Vanillic acid metabolism by selected soft-rot,brown-rot,and white-rot fungi

Metabolism of vanillic acid, a product of lignin degradation, has been studied in selected representatives of soft-rot, brown-rot and white-rot fungi. All of the brown-and white-rot species examined decarboxylated vanillate to methoxyhydroquinone oxidatively. Mycelium extracts of all these fungi, except Pleurotus ostreatus contained high levels of an NAD(P)H-dependent vanillate hydroxylase. P. ostreatus also released ¹⁴CO₂ from ¹⁴COOH-vanillate but by a different mechanism possibly involving phenoloxidases. Most of these fungi also contained a dioxygenase which catalysed the intra-diol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to form maleylacetate. No 3-O-demethylase activity was detected, and data indicate that in some of the fungi examined cleavage of the aromatic ring occurs without prior removal of the methoxyl group. None of the soft-rot fungi tested contained vanillate hydroxylase or hydroxyquinol 1,2-dioxygenase, but very low levels of protocatechuate 3,4-dioxygenase were detected in mycelium extracts. Vanillate catabolism among members of this group occurs via a different route which may involve ring demethylation although no 3-O-demethylase activity was detected in this study. The enzyme NAD(P)H-quinone oxidoreductase was demonstrated to exist in all the studied groups of fungi.     

Ligninolytic activity and levels of ammonia assimilating enzymes in Sporotrichum pulverulentum

Levels of ammonia-assimilating enzymes (glutamate dehydrogenase, glutamine synthetase, glutamate synthase) were determined in extracts of Sporotrichum pulverulentum grown under different conditions with respect to both nitrogen source and concentration. Evolution of ¹⁴CO₂ from ¹⁴C-synthetic lignin by fungal cultures grown under parallel conditions was also determined as a measure of lignin decomposition and the suppressive effect of nitrogen on ligninolysis confirmed. Under low nitrogen conditions, fungal extracts exhibited relatively high levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase dehydrogenase. Conversely, in high nitrogen extracts, lower levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase activity, and higher levels of NAD-dependent glutamate dehydrogenase, were recorded. Possible effects of enzyme activities on intracellular pool concentrations of glutamate/glutamine, and the implications for the regulation of lignin metabolism, are discussed.A preliminary report was presented at The Ekman Days 1981, International Symposium on Wood and Pulping Chemistry, Stockholm, Sweden, June 9–12, 1981.     

Animal Welfare in Relation to Standards in Organic Farming

The new EU-regulations on organic farming (1804/1999) are also influencing the animal welfare. A lot of positive regulations is to find, but also regulations that seen to mind more about the general public and customer and their view on organic farming, than the health and welfare of the animals.The paper specially focus on the impact of the regulations and the recommendations that phytotherapeutic essences and homeopathic products take precedence over the so called chemically-synthesised allopatic veterinary medical products, and that the use of the same is prohibited for preventive treatments. Key questions here are the lack of scientific evidence concerning homeopathy in animals, and that Swedish veterinarians are not allowed to work with homeopathy.Differences in interpretation of the regulations between animal owners and veterinarians will also be discussed. What is a disease that needs treatment? Who is to decide about the treatment? Parasitic infections are discussed as an illustrative example.Other consequences of the regulations concerning the animal welfare are problems in certain geographical zones, for instance subarctic areas where necessary crops are impossible to grow.Animal transports and splitting mother-offspring are briefly discussed as future problems to be handled in the regulations, and the paper ends by presenting the need of regulated herd health control programs in organic husbandry, which can detect and focus on welfare and production problems. The organic movement is not static, and must not be so.     

Degradation of missense mutant beta-galactosidase proteins in Escherichia coli K-12.

Summary Ten out of 43 missense mutations in the lacZ gene of Escherichia coli gave rise to polypeptide chains that were degraded in vivo. While many of the mutants appeared to be fully or partially CRM^–, there appeared to be no obvious correlation between degradation, map position, altered subunit association and the half-life of the mutant proteins.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

The impact of nandrolone decanoate and growth hormone on biosynthesis of steroids in rats

Growth hormone (GH) and anabolic androgenic steroids (AAS) are commonly used in sports communities. Several studies have suggested an association between GH and AAS. We have investigated the impact of GH in rats treated with nandrolone decanoate (ND). Male Wistar rats received ND (15 mg/kg) every third day during three weeks and were subsequently treated with recombinant human GH (1.0 IU/kg) for ten consecutive days. Plasma samples were collected and peripheral organs (i.e. heart, liver, testis and thymus) were dissected and weighed. Concentration of thirteen endogenous steroids was measured in the rat plasma samples using high specificity LC–MS/MS methods. Seven steroids were detected and quantified, and concentrations of estrone, testosterone, and androstenedione were significantly different among the groups, while concentrations of pregnenolone, DHEA, 17-hydroxyprogesterone and corticosterone were not altered. Administration of rhGH alone altered the plasma steroid distribution, and the results demonstrated significantly increased concentrations of plasma estrone as well as decreased concentrations of testosterone and androstenedione in the ND-treated rats. Administration of rhGH to ND-pretreated rats did not reverse the alteration of the steroid distribution induced by ND. Administration of ND decreased the weight of the thymus, and addition of rhGH did not reverse this reduction. However, rhGH administration induced an enlargement of thymus. Taken together, the plasma steroid profile differed in the four groups, i.e. control, AAS, rhGH and the combination of AAS and rhGH treatment.     

Quantitative analysis of tryptic protein mixtures using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

For the first time, quantitative analysis of tryptic protein mixtures, labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers, were performed with electrospray ionization and a 9.4 T Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer. Coupling a High-Pressure Liquid Chromatography (HPLC) separation step prior to mass analysis resulted in an increased amount of identified labeled tryptic peptides. The range for the determined intensity ratios of two peptides in a labeled pair was large, but the obtained median intensity ratio correlated very well with the corresponding concentration ratio. This method can be used for observing protein dynamics in a specific cell type, tissue, or in body fluids.     

Proteins associated with the cell envelope of Trichoderma reesei: a proteomic approach

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.     

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei

AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.