微生物法制备抗抑郁药R-托莫西汀中间体

从 2 0株酵母菌中筛选出了两种还原 3 氯 1 苯丙酮生成高光学纯度R 3 氯 1 苯丙醇的较好菌株SaccharomycesB5和Candida 10 4 ,R 3 氯 1 苯丙醇的对映体过剩值为 10 0 % .研究了温度、pH、反应时间、底物质量浓度、菌体添加量等微生物培养条件及反应条件对两种菌种还原 3 氯 1 苯丙酮的影响 .以体积分数 5 %的乙醇为共底物 ,可使SaccharomycesB5和Candida 10 4转化底物的产率分别从 17%和 9%提高到 5 0 %和 4 8%     

重组大肠杆菌BL21（DE3）-pET28a（＋）-bgl诱导表达β-葡聚糖酶的条件优化

研究自行构建的产β-葡聚糖酶的工程菌E.coli BL21(DE3)-pET28a(+)-bgl在LB培养基中的生长特性,考察种子液的菌龄、培养基起始pH、接种量及诱导起始时发酵液菌浓度等对β-葡聚糖酶产生水平的影响;通过正交试验确定诱导剂IPTG及乳糖添加量、诱导温度及诱导剂作用时间.结果表明:培养基起始pH 7.0,对数生长中期的种子液(OD600为0.35)以接种量(体积分数)10%接入摇瓶发酵培养,37 ℃,200 r/min培养约3 h,菌液OD值达到1.0左右,添加终浓度分别为0.033 6 mmol/L的IPTG及10 mmol/L乳糖,24℃诱导6 h,发酵液清液中酶活达到最高(336.33 U/mL),菌体生长量为1.12 g/L,发酵液中总酶活达到459.32 U/mL,是原始菌株在相同条件下所产酶活的6.62倍.采用优化培养条件及诱导剂作用条件,重组菌在TB培养基中酶活水平进一步提高,诱导剂作用10 h,发酵清液中酶活为1 090.31 U/mL,总酶活1 570.83 U/mL,是原始菌在该条件下酶活的19.73倍,显示出重组菌具有广阔的工业化应用前景.     

利用his-tag纯化和复性基因工程产物肠激酶

构建了融合表达肠激酶轻链（EKLC）的基因工程大肠杆菌，将该菌于37℃在含30mg／L卡那霉素的LB培养基中培养，当细胞密度（OD600）达到0．5～0．6时加入最终浓度为0．4mmol／L的IPTG并降温至26℃进行诱导表达，4h后目的蛋白质的表达量可达菌体总蛋白质的40％以上，但所表达的重组产物大多以包涵体形式存在于菌体中。利用产物N端携带6个组氨酸标签与金属螯合层析介质中镍离子的亲和性，应用镍离子金属螯和层析对样品包涵体进行了纯化和复性研究。实验结果表明，在变性条件下纯化的样品在柱上不经洗脱，直接用复性液洗涤6h，可以使EKLC在得到提纯的同时实现复性，得到了电泳纯度为96％的具有生物活性的EKLC，最高活性为712U。     

好氧同时硝化-反硝化脱氮微生物的混合培养

筛选和分离得到的多株脱氮微生物 ,能在完全好氧条件下将氨氮转化为NO2 - ,随即在好氧反硝化菌的作用下还原为N2 排放 ,整个生物脱氮过程历时较短 ,30h内对 2 0 0mg/L的氨氮去除率达 99% ,而且无中间产物NO2 - 的积累 .混合脱氮微生物菌群生长的适宜 pH范围为 7～ 10 ,探索实现混合脱氮微生物菌群高密度培养的 pH :发酵前期补酸控制 pH≤ 8,发酵中后期不控制 pH值 ,可缩短菌体的生长周期 ,提高菌体的氨氮降解速率 ,细胞质量浓度达 3.9g/L ,比自然 pH条件下提高了 6 2 .5 % .     

