Effect of temperature on the extraction kinetics and diffusivity of Cyclosporin A in the fungus Tolypocladium inflatum

The influence of temperature on the extraction kinetics of Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum was examined in this study. The extraction of CyA from mycelia was performed in a 2-L stirred, baffled vessel using 30% v/v aqueous methanol. The temperature range used was from 5 to 45 degrees C. A linear relationship was found between the extraction yield of CyA and temperature. As the temperature increased, the yield of CyA increased with a maximum CyA yield of 18.3% obtained at 45 degrees C, which is 21.3% higher than the yield at 25 degrees C. The activation energy for the extraction of CyA from T. inflatum was found to be 36.7 kJ/mol, which indicates that the extraction of CyA from T. inflatum is controlled by both solubilization of CyA and diffusion of CyA through the solid phase of mycelia. The overall mass transfer coefficient, k(L)a(S), was found to increase from 1.02 x 10(-3) to 1.34 x 10(-2) s(-1) as the temperature increased from 5 to 45 degrees C. The effective diffusivity of CyA in the solid matrix of mycelia was found to increase from 1.05 x 10(-15) to 1.43 x 10(-14) m(2)/s as the temperature increased from 5 to 45 degrees C. A mathematical diffusion model was developed and was used to fit the experimental kinetic data of CyA extraction and determination of CyA effective diffusivities at different temperatures. This is the first time CyA diffusivities as a function of extraction temperature are reported in the literature.     

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     

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。     

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。     

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.     

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.     

薏苡仁油回流提取工艺参数优化及其动力学研究

探索加热回流法提取薏苡仁油的最佳工艺条件,并对提取过程进行深入探讨。采用单因素法考察溶剂、料液比、药材粒径、提取时间等对薏苡仁油提取率的影响,在此基础上研究其提取动力学。结果表明薏苡仁油较佳的提取条件为采用丙酮作为溶剂,液固比7倍,药材平均粒径0.25 mm,提取3 h。动力学研究表明,粒径较小时(0.25~0.83 mm),薏苡仁油的提取动力学符合二阶溶出动力学模型,即减小药材粒径,可明显增加有效成分的提取率。     

Estimation of ethanol in fermentation broth by solvent extraction and gas chromatography

Ethanol from fermentation is usually estimated by gas chromatography after centrifuging or distilling the broth. In this paper a more efficient and rapid method is described in which ethanol is extracted by an organic solvent such as n-butanol and the extract is analysed by gas chromatography. The distribution factor determined has a value close to unity and is dependent on ethanol concentration, but independent of sugar concentration.     

Cyclosporin A does not protect the disruption of the inner mitochondrial membrane potential induced by potassium ionophores in intact K562 cells

Mitochondrial dysfunction has been widely associated with programmed cell death. Studies of intact cells are important for the understanding of the process of cell death and its relation to mitochondrial physiology. Using cytofluorometric approaches we studied the mitochondrial behavior in an erythroleukemic cell line. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), potassium exchanger (nigericin), potassium ionophore (valinomycin), Na+K+-ATPase inhibitor (ouabain) and mitochondrial permeability transition pore inhibitor (cyclosporin A) were evaluated. Cyclosporin A (CSA) was very effective in attenuating the disruption of inner mitochondrial membrane potential induced by CCCP. However, CSA failed to protect the loss of inner mitochondrial membrane potential induced by potassium intracellular flux manipulation. Our findings suggest that mitochondrial cyclophilin is not involved in the cell events mediated by deregulation of potassium flux, underlining the need for further studies in intact tumor cells for a better understanding of the involvement of mitochondria physiology in cell death events.     

Early Treatment with Cyclosporin A Ameliorates the Reduction of Muscarinic Acetylcholine Receptors in Gerbil Hippocampus after Transient Forebrain Ischemia

Recent evidence has suggested that cyclosporin A (CsA), an immunosuppressive agent, has neuroprotective properties. However, its mechanisms associated with this activity remain unclear. We have previously shown that post-ischemic administration of CsA daily for 14 days prevented the decrease of muscarinic acetylcholine receptor binding in the hippocampus in the gerbil model of 5-min transient forebrain ischemia. In the present study, CsA (5 mg/kg, subcutaneously) was administered to each animal just after, 2 and 6 h after ischemia so as not to exert its immunosuppressive effect. Initial CsA treatment significantly restored the declined muscarinic acetylcholine receptor binding of the hippocampus 14 days after ischemia similar to the previous report. However, CsA did not alter reactive changes of astrocytes and microglia in the CA1 area of the hippocampus, which had been suppressed by daily administration. These results indicate that CsA could positively modulate the hippocampal acetylcholine neurotransmission system broken down through the ischemia-induced pyramidal cell death and its action mechanism may have no relation to the immunosuppressive properties.     

On the Approximation of Solvent Effects on the Conformation and Dynamics of Cyclosporin A by Stochastic Dynamics Simulation Techniques

Abstract

The molecular simulation technique of stochastic dynamics (SD) is tested by application to the immunosuppressive drug cyclosporin A (CPA). Two stochastic dynamics simulations are performed, one (SD_CCl4 ) with atomic friction coefficients proportional to the viscosity of the nonpolar solvent CCl₄, and one (SD_H2O) with atomic friction coefficients corresponding to an aqueous solution. The atomic friction coefficients are also taken proportional to an approximate expression for the atomic accessible surface area. The properties of both stochastic dynamics simulations are compared to those of two full molecular dynamics (MD) simulations of cyclosporin A, one in a box with 591 CCl₄ molecules, and one in a box with 632 H₂O molecules.

