Cyclosporine A nephropathy, its pathogenesis and management

CsA, obtained from a fungus called Tolypocladium inflatum came into medical use in 1983. Organ transplants have shown great success after the use of Cyclosporine, especially in 3- and 5-year graft survival. However, nephrotoxicity seen in the early and late periods complicates its use. It is very important to distinguish especially early toxicity from rejection attacks; because the treatments of both processes are completely different. While vasocostriction in the renal artery system is prominent in the early period, the underlying factor for late toxicity is the thickening of the arteriolar intima and the consequent decrease in tissue oxygenation. The article discusses the variants of toxicity caused by the use of cyclosporin A. Morphological changes with the use of cyclosporin A are shown in rat models. The results of our own observations on the use of prostaglandin, which demonstrated the effect of vasodilation, are also presented, which can probably be used for further studies in order to reduce the nephrotoxicity of cyclosporin A. In particular, we found that PGE2 significantly reduced vasoconstriction and reduced the toxic effect due to CsA. The limitations was the usage of these agents once, so we couldn’t continue and only gave them intravenously. However, the results obtained were found to be significant.

Destructive activity of hydrocarbon-oxidizing micromycetes extracted from the substrates of the coastal areas, the Barents and White Seas

We analyzed the hydrocarbon-oxidizing activity of 33 micromycetes strains isolated from water and soils of the White Sea and Barents Sea coastal territories. The microscopic fungi studied were represented by the following genera: Acremonium, Aspergillus, Meyerozyma, Oidiodendron, Paradendryphiella, Penicillium, Pseudoqymnoascus, Tolypocladium, Trichoderma, Sarocladium, Talaromyces, Umbelopsis. Hydrocarbon-oxidizing activity of fungi was carried out in a laboratory experiment for two time periods: 14 and 28 days. The residual concentration of oil hydrocarbons in the medium was determined by the infrared spectrometry method. We revealed that micromycetes had different oil-destructive activity. The decrease in oil content for the strains over 14 days ranged from 11 to 83 %. Tolypocladium inflatum st.1, T. inflatum st.2, Penicillium thomii, Meyerozyma guilliermondii, P. simplicissimum, P. camemberti, P. solitum and Trichoderma minutisporum strains had the greatest oil destructive activity, reducing the concentration in the medium by 57-83 %. These micromycetes were isolated from coastal substrates, the content of oil products in which did not exceed the MPC. We found that some individual strains of microscopic fungi had high oil-destructive activity for a longer period of time: Tolypocladium cylindrosporum reduced the content of oil hydrocarbons for 28 days by 95 %. No clear relationship between the fungal biomass and the degree of decomposition of oil products was found, but some tolerant species of micromycetes can actively decompose oil hydrocarbons without significantly increasing their biomass. The identified active strains of hydrocarbon-oxidizing micromycetes can be used to create biopreparations with the prospect of their further use for bioremediation of oil spills in the northern marine ecosystems.

A Comparative Analyses of the Complete Mitochondrial Genomes of Fungal Endosymbionts in Sogatella furcifera, White-Backed Planthoppers

Sogatella furcifera Horvath, commonly known as the white-backed planthoppers (WBPH), is an important pest in East Asian rice fields. Fungal endosymbiosis is widespread among planthoppers in the infraorder Fulgoromorpha and suborder Auchenorrhyncha. We successfully obtained complete mitogenome of five WBPH fungal endosymbionts, belonging to the Ophiocordycipitaceae family, from next-generation sequencing (NGS) reads obtained from S. furcifera samples. These five mitogenomes range in length from 55,390 bp to 55,406 bp, which is shorter than the mitogenome of the fungal endosymbiont found in Ricania speculum, black planthoppers. Twenty-eight protein-coding genes (PCGs), 12 tRNAs, and 2 rRNAs were found in the mitogenomes. Two single-nucleotide polymorphisms, two insertions, and three deletions were identified among the five mitogenomes, which were fewer in number than those of four species of Ophiocordycipitaceae, Ophiocordyceps sinensis, Hirsutella thompsonii, Hirsutella rhossiliensis, and Tolypocladium inflatum. Noticeably short lengths (up to 18 bp) of simple sequence repeats were identified in the five WBPH fungal endosymbiont mitogenomes. Phylogenetic analysis based on conserved PCGs across 25 Ophiocordycipitaceae mitogenomes revealed that the five mitogenomes were clustered with that of R. speculum, forming an independent clade. In addition to providing the full mitogenome sequences, obtaining complete mitogenomes of WBPH endosymbionts can provide insights into their phylogenetic positions without needing to isolate the mtDNA from the host. This advantage is of value to future studies involving fungal endosymbiont mitogenomes.

