简讯

利用解脂假丝酵母B_(74)发酵废糖蜜产生柠檬酸沈阳市食品发酵所,利用中国科学院微生物研究所提供的解脂假丝酵母民B_(74),以预处理过的甜菜废糖蜜做为主要原料,进行了发酵产生     

三颗针对白假丝酵母的抑制作用

目的研究三颗针对白假丝酵母的抑制作用。方法采用溴化噻唑蓝四唑法(MTT)和琼脂平板法测定三颗针对白假丝酵母的最低抑菌浓度(MIC)和最低杀菌浓度(MFC);利用倒置荧光显微镜观察三颗针对白假丝酵母菌丝形成的影响;采用MTT法测定三颗针对白假丝酵母菌丝萌发不同时期的抑制率。结果三颗针对白假丝酵母具有较强的抑制作用。其中,采用琼脂平板法测得三颗针抑制该菌的MIC为2mg/mL,MFC为4mg/mL。三颗针能减缓该菌的生长速度或使其停止生长,且浓度越高作用效果越明显。其中4mg/mL的三颗针作用白假丝酵母6h后,能够完全抑制菌丝形成。对于24h后已经萌发为菌丝态的白假丝酵母,三颗针可抑制菌丝的继续生长,与对照组相比,4mg/mL的三颗针对于萌发2h后的白假丝酵母的菌丝抑制率为76.7%(P0.01)。结论三颗针能抑制白假丝酵母菌丝的生长和萌发。     

香叶挥发油成分分析及其对 小鼠白色假丝酵母菌阴道病的影响

目的 研究香叶挥发油对白色假丝酵母生物膜形成的影响。方法 水蒸气蒸馏法提取香叶挥发油,GC-MS分析其成分,测定其对白色假丝酵母的MIC、MBC,激光共聚焦显微镜下观察白色假丝酵母生物膜的形成,制备小鼠白色假丝酵母菌阴道病模型,观察香叶油治疗效果。结果 香叶挥发油提取率为1.83%,分析出22个成分,主要成分为5-甲基-2-异丙烯基环己酮、3,7-二甲基-2-辛烯-1-醇、(顺)香叶醇、异香叶醇、香叶醇和甲酸香茅酯,MIC值为0.3625%,MBC值为0.6250%,激光共聚焦结果显示不同浓度香叶油均可显著抑制白色假丝酵母生物膜形成,动物实验结果显示香叶油可抑制小鼠阴道炎症改变。结论 香叶油用于治疗白色假丝酵母引起的阴道炎机制可能与其影响细菌生物膜形成有关。     

褐飞虱共生解脂假丝酵母抗吡虫啉菌株的驯化

为进一步研究共生菌在褐飞虱对吡虫啉产生抗性中的生理生化机制,在稻田杀虫剂对褐飞虱共生解脂假丝酵母生长影响的基础上,选用不同吡虫啉浓度进行抗药性菌株的驯化。结果表明,褐飞虱共生解脂假丝酵母在不同吡虫啉浓度(2 000、1 000和500 mg/L)的固体培养基上继代培养,经过20代后2 000 mg/L培养基上的共生菌菌落数量,与未加吡虫啉的培养基上的菌落数量差异不明显,并且连续3代稳定后定为抗2 000 mg/L吡虫啉的共生菌菌株。在光镜下比较不同抗感吡虫啉菌株假菌丝的形态变化,发现抗吡虫啉菌株的假菌丝出现畸形,而且假丝变短,部分出现了膨大。     

利用正癸烷发酵产生癸二酸的酵母19—2菌株的鉴定

对分离筛选所得的产酸较高,酸质较纯的酵母19—2菌株进行了鉴定,根据其具有较发达的假菌丝,不产生子囊孢子,利用葡萄糖,赤藓醇,不同化硝酸盐;冻化牛奶等主要特征,定为解脂假丝酵母(Candidalipolytica)。该菌对直链烷烃能很好利用,具有较强的趋化性。酵母19—2菌株是一株利用石油正癸烷产生癸二酸的优良菌株。本文报道了对酵母19—2菌株的鉴定     