The properties of cyclosporin A as found in the molecular dynamics simulation in CCl₄ are well reproduced by the SD_CCl4 simulation. This indicates that the neglect of a mean force reresenting the average solvent effects on the solute is justified in the case of nonpolar solvents. For polar solvents, like water, this mean force may not be neglected. The SD_H2O simulation of cyclosporin A clearly fails to reproduce the amount of hydrogen bonding found in the molecular dynamics stimulation of cyclosporin A in water.

A comparison with a molecular dynamics simulation of cyclosporin A in vacuo shows that both the SD_CCl4 and the SD_H2O simulation come closer to the properties of the molecular dynamics simulations in CCl₄ and in H₂O than a molecular dynamics simulation in vacuo.     

Effect of extraction method on the yield of furanocoumarins from fruits of Archangelica officinalis Hoffm

Optimal conditions for the extraction and analysis of furanocoumarins from fruits of Archangelica officinalis Hoffm. have been determined. The following extraction methods were used: exhaustive extraction in a Soxhlet apparatus, ultrasonication at 25 and 60 degrees C, microwave-assisted solvent extraction in open and closed systems, and accelerated solvent extraction (ASE). In most cases the yields of furanocoumarins were highest using the ASE method. The effects of extracting solvent, temperature and time of extraction using this method were investigated. The highest yield of furanocoumarins by ASE was obtained with methanol at 100-130 degrees C for 10 min. The extraction yields of furanocoumarins from plant material by ultrasonication at 60 degrees C and microwave-assisted solvent extraction in an open system were comparable to the extraction yields obtained in the time- and solvent-consuming exhaustive process involving the Soxhlet apparatus.     

Comparative studies of physiological and environmental effects on the production of cyclosporin A in suspended and immobilized cells of Tolypocladium inflatum

Process improvement of the production of cyclosporin A (Cy A), a powerful immunosuppressive fungal metabolite, has been undertaken by analyzing suspended and immobilized cell cultures in parallel. Conidiospores of the producer microorganism, Tolypocladium inflatum, were entrapped into porous celite particles. Easier germination of the entrapped spores and more active growth of the immobilized cells were manifested when compared with free cell cultures initiated with spores or with mycelial inocula. Significant differences in precursor flow between the immobilized and free cell systems were evident when the effects of L-valine (a constituent amino acid of the Cy A molecule) on Cy A biosynthesis were compared in the two systems. For the freely suspended cells, L-valine supplemented early in the fermentation served as a possible precursor or stimulator of Cy A biosynthesis. A significant increase in specific production and Cy A yield on carbon source was observed in this system relative to suspended cultures supplemented with L-valine during or after exponential growth. In contrast to the free cell cultures, the addition of L-valine during the initial stage of immobilized cell growth had a negative effect on Cy A production but resulted in somewhat increased cell growth. This suggests an incompatibility between primary and secondary metabolic networks involved in Cy A biosynthesis in the immobilized state upon external addition of the amino acid.     

Reversed micellar extraction of trypsin: Effect of solvent on the protein transfer and activity recovery

By using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid-liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT-reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. (c) 1995 John Wiley & Sons, Inc.     

Cyclosporin A Impairs the Secretion and Activity of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeat)

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.     

Effect of the concentration of DODMAC and 1-decanol on the behavior of reverse micelles in the extraction of amino acids

The concentrations of dioctyldimethyl ammonium chloride (DODMAC) and 1-decanol in isooctane needed to form reverse micelles by phase contact have been determined. The behavior of these reverse micelles in the extraction of aspartic acid, glutamic acid, and threonine was studied by analyzing all of the ionic species in the aqueous phase. The amino acid is extracted from the aqueous phase by exchanging with the Cl(-) counterions of DODMAC in the reverse micelles. The ionic species in the reverse micelles tend toward their undissociated states as the water uptake by the reverse micelles decreases. The effect of 1-decanol on the extraction of the amino acids with two negative charges is due to the change in the water uptake of the reverse micelles. The concentration of DODMAC has no effect on the ion exchange of the amino acid with one negative charge with the Cl(-) counterions of DODMAC in the reverse micelles. Higher molar ratios of decanol to DODMAC favor the selective separation of amino acids with different charge numbers. (c) 1995 John Wiley & Sons, Inc.     

Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

A method to construct cDNA library of the entomopathogenic fungus, <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>, in the hemolymph of the infected locust

A method was developed to construct cDNA library of pathogenic fungus in the blood of the infected insect for cloning the fungal genes expressed in the host. This method is designed to take advantage of the obvious difference between the cell structures and components of the pathogen cells and that of the host cells. The host blood cells only have cell membrane, which can be disrupted by using SDS/proteinase K (PK). The fungal cells grown in the animal blood have cell wall, which can protect the fungal cell from the disruption of SDS/proteinase K (PK). By this method, the blood cells were disrupted by SDS/proteinase K (PK) and then the released animal RNA and DNA were digested completely with RNase and DNase. Therefore, the fungi grown in the blood were harvested without any contamination of host RNA and DNA. The pure fungi harvested from the infected blood can be used for mRNA extraction and cDNA library construction. The purity of the fungal mRNA was confirmed by PCR and RT-PCR with specific primer pairs for the host and specific primer pairs for the fungus, respectively, and the clones of cDNA library constructed by using the fungal mRNA was also analyzed. The results showed that there was no detectable contaminated insect DNA or RNA existing in the fungal mRNA. Randomly selected cDNA clones from cDNA library were sequenced and analyzed against GenBank using Blastx; no selected sequences had significant similarity with insects’ genes in comparison with the data of GenBank. The results further confirmed that the method to purify the pathogenic fungus from the host animal is reliable and the mRNA extracted from the fungus is eligible for cDNA library construction, and other molecular analysis including RT-PCR. This method may be applied to other pathogenic fungi and their host animals.