Cyclosporin A--a review on fermentative production, downstream processing and pharmacological applications

In present times, the immunosuppressants have gained considerable importance in the world market. Cyclosporin A (CyA) is a cyclic undecapeptide with a variety of biological activities including immunosuppressive, anti-inflammatory, antifungal and antiparasitic properties. CyA is produced by various types of fermentation techniques using Tolypocladium inflatum. In the present review, we discuss the biosynthetic pathway, fermentative production, downstream processing and pharmacological activities of CyA.     

环孢菌素8562的研究

中国弯颈霉Tolypocladium sinense C.L.Li 8562培养物用乙酸乙酯萃取,减压浓缩,获粗提品,经硅胶柱层析分离,葡聚糖凝胶LH-20纯化,活性炭脱色,得到2个成份,8562-成份1,8562-成分2,以成分1为主。经紫外光谱、红外光谱、高效液相色谱、质谱、核磁共振氢谱等分析表明8562-成份1、8562-成份2与环孢菌素A、环孢菌素C同质。8562-成分1的抗真菌试验显示了对半知菌和酵母菌具有明显拮抗活性,最低抑菌浓度(MIC)为2—150μg/ml。     

Antifungal activity in vitro of Baccharis glutinosa and Ambrosia confertiflora extracts on Aspergillus flavus, Aspergillus parasiticus and Fusarium verticillioides

The aims of this study were to evaluate the antifungal properties of Baccharis glutinosa and Ambrosia confertiflora extracts against Aspergillus flavus, A. parasiticus and Fusarium verticillioides, and to isolate the group of compounds that are responsible for the antifungal activity. Samples of aerial parts from each plant were extracted with 70% methanol and sequentially partitioned with hexane, ethyl acetate, and n-butanol. The partitioned fractions were evaluated in their capacity to inhibit the radial growth of the three species of fungi. The active fraction was used for an assay-guided chromatography of antifungal extracts. The results showed that the extract from B. glutinosa partitioned in ethyl acetate (Bea) showed the highest antifungal activity against the three fungi. Bea completely inhibited the growth of F. verticillioides at 0.8 mg/ml, whereas the radial growth of A. flavus and A. parasiticus was inhibited 70% at 1.5 mg/ml. The purified antifungal fraction from Bea showed 72, 54, and 52% of antifungal activity, respectively.     

Cyclosporin C is the main antifungal compound produced by Acremonium luzulae.

A strain of Acremonium luzulae (Fuckel) W. Gams was selected in screening new microorganisms for biological control of fruit postharvest diseases, especially gray and blue mold diseases on apples and strawberries. This strain manifests a very strong activity against a large number of phytopathogenic fungi. In this work, the product responsible for this antifungal activity was isolated from modified Sabouraud dextrose broth cultures of A. luzulae. It was purified to homogeneity by reverse-phase column chromatography. On the basis of UV, infrared, and 1H and 13C nuclear magnetic resonance spectra, mass spectral analysis, and the amino acid composition of the acid hydrolysates, the antibiotic was determined to be cyclosporin C. Cyclosporin C showed a broad-spectrum activity against filamentous phytopathogenic fungi but no activity against bacteria or yeasts. Its antifungal activity is only fungistatic. In contrast to Tolypocladium inflatum, another cyclosporin-producing strain, A. luzulae, did not produce additional cyclosporins. This was confirmed by in vivo-directed biosynthesis.     

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca⁺⁺ and inhibited by Mg⁺⁺, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol. 258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.Abbreviations used: PT: permeability transition; PTP: permeability transition pore; MMC: mitochondrial megachannel; IMAC: inner membrane anion channel. P_A: permeability of ion A. CSP: Cyclosporin A.     

Antifungal activity of a food‐grade dilution‐stable microemulsion against Aspergillus niger