大鼠外阴阴道假丝酵母菌病模型中局部与全身免疫机制的研究

外阴阴道假丝酵母菌病(VVC)是妇女常见病及多发病,白假丝酵母(白色念珠菌)是其主要致病菌。我们通过在大鼠皮下注射大剂量雌激素建立大鼠的假发情模型,然后在大鼠阴道内接种白假丝酵母(白色念珠菌)SC5314悬液,建立大鼠的外阴阴道假丝酵母菌病模型。在不同时间点检测大鼠血浆中及阴道局部白细胞介素‐2(IL2)、甘露糖结合凝集素(MBL)、Toll样受体‐4(TLR4)的值,探讨VVC及复发性外阴阴道假丝酵母菌病(RVVC)的免疫发病机制。我们发现大鼠血清中只有TLR4在第2周出现显著性上升,而在阴道灌洗液中,IL2在第3周显著下降,TLR4在第2和第3周显著上升,MBL在第1至第3周均显著下降。由此我们推断在外阴阴道假丝酵母菌发病过程中,阴道局部免疫功能异常可能与VVC和RVVC的发生密切相关。     

白假丝酵母天冬氨酸蛋白酶研究进展

白假丝酵母是免疫功能低下宿主条件致病感染中最常见的病原真菌之一,目前基本已肯定分泌型天冬氨酸蛋白酶(s印)在其致病过程中具有重要作用。现将近年来有关白假丝酵母Sap的研究从其编码基因、分子特性和表达以及在白假丝酵母致病中的作用等作一综述。     

克鲁斯假丝酵母及其近似种的脉冲电泳核型分析

用钳位均匀电场脉冲电泳(CHEF)系统分析了克鲁斯假丝酵母(Candida krusei),郎比可假丝酵母(C. lambica)和粗状假丝酵母(C. valiad)的模式菌株的电泳核型,发现这三种表型相似的假丝酵母却具有互不相同的染色体DNA分子带型,为其分类学研究提供了可靠的鉴别依据。在常规分类学研究的基础上,测定了AS 2.75(原定种名为(C. incospicua),AS2.1182(原定种名为 C. lambica)和AS 2.1772(未定种)等三株假丝酵母的G+C含量和脉冲电泳核型。通过对已报道的C. inconspicu的G+C含量及上述三种假丝酵母模式菌株的脉冲电泳核型的比较分析证明,AS 2.75和AS 2.1772为粗状假丝酵母(C. valida),AS 2.1182为克鲁斯假丝酵母(C. krusei)。     

新一代高产甘油重组菌的构建及其初步发酵

产甘油假丝酵母(Candida glycerinogenes WL2002-5)是一株发酵生产甘油的工业化菌株。为进一步提高其产甘油能力,本研究利用前期研究中成功克隆的产甘油假丝酵母中甘油合成关键酶3-磷酸甘油脱氢酶基因CgGPD1,构建根癌农杆菌双元载体pCAM3300-zeocin-CgGPD1后,电击转化根癌农杆菌LBA4404,通过根癌农杆菌介导法(ATMT)转化产甘油假丝酵母,构建了产甘油假丝酵母重组菌。并从中筛选出一株酶活力和产甘油性能较好的产甘油假丝酵母重组菌株C.g-G8。以葡萄糖为底物摇瓶发酵96h后,重组菌C.g-G8的甘油产量比野生型菌株Candida glycerinogene提高18.06%,平均耗糖速率提高12.97%,平均酶活力提高27.55%。本研究成功利用ATMT法转化产甘油假丝酵母构建新一代高产甘油菌株。     

山苍子油对白假丝酵母抗菌活性研究

研究山苍子油对白假丝酵母菌的抗菌活性,并阐明其可能的抗菌机制。通过水蒸馏法提取山苍子油,并利用气相色谱-质谱联用仪(GC/MS)分析其成分。通过琼脂平板稀释法测定山苍子油对白假丝酵母的最低抑菌浓度(MIC)和最低杀菌浓度(MFC),并研究山苍子油对白假丝酵母的抗菌动力学;同时利用扫描电子显微镜(SEM)和透射电子显微镜(TEM)观察山苍子油对白假丝酵母细胞超微结构的影响。结果表明,自提山苍子油主成分为柠檬烯(26.51%),柠檬醛(11.94%)和马鞭烯醇(11.84%)。山苍子油对白假丝酵母菌的MIC和MFC均为1.25μL/m L;其抗菌动力学研究表明浓度低于其MIC时,山苍子油仅延长白假丝酵母的生长适应期,并不能彻底杀死细胞;SEM结果表示,山苍子易破坏正在出芽的细胞;TEM显示出山苍子破坏细胞壁,细胞膜,使细胞裂解。山苍子油具有优良的抗白假丝酵母活性,且白假丝酵母在芽痕处对山苍子油比较敏感,高浓度的精油(5.0μL/m L)对细胞产生不可逆破坏;山苍子油杀菌的靶标可能是细胞壁和细胞外膜,使细胞内大分子外泄,细胞器变形,最终导致细胞死亡。     