耐热过氧化氢酶基因工程菌的构建及其发酵条件

利用PCR扩增技术以嗜热脂肪芽孢杆菌IAM11001染色体DNA为模板，扩增得到嗜热脂肪芽孢杆菌过氧化氢酶编码基因perA。再与经EcoRⅠ酶切的温控表达载体pBV220连接，构建重组质粒，并转化宿主大肠杆菌JM109，得到耐热过氧化氢酶基因工程菌，然后对该工程菌在LB培养基和半合成培养基中的发酵条件进行了优化。结果表明：在LB培养基中，诱导时期和酶合成最佳诱导时间均为5h，最大产酶量达到72．9U／mL；在半合成培养基中，于30℃培养4h，再于42℃诱导培养6h，产酶量最高达到131U／mL。     

法夫酵母产虾青素的补料发酵

以法夫酵母 (Phaffiarhodozyma)WSS FF6为产生菌进行产虾青素的补料发酵 ,在通气量为 2 5 0L/h、pH =6.0± 0 .5的条件下 ,先流加高糖浓度的培养基 ,后添加 0 .1%乙醇 ,进行分批补料发酵 ,经 130h发酵后 ,生物量与类胡萝卜素产量分别为 2 7.4mg/mL、2 6.12 μg/mL ,生长得率、产物得率及酵母色素质量分数分别为 0 .4 6、0 .4 4和 0 .95     

大肠杆菌60Co诱变育种及其发酵生产聚唾液酸条件

对产聚唾液酸菌株大肠杆菌WX J11进行6 0 Co诱变 ,筛选出高产突变株WX J4 ,其聚唾液酸产量可达 10 0 1mg/L ,通过优化发酵条件即在 2 50mL的摇瓶中 ,2 50r/min转速下 ,37℃恒温培养 6 5h ,起始 pH值为 7.8,装液量为 4 0mL ,使聚唾液酸的产量提高到 150 0mg/L .聚唾液酸在菌体生长停止后释放到培养基中 ,动力学上表现为次级代谢产物 .此外 ,还对聚唾液酸的提取、水解和唾液酸的纯化作了初步研究 .     

雌二醇对人前列腺间质细胞转化生长因子β与平滑肌细胞特异性抗原表达的影响

目的　探讨人前列腺组织中雌激素、转化生长因子 β(TGFβ)、平滑肌细胞三者的关系。　方法　成功培养 11株良性前列腺增生患者的前列腺间质细胞 ,给予雌二醇 (E2 )刺激 ,MTT法观察细胞增殖 ,RT PCR法观察TGFβ1和TGFβ2 以及平滑肌细胞特异性抗原 (smoothelin)的mRNA表达。　结果　E2 作用 4 8h后 ,人前列腺间质细胞TGFβ2 和smoothelin相对表达量分别为 0 .194 6～1.2 4 0 3和 0 .15 5 4～ 0 .4 2 34,与对照组相比差异均有显著性意义 (P <0 .0 5 ) ,且两者表达量呈正相关 (P<0 .0 5 ) ;TGFβ1相对表达量与对照组差异无显著性意义 (P >0 .0 5 )。　结论　E2 可能通过诱导TGFβ2 表达而影响前列腺平滑肌细胞生长分化。     

重组大肠杆菌生产人表皮生长因子的发酵条件

通过质粒转化实验,获得了较好的产hEGF重组菌,对重组菌发酵条件进行优化,获得最佳初始糖质量浓度为5g/L、蛋白胨20g/L、酵母抽提物10g/L、(NH4)2HPO43.5g/L、Amp100mg/L;最佳接种时间为种子液生长到5～6h,即菌体OD值在1.0～2.0;诱导剂最佳添加时间为8h,即菌体OD值在8.0左右.通过流加发酵进行高密度培养,可使重组菌的hEGF的产率达102mg/L,比优化前提高了近30%.     

米曲霉腺苷酸脱氨酶的产酶条件

通过实验获得了米曲霉 3.80 0固态发酵产腺苷酸 (AMP)脱氨酶的适宜培养基 ,麸皮为主原料 ,成分质量分数为蔗糖 2 % ,鱼粉 2 % ,(NH4 ) 2 SO4 0 .1% ,吐温 80 0 .1% ,含水量 5 0 % .最佳的培养条件为 :2 5 0mL的三角瓶装 2 0 g培养基 ,在 2 8～ 30℃培养 6 0h .发酵结束称取一定量的麸曲加 10倍质量的蒸馏水 ,调节 pH 6 .0 ,在 35℃条件振荡水浴 2h .酶的浸提液经盐析、透析、DEAE柱层析分离后 ,可收集到较单一的活性峰酶 .     

