Genetic variation, real-time PCR, metabolites and mycotoxins ofFusarium avenaceum and related species

In this paper the latest studies dealing with genetic variation and mycotoxins ofF. avenaceum and related species are reviewed and compared to the data from chromatographic image analyses. Forty-three European strains ofFusarium avenaceum and related species were classified by chromatographic image analysis on full chromatographic matrices. The results were in most cases in agreement with those from morphological and molecular analyses and supported the separation betweenF. avenaceum, F. arthrosporioides andF. tricinctum and betweenF. avenaceum groups I and II. The mycotoxin profiles of the FinnishF. avenaceum, F. arthrosporioides andF tricinctum strains were very similar to each other. Moniliformin and enniatins were the main mycotoxins produced. A fluorogenic TaqMan PCR assay (qPCR) was used for the detection ofF. avenaceum/ F. arthrosporioides DNA in Finnish barley and wheat. The qPCR results obtained from grain samples were compared to mycotoxin levels. A correlation was found betweenF. avenaceum/F. arthrosporioides DNA and moniliformin (MON) and enniatin (ENNs) levels in barley. A correlation was also found between the combinedF. avenaceum/F. arthrosporioides/F. tricinctum contamination and MON and ENNs levels in barley in 2002, but not in 2003. This was probably due to the higher MON and ENNs levels in 2002 than in 2003. It was possible to use the DNA levels ofF. avenaceum/F. arthrosporioides to distinguish between most barley samples containing high amounts of MON and ENNs from those containing low levels of the mycotoxins. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Grants from the National Technology Agency of Finland (No. 40168/03) and the Academy of Finland (No. 52104); travel grants from NorFA and the European Commission to the Laboratory of Dr. Ulf Thrane     

Sexual dimorphism in the pyrgomorphid grasshopper Phymateus morbillosus: from wing morphometry and flight behaviour to flight physiology and endocrinology

Abstract In the field, adult males of the grasshopper Phymateus morbillosus are able to fly for up to 1 min and cover up to c. 100 m, whereas females, although fully winged, are apparently unable to get airborne. Morphometric data indicate that the males are lighter, have longer wings, a higher ratio of flight muscles to body mass, and a lower wing load value than females. It was investigated whether this inability of females to fly is related to fuel storage, flight muscle enzymatic design and/or the presence and quantitative capacity of the endocrine system to mobilize fuels. In both sexes, readily available potential energy substrates are present in the haemolymph in similar concentrations, and the amount of glycogen in flight muscles and fat bodies does not differ significantly between males and females. Mass-specific activities of the enzymes GAPDH (glycolysis), HOAD (fatty acid oxidation) and MDH (citric acid cycle) in flight muscles are significantly lower in females compared with males, and mitochondria are less abundant in the flight muscles of females. There is no significant difference between the ability of the two sexes to oxidize various important substrates. Both sexes contain three adipokinetic peptides in their corpora cardiaca; the amount of each peptide in female grasshoppers is higher than in males.
Thus, despite some differences listed above, both sexes appear to have sufficient substrates and the necessary endocrine complement to engage in flight. It seems more likely, from the morphometric data above, that the chief reason for flightlessness is that P. morbillosus females cannot produce sufficient lift for flight; alternatively, the neuronal functioning associated with the flight muscles may be impaired in females.     

Betulachrysoquinone hemiketal: A p-benzoquinone hemiketal macrocyclic compound produced by Phanerochaete chrysosporium

Betulachrysoquinone hemiketal was isolated from pre-extracted wood of Betula lutea Michx. inoculated with Phanerochaete chrysosporium Burds. Acid-catalysed hydrolysis of betulachrysoquinone hemiketal produced betulachrysoquinone which was shown to be 2-hydroxy-6-(13′-hydroxytetradecanyl)-p-benzoquinone.     

Identification and Distribution of Phenylacetic Acid in the Brain of the Rat

Abstract: Phenylacetic acid, the major metabolite of phenylethylamine, has been identified and quantitated in rat brain regions by capillary column high-resolution gas chromatography mass spectrometry. Its distribution is heterogeneous and correlates with that of phenylethylamine. The values obtained were (ng/g ± SEM): whole brain, 31.2 ± 2.7; caudate nucleus, 64.6 ± 6.5; hypothalamus, 60.1 ± 7.4; cerebellum, 31.3 ± 2.9; brainstem, 33.1 ± 3.3, and the "rest," 27.6 ± 3.0.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8-¹⁴C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.     

Natural product discovery through microbial genome mining

The advent of the genomic era has opened up enormous possibilities for the discovery of new natural products. Also known as specialized metabolites, these compounds produced by bacteria, fungi, and plants have long been sought for their bioactive properties. Innovations in both DNA sequencing technologies and bioinformatics now allow the wealth of sequence data to be mined at both the genome and metagenome levels for new specialized metabolites. However, a key problem that remains is rapidly and efficiently linking these identified genes to their corresponding compounds. Within this review, we provide specific examples of studies that have used the power of genomic or metagenomic data to overcome these problems and identify new small molecules and their biosynthetic pathways.     

Subcellular compartmentation of pyrophosphate and dark/light kinetics in comparison to fructose 2,6-bisphosphate

Subcellular compartmentation of pyrophosphate (PP₁) was determined by rapid membrane filtration of evaeuolated oat mesophyll protoplasts. By improving both the extraction procedure and its assay via bioluminescence, PP₁ recovery from samples was quantitative and linear down to below 200 fmol. Based on the content of the different fractions obtained after membrane filtration and compared to the respective pools of marker metabolites [cytosol, fructose 2,6-bisphosphate (F26BP); chloroplast stroma, ribulose bisphosphate] rather than enzymes, we found ca 2/3 of the total cellular content to be chloroplast-assotiated. Referred to compartmental volumes, cytosolic and stromal concentrations of PP₁ were nearly equal (70–100 μ M ). PP₁ was higher in evacuolated compared to racuotated protoplasts which indicates a possible role of the tonoplast-located H⁺ pumping PP₁ase in regulating the cellular pool size of PP₁. During dark-light-transition the pool sizes of PP₁ changed only marginally in both vacuolated and evacuolated protoplasts, while there were pronounced changes in those of F26BP, starch and sucrose. Thus our findings support the notion that the cellular pool size of PP₁ is kept rather constant. They are, however, in contrast to the assumption that appreciable PP₁ levels only exist in the cytosol.