Aims: To study the antifungal activities of a prepared food‐grade dilution‐stable microemulsion against Aspergillus niger. Methods and Results: Results from the antifungal activity on solid medium by agar dilution method showed that the microemulsion caused complete growth inhibition at 2000 ppm, and at 1000 ppm, showed 55% growth inhibition after 4 days of incubation and a delay of conidiation by 1 day compared with controls. Results from the antifungal activity in liquid medium by broth dilution method showed that the growth of A. niger was completely inhibited when a liquid medium containing 10⁶ spores per ml was treated with 500 ppm of microemulsion, which was determined by minimum fungicidal concentration. Study of fungicidal kinetics showed that more than 99% of viable spores were killed within 15 min. These antifungal activities were confirmed by scanning electron microscopy, light microscopy and increased Ca⁺², K⁺ and Mg⁺² leakages. Conclusions: The results suggest that the prepared microemulsions are effective antifungal systems with excellent growth inhibition and sporicidal activities, and indicate that their antifungal activity may be to the result of the disruption and dysfunction of A. niger cell walls and biological membranes. Significance and Impact of the Study: This study suggests the potential use of food‐grade dilution‐stable microemulsions for antifungal use in the food and pharmaceutical industries.     

Development and antifungal evaluation of a food-grade U-type microemulsion

Aims: Food-grade microemulsions have been of increasing interest to researchers as potential delivery systems for bioactive compounds. However, food-grade microemulsions are difficult to formulate and no microemulsion has been documented for antifungal purpose. The physicochemical characterization of a food-grade glycerol monolaurate (GML)/ethanol (EtOH)/Tween 80/potassium sorbate (PS)/water microemulsion system and the antifungal activities against Aspergillus niger and Penicillium italicum have been studied in this paper. Methods and Results: The influence of EtOH and PS on oil solubilization capability was clearly reflected in the phase behaviour of U-type microemulsion systems. One dilution-stable formulation ME (GML/EtOH/Tween 80/PS/water = 3 : 3 : 3·5 : 10·5 : 16) was selected. After 4 days of incubation, ME showed 80%A. niger growth inhibition at 0·2% and 72%P. italicum growth inhibition at 0·1%, respectively, and a delay of conidiation of 2 days compared with the control. In the antifungal activities of the microemulsion, GML and PS made major contributions with similar antifungal activities at a GML/PS weight ratio of 1: 3·5. Conclusions: Food-grade dilution-stable microemulsions prepared with GML as oil phase for antifungal purpose are feasible and solubilization of a hydrotrope contributes to the formation of microemulsions and enhanced antifungal activities. Significance and Impact of the Study: The present report represents the first to develop a food-grade microemulsion system for antifugal purpose.     

Heteronuclear two-and three-dimensional nmr spectroscopy of 13C in natural abundance by multiplicity selection and editing: Application to peptides

Several new proton-detected heteronuclear two-dimensional (2D) nmr techniques are presented that exhibit the features of selection and editing of multiplicity. The theoretical basis of the new features is briefly discussed and the application of the 2D techniques to a thioanalogon of Cyclosporin A [Thio-(1)-cyclosporin A] is shown. In addition, an extension of the experiments to three-dimensional (3D) techniques is demonstrated and the application of two 3D techniques to Thio-(1)-cyclosporin A with ¹³C in natural abundance is presented. The new techniques can help simplify heteronuclear shift correlations and increase the resolution in the carbon dimension.     

Characterization of fungal antagonistic bacilli isolated from aerial roots of banyan (Ficus benghalensis) using intact‐cell MALDI‐TOF mass spectrometry (ICMS)

Aims

To characterize fungal antagonistic bacilli isolated from aerial roots of banyan tree and identify the metabolites responsible for their antifungal activity.

Methods and Results

Seven gram positive, endospore‐forming, rod‐shaped endophytic bacterial strains exhibiting a broad‐spectrum antifungal activity were isolated from the surface‐sterilized aerial roots of banyan tree. The isolates designated as K1, A2, A4 and A12 were identified as Bacillus subtilis, whereas isolates A11 and A13 were identified as Bacillus amyloliquefaciens using Biolog Microbial Identification System. The antifungal lipopeptides, surfactins, iturins and fengycins with masses varying in the range from m/z 900 to m/z 1550 could be detected using intact‐cell MALDI‐TOF mass spectrometry (ICMS). On the basis of mass spectral and carbon source utilization profile, all seven endophytes could be distinguished from each other. Furthermore, ICMS analysis revealed higher extent of heterogeneity among iturins and fengycins produced by B. subtilis K1, correlating well with its higher antifungal activity in comparison with other isolates.

Conclusion

Seven fungal antagonistic bacilli were isolated from aerial roots of banyan tree, exhibiting broad spectrum of antifungal activity, among which B. subtilis K1 isolate was found to be most potent. The ICMS analysis revealed that all these isolates produced cyclic lipopeptides belonging to surfactin, iturin and fengycin families and exhibited varying degree of heterogeneity.