Highly efficient release of lovastatin from poly(lactic‐co‐glycolic acid) nanoparticles enhances bone repair in rats

Lovastatin exhibits higher thermal stability and lower degradation rate than simvastatin. However, the amount of research studying a lovastatin delivery device has been far less than similar research on simvastatin. As a consequence, a high lovastatin release rate system has not been developed. We hypothesized that highly efficient release of lovastatin from poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles in a short‐term release (7 days) could provide an effective delivery system for bone repair. This study optimized the emulsion (o/w) technique in the fabrication process for PLGA nanoparticles, thereby producing the first recorded case of a high release rate (97%) of lovastatin. We also calculated the calibration curve of lovastatin using a UV spectrometer. The results demonstrated that the ALPase activity in human osteoblasts could be significantly stimulated by lovastatin carried in PLGA nanoparticles, but was prominently decreased by free lovastatin with the concentration higher than 4 µg/ml. Animal studies showed that the amount of lovastatin contained in 1 mg PLGA was the optimum dosage. These results suggest the new lovastatin‐releasing PLGA delivery device exhibits potential for clinical treatment of bony defects. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1504–1510, 2011     

Lovastatin in the treatment of multifactorial hyperlipidemia associated with proteinuria

The efficacy and safety of lovastatin as a hypolipidemic agent were evaluated in ten adult patients with secondary hypercholesterolemia due to proteinuria (greater than 2 g/d) and (in seven patients) concurrent corticosteroid therapy. Patients were on a low-cholesterol diet throughout the study. After a 4-week baseline period, patients were randomized to receive either placebo or 10 mg lovastatin twice daily for a period of 6 weeks. The dose of lovastatin was increased to 20 mg twice daily for 6 weeks, and 40 mg twice daily for 6 weeks in the latter group. Those patients who received placebo for the first 6 weeks subsequently received 10, 20, and 40 mg of lovastatin twice daily in a stepped dose regimen, with each dose given for 6 weeks. Lovastatin was well tolerated by all patients and none withdrew from the study. Baseline plasma cholesterol concentrations (390 +/- 20 mg/dL; mean +/- SEM) decreased 22% (P less than 0.003) at the lowest dose of 10 mg twice daily, 27% at 20 mg twice daily, and 33% at 40 mg twice daily. Baseline plasma triglycerides decreased by 25% (P less than 0.05) at the highest dosage. Concentrations of low-density lipoprotein (LDL) cholesterol fell by 29%, 34%, and 45% on doses of 10, 20, and 40 mg of lovastatin twice daily. Concentrations of high-density lipoprotein (HDL) cholesterol increased slightly. Serum creatinine concentrations and proteinuria were not affected by lovastatin therapy. We conclude that lovastatin was a well-tolerated and extremely effective hypocholesterolemic agent in patients with persistent secondary hypercholesterolemia associated with proteinuria or proteinuria and concurrent corticosteroid therapy.     

Lovastatin treatment of hypercholesterolemia in renal transplant recipients

The treatment of hypercholesterolemia in renal transplant recipients has been problematic. In the present double-blind study, 11 patients were treated with diet for at least 4 weeks. They were then randomized to placebo or the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, lovastatin (20 mg/day) for 6 weeks, followed by crossover to an additional 6 weeks of lovastatin or placebo. All patients had stable allograft function 8.4 +/- 1.2 years (mean +/- SEM) after transplantation, and received low-dose prednisone and azathioprine immunosuppression. Compared with diet alone, lovastatin caused a 21% reduction in total cholesterol from 307 +/- 14 mg/dL to 244 +/- 13 mg/dL (P less than 0.05). Lovastatin reduced LDL cholesterol 28% from 214 +/- 12 mg/dL to 155 +/- 11 mg/dL (P less than 0.05). Trends toward favorable changes in HDL cholesterol, serum triglycerides, and apolipoproteins were not statistically significant. Liver enzymes, creatine phosphokinase, and renal function remained stable. With lovastatin there was a 27% increase in the WBC (from 6220 +/- 530 cells/mm3 to 7780 +/- 510 cells/mm3, P less than 0.05) that was attributable to a 45% increase in neutrophils (P less than 0.05). This effect of lovastatin, possibly the result of reduced azathioprine bone marrow suppression, could have important implications for immunosuppressive therapy in this patient population. Altogether, these results suggest that lovastatin may be a safe and effective treatment for hypercholesterolemia in renal transplant recipients receiving conventional immunosuppression.     