Role of protein kinase C and mitochondrial permeability transition pore in the neuroprotective effect of ceramide in ischemia-induced cell death

In this study, we investigated the role of protein kinase C (PKC) and mitochondrial permeability transition pore (mPTP) on the effect of ceramide in an in vitro model of ischemia in SH-SY5Y neuroblastoma cells. In ischemic cell viability studies, a dual effect of ceramide was observed, depending on ceramide concentration. PKC isoforms are involved in the protective effect of low concentrations of ceramide. During ischemia, ceramide treatment leads to an increase in the formation of reactive oxygen species (ROS), which induces a controlled opening of mPTP. This fact prevents mitochondrial Ca²⁺ overload, which is clearly protective.     

Lea-active heptaglycosylceramide, a hybrid of type 1 and type 2 chain, and the pattern of glycolipids with Lea, Leb, X (Lex), and Y (Ley) determinants in human blood cell membranes (ghosts). Evidence that type 2 chain can elongate repetitively but type 1 chain cannot

Two major glycolipids reactive with the monoclonal anti-Lea antibody have been isolated from human blood cell membranes. One component was identified as lactofucopentaosyl(II)ceramide and the other as a ceramide heptassaccharide with the structure described below: (formula; see text) The structure includes the Lea determinant (type 1 chain) linked to lactoneotetraosylceramide (type 2 chain); thus, it is regarded to be a hybrid between type 1 and 2 chain. In addition, a minor component having the thin-layer chromatographic mobility of a ceramide nonasaccharide, which was reactive to anti-Lea antibody, was detected. No other component with a thin-layer chromatographic mobility slower than the above components and reactive to the anti-Lea antibody was detected. In contrast, a series of slowly migrating glycolipids having X (Lex) determinant (Gal beta 1----4(Fuc alpha 1----3)GlcNAc) was detected. A similar series of long chain glycolipids having Y (Ley) determinant (Fuc alpha 1----2Gal beta 1----4(Fuc1----3)GlcNAc) was detected in human blood cells; in contrast, only one major Leb glycolipid was found with the mobility of a ceramide hexasaccharide. No glycolipid with a long carbohydrate chain composed exclusively of type 1 chain was detected. Thus, chain elongation may proceed through type 2 chain, but not through type 1 chain. Lea and X (Lex) haptens are distributed equally among blood group A, B, and O red blood cells, whereas the quantity of Leb and Y (Ley) haptens is much lower in A and B blood cells than in O blood cells.     

Imipramine blocks ethanol-induced ASMase activation, ceramide generation, and PP2A activation, and ameliorates hepatic steatosis in ethanol-fed mice

Our previous data showed the inhibitory effect of ethanol on AMP-activated protein kinase phosphorylation, which appears to be mediated, in part, through increased levels of hepatic ceramide and activation of protein phosphatase 2A (Liangpunsakul S, Sozio MS, Shin E, Zhao Z, Xu Y, Ross RA, Zeng Y, Crabb DW. Am J Physiol Gastrointest Liver Physiol 298: G1004-G1012, 2010). The effect of ethanol on AMP-activated protein kinase phosphorylation was reversed by imipramine, suggesting that the generation of ceramide via acid sphingomyelinase (ASMase) is stimulated by ethanol. In this study, we determined the effects of imipramine on the development of hepatic steatosis, the generation of ceramide, and downstream effects of ceramide on inflammatory, insulin, and apoptotic signaling pathways, in ethanol-fed mice. The effect of ethanol and imipramine (10 μg/g body wt ip) on ceramide levels, as well as inflammatory, insulin, and apoptotic signaling pathways, was studied in C57BL/6J mice fed the Lieber-DeCarli diet. Ethanol-fed mice developed the expected steatosis, and cotreatment with imipramine for the last 2 wk of ethanol feeding resulted in improvement in hepatic steatosis. Ethanol feeding for 4 wk induced impaired glucose tolerance compared with controls, and this was modestly improved with imipramine treatment. There was a significant decrease in total ceramide concentrations in response to imipramine in ethanol-fed mice treated with and without imipramine (287 ± 11 vs. 348 ± 12 pmol/mg tissue). The magnitude and specificity of inhibition on each ceramide species differed. A significant decrease was observed for C16 (28 ± 3 vs. 33 ± 2 pmol/mg tissue) and C24 (164 ± 9 vs. 201 ± 4 pmol/mg tissue) ceramide. Ethanol feeding increased the levels of the phosphorylated forms of ERK slightly and increased phospho-p38 and phospho-JNK substantially. The levels of phospho-p38 and phospho-JNK were reduced by treatment with imipramine. The activation of ASMase and generation of ceramide in response to ethanol feeding may underlie several effects of ethanol. ASMase inhibitors may be considered as a therapeutic target for alcohol-induced hepatic steatosis and activation of stress kinases.     

