The in vitro effect of lactose on Clostridium perfringens alpha toxin production and the implications of lactose consumption for in vivo anti-alpha toxin antibody production.

Necro-hemorrhagic enteritis in calves, caused by Clostridium perfringens type A, is a fatal disease, mostly affecting calves in intensive rearing systems. The lack of development of active immunity against α toxin, an essential virulence factor in the pathogenesis, has been proposed as a main trigger. In this experimental study, the effect of a set of milk replacer components on α toxin production, and the effect of lactose on in vivo antibody production, were investigated. For the latter, Holstein-Friesian bull calves (n = 18) were fed an all liquid diet that contained either a milk replacer with high-lactose content (45% DM) or the same milk replacer that was lactase treated, resulting in a lactose-free equivalent. Antibody levels against α toxin were monitored from 2 to 12 wk of age. In the in vitro part of the study, a concentration-dependent inhibitory effect of lactose on in vitro C. perfringens α toxin activity was observed, whereas protein did not influence α toxin activity. The in vivo experiment then showed from the age of 10 wk onwards, that anti-α toxin antibody levels of high-lactose animals declined, whereas antibody levels of the animals consuming lactose-free milk replacer remained the same throughout the trial. This points to a natural decline in maternal immunity of lactose-consuming animals, that is not compensated by the development of an active immunity, resulting in inferior protection. This study suggests that dietary lactose reduces C. perfringens α toxin production in vivo, which may lead to a decreased antigen presentation and thus lower serum antibody levels against the toxin. Consequently, any event causing massive α toxin production puts lactose-consuming calves at higher risk of developing necro-hemorrhagic enteritis.

2-D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum DeltaTri5 infection and grain development.

Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase-inhibiting proteins (XIPs), and thaumatin-like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a DeltaTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2-D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum DeltaTri5. Some (iso)forms were up-regulated, whereas others were down-regulated. This pathogen-specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI- and XIP-type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non-glycosylated XIP forms increased more strongly than their glycosylated counterparts.

The three classes of wheat xylanase-inhibiting proteins accumulate in an analogous way during wheat ear development and germination.

Wheat contains high levels of the three classes of xylanase inhibitors (XIs), Triticum aestivum xylanase inhibitor (TAXI), xylanase-inhibiting protein (XIP) and thaumatin-like xylanase inhibitor (TLXI). These proteins have been linked to plant defense. In this study, expression of XIs during wheat ear development and germination was examined using immunoblotting. The three types of XIs accumulated at high levels between the milky and the soft dough stages of ear development, and reached the highest levels at the hard kernel stage. From the hard kernel stage to harvest ripeness, a slight drop in inhibitor levels was observed, which was more marked for TAXI and TLXI than for XIP. During germination, the levels of the three types of XIs initially decreased, but XIs accumulated again after 1-2d, reaching maximum levels between 5 and 9d after imbibition. The levels of TAXI, XIP and TLXI in the seedlings then gradually and continuously declined as a function of time. 1D- and 2D-immunoblotting indicated that the three types of XIs occur in a wide variety of forms. This polymorphism is maintained throughout ear development and germination, although the proportions of the different (iso)forms vary with time. A differential temporal profile was observed for the unprocessed and processed forms of TAXI-type proteins. Finally, the occurrence of TAXI and XIP, but not TLXI, in roots and shoots of young seedlings was demonstrated. No XIs were detected in roots, leaves or stems at later stages of ear development. Overall, the three classes of XIs show remarkable similarities in their temporal distribution, indicating a related function within the wheat plant.

A quantitative portrait of three xylanase inhibiting protein families in different wheat cultivars using 2D-DIGE and multivariate statistical tools.

