Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

The Synthesis of Collagen and Glycosaminoglycans by Dedifferentiated Chondroblasts in Culture

Marked changes occur in the morphology of chick chondroblasts grown for 5 days in F-10 medium containing either 5-bromo-2'-deoxyuridine or embryo extract. The cells lose the characteristic polygonal morphology and assume a flattened 'fibroblastic' appearance. To determine whether the morphological changes reflect a biochemical transformation toward frank fibroblasts, changes in collagen and glycosaminoglycan synthesis were examined in these 'dedifferentiated' cells. Growth in either medium did not significantly affect the total amount of collagen synthesized. However, the subunit composition of the collagen chains was different. Freshly isolated cartilage trunks or control chondroblast cultures synthesized only α1 subunits (suggesting exclusive synthesis of α1(ll)₃-type collagen), whereas dedifferentiated cultures synthesized both α1 and, in addition, some α2 subunits (suggesting synthesis of fibroblasttype α1(l)₂α2-type collagen). Incorporation of labelled glucosamine in F-10 medium showed that the major glycosaminoglycan synthesized by either cartilage trunks or chondroblast monolayers is chondroitin sulphate; little, if any, hyaluronic acid could be detected. With growth in embryo extract (EE) glucosamine was incorporated equally into chondroitin sulphate and hyaluronic acid, whereas in BUdR, chondroitin sulphate synthesis was completely inhibited. Distinct biochemical differences were therefore found for both collagen and glycosaminoglycan synthesis during growth in either BUdR or EE. Such changes were not identical but both demonstrate changes in synthetic programme tending to approach that of frank fibroblasts.     

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis)

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.     

Optimization of the dilute maleic acid pretreatment of wheat straw

Background  

In this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables.     

Induced plant responses to pathogen attack. Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63).

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.     

Characterization of Porin from Roseobacter denitrificans

Porin from Roseobacter denitrificans was isolated and purified to homogeneity. The pore characteristics from this marine bacterium were compared to those of its phylogenetically closely related freshwater bacteria Rhodobacter capsulatus 37b4, Rhodobacter sphaeroides and Rhodopseudomonas blastica. The porin formed weakly cation-selective, general diffusion pores in lipid bilayer membranes. High transmembrane potentials caused channel closing in steps that were of one or two thirds of the initial on-steps indicating that the porin of R. denitrificans comprised three more or less independent channels similar to PhoE and OmpC of Escherichia coli and the porin of Rhodobacter capsulatus. Prediction of the secondary structure of the 36 N-terminal amino acid residues indicated two transmembrane -strands similar to those of the porins of Rhodobacter capsulatus 37b4 and Rhodopseudomonas blastica. Differences of the single channel conductivities between the porin of R. denitrificans and those of the related freshwater bacteria show that R. denitrificans evolved porin channels that are well adapted to the marine habitat.     

The structure of cat muscle pyruvate kinase.

The complete amino acid sequence of cat muscle pyruvate kinase has been determined and fitted to the 2.6 A resolution electron density map. Residues in the active site region are highly conserved in the cat muscle, chicken muscle, rat liver and yeast enzymes. The enzyme-bound magnesium, which is essential for activity, interacts with the side chain of glutamate-271 and with two main carbonyl groups. Lysine-269 is the probable acid/base catalyst responsible for the interconversion of pyruvate and enolpyruvate. A possible binding site for the essential monovalent cation is proposed.