An inexpensive insoluble chromogenic substrate for the determination of proteolytic activity

Summary A new insoluble chromogenic substrate for the determination of proteolytic activity was developed. This substrate was prepared by incorporating black drawing ink into casein and heating this complex at 200°C for 4 h. It is especially suitable for determining the activity of alkaline bacterial proteinases.     

The interaction of the vanadyl(IV) cation with phytic acid

The interactions of VO²⁺ with phytate to form both soluble and insoluble complexes, have been studied by electronic absorption spectroscopy. A soluble 1∶1 VO²⁺: phytate complex is formed at pH <1. At higher pH-values insoluble complexes are produced. Two different solid complexes, obtained respectively at pH=2 and 4, were isolated and characterized. The maximal bonding ratio of VO²⁺: phytate was found to be 4, on the basis of a pH binding profile.     

Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour

Background

Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity.

Methods

SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC–ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour.

Results

Gliadin and glutenin yields decreased to 7.6 ± 0.5% and 7.5 ± 0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC–ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31–49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31–49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics.

Conclusions

The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties.

General significance

Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients.     

Diary

Abstract

The formation of sparingly soluble Ca²⁺-phytate (Phy) species was studied using several techniques (potentiometry, calorimetry, thermoanalysis, ICP, SEM). Species with empirical formula Ca_xH(₁₂__2x)Phy, (6 ≤ x ≤ 4.5) were formed in experiments performed at pH = 5.0, 7.5 and 10.5; at pH = 10.5 and in excess of Na+ the species Ca₅Na₂Phy was also found. Solubilities (s = 1.0 x 10^-9and 1.1 x 10^-10mol L^-1 for the species Ca₆Phy and Ca₅Na₂Phy respectively, at l = 0 mol L^-1), thermogravimetric profiles (dehydratation, monohydrogen-phosphate and final pyrophosphate formation), and formation enthalpies (ΔΗ = 7.6 ± 1 and 3.7 ± 0.8 kJ mol^-1for the equilibria 6Ca²⁺ + H₃L^9- = Ca₆L⁰ + 3 H⁺ and 5Ca²⁺ + 2Na⁺ + H₃L^9- = Ca₅Na₂L⁰ + 3 H⁺, respectively), were obtained.     

PrPC is sorted to the basolateral membrane of epithelial cells independently of its association with rafts

PrP(C) is a glycosylphosphatidylinositol-anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrP(C) remains unknown, a conformational isoform called PrP(Sc) (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrP(C) to PrP(Sc) conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild-type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrP(C) in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol-anchored proteins, we found that PrP(C) is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol-proteins, PrP(C) associates with detergent-resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol-proteins, as well as in the conversion process of PrP(C) to PrP(Sc). In order to discriminate whether detergent-resistant microdomains have a direct role in PrP(Sc) conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent-resistant microdomain association on PrP(C) intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol-linked protein, our data exclude a classical role for detergent-resistant microdomains in the post-trans-Golgi network sorting and transport of PrP(C) to the plasma membrane.     

Carbohydrate metabolism before and after dehiscence in the recalcitrant pollen of pumpkin (Cucurbita pepo L.)

Pumpkin (Cucurbita pepo L.) pollen is starchy, sucrose‐poor and recalcitrant, features opposite to those of several model species; therefore, some differences in carbohydrate metabolism could be expected in this species. By studying pumpkin recalcitrant pollen, the objective was to provide new biochemical evidence to improve understanding of how carbohydrate metabolism might be involved in pollen functioning in advanced stages. Four stages were analysed: immature pollen from 1 day before anthesis, mature pollen, mature pollen exposed to the environment for 7 h, and pollen rehydrated in a culture medium. Pollen viability, water and carbohydrate content and activity of enzymes involved in carbohydrate metabolism were quantified in each stage. Pollen viability and water content dropped quickly after dehiscence, as expected. The slight changes in carbohydrate concentration and enzyme activity during pollen maturation contrast with major changes recorded with ageing and rehydration. Pumpkin pollen seems highly active and closely related to its surrounding environment in all the stages analysed; the latter is particularly evident among insoluble sucrolytic enzymes, mainly wall‐bound acid invertase, which would be the most relevant for sucrose cleavage. Each stage was characterised by a particular metabolic/enzymatic profile; some particular features, such as the minor changes during maturation, fast sucrolysis upon rehydration or sharp decrease in insoluble sucrolytic activity with ageing seem to be related to the lack of dormancy and recalcitrant nature of pumpkin pollen.     