Significance and Impact of the study

The endophytes are considered as a potential source of novel bioactive metabolites, and this study describes the potent fungal antagonistic bacilli from aerial roots of banyan tree. The isolates described in this study have a prospective application as biocontrol agents. Also ICMS analysis described in this study for characterization of antifungal metabolites produced by banyan endophytic bacilli may be used as a high throughput tool for screening of microbes producing novel cyclic lipopeptides.     

Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides1

A new antifungal compound was isolated from peel and flesh of unripe avocado fruits and identified as 1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene. The maximal concentration of the anti-fungal monoene in unripe fruits was about 800 μg. g^?1 fr.wt. During ripening the monoene decreased to 40 μg. g^?1 fr.wt. concomitantly with the appearance of disease symptoms. The concentration of the previously described antifungal diene, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (Prusky et al. 1982), in avocado peel was 1,600 μg. g^?1 fr.wt. in unripe fruits, decreasing during ripening to 120 μg. g^?1 fr.wt. At 750 μg. ml^?1 the inhibition of germ tube elongation of germinated conidia by the antifungal monoene and the antifungal diene was 15 % and 44 %, respectively. A 1: 1 mixture of both antifungal compounds in concentrations ranging from 50 to 750 μg. ml^?1, showed synergistic activity and increased the percent of inhibited germ tubes of germinated conidia up to 15 % over the sum of activities of the separate compounds. The results are discussed in relation to the hypothesis that the antifungal diene and the antifungal monoene are involved in the quiescence of the germinated appressoria of Colletotrichum gloeosporioides in unripe avocado fruits.     

玉米大斑病菌钙调磷酸酶A亚基的克隆与特征分析

根据已知病原真菌钙调磷酸酶A亚基(Calcineurin A, CNA)丝苏蛋白磷酸酶保守结构域设计引物, 从玉米大斑病菌cDNA中扩增出CNA基因片段。利用cDNA末端快速克隆(Rapid amplification of cDNA ends, RACE)手段获得该基因全长cDNA序列(GenBank登录号: EF 407562)。Southern杂交结果显示, 该基因在玉米大斑病菌基因组只有1个拷贝。CNA特异性抑制剂Cyclosporin A对玉米大斑病菌分生孢子和附着胞发育有抑制作用, 抑制作用与抑制剂浓度呈正相关, 相同浓度的抑制剂对附着胞形成的抑制作用大于对孢子萌发的抑制。经CsA(5µg/mL)处理后, 玉米大斑病菌分生孢子不能侵入玉米叶片, 初步表明CNA基因参与玉米大斑病菌的致病过程。     

Nitric oxide (NO) donor 3-morpholinosydnonimine antagonizes cyclosporin A-induced contraction in two in vitro glomerular models

Cyclosporin A induces in vivo a severe nephrotoxicity characterized by a large decrease in renal hemodynamics. The aim of this study is to establish the ability of the known NO donor 3-morpholinosydnomine (SIN-1) to prevent the cyclosporin A-induced contraction by using rat isolated glomeruli and cultured glomerular mesangial cells. Isolated rat glomeruli are obtained from the renal superficial cortex by a sieving method. Mesangial cells are cultured in RPMI 1640 with 15% fetal calf serum. The planar surface area (PSA) of either isolated glomeruli or mesangial cells is assessed using anage analyzer. Each glomerusus or mesangial cell serves as its own control through calculation of the area before any drug incubation and after incubation for 10, 20 and 30 min either in control solution or in control solution with cyclosporin A alone or cyclosporin A and SIN-1. Cyclosporin A (10^–6 mol/L) induces an important time-dependent contraction of either glomerulus or mesangial cell. When pretreated with different concentrations of SIN-1 (10^–4 to 10^–9 mol/L), only a slight size decrease is noted. In conclusion, a direct constrictive effect of cyclosporin A in isolated glomeruli and mesangial cells can be prevented bythe NO donor SIN-1, suggesting an important involvement of the nitric oxide pathway in the cyclosporin A-induced nephrotoxicity.Abbreviations CyA cyclosporin A - SIN-1 3-morpholinosydnonimine - FCS fetal calf serum - PSA planar surface area     

Isolation and biochemical characterization of a novel leguminous defense peptide with antifungal and antiproliferative potency