Apoptosis induced by doxorubicin in neurotumor cells is divorced from drug effects on ceramide accumulation and may involve cell cycle-dependent caspase activation

Doxorubicin (0.5 microgram/ml) induced caspase-dependent apoptosis in SH-SY5Y neuroblastoma and CHP-100 neuroepithelioma cells. The apoptotic response started to be evident approximately 15 h after drug administration and, as monitored over a 48-h period, was more pronounced in CHP-100 than in SH-SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B(1) (25 microM); this compound, however, did not prevent drug-induced apoptosis. Untreated cells from both lines expressed negligible p53 levels; on the other hand, whereas p53 and p21(Cip1/Waf1) were rapidly up-regulated in doxorubicin-treated SH-SY5Y cells, such a response was not observed in CHP-100 cells. Doxorubicin induced a G(2)/M phase block in both cell lines, but whereas the G(1) phase was markedly depleted in CHP-100 cells, it was substantially retained in SH-SY5Y cells. In the latter system, double G(1) and G(2)/M block largely preceded cell death; however, as apoptosis underwent completion, it selectively targeted late S and G(2)/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G(1) cell population. These results support the notion that doxorubicin-induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G(1) cells appear to be refractory to doxorubicin-induced apoptosis, p53 up-regulation and p21(Cip1/Waf1) expression may provide an unfavorable setting for the apoptotic action of the drug.     

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.     

Regulation of the selective uptake of cholesteryl esters from high density lipoproteins by sphingomyelin

Although sphingomyelin (SM) is a major phospholipid in lipoproteins as well as in the membrane rafts where the scavenger receptor class B type I (SR-BI) is localized, its possible role in the selective uptake of cholesteryl ester (CE) by the SR-BI-mediated pathway is unknown. We investigated the effect of SM in lipoproteins and cell membranes on the selective uptake in three different cell lines: SR-BI-transfected CHO cells, hepatocytes (HepG2), and adrenocortical cells (Y1BS1). Incorporation of SM into recombinant high density lipoprotein (rHDL) containing labeled CE resulted in up to 50% inhibition of the selective uptake of CE in all three cell lines. This inhibition was completely reversed by treatment of rHDL with sphingomyelinase (SMase). Selective uptake from plasma HDL was activated by 22-72% after treatment of HDL with SMase. In addition, pretreatment of the cells with SMase resulted in stimulation of CE uptake from rHDL by CHO and Y1BS1, although not by HepG2. Incorporation of ceramide into rHDL resulted in up to 2-fold stimulation of CE uptake, although pretreatment of cells with egg ceramide had no significant effect. These results show that SM and ceramide in the lipoproteins and the cell membranes regulate the SR-BI-mediated selective uptake of CE, possibly by interacting with the sterol ring or with SR-BI itself.     

Mobile phase compositions for ceramide III by normal phase high performance liquid chromatography

Ceramide III was prepared by the cultivation ofSaccharomyces cerevisiae. Ceramide III was partitioned from the cell extracts by solvent extraction and analyzed by Normal Phase High Performance Liquid Chromatography (NP-HPLC) using Evaporative Light Scattering Detector (ELSD). We experimentally determined the mobile phase composition to separate ceramide III with NP-HPLC. Three binary mobile phases of n-hexane/ethanol,n-hexane/Isoprophyl Alcohol (IPA) andn-hexane/n-butanol and one ternary mobile phase ofn-hexane/IPA/methanol were demonstrated. For the binary mobile phase ofn-hexane/ethanol, the first mobile phase composition, 95/5 (v/v), was step-increased to 72/23 (v/v) at 3 min. In the binary mobile phase, the retention time of ceramide III was 7.87 min, while it was 4.11 min respectively in the ternary system, where the mobile phase composition ofn-hexane/IPA/methanol, 85/7/8 (v/v/v), was step-increased to 75/10/15 (v/v/v) at 3 min. However, in the ternary mobile phase, the more peak area of ceramide III was observed.     

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.