利用微波诱变技术提高菌株B1抑菌活性的研究

目的研究微波辐射技术对具强抑菌活性的海洋微生物紫外突变株B1的诱变效应,并获得诱变处理的合适剂量。方法对处于对数生长期的菌悬液进行不同时间微波辐射处理,基于微生物诱变育种随机性,利用数理统计方法对诱变结果分析评估,并对筛选的高活性菌株进行传代。结果60s微波辐射为最适诱变剂量,在此处理上获得的1株高活性橙色菌株B1-413,其最小抑菌浓度(MIC)由亲代B1的125μl.mL-1,降低至突变株B1-413的32μl.mL-1,抑菌活性提高了近4倍,并经传代实验证实其抑菌活性稳定。结论菌株B1对微波辐射敏感,诱变效应明显。     

微波诱变及添加氯化镧筛选阿扎霉素B高产菌株

目的 拟通过微波诱变和添加氯化镧对阿扎霉素B产生菌N98-1634进行选育,获得高产菌株.方法 对菌株进行40～240s的微波诱变,采用金黄色葡萄球菌抑菌圈法获得初筛通过株,并进行HPLC复筛获得高产菌株;在高产菌株发酵培养基中加入5～30mg/L的氯化镧考查其对阿扎霉素B合成的影响.结果 通过微波诱变选出l株阿扎霉素B产量达到945.0μg/mL的突变菌株MW-638,较出发菌株N98-1634的752.4μg/mL提高了25.6％,随后经过添加15mg/L氯化镧阿扎霉素B单位最终达到1413.8μg/mL.结论 微波诱变对提高菌株N98-1634阿扎霉素B发酵单位效果明显,诱变菌株添加适量氯化镧可以显著提高其产量.     

微波诱变在药源微生物菌株选育中的应用

微波诱变是一种较新兴非电离电磁辐射物理诱变育种技术,已开始应用于农作物、植物以及微生物育种研究中,并在药源微生物产能提高以及拓展活性菌株新资源相关研究中展露出很好的应用发展前景。本文结合本课题组新近研究进展,综述了微波的生物效应与诱变机制、诱变实验方法与影响诱变效果的因素、微波诱变技术在药源微生物菌株选育研究中的应用进展等,并对微生物微波诱变育种研究现状、优势和不足以及应用发展前景进行了讨论。     

紫外分光光度法快速测定庆大霉素 C1a 含量在高通量筛选中的应用

目的 探索庆大霉素 C1a 含量的快速检测方法,实现诱变菌株的高通量筛选。方法 以紫外分光光度法作为检测庆 大霉素 C1a 含量的手段,对庆大霉素 C1a 单组分生产菌株进行 ARTP、LiCl、微波和 ARTP+LiCl 诱变,通过高通量筛选获得高产 菌株,并在 5L 发酵罐中对高产稳定性菌株进行验证。结果 紫外分光光度法和高效液相色谱法对发酵液中庆大霉素 C1a 含量进 行测定,最大相对误差为 3.98%;筛选获得了 10 株高产菌株,其中 AL324 菌株与出发菌株相比,摇瓶效价提高了 52%;通过稳 定性传代实验,获得了 4 株高产稳定性菌株。5L 发酵罐验证实验表明 AL324 与出发菌株相比效价提高了 81.3%。结论 紫外分 光光度法可以快速检测发酵液中庆大霉素 C1a 含量,该方法应用于高通量筛选中,获得了高产菌株,筛选结果表明 ARTP+LiCl 复合诱变方法的正突变率较高,是一种有效的庆大霉素 C1a 高产菌株诱变方式。     

复合诱变选育乳链菌肽高产菌株

目的选育乳链菌肽高产菌株。方法以乳酸链球菌ATCC11454为出发菌株,采用紫外线、微波和亚硝酸钠对其进行复合诱变处理,并结合乳链菌肽抗性筛选选育出高产乳链菌肽的菌株。结果通过选育获得一株乳链菌肽的高产菌株Nis-123,乳链菌肽产量为5 250 U/mL,较出发菌株提高了4.3倍。传代实验证明该菌株遗传性稳定。结论紫外线、微波和亚硝酸钠复合诱变结合乳链菌肽抗性选育可以提高乳酸链球菌的产肽能力。     

