated with acute myocardial infarction associated with inappropriate blood donation.

Case presentation

A 33 year-old-man was admitted to our emergency with acute anteroseptal myocardial infarction after a blood donation. The electrocardiography showed sinus rhythm and was consistent with an acute anteroseptal myocardial infarction. We decided to perform primary percutanous intervention (PCI). Myocardial bridging was observed in the mid segment of the left anterior descending coronary artery on coronary angiogram. PCI was canceled and medical follow up was decided. Blood transfusion was made because he had a deep anemia. A normal hemaglobin level and clinical reperfusion was achieved after ten hours by blood transfusion. At the one year follow up visit, our patient was healthy and had no cardiac complaints.

Conclusions

Myocardial bridging may cause acute myocardial infarction in various clinical conditions. Although the condition in this case caused profound anemia related acute myocardial infarction, its treatment and management was unusual.     

In muro fragmentation of the rhamnogalacturonan I backbone in potato (Solanum tuberosum L.) results in a reduction and altered location of the galactan and arabinan side-chains and abnormal periderm development

Rhamnogalacturonan (RG) I is a branched pectic polysaccharide in plant cell walls. Rhamnogalacturonan lyase (eRGL) from Aspergillus aculeatus is able to cleave the RG I backbone at specific sites. Transgenic potato (Solanum tuberosum L.) plants were made by the introduction of the gene encoding eRGL, under the control of the granule-bound starch synthase promoter. The eRGL protein was successfully expressed and translated into an active form, demonstrated by eRGL activity in the tuber extracts. The transgenic plants produced tubers with clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis of the isolated cell walls showed a large reduction in galactosyl and arabinosyl residues in transgenic tubers. Immunocytochemical studies using the LM5 (galactan) and LM6 (arabinan) antibodies also showed a large reduction in galactan and arabinan side-chains of RG I. Most of the remaining LM5 epitopes were located in the expanded middle lamella at cell corners of eRGL tubers, which is in contrast to their normal location in the primary wall of wild type tubers. These data suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm.