中国弯颈霉新种及产环孢菌素的研究

对云南省迪庆藏族自治州产的冬虫夏草 Cordyceps sinensis(Berk.)Sacc.进行分离获一纯种,经培养鉴定确为弯颈霉属 Tolypocladium w.Gams 一新种,命名为中国弯颈霉 To-lypocladium sinense c.L.Li sp.nov.。它在几种琼脂培养基上生长形成瓶梗及瓶梗孢于,瓶梗基部呈球形或椭圆形膨大,瓶颈细长而弯曲,瓶梗孢子球形至卵形。其形态特征及生活习性与弯颈霉属其它种类显然不同,故定为一新种。它在蛋白胨葡萄糖培养液中,温度26℃,pH5.2,摇瓶振荡培养12天。发酵液用乙酸乙酯萃取,减压蒸馏,从每升发酵液中获50—80mg 环孢菌素粗品。对几种半知真菌显示了明显的拮抗作用。     

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.     

Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless

Restless is an endogenous hAT transposon found in the cyclosporin-producing fungus Tolypocladium inflatum. This element is present in about 15 copies in a particular strain (ATCC34921) which was used for successful gene tagging. We have isolated a T. inflatum mutant with a defect in nitrogen metabolism. This mutant carries a copy of the Restless element in a gene encoding a C6 zinc-finger protein. The deduced amino acid sequence of the gene product shows a significant similarity to the NIT4 protein of Neurospora crassa, which is a regulator of nitrogen metabolism. The wild-type T. inflatum gene was shown to complement a nit-4 mutant of N. crassa. From these data, we conclude that the T. inflatum gene also encodes a regulator of nitrogen metabolism, which was named tnir1 (Tolypocladium nitrogen regulator 1). To the best of our knowledge, this is the first fungal gene to be identified by transposon-directed gene tagging. A general method for gene tagging using an endogenous fungal transposon is presented. Received: 20 October 1999 / Accepted: 15 December 1999     

Physiological and genetic factors for process development of cyclosporine fermentations

Summary The new immunosuppressive agent cyclosporine (Cyclosporin A, Cy) is the most prominent member of a group of cyclic peptide fungal metabolites (cyclosporins) produced byTolypocladium inflatum in submerged fermentations. In the present study, kinetics and physiology of mycelial growth and Cy production byT. inflatum were examined. A new semi-synthetic medium was formulated, consisting of a single carbon/energy source, Bacto-peptone, potassium phosphate and potassium chloride. A wide variety of carbon sources supported growth and Cy production. 3% (w/v) sorbose gave the highest final Cy titer (105.5 mg/l), based on 10-day fermentations. The best specific Cy production was observed with 2% sorbose (14.3 mg Cy/g biomass) followed by 5%myo-inositol (13.4 mg Cy/g biomass). A feeding strategy consisting of sequential addition of two carbon sources such as sorbose and maltose was developed in order to reach higher volumetric production. Genetic studies were also conducted, focussing on the development of mutants for increased Cy production and for the synthesis of novel cyclosporins. In the course of these studies, viable protoplasts ofT. inflatum have been isolated and regenerated.     

Tolypocladium cylindrosporum gams (deuteromycetes,moniliaceae) as a biological control agent against the window midge Sylvicola cinctus Fabr. (diptera: Anisopodidae)

Larvae of Sylvicola cinctus Fabr. were found killed by an entomopathogenic fungus Tolypocladium cylindrosporum Gams, in Vetlanda, Sweden. This is the first report of this fungus as an insect pathogen from Sweden. The fungus was cultured and sprayed on aspen log piles in an attempt to reduce population levels. In September 1992, when maximum abundance is expected, the area treated with T. cylindrosporum had significantly fewer midges than an untreated area. Possibilities for biological control are discussed.     