微波在顶头孢霉菌选育中的应用

头孢菌素 C 是半合成头孢菌素类抗生素的重要原料,通过对其产生菌顶头孢霉菌的诱变研究,能使发酵单位不断提高。本实验通过用微波诱变顶头抱霉菌 B_(42)节孢子,得到变株 B42—V2,单位提高16.03%,且遗传性状非常稳定。实验表明:微波诱变育种效果好、操作简便、所选育菌株遗传性状稳定。     

达托霉素高产菌株选育及发酵条件优化

目的 通过对达托霉素产生菌进行诱变选育,并对其发酵条件优化,以提高其达托霉素发酵水平,降低生产成本。方法 以玫瑰孢链霉菌(DT-9#F2)为出发菌株,分别采用紫外诱变、微波诱变、LiCl诱变及复合诱变并结合链霉素抗性筛选进行菌株选育,同时,对其发酵培养基中氮源、碳源及生长因子进行优化,以进一步提高其发酵产量。结果 得到一株达托霉素高产突变株DT-37,其摇瓶发酵产量达到12.2mg/L,较出发菌株提高20.79%;优化后的发酵培养基为：酵母粉(YP300)1.65%、FeSO4 0.043%、葡萄糖1.50%、玉米淀粉7.20%、糖蜜0.72%,VB12 1.50μg/mL、硫辛酸5.00μg/mL。其摇瓶发酵产量达到30.56mg/L,较初始培养基提高了297.92%。经100L发酵罐上放大验证,达托霉素的产量达到了2872mg/L,较优化前提高了14.88%。结论 紫外、微波及LiCl复合诱变后经抗生素抗性筛选,结合发酵培养基优化,可有效提高菌株的达托霉素发酵产量。     

产抗生素角毛壳菌CH21的诱变育种

以角毛壳菌(Chaetomium cupreum)CH21的萌发孢子作为诱变材料,采用微波辐照和紫外线照射进行诱变处理,用辣椒炭疽菌(Colletotrichum capsici)作为测试菌株,结合PDA双层平板法初筛和管碟法复筛,选育高产抗生素的突变株.结果表明:最佳微波诱变条件为500W、2450MHz、15s,紫外诱变条件为15W、30cm、300s.对出发菌株CH21进行一轮微波诱变和一轮紫外诱变,筛选得到5株高产菌株,其中CH21-2-20抑菌圈直径最大,为20.20mm,较抑菌圈直径为12.50mm的出发菌株增大了62％.连续传代5次,CH21-2-20抑菌圈直径稳定在19.40～20.38mm.     

纳他霉素产生菌的诱变育种及其发酵条件的优化

目的 以纳塔尔链霉菌(Streptomyces natalensis)SIPI-NHF-09为出发菌株进行诱变选育和培养条件优化,以提高其纳他霉素的产量.方法 采用紫外和微波结合对纳塔尔链霉菌SIPI-NHF-09进行诱变选育,并对获得的高产突变株SIPI-UW-10-22的发酵条件进行了初步优化.结果 经过连续3轮的紫外和微波诱变,获得高产突变株SIPI-UW-10-22,其纳他霉素(Natamycin)产量达到3.15g/L,是出发菌株的3.5倍,且该菌株经多次传代,遗传性能稳定.对SIPI-UW-10-22发酵条件优化后获得其发酵条件为:以5.5％玉米淀粉加0.5％葡萄糖为碳源,2％黄豆饼粉加1％酵母粉为氮源,种龄46h,初始pH7.0,接种量8％,摇床转速220r/min,28℃培养126h.此条件下,纳他霉素的产量达到4.07g/L,较优化前提高了1.29倍.结论 紫外与微波诱变相结合的方法简便有效,所获得高产菌株遗传性状稳定.     

