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.     

Mutagenicity of 7,12-dimethyl[a]anthracene and some other aromatic mutagens in Drosophila melanogaster

The optimal conditions for mutagenesis studies with DMBA and some other aromatic carcinogens in Drosophila were investigated in detail. The results presented in this paper indicate the following.The mutagenic effectiveness of DMBA is dependent on the route of administration, injection being far more effective when compared with feeding.The choice of the solvent is a crucial experimental condition. DMBA, when dissolved in oil/DMF, is ineffective whereas a special fat emulsion of DMBA gives high mutation frequencies.There appears to be an extreme strain dependence in the mutagenicity of DMBA. Mutagenic effectiveness in strain Berlin-K was rather low, whereas Oregon-K and Karsnäs-60 proved to be very susceptible to DMBA.Under the conditions of test, DMBA did not induce loss of a ring-X chromosome and did not produce recessive lethal mutations in such a chromosome.DMBA did not produce 2–3 translocations to any significant extent.An increase in DMBA-induced recessive lethal mutations was found upon storage of treated sperm with an optimal storage time of 4–10 days.DMBA is efficient in the production of delayed recessive lethal mutations in strain Berlin-K. Twice as many lethals were recovered with the F3 generation as compared with those in F2. In strain Oregon-K, where the frequency of F2 lethals was much higher than in strain Berlin-K, the ratio of F3/F2 lethals was clearly lower.Enzyme induction with phenobarbital reduces the mutagenic effectiveness of DMBAWith TMBA, similar strain differences in sensitivity were observed as those found for DMBA. Whereas TMBA was not mutagenic in Berlin-K, considerable mutagenicity was observed in Oregon-K and Karsnäs-60.Injection of carcinogenic polycyclic aromatic hydrocarbons and aromatic amines, when dissolved in special fat emulsions, enhances the mutagenic effectiveness of some compounds (DMBA, TMBA, DA and AcO-AAF), but this procedure does not always solve the problems-pertinent to these classes of promutagens in Drosophila.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Species-specific aggregation factor in sponges. XIII. Entire and core structure of the large circular proteid particle from Geodia cydonium

High-molecular weight particles have been isolated from the sponge Geodica cydonium. In the "native" from these particles consist of a spherical center and have 25-30 filaments attached to it. The core structure of the particles is assembled of a central circle and 25 radially-arranged filaments. The core structure is obtained from the entire structure by incubation in a medium, containing a non-ionic detergent and EDTA. The molecular weight of the enitre structure was in the range of 1.4 X 10(9) daltons or more and of the core structure 6.1 x 10(8) daltons. Two functional proteins are released from the "native" particles: the aggregation factor and the sialytransferase.     

Age-related changes in mutagen activation by rat tissues

Age-related changes in drug metabolism of the liver, lung and kidney of adult female Long-Evans rats were determined by measuring changes in mutagen formation. Activation of aflatoxin B1 (AFB1), 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF) to mutagenic derivatives was assayed using the Ames Salmonella test system. The promutagens were incubated with tissue fractions from rats ranging in age from 2.5 to 25 months. With all three compounds, hepatic, renal and pulmonary activation was lower in the senescent than in the young adult animals. The largest decrease, however, occurred prior to middle-age, i.e. before 9-13 months. In liver and kidney, little change was detectable between the middle-aged and the old (20-25 months) animals. However, pulmonary metabolism in the oldest animals was slightly higher than in the extracts from the middle-aged rats. The observed decline in mutagen activation may thus be a function of maturation rather than senescence.     

Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven,auxin-dependent patterning

Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP₂) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

Design and bio-evaluation of indole derivatives as potent Kv1.5 inhibitors

Atrial fibrillation (AF) is one of the common arrhythmias that threaten human health. Kv1.5 potassium channel is reported as an efficacious and safe target for the treatment of AF. In this paper, we designed and synthesized three series of compounds through modifying the lead compound RH01617 that was screened out by the pharmacophore model we reported earlier. All of the compounds were evaluated by the whole-patch lamp technology and most of them possessed potent inhibitory activities against Kv1.5. Compounds IIIi and IIIl were evaluated for the target selectivity as well as the pharmacodynamic effects in an isolated rat model. Due to the promising pharmacological behavior, compound IIIl deserves further pharmacodynamic and pharmacokinetic evaluations.