Effects of Tolypocladium cylindrosporum and its secondary metabolites, efrapeptins, on the immune system of Galleria mellonella larvae

Interactions between Tolypocladium cylindrosporum (Deuteromycetes), its metabolite, efrapeptins, and the insect immune defense were investigated in vivo and in vitro. In the different phagocytosis studies, Bacillus cereus spores which had been labelled with fluorescein-isothiocyanate (FITC) were used. In vitro studies showed that efrapeptins inhibit phagocytic activity of Galleria mellonella (Lepidoptera: Pyralidae) haemocytes. The response was dose-related. Efrapeptins significantly reduced the number of nodules formed in response to an injection of zymosan supernatant. Phenoloxidase (PO) activation system contained in haemocyte lysate (HLS) was not affected by efrapeptins. In vivo studies when larvae were injected with efrapeptins also revealed that efrapeptins did not affect PO activities and total haemocyte count (THC) after 1 and 6 h post-injection. However, 12 h post-injection there was a significant inhibition of PO activities in HLS. There was also no significant reduction of PO activities and THC when larvae were injected with Tolypocladium cylindrosporum spores until 24 h post-injection. However, PO activities were suppressed and THC reduced 48 h post-treatment of larvae with spores. This study suggests that efrapeptins may interfere with the ligand-receptor interactions that are likely to occur at the plasma membrane of specific haemocytes.     

植物细胞亲环素研究进展

亲环素（cyclophilin,CyP）是能够与免疫抑制药物环孢霉素A（cyclosporine A,CsA）特异结合的、高度保守的一个蛋白家族,广泛存在于细菌、酵母、植物和动物等各种有机体中。在高等植物中存在多种同工型亲环素,它们位于细胞浆、细胞核、线粒体和叶绿体等不同亚细胞分室中,参与多种重要的生理生化过程。本文从亲环素的发现、结构特点、生化特性及生物学功能等方面进行综述,并对植物亲环素研究前景进行了讨论。     

Solubilization of cyclosporin A

This study investigated the solubilization of cyclosporin A (CsA), a neutral undecapeptide, by cosolvency, micellization, and complexation. Cosolvents (ethanol, propylene glycol, polyethylene glycol, tetrahydrofurfuryl alcohol polyethyleneglycol ether, and glycerin), surfactants (polyoxyethylene sorbitan monooleate [(Tween 80)], polyoxyethylene sorbitan monolaurate [(Tween 20)], and Cremophor EL), and cyclodextrins (α-cyclodextrin [(αCD)] and hydroxypropyl-β-cyclodextrin[(HP\CD)] were used as solubilizing agents in this study. Surfactants had a noticeable effect in increasing CsA solubility. Twenty percent solutions of Tween 20, Tween 80, and Cremophor EL increased the solubility by 60 to 160 fold. Cyclodextrins can increase the CsA solubility, but αCD was more effective than HP\CD. Cosolvents on the other hand did not increase the solubility of CsA as much as expected from the LOGP (logrithm of wateroctanol partition coefficent) value of CsA.     

Effects of cyclosporin A on model lipid membranes

Cyclosporin A (CSA) is a widely used immunosuppressant drug for transplant therapy, however its limitation is its toxicity. The effect of CSA on model membranes such as dimyristoyl phosphatidylcholine (DMPC) bilayers was studied using small-angle X-ray diffraction and differential scanning calorimetry (DSC). CSA abolishes the pretransition and affects the transition of DMPC model membranes in a concentration-related manner as is shown by DSC. CSA induces a second peak at the high temperature side of the main transition, which is interpreted as a phase separation between areas rich and poor in CSA concentration. Small angle X-ray diffraction shows that the repeat distance of the DMPC bilayers in the lamellar Lalpha state increases as a function of concentration up to 10 mol% and remains constant thereafter. Furthermore, CSA affects the fatty acyl chains of the bilayer, especially the part of the chain proximal to the head group. In conclusion, CSA, as both small-angle X-ray diffraction and DSC show, affects in a concentration-wise manner the DMPC model membranes and perturbs the bilayer, in particular the acyl chain region.     