(R)-(-)-扁桃酸的不对称生物合成

研究了生物不对称催化苯乙酮酸合成（R）-（-）-扁桃酸的方法。通过对38株菌进行初筛,获得对底物苯乙酮酸具有较高转化活性的出发菌株假丝酵母Ca.3。进一步紫外与微波诱变,得到具有高转化率与高对映体选择性的突变菌株Ca.3.37.48。在转化培养中,选择初始底物浓度30mmol/L、pH7.0、温度32℃的条件,（R）-（-）-扁桃酸得率86.5%,对映体过量值为99.5%。     

顶空气相色谱法测定头孢哌酮钠他唑巴坦钠中残留有机溶剂

目的建立头孢哌酮钠他唑巴坦钠中甲醇、丙酮溶剂残留量的测定方法。方法采用顶空气相色谱法，以去离子水为溶剂，氮气为载气。进样口温度为200℃，检测器温度为250℃。结果甲醇、丙酮的质量浓度分别在37．97～189．84mg／L和63．68—318．4mg／L的范围内与峰面积线性关系良好，r分别为0．9985和0．9991，平均回收率分别为104．20％（RSD=1．6％，n=6）和104．13％（RSD=1．3％，n=6）．最低检测限分别为0．21mg／L和0．32mg／L。结论顶空气相色谱法简单、准确，可用于头孢哌酮钠他唑巴坦钠的有机溶剂残留测定。     

吴茱萸次碱对棕榈酸诱导的脂质损伤肝细胞的影响

目的:研究吴茱萸次碱(Rut)对棕榈酸(PA)诱导的脂质损伤肝细胞(LO2)的细胞活力、活性氧(ROS)以及缝隙连接蛋白32(Cx32)和缝隙连接蛋白26(Cx26)表达水平的影响。方法:加入不同浓度的Rut溶液预处理LO2细胞20 min后,每个孔加入PA(0.2 mmol/L)溶液,共同孵育24 h,分别应用细胞增殖及毒性检测试剂盒(CCK-8)检测细胞活力,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光探针检测细胞内活性氧的表达、划痕负载实验检测缝隙连接细胞间通讯(GJIC)的功能变化及免疫印迹实验(Western blotting)检测缝隙连接蛋白Cx32和Cx26的表达水平。结果:CCK-8实验结果显示,Rut(0.1μmol/L,0.5μmol/L,1μmol/L)能抑制PA诱导的LO2细胞活力的下降;DCFH-DA荧光探针实验显示PA诱导细胞后ROS的表达增强,Rut预处理细胞后能够减少ROS的表达;划痕负载实验结果显示,PA诱导细胞后能够抑制细胞的GJIC功能,预先给予Rut处理能够改善GJIC的功能障碍;Western blotting显示PA诱导细胞后缝隙连接蛋白Cx32和Cx26的表达水平显著降低,给予Rut预处理后能够恢复Cx32和Cx26的表达。结论:PA能够诱导LO2细胞的脂质损伤,导致细胞活性降低、细胞内活性氧增加以及GJIC的功能障碍;Rut预处理LO2细胞后可减轻PA诱导的细胞损伤,并且恢复细胞的缝隙连接蛋白Cx32和Cx26的表达和GJIC的功能。     

青霉素G酰化酶工程菌培养条件的研究

以产生青霉素G酰化酶的工程菌株LRN075为研究对象,通过正交试验优化了培养条件,研究了不同比例的培养基成分对酶产量的影响.结果显示在最优条件下,进行了2000L发酵罐的发酵试验,该菌株在培养36h酶活最高达到2.28×104U·L-1.细胞破碎实验表明,高压均质法效果最佳,可以使酶达到最大程度的释放.     

Urinary arsenic and porphyrin profile in C57BL/6J mice chronically exposed to monomethylarsonous acid (MMAIII) for two years

Arsenicals are proven carcinogens in humans and it imposes significant health impacts on both humans and animals. Recently monomethylarsonous acid (MMA(III)), the toxic metabolite of arsenic has been identified in human urine and believed to be more acutely toxic than arsenite and arsenate. Arsenic also affects the activity of a number of haem biosynthesis enzymes. As a part of 2-year arsenic carcinogenicity study, young female C57BL/6J mice were given drinking water containing 0, 100, 250 and 500 microg/L arsenic as MMA(III)ad libitum. 24 h urine samples were collected at 0, 1, 2, 4, 8 weeks and every 8 weeks for up to 104 weeks. Urinary arsenic speciation and porphyrins were measured using HPLC-ICP-MS and HPLC with fluorescence detection respectively. DMA(V) was a major urinary metabolite detected. Significant dose-response relationship was observed between control and treatment groups after 1, 4, 24, 32, 48, 56, 88, 96 and 104 weeks. The level of uroporphyrin in 250 and 500 microg As/L group is significantly different from the control group after 4, 8, 16, 32, 56, 72, 80, 96 and 104 weeks. Coproporphyrin I level in 500 microAs/L group is significantly different from control group after 8, 24, 32, 40, 56, 72, 80, 88 and 104 weeks. After 4 weeks the level of coproporphyrin III concentration significantly increased in all the treatment groups compared to the control except week 16 and 48. Our results show urinary DMA(V) and porphyrin profile can be used as an early warning biomarker for chronic MMA(III) exposure before the onset of cancer.     

