Measurement of ß-Glucan in Mushrooms and Mycelial Products.

A robust and reliable method has been developed for the measurement of ß-glucan in mushroom and mycelial products. Total glucan (plus free glucose and glucose from sucrose) was measured using controlled acid hydrolysis with H2SO4 and the glucose released specifically was measured using glucose oxidase/peroxidase reagent. α-Glucan (starch/glycogen) plus free glucose and glucose from sucrose were specifically measured after hydrolysis of starch/glycogen to glucose with glucoamylase and sucrose to glucose plus fructose with invertase and the glucose specifically measured with GOPOD reagent. ß-Glucan was determined by the difference. Several acid and enzyme-based methods for the hydrolysis of the ß-glucan were compared, and the best option was the method using H2SO4. For most samples, similar ß-glucan values were obtained with both the optimized HCl and H2SO4 PROCEDURES: However, in the case of certain samples, specifically Ganoderma lucidum and Poria cocus, the H2SO4 procedure resulted in significantly higher values. Hydrolysis with 2 N trifluoroacetic acid at 120°C was found to be much less effective than either of the other two acids evaluated. Assays based totally on enzymatic hydrolysis, in general, yielded much lower values than those obtained with the H2SO4 procedure.

Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by ß-xylanase, α-L-arabinofuranosidase and ß-xylosidase.

A range of α-L-arabinofuranosyl-(1-4)-ß-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 ß-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and ß-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum ß-xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 3(2)-α-L-Araf-(1-4)-ß-D-xylobiose (A(3)X), 2(3)-α-L-Araf-(1-4)-ß-D-xylotriose (A(2)XX), 3(3)-α-L-Araf-(1-4)-ß-D-xylotriose (A(3)XX), 2(2)-α-L-Araf-(1-4)-ß-D-xylotriose (XA(2)X), 3(2)-α-L-Araf (1-4)-ß-D-xylotriose (XA(3)X), 2(3)-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(2)XX), 3(3)-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(3)XX), 2(3),3(3)-di-α-L-Araf-(1-4)-ß-D-xylotriose (A(2+3)XX), 2(3),3(3)-di-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(2+3)XX), 2(4),3(4)-di-α-L-Araf-(1-4)-ß-D-xylopentaose (XA(2+3)XXX) and 3(3),3(4)-di-α-L-Araf-(1-4)-ß-D-xylopentaose (XA(3)A(3)XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A(2,3)XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with ß-xylosidase and ß-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

Measurement of endo-1,4-ß-glucanase.

Several procedures are available for the measurement of endo-1,4-ß-glucanase (EG). Primary methods employ defined oligosaccharides or highly purified polysaccharides and measure the rate of hydrolysis of glycosidic bonds using a reducing-sugar method. However, these primary methods are not suitable for the measurement of EG in crude fermentation broths due to the presence of reducing sugars and other enzymes active on these substrates. In such cases, dyed soluble or insoluble substrates are preferred as they are specific, sensitive, easy to use, and are not affected by other components, such as reducing sugars, in the enzyme preparation.

Aggregation of spectrin and PKCtheta is an early hallmark of fludarabine/mitoxantrone/dexamethasone-induced apoptosis in Jurkat T and HL60 cells.

It has been shown that changes in spectrin distribution in early apoptosis preceded changes in membrane asymmetry and phosphatidylserine (PS) exposure. PKCtheta was associated with spectrin during these changes, suggesting a possible role of spectrin/PKCtheta aggregation in regulation of early apoptotic events. Here we dissect this hypothesis using Jurkat T and HL60 cell lines as model systems. Immunofluorescent analysis of alphaIIbetaII spectrin arrangement in Jurkat T and HL60 cell lines revealed the redistribution of spectrin and PKCtheta into a polar aggregate in early apoptosis induced by fludarabine/mitoxantrone/dexamethasone (FND). The appearance of an alphaIIbetaII spectrin fraction that was insoluble in a non-ionic detergent (1% Triton X-100) was observed concomitantly with spectrin aggregation. The changes were observed within 2 h after cell exposure to FND, and preceded PS exposure. The changes seem to be restricted to spectrin and not to other cytoskeletal proteins such as actin or vimentin. In studies of the mechanism of these changes, we found that (i) neither changes in apoptosis regulatory genes (e.g., Bcl-2 family proteins) nor changes in cytoskeleton-associated proteins were detected in gene expression profiling of HL60 cells after the first hour of FND treatment, (ii) caspase-3, -7, -8, and -10 had minor involvement in the early apoptotic rearrangement of spectrin/PKCtheta, and (iii) spectrin aggregation was shown to be partially dependent on PKCtheta activity. Our results indicate that spectrin/PKCtheta aggregate formation is related to an early stage in drug-induced apoptosis and possibly may be regulated by PKCtheta activity. These findings indicate that spectrin/PKCtheta aggregation could be considered as a hallmark of early apoptosis and presents the potential to become a useful diagnostic tool for monitoring efficiency of chemotherapy as early as 24 h after treatment.