Wheat grains contain three classes of xylanase inhibitors (XIs), i.e. TAXI (Triticum aestivum xylanase inhibitor), XIP (xylanase inhibiting protein) and TLXI (thaumatin-like xylanase inhibitor). These proteins are involved in plant defence and strongly affect cereal-based processes in which inhibitor-sensitive xylanases are used. This paper reports on the successful use of 2D-DIGE and tandem MS to discriminate XI (iso)forms and measures their qualitative and quantitative variation in six different wheat cultivars. In total, 18 TAXI-, 27 XIP- and 3 TLXI-type XI spots were identified. The multiple members of the large TAXI-gene family make a considerable contribution to the total TAXI population. For XIP-type XIs, XIP-I is expressed as the predominant form, albeit under variable degrees of PTMs. Only one TLXI genetic variant was identified, showing different degrees of glycosylation. Multiple comparison analysis revealed up to 5-fold intercultivar differences in protein level of XI (iso)forms. Evaluation of abundance patterns using multivariate statistical tools revealed highly distinctive as well as correlated levels of different XI forms among the six cultivars. As the constitutive (and induced) levels of the different XI (iso)forms, which are differentially regulated in response to various forms of stress in other wheat plant parts, considerably vary between the cultivars, it can be assumed that their degree of resistance against pathogenic attack differs. Similarities in abundance profiles between XI (iso)forms and pathogenesis-related chitinases are also in line with a role in plant defence.

Immunoblot quantification of three classes of proteinaceous xylanase inhibitors in different wheat ( Triticum aestivum ) cultivars and milling fractions.

In wheat ( Triticum aestivum ) grains, TAXI- (T. aestivum xylanase inhibitor), XIP- (xylanase inhibiting protein), and TLXI-type (thaumatin-like xylanase inhibitor) xylanase inhibitors (XIs) are expressed in considerable levels and under different forms. As these proteins have a significant impact on microbial xylanases frequently used in cereal-based biotechnological processes, knowledge of their quantitative and qualitative variability in wheat is of great interest. This paper reports the successful use of immunoquantification by Western blotting to determine the intercultivar variation in the three structurally different classes of XIs, as well as their distribution among various industrial milling fractions. TAXI and XIP protein levels in eight wheat cultivars ranged from 81 to 190 ppm and from 156 to 371 ppm, with average values of 133 and 235 ppm, respectively. Using immunoblotting, TLXI protein levels could be measured directly for the first time. They ranged from 51 to 150 ppm and amounted to 112 ppm on average. The three classes of XIs were distributed among different wheat milling fractions in a similar way, with 4 and 10 times higher concentrations in the aleurone-enriched fraction than in white flour and pericarp fractions, respectively. Immunoblot patterns suggested that the observed intercultivar and spatial variabilities within the wheat grain are not due to the presence or absence of specific members of the large polymorphic XI families but to differences in the overall level and/or proportions of the specific members.

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome.

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.

Enzymic degradability of hull-less barley flour alkali-solubilized arabinoxylan fractions by endoxylanases.

The impacts of the arabinose to xylose (A/X) ratio of arabinoxylans (AX) and the endoxylanase substrate specificity on the enzymic degradability of hull-less barley flour AX by endoxylanases were studied by using alkali-solubilized AX (AS-AX) fractions with different A/X ratio, on the one hand, and glycoside hydrolase family 10 and 11 endoxylanases of Aspergillus aculeatus (XAA) and Bacillus subtilis (XBS), respectively, on the other hand. AS-AX were obtained by saturated barium hydroxide treatment of hull-less barley flour water-unextractable AX. Fractionation of AS-AX by stepwise ethanol precipitation resulted in structurally different hull-less barley flour AS-AX fractions. Their A/X ratios increased with increasing ethanol concentration, and this increase in A/X ratio was reflected in their xylose substitution levels. For both XAA and XBS, the enzymic degradability of AX and apparent specific endoxylanase activity decreased with increasing A/X ratio of the AS-AX substrates, implying that both endoxylanases were sterically hindered by arabinose substituents. However, for all AS-AX fractions, hydrolysis end products of lower average degree of polymerization were obtained after incubation with XAA than with XBS, indicating that the former enzyme has a lower substrate specificity toward hull-less barley flour AS-AX than the latter. In addition, apparent specific endoxylanase activities indicated that XBS was approximately 2 times more sensitive to variations in the A/X ratio of AS-AX fractions than XAA. Furthermore, AS-AX with higher A/X ratio were relatively resistant to degradation by XBS.