Cholesterol-lowering effect of NK-104, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, in guinea pig model of hyperlipidemia

Although benefits of statins have been demonstrated even in normolipidemic patients at high risk, the main target of statin therapy is the hypercholesterolemic patient. The aim of this study was to examine the hypocholesterolemic effect of NK-104 ((+)-monocalcium bis((3R,5S,6S)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]- 3,5-dihydroxy-6-heptenoate), CAS 147526-32-7), a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and its mechanism of action in hypercholesterolemic animals. In guinea pigs fed a diet containing 15% (w/w) fat rich in laurate for 6 weeks, the liver cholesterol content was markedly increased and plasma total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and LDL-apoB were elevated 4.8, 5.2 and 1.7 times, respectively, compared with normal diet fed animals. These changes were maintained by reduced LDL clearance in the presence of markedly cholesterol-enriched LDL in the plasma. In this model, the LDL-C reduction rates by 0.1, 0.3 and 1 mg/kg of NK-104 orally administered for 2 weeks (from week 4 to week 6), were 11, 27 and 32%, respectively, from controls, being similar in normal guinea pigs previously examined. Those for 3 and 10 mg/kg of atorvastatin (CAS 134523-00-5) were 25 and 39%, respectively. Thus about 10 times higher doses of atorvastatin were required than of NK-104 to cause a similar cholesterol-lowering effect. This reduction of plasma cholesterol was accompanied by an improvement of LDL clearance (24 and 47% increase in fractional catabolic rate by 1 mg/kg of NK-104 and 10 mg/kg of atorvastatin, respectively) and LDL composition. In conclusion, in guinea pig hypercholesterolemia caused by high-laurate diet, NK-104 and atorvastatin lowered plasma cholesterol levels with an improvement of LDL composition and with an increase in LDL clearance, presumably through reduction of the liver cholesterol content, although hepatic cholesterol synthesis might have been markedly suppressed in this model.     

Hypolipidemic effect of NK-104 and other 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in guinea pigs

Hypolipidemic effects of various HMG-CoA reductase inhibitors (statins) were examined in guinea pigs. After 2-week administration of NK-104 ((+)-monocalcium bis((3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]-3, 5-dihydroxy-6-heptenoate), CAS 147526-32-7), simvastatin (SV), pravastatin (PV), fluvastatin (FV), cerivastatin (CV) and atorvastatin (AV) to guinea pigs at various doses, which did not affect body weight, plasma and liver lipids were measured. Liver endoplasmic reticulum (ER) was isolated and measured for lipid content and activity of ER enzymes (NK-104: 3 mg/kg, SV: 30 mg/kg). Each statin except CV dose-dependently lowered total cholesterol (TC), with NK-104 showing the greatest effect. Only NK-104 and AV, long-acting statins, significantly lowered triglycerides (TG). A significant reduction of the liver cholesterol content was observed for NK-104 and AV. The liver TG content increased with dose for SV or PV, but not for NK-104 or AV. The TG content in ER and activity of diacylglycerol acyltransferase of ER, increased with SV and slightly with NK-104. Activity of microsomal triglyceride transfer protein, which is necessary for both apoB particle formation at rough ER and TG droplet production at smooth ER, increased by SV but not by NK-104. It is suggested that statins with prolonged action lower TG by decreasing cholesterol ester supply necessary for very low density lipoprotein formation and by attenuating an increase in TG production at ER after HMG-CoA reductase inhibition.     