Percutaneous lovastatin accelerates bone healing but is associated with periosseous soft tissue inflammation in a canine tibial osteotomy model

Experimental studies have shown the ability of statins to stimulate bone formation when delivered locally or in large oral doses, however most have been studied in rodents. This anabolic effect is through the selective activation of BMP‐2. Our purpose was to determine the effects of local treatment with lovastatin on bone healing in mammals as a preclinical animal model. We administered lovastatin (6 mg/kg) by percutaneous injection to a canine tibial osteotomy stabilized with external fixation. We found that lovastatin improved bone healing after a single injection into the fracture site assessed by serial radiography and histology at bone union. However, lovastatin treatment resulted in adverse local soft tissue inflammation. These results suggest that percutaneous lovastatin injection may be a useful adjuvant treatment over the course of bone healing to augment fracture repair, although further investigation into the mechanism of soft tissue adverse effects is warranted. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:210–216, 2014.     

Lovastatin inhibits transforming growth factor-beta1 expression in diabetic rat glomeruli and cultured rat mesangial cells

Diabetic nephropathy is a leading cause of end-stage renal disease and is characterized by excessive deposition of extracellular matrix (ECM) proteins in the glomeruli. Transforming growth factor-beta (TGF-beta) is the major mediator of excessive accumulation of ECM proteins in diabetic nephropathy through upregulation of genes encoding ECM proteins as well as downregulation of genes for ECM-degrading enzymes. It has been shown that lovastatin, an inhibitor of 3-hydroxy3-methylglutaryl CoA reductase, delays the onset and progression of different models of experimental nephropathy. To evaluate the effect of lovastatin on the development and progression of diabetic nephropathy, streptozotocin-induced diabetic rats were studied for 12 mo. In untreated diabetic rats, there were significant increases in blood glucose, urine albumin excretion, kidney weight, glomerular volume, and TGF-beta1 mRNA expression in the glomeruli compared with normal control rats treated with citrate buffer only. Treatment with lovastatin in diabetic rats significantly suppressed the increase in urine albumin excretion, kidney weight, glomerular volume, and TGF-beta1 mRNA expression despite high blood glucose levels. To elucidate the mechanisms of the renal effects of lovastatin, rat mesangial cells were cultured under control (5.5 mM) or high (30 mM) glucose with lovastatin alone, mevalonate alone, or with both. Under high glucose, TGF-beta1 and fibronectin mRNA and proteins were upregulated. These high glucose-induced changes were suppressed by lovastatin (10 micro/M) and nearly completely restored by mevalonate (100 microM). These results suggest that lovastatin has a direct cellular effect independent of a cholesterol-lowering effect and delays the onset and progression of diabetic nephropathy, at least in part, through suppression of glomerular expression of TGF-beta1.     

Lovastatin prevents discography-associated degeneration and maintains the functional morphology of intervertebral discs

Background context

Discography is an important diagnostic approach to identify the painful discs. However, the benefit of discography, a procedure involving needle puncture and injection of the diagnostic agent into the intervertebral disc, is controversial and has been reported to be associated with accelerated degeneration.

Purpose

To investigate the effect of lovastatin on the prevention of degeneration caused by a discography simulation procedure in rat caudal discs.

Study design

In vivo study using rat caudal discs.

Methods

A single flexible 27-gauge needle puncture into rat caudal discs was performed under fluoroscopic monitoring. Different concentrations (0.1, 1, 5, and 10 μM) of lovastatin were prepared and injected into randomly chosen caudal discs. RNA expression of selected genes, histologic, and immunohistochemical staining were performed to evaluate the phenotypic effects of lovastatin on rat caudal discs.