环孢素A对异种脱细胞神经移植的影响

目的研究脱细胞异种面神经移植中环孢素A（cyclosporin A,CSA）的作用效果。方法 48只Wistar大鼠随机选择一侧作为实验侧,解剖面神经后于颊支制造1cm的神经缺损。按神经移植的种类分为4组,A组：异种面神经移植组;B组：异种面神经移植应用CSA免疫抑制组;C组：异种脱细胞神经移植组;D组：异种脱细胞神经移植应用CSA免疫抑制组。各组实验动物分别于术后5周,12周进行电生理学测试（潜伏期、运动神经传导速度）,并进行光镜、电镜观察形态学变化。结果术后5周和12周时,C,D组动物在电生理指标及光镜、电镜指标上均优于A,B组,术后5周和12周,D组面神经颊支传导速度均高于其它各组。结论 1.0 cm缺损的异种脱细胞面神经移植动物实验中,应用CSA5mg/（kg.d）5周可以降低排斥反应,提高神经再生能力。     

Papain kinetics in the presence of a water-miscible organic solvent

The effects of various concentrations of a water-miscible organic solvent [a 7:3 (v/v) mixture of N, N dimethylformamide and dimethylsulfoxide] on the kinetics of papain have been investigated. The parameters k(cat) and K(m) for the amidase and esterase activity of papain using N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as substrates were determined. For both types of activity, k(cat) initially increased (up to about 15% solvent), and then decreased with increasing concentrations of organic solvent. In contrast, K(m) increased sharply with the organic solvent concentration. Active site titration at 0 and 50% solvent indicated no change in the amount of active enzyme. Fluorometric measurements of the emission spectrum of papain did not indicate any major conformational changes with increasing concentrations of organic solvent.     

环孢霉素A治疗儿童再生障碍性贫血的临床疗效研究

目的:探讨环孢霉素A治疗儿童再生障碍性贫血患者的临床疗效。方法:选择在我院就诊或住院治疗的50例儿童再生障碍性贫血患者,随机分为实验组和对照组,每组25例。对照组患者给予司坦唑醇治疗,实验组患者在对照组基础上给予环孢素治疗。治疗结束后,检测并比较两组患者的血清雌二醇、睾酮水平、网织红细胞计数以及临床疗效。结果:与治疗前相比,两组患者治疗后的血清雌二醇水平均显著下降(P0.05),血清睾酮水平以及网织红细胞计数水平均明显升高(P0.05);与对照组相比,实验组患者的血清雌二醇水平较低(P0.05),血清睾酮水平以及网织红细胞计数水平较高(P0.05)。此外,实验组的临床治疗总有效率较对照组明显升高(P0.05)。结论:环孢霉素A可提高儿童AA患者的疗效,可能与其降低血清雌二醇水平,升高血清睾酮水平以及网织红细胞计数水平有关。     

Diffusion of lactose in acidogenic biofilms

Effective diffusivity of lactose in active acidogenic biofilms was measured at 35 degrees C and pH 4.6 with a specially designed diffusion cell. The diffusion cell was designed and operated in such a way that the lactose concentrations on the surface and at the center of a living bacterial aggregate could be measured at steady state. As a model parameter in a widely accepted reaction-diffusion equation which describes lactose distribution in living biofilms, the effective diffusivity of lactose in the biofilms was found to be about 65% of the lactose diffusivity in free solutions. It was experimentally determined that the active biofilms had about 66% void volume made up of channels through which the lactose molecules were transported into the bacterial aggregates. Therefore, the decrease in lactose diffusivity was mainly caused by the biofilm's solid biomass fraction rather than the tortuosity of the channels. (c) 1993 John Wiley & Sons, Inc.     

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.     

Cyclosporin A—cyclophilin complex formation A model based on X-ray and NMR data

The previously determined 3D NMR solution structure of cyclophilin-bound cyclosporin A (CsA) was docked onto the X-ray crystal structure of cyclophilin. Intermolecular nuclear Overhauser effects (NOE) between CsA and cyclophilin were used as constraints in a restrained energy minimization to generate a model of the complex which satisfied all the NOE distance constraints. The model shows that the residues 9 to 11 and 1 to 5 of the cyclic CsA molecule are in contact with cyclophilin. Comparing the model of the CsA—cyclophilin complex to the X-ray crystal structure of a complex of cyclophilin with a substrate for peptidyl-proline cis-trans isomerase activity, i.e. the linear tetrapeptide substrate ae-Ala-Ala-Pro-Ala-amc (ac. acetyl; amc. amidomethylcoumarin), one notices that the contacting peptide segments in the two ligands are oriented in opposite directions, and that the side chain or MeVal-11 of CsA superposes rather precisely with the position of the prolyl residue in ae-Ala-Ala-Pro-Ala-amc.