麦迪霉素与乙酰螺旋霉素治疗104例感染随机对照观察

应用麦迪霉素和乙酰螺旋霉素随机对照治疗革兰阳性球菌和厌氧菌感染104例,包括口腔感染、耳鼻喉感染、呼吸系感染和皮肤软组织感染等,临床有效率各为90.6％和90.2％,细菌清除率各为89.4％和90.7％;临床应用中两组不良反应均低微。上述结果统计学处理皆无显著差异,提示西药的疗效和安全性相仿。麦迪霉素和乙酰螺旋霉素对本系列24株葡萄球菌的MIC_(50)分别为2mg/L和32mg/L,敏感率分别为67％和46％;纸片法药敏结果显示,37株临床分离厌氧菌对两药呈现敏感者分别为30株和31株。     

荧光光谱法研究左氧氟沙星与酪蛋白的相互作用

目的:研究左氧氟沙星与酪蛋白的相互作用,为制备以酪蛋白为载体的左氧氟沙星新型药物传输系统提供依据。方法:应用荧光光谱技术研究了各组分的荧光发射光谱;并且在不同温度下(25℃、30℃)进行了2组分相互作用的猝灭试验,计算得到相关热力学参数和结合常数等。结果:左氧氟沙星对酪蛋白的内源性荧光有较强的猝灭作用;推断猝灭机制为静态猝灭;计算得到静态猝灭结合常数为2.83×104L·mol-1(25℃)和2.17×104L·mol-1(30℃),平均结合位点数为1.38。结论:左氧氟沙星与酪蛋白2种分子以接近1∶1的比例,通过氢键和范德华力发生相互作用;本研究为制备以酪蛋白为载体的左氧氟沙星传输系统提供了药载比等相关信息。     

阿维拉霉素高产菌株的选育

目的诱变选育阿维拉霉素高产菌株。方法紫外线随机诱变结合阿维拉霉素、2-氧-D-葡萄糖和高浓度CaCl2抗性筛选的推理选育方案。结果通过选育获得了抗CaCl2的高产菌株Avl-104,其阿维拉霉素产量为800mg/L,产量较出发菌株提高了217.4%。稳定性研究发现Avl-104具有遗传不稳定的特性,菌种高产特性退化严重,但通过自然分纯的方法,得到菌株Avl-104-312,其阿维拉霉素产量达1 222 mg/L。结论紫外随机诱变结合高浓度CaCl2抗性推理选育辅以自然分纯可以提高阿维拉霉素的产素能力。利用阿维拉霉素高产菌株的遗传不稳定性,通过自然分纯,不但可以维持其原有的高产特性,也可以进一步提高高产菌株的产素能力。     

Tissue-specific expression of human lipoprotein lipase in the vascular system affects vascular reactivity in transgenic mice.

1. The role of smooth muscle-derived lipoprotein lipase (LPL) that translocates to the endothelium surface on vascular dysfunction during atherogenesis is unclear. Thus, the role of vascular LPL on blood vessel reactivity was assessed in transgenic mice that specifically express human LPL in the circulatory system. 2. Aortic free fatty acids (FFAs) were increased by 69% in the transgenic mice expressing human LPL in aortic smooth muscle cells (L2LPL) compared with their non-transgenic littermates (L2). 3. Contractility to KCl was increased by 33% in aortae of L2LPL mice. Maximal contraction to phenylephrine (PE) was comparable in L2 and L2LPL animals, while the frequency of tonus oscillation to PE increased by 104% in L2LPL mice. 4. In L2LPL animals, *NO mediated relaxation to acetylcholine (ACh) and ATP was reduced by 47 and 32%, respectively. In contrast, endothelium-independent relaxation to sodium nitroprusside (SNP) was not different in both groups tested. 5. ATP-initiated Ca(2+) elevation that triggers *NO formation was increased by 41% in single aortic endothelial cells freshly isolated from L2LPL animals. 6. In aortae from L2LPL mice an increased *O(2)(-) release occurred that was normalized by removing the endothelium and by the NAD(P)H oxidase inhibitor DPI and the PKC inhibitor GF109203X. 7. The reduced ACh-induced relaxation in L2LPL animals was normalized in the presence of SOD, indicating that the reduced relaxation is due, at least in part, to enhanced *NO scavenging by *O(2)(-). 8. These data suggest that despite normal lipoprotein levels increased LPL-mediated FFAs loading initiates vascular dysfunction via PKC-mediated activation of endothelial NAD(P)H oxidase. Thus, vascular LPL activity might represent a primary risk factor for atherosclerosis independently from cholesterol/LDL levels.