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.     

A neutron activation technique for manganese measurements in humans

Manganese (Mn) is an essential element for humans, animals, and plants and is required for growth, development, and maintenance of health. Studies show that Mn metabolism is similar to that of iron, therefore, increased Mn levels in humans could interfere with the absorption of dietary iron leading to anemia. Also, excess exposure to Mn dust, leads to nervous system disorders similar to Parkinson's disease. Higher exposure to Mn is essentially related to industrial pollution. Thus, there is a benefit in developing a clean non-invasive technique for monitoring such increased levels of Mn in order to understand the risk of disease and development of appropriate treatments.To this end, the feasibility of Mn measurements with their minimum detection limits (MDL) has been reported earlier from the McMaster group. This work presents improvement to Mn assessment using an upgraded system and optimized times of irradiation and counting for induced gamma activity of Mn. The technique utilizes the high proton current Tandetron accelerator producing neutrons via the ⁷Li(p,n)⁷Be reaction at McMaster University and an array of nine NaI (Tl) detectors in a 4π geometry for delayed counting of gamma rays. The neutron irradiation of a set of phantoms was performed with protocols having different proton energy, current and time of irradiation. The improved MDLs estimated using the upgraded set up and constrained timings are reported as 0.67 μgMn/gCa for 2.3 MeV protons and 0.71 μgMn/gCa for 2.0 MeV protons. These are a factor of about 2.3 times better than previous measurements done at McMaster University using the in vivo set-up. Also, because of lower dose-equivalent and a relatively close MDL, the combination of: 2.0 MeV; 300 μA; 3 min protocol is recommended as compared to 2.3 MeV; 400 μA; 45 s protocol for further measurements of Mn in vivo.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997     

Chloroplast DNA phylogeography of Fagus crenata (Fagaceae) in Japan

 CpDNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 45 populations distributed throughout the species' range. Two cpDNA regions were sequenced: the non-coding region between the trnL (UAA) 5′exon and trnF (GAA), and the trnK region (including matK). Thirteen distinct cpDNA haplotypes were recognized and each haplotype was found to be geographically structured. Two major clades (I and II+III) were revealed in phylogenetic analyses among the haplotypes using F. sylvatica as an outgroup. The haplotypes of Clade I were distributed mainly along the Japan Sea side of the Japanese Archipelago, while those of Clade II+III occurred chiefly along the Pacific Ocean side. Consequently, the distribution of the two major cpDNA clades suggests that there were two migration routes in the history of F. crenata; one along the Japan Sea and the other along the Pacific Ocean side of the Japanese Islands. Received March 19, 2001 Accepted November 22, 2001     

Conversion of 5′-Methylthioadenosine into S-adenosylmethionine by yeast cells

5′-Methylthio[U-¹⁴C]adenosine was used as a culture supplement for Candida utilitis. The resulting S-adenosylmethionine was hydrolyzed into its structural components. Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine. Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound. The U-¹⁴C)-labeled adenine of 5′-methylthio[U-¹⁴C]adenosine did not contribute to the labeling of the amino acid component of the sulfonium compound.