Results

Simulation of the discography procedure by puncturing the rat caudal discs with a 27-gauge needle and injection of saline solution induced degenerative changes in the nucleus pulposus with minimal damage to the annulus fibrosus. Aggrecan, Type II collagen, and SOX9 expressions were upregulated, whereas Type I collagen expression was significantly suppressed in discs treated with 5 and 10 μM lovastatin. Discs treated with 5 and 10 μM lovastatin were subjected to alcian blue staining and immunohistochemistry that revealed higher levels of glycosaminoglycans and an increase in the number of cells producing S-100 proteins, Type II collagen, and bone morphogenetic protein-2 (BMP-2), respectively. The most effective phenotypic repair was observed in discs treated with 10 μM lovastatin.

Conclusions

Intradiscal administration of lovastatin solution upregulated the expressions of BMP-2 and SOX9 and promoted chondrogenesis of rat caudal discs after needle puncture and substance injection. Therefore, we suggest that lovastatin promotes disc repair and can be used as a potential therapeutic agent for biological repair of disc degeneration after the diagnostic discography procedure.     

Lovastatin downregulates renal myofibroblast function in vitro

Interstitial fibrosis is recognised as the best histological predictor of progressive renal disease. Myofibroblasts contribute to this process through several functions including hyperproliferation, collagen and collagenase synthesis and reorganisation of extracellular matrix. Recent limited in vitro studies suggest that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors may reduce renal injury not only through their lipid-lowering effects but also by antagonising myofibroblast function. This study therefore examined the effects of lovastatin on the above interstitial myofibroblast behaviours in vitro. Primary cultures of rat renal cortical myofibroblasts were grown by explantation and characterised by immunohistochemistry. Dose response effects of lovastatin (0, 15, 30 microM) in DMEM and 10% FCS were examined on myofibroblast kinetics, total collagen synthesis, collagen I lattice contraction and actin filament rearrangement. Lovastatin decreased myofibroblast proliferation and growth. Likewise, collagen I lattice contraction and actin filament rearrangement were partially inhibited when lovastatin was added at 30 microM. In addition, lovastatin decreased both collagen and collagenase synthesis. Our results suggest that myofibroblast function may be downregulated by lovastatin in vitro. Although a decrease in myofibroblast activity may offer potential benefit in the prevention of progressive scarring, further studies will be necessary to determine the relative importance of these functions.     

Low plasma lipid levels and increased cholesterol synthesis after partial ileal bypass plus lovastatin in hypercholesterolemic rabbits

Partial ileal bypass (PIB) lowers the plasma total cholesterol (C) level, thereby increasing hepatic C synthesis to replenish bile acid and C stores. Lovastatin, a C synthesis inhibitor, may act as a potential adjuvant to PIB for lipid lowering. In this study, the effects of PIB and lovastatin, alone and in combination, were examined in plasma and tissue. For 14 weeks, 32 New Zealand White rabbits received a C-free, alfalfa-free, natural-ingredients diet previously shown to induce hypercholesterolemia. The rabbits were divided into control, lovastatin, PIB, and PIB plus lovastatin groups. Lovastatin was administered at a dose of 0.35 mg/kg twice daily. Compared with the control group, PIB alone decreased the plasma total C level by 75% (p less than 0.005), the low-density lipoprotein (LDL)-C level by 79% (p less than 0.025), and hepatic C content by 50% (p less than 0.05), while increasing hepatic C synthesis by 176% (p less than 0.05). Compared with the control group, lovastatin alone decreased the plasma total C value by 36% (p = NS), the LDL-C level by 35% (p = NS), hepatic C content by 29% (p = NS), and hepatic C synthesis by 52% (p = NS). Compared with the control group, the combination of PIB and lovastatin decreased the plasma total C level by 78% (p less than 0.005), the LDL-C level by 74% (p less than 0.025), and hepatic C content by 58% (p less than 0.05); however, the hepatic C synthesis increased by 490% (p less than 0.005) compared with the control group and by 110% (p less than 0.05) compared with PIB alone. This is the first demonstration of a metabolic reversal of the cholesterol synthesis inhibition engendered by lovastatin. We conclude that both PIB and lovastatin lower plasma total C and lipoprotein C fractions. Their combination has an additive C-lowering effect in plasma and decreases tissue C content by increasing cellular C demand. This latter effect overcomes the inhibitory effect of lovastatin on hepatic C synthesis, resulting in an augmented compensatory increase in hepatic C synthesis.