贝壳的有机质及生物矿化机制分析

以腹足纲贝壳香螺壳为研究对象,用弱酸去钙法进行蛋白提取,采用280纳米(A280)光吸收法测定蛋白含量,并通过聚丙烯酰胺(SDS-PAGE)凝胶电泳法对蛋白按照分子量大小的区别进行分离.实验结果表明香螺壳中蛋白含量和种类较少,其文石层比方解石层蛋白含量高的多,总量分别为0.89%和0.0533%;文石层分离出五种分子量的可溶性蛋白和四种分子量的不可溶性蛋白;而方解石层中分离出三种分子量的可溶性蛋白和三种分子量的不可溶性蛋白,且分子量不相同.正是这少量的蛋白质对贝壳的生物矿化过程和不同晶型的形成起着决定性作用.     

Purification of xylanase from alkaliphilic Bacillus sp. K-8 by using corn husk column

The objective of this work was to apply low cost materials, agricultural residues, to the purification of xylanase. The results showed that crude extracellular, cellulase-free xylanase of an alkaliphilic Bacillus sp. strain K-8 could be purified in a single step by affinity adsorption–desorption on a corn husk column using a high flow rate, under the conditions 25 mM acetate buffer, pH 4.0, 4 °C, which prevented the hydrolysis of xylan by xylanase. After adsorption, the xylanase was eluted from the enzyme–corn husk complex with 500 mM Urea. The enzyme was purified 5.3-fold to homogeneity from culture supernatant. The molecular weight of the purified enzyme was 24 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity and recovery yield after purification were 25.4 U/mg protein and 42.3%, respectively.     

Chemical composition, intake by sheep, and in situ disappearance in cannulated cows of bermudagrass hayed at two moisture concentrations and treated with a non-viable Lactobacillus-lactic acid preservative

Bermudagrass [Cynodon dactylon (L.) Pers.] is commonly used for grazing and haying in the southern USA, but hay curing can be challenging due to frequent rainfall events during spring and early summer. An existing stand of ‘Greenfield’ bermudagrass was divided into 12 plots using a randomized complete block design with a 2×2 factorial treatment arrangement to evaluate the influence of a non-viable Lactobacillus-lactic acid preservative and moisture concentration at baling on chemical composition, intake by sheep, and in situ disappearance in cattle. At time of mowing, half of the plots in each block were either spray-treated (T) or not treated (U) with 81 mL/t forage dry matter (DM) of the preservative solution. Hay was then baled at target moisture concentrations of either 174 g/kg DM (L) or 267 g/kg DM (H). Maximum temperature and heating degree days were greater (P<0.05) from H compared with L during the 42-d storage period. An interaction between spray and moisture treatments tended (P<0.10) to affect recovery of DM; recoveries for LT (0.992) differed (P<0.10) from HT (0.913), but LU and HU were intermediate between the spray-treated hays, and did not differ from either (P>0.10). Post-storage nutritive value was largely influenced by moisture treatments only. Intake and digestibility, and in situ DM disappearance of these same hays were determined using 16 wether lambs (43 ± 3.7 kg initial BW), or six ruminally cannulated cows (617 ± 3.5 kg initial BW), respectively. Dry matter intake by sheep was not affected by either treatment factor (P>0.05), but DM digestibility and digestible DM intake were greater (P<0.05) from U compared with T. The in situ immediately soluble DM portion was greater from (P<0.05) L compared with H, but the reverse was true for the potentially degradable DM fraction. The lag time tended (P<0.10) to be greater from H compared with L. Treating bermudagrass with a non-viable Lactobacillus acidophilus-lactic acid spray product at time of baling may not offset the negative effects on forage quality and digestibility of baling bermudagrass hay at excessive moisture concentrations.     

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP^C into its pathogenic isoform PrP^{Sc 1}. PrP^C is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP^Sc forms detergent-insoluble aggregates and is partially resistant to PK^2-6. The conversion of PrP^C to PrP^Sc is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP^Sc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain⁷.Using a combination of biophysical and biochemical approaches, we identified insoluble PrP^C aggregates (designated iPrP^C) from uninfected mammalian brains and cultured neuronal cells^{8, 9}. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms¹⁰, and PK-treatment. The combination of these approaches isolates not only insoluble PrP^Sc and PrP^C aggregates but also soluble PrP^C oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrP^Sc from infected brains and iPrP^C from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrP^C are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrP^Sc that shares the immunoreactive behavior and fragmentation with iPrP^{C 11, 12}. Moreover, we recently demonstrated that iPrP^C is the main species that interacts with amyloid-β protein in Alzheimer disease¹³. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer''s disease¹³, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.