Doubly Protonated Species Collision Induced Dissociation for Identification of Isocyclosporins

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus <i>Tolypocladium inflatum</i>, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by <i>Tolypocladium inflatum </i>strains of marine origin (White Sea). Cyclosporins A–C contain an unusual amino acid, (4<i>R</i>)-4-((<i>E</i>)-2-butenyl)-4,<i>N</i>-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporine by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]⁺ ions due to the rapid [CsA+H]⁺→[isoCsA+H]⁺ conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]²⁺ ions, and their MS/MS fragmentation can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. The fragmentation patterns of [CyA+2H]²⁺ and [isoCyA+2H]²⁺ ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing β-hydroxy-α-amino acids.

Doubly Protonated Species Collision Induced Dissociation for Identification of Isocyclosporins

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus <i>Tolypocladium inflatum</i>, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by <i>Tolypocladium inflatum </i>strains of marine origin (White Sea). Cyclosporins A–C contain an unusual amino acid, (4<i>R</i>)-4-((<i>E</i>)-2-butenyl)-4,<i>N</i>-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporine by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]⁺ ions due to the rapid [CsA+H]⁺→[isoCsA+H]⁺ conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]²⁺ ions, and their MS/MS fragmentation can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. The fragmentation patterns of [CyA+2H]²⁺ and [isoCyA+2H]²⁺ ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing β-hydroxy-α-amino acids.

Sintesis Tetrapeptida Antioksidan TKPS dengan Metode Fasa Padat

Senyawa tetrapeptida antioksidan threonin-lysin-prolin-serin (TKPS) merupakan salah satu derivat hasil isolasi dari jamur Tolypocladium inflatum dan telah berhasil disintesis menggunakan metode solid-phase peptide synthesis (SPPS). Penelitian ini bertujuan untuk menyintesis senyawa tetrapeptida TKPS dengan metode SPPS dan mengetahui aktivitas antioksidan senyawa tetrapeptida hasil sintesis dengan uji DPPH. Tetrapeptida TKPS disintesis menggunakan strategi SPPS pada resin 2-klorotritilklorida sebagai padatan penyangga, strategi gugus pelindung Fmoc, dan penggunaan reagen kopling HBTU/HOBt. Padatan TKPS berhasil dimurnikan menghasilkan TKPS sebanyak 111,4 mg. Setelah dimurnikan, TKPS dikarakterisasi dengan HR-TOF-MS yang memberikan nilai m/z [M+H]+ 431,2588 yang sesuai untuk TKPS. Uji aktivitas antioksidan dilakukan pada TKPS dengan nilai IC50 sebesar 4,039 mg/mL.

Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment

ABSTRACT The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate d-alanine is catalyzed from l-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL. We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions. IMPORTANCE The cyclopeptide cyclosporin A was first isolated from the filamentous fungus Tolypocladium inflatum showing antifungal activity and was later developed as an immunosuppressant drug. We report the biosynthetic mechanism of cyclosporines that are mediated by a cluster of genes encoding NRPS and PKS controlled by a bZIP-type transcriptional regulator. The two unusual amino acids Bmt and d-Ala are produced by the PKS pathway and alanine racemase, respectively. The cyclophilin and transporter genes jointly contribute to fungal self-protection against cyclosporines. Cyclosporine confers on T. inflatum the abilities to outcompete other fungi in competitive interactions and to facilitate fungal infection of insect hosts, which therefore benefits fungal adaptations to different environments.