Leguminous plants have formed a popular subject of research owing to the abundance of proteins and peptides with important biological activities that they produce. The antifungal proteins and peptides have been purified from a number of leguminous species. However, research continues to discover novel antifungal plant-produced peptides and proteins are being needed, specially those novel ones with both antifungal activity and other significant bioactivities. The objective of this study was to isolate a novel peptide from Phaseolus limensis. A 6.8 kDa peptide designated Limyin, with both antifungal and antiproliferative activity, was isolated from the large lima bean (P. limensis) legumes. The isolation procedure consisted of extraction, precipitation, affinity chromatography on Affi-gel blue gel, ion chromatography on SP-Toyopearl, and gel filtration on Superdex 75. Its N-terminal sequence was determined to be KTCENLATYYRGPCF, showing high homology to defensin and defensin precursors from plants. It potently suppressed mycelial growth in Alternaria alternata, Fusarium solani, and Botrytis cinerea. Its antifungal activity was stable up to 80°C. It showed antiproliferative activity towards tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. However, it had no effect on bacteria Staphylococcus aureus and Salmonella. The present findings make a significant addition of the research on leguminous plants.     

Resistant Microascus cirrosus pneumonia can be treated with a combination of surgery, multiple anti-fungal agents and a growth factor

A 49-year old male with acute myelogenous leukemia relapsed eight years post allogeneic bone marrow transplantation. The patient received induction chemotherapy causing prolonged neutropenia. The patient developed pneumonia for which empirical antibacterial and antifungal therapy were started. The patient underwent a video-assisted thorocascopy with near complete resection of the lesion because of poor response to treatment. Microascus cirrosus was identified in the tissue. In vitro susceptibility test to different antifungal agents showed M. cirrosus was very resistant. The patient is undergoing second allogeneic transplant with improved pneumonia resulting from a combination of treatment for fungal infection, which included surgery, antifungal agents, and granulocyte-colony stimulating factor. The Microascus genus rarely causes invasive fungal infection in humans and can be very difficult to treat because of the resistance to available antifungal agents.     

Antifungal effect of Penicillium metabolites against some fungi

Microorganisms are increasingly exploited as a source of new biological control agents. Genus Penicillium is a source of novel bioactive molecules which can be used as antifungal agents. The objective of this study was to evaluate the antifungal potential of Penicillium strains. Culture filtrates of two Penicillium species were tested for their antifungal potential by well diffusion assays. Filtrate of Penicillium isolates showed high antifungal effects on mycelial growth of Fusarium oxysporum, Fusarium solani, Macrophomina phaseolina, Aspergillus japonicus var aculeatus and Cladosporium cladosporioides. But Penicillium italicum inhibit the fungal growth from 45 to 68% as compared to Penicillium simplissimum (25–68%). However in case of A. japonicus var aculeatus, Penicillium spp. extracts were equally effective and reduce the colony growth up to 68%. However, P. simplissimum extract was least effective in case of M. phaseolina, where it decreased the colony growth only 25%.     

Antifungal Activity of Volatile Compounds Produced by an Edible Mushroom Hypsizygus marmoreus against Phytopathogenic Fungi

Volatile compounds with antifungal activity produced by edible mushrooms have potential as biological control agents to combat fungal diseases and reduce fungicide use in agriculture. Here we investigated the antifungal activity of volatile compounds produced by the edible mushroom Hypsizygus marmoreus (TUFC 11906) against eight phytopathogenic fungi. The results showed that volatile compounds from the mycelia and culture filtrates (CFs) of H. marmoreus had antifungal activity against some phytopathogenic fungi. Among them, the mycelial growth and conidial germination of Alternaria brassicicola were significantly inhibited by 60 and 100%, respectively. Moreover, the volatile compounds from CFs inhibited the lesion formation of A. brassicicola on detached cabbage leaves by 94%. The volatile compounds had higher antifungal activity against A. brassicicola than other fungi. With the removal of the volatile compounds from conidia of A. brassicicola, the conidia began to germinate, which indicates fungistatic activity of the compounds. The volatile compounds were isolated from the CFs of H. marmoreus, and the major volatile compound with antifungal activity was estimated to be 2‐methylpropanoic acid 2,2‐dimethyl‐1‐(2‐hydroxy‐1‐methylethyl)propyl ester. As the volatile compound produced by H. marmoreus is a product of an edible mushroom and has fungistatic activity against some phytopathogenic fungi, especially A. brassicicola, it may be possible to use the compounds as a novel safe agent for protecting crops in the field and during storage.     

Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides

Several polyene macrolides are potent antifungal agents that have severe side effects. Increased glycosylation of these compounds can improve water solubility and reduce toxicity. Three extending glycosyltransferases are known to add hexoses to the mycosaminyl sugar residues of polyenes. The Actinoplanes caeruleus PegA enzyme catalyses attachment of a D-mannosyl residue in a β-1,4 linkage to the mycosamine of the aromatic heptaene 67-121A to form 67-121C. NppY from Pseudonocardia autotrophica adds an N-acetyl-D-glucosamine to the mycosamine of 10-deoxynystatin. NypY from Pseudonocardia sp. P1 adds an extra hexose to a nystatin, but the identity of the sugar is unknown. Here, we express the nypY gene in Streptomyces nodosus amphL and show that NypY modifies 8-deoxyamphotericins more efficiently than C-8 hydroxylated forms. The modified heptaene was purified and shown to be mannosyl-8-deoxyamphotericin B. This had the same antifungal activity as amphotericin B but was slightly less haemolytic. Chemical modification of this new disaccharide polyene could give better antifungal antibiotics.

     

Treatment options in emerging mold infections

Although Zygomycetes, Fusarium spp, and Scedosporium spp are far less frequent causes of invasive fungal disease than Aspergillus and Candida, they are emerging. These types of infections in severely immunocompromised patients have a common feature: a poor clinical response to antifungal therapy. Infection is usually airborne, although local infections in cases of skin trauma are also possible. These fungi are resistant to some common antifungal agents; therefore, surgical debridement of the necrotic tissue, when possible, should be combined with specific systemic antifungal treatment in immunocompromised patients. In the absence of randomized clinical trials, most experience in the treatment of these infections is with amphotericin B. Experience with new antifungal agents is still limited, and recovery from neutropenia remains the main predictor of a favorable outcome.     

Production and Chemical Characterization of Antifungal Metabolites From Micromonosporasp. M39 Isolated From Mangrove Rhizosphere Soil

Micromonospora sp. M39 was selected as a producer of antifungal substances. Cell mass and antifungal activity of the organism was dependent on the carbon and nitrogen sources used. A combination of glucose, starch and glycerol as carbon sources at 1%(w/v) and corn steep powder as a nitrogen source at 0.25%(w/v) concentration in basal medium gave a maximum growth of 17.2 PCV/50 ml and in vitro an antifungal activity of 19.0 mm against the rice blast pathogen Pyricularia oryzaeMPO 293. In the selected production medium, optimum antifungal activity of the crude extract of the strain M39 was on day 16 of fermentation at 28 ± 2 °C. The crude extract of the strain M39 was analyzed and characterized by HPLC-DAD-UV–visible spectra. The peaks with a retention time of 4.88 min had a u.v. spectrum identical to that of 2,3-dihydroxybenzoic acid. The peak with a retention time of 6.08 min was confirmed by an automated spectral library search as phenylacetic acid. Two other peaks in the chromatogram were identified as cervinomycin A1 with a retention time at 12.78 min and cervinomycin A2 with a retention time at 13.46 min.     

<Emphasis Type="Italic">In vitro</Emphasis> antifungal activity of propolis samples of Czech and Slovak origin

Propolis has been used in traditional folk medicine for ages owing to a number of biological effects. Four propolis samples of Czech and one of Slovak origin were extracted using Soxhlet apparatus and analysed by thin-layer chromatography. Raw propolis samples and their extracts were tested by microdilution broth method to determine minimal inhibitory concentration (MIC) in eight strains of human pathogenic fungi. Raw propolis samples showed a lower in vitro antifungal activity than their extracts. In general, the petroleum ether extracts exhibited the highest in vitro antifungal activity (MIC range of 16–64 μg/ml). The content of flavonoids in the samples varied according to region. The highest amount of flavonoids was found in sample A that originated from Broumov (4%). The most susceptible to the propolis extracts were Trichophyton mentagrophytes and Candida albicans. The propolis samples of Czech and Slovak origin and their extracts showed a considerable in vitro antifungal effect which was associated especially with nonpolar petroleum ether and toluene extracts. There was only a partial correlation between flavonoids content and in vitro antifungal activity.