应用双亲株灭活原生质体融合法选育林可霉素高产菌株

目的：选育高产优质林可霉素产生菌。方法：将林可霉素产生菌S78－1^#,N65-2^#的原生质体分别热灭活和紫外灭活，并将两种灭活原生质体用PEG融合，从融合株中筛选高产优质菌种。结果：获得82－201^#融合株，其摇瓶效价分别较N65－2^#,S78-2^#提高17％和52％，且色素分泌少，传代稳定性强。结论：双亲株灭活原生质体融合选育林可霉素高产菌株是值得推广的一种选育方法。     

应用原生质体融合法选育林可霉素高产菌株

将林可霉素产生菌N65—2^#、S78—1^#的原生质体分别热灭活和紫外灭活，并将灭活原生质体用PEG融合，从中获得92-201^#融合株，其摇瓶效价分别较N65—2^#、S78—1^#提高17％、52％，且色素分泌少，传代稳定性强。     

非对称灭活原生质体融合法选育头孢菌素C高产菌株

目的选育高产头孢菌素C产生菌。方法分别以紫外线和加热灭活头孢菌素C产生菌Cephalosporium acremonium 18-U5和Cephalosporium acremonium 18-M6原生质体,并将两种灭活的原生质体经PEG4000融合,从融合株中筛选头孢菌素C高产菌株。结果获得高产头孢菌素C的顶头孢霉融合株Cephalosporium acremonium F20,发酵单位达4 655 mg.L-1。与双亲菌株相比分别提高了42.4%和34.9%。结论非对称灭活原生质体融合法选育头孢菌素C高产菌株的方法值得推广。     

基因组重排选育麦迪霉素高产菌株

目的以生米卡链霉菌(Streptomyces mycarofaciens)突变株为研究对象,选育麦迪霉素高产菌株。探索并验证基因组重排技术在菌种选育中的重要作用。方法通过2轮多亲株灭活原生质体融合技术实现基因组重排。将来自不同育种方法的5株麦迪霉素高产菌株B64-5、01-GM-1、H-101、H-106和H-108作为第一轮亲本,在质量浓度为3 g.L-1溶菌酶3、2℃水浴60 min条件下制备原生质体,分别采用紫外线照射148 s和52℃加热60 min灭活原生质体,然后采用质量分数35%PEG 4000、37℃保温2 min诱导原生质体融合。融合子经过初筛和复筛,获得产量进一步提高的重组子作为亲本进行第二轮的多亲株灭活原生质体融合实验。用生物学方法测定麦迪霉素效价,用HPLC方法测定麦迪霉素A1含量。结果与结论经过2轮基因组重排实验成功选育出3株高产且遗传稳定的麦迪霉素生产菌株。其中1株菌株GSZ2-32发酵前补加前体X-1后摇瓶产量比原始出发菌株Streptomyces mycarofaciens var.464提高1.1倍,麦迪霉素A1(MDMA1)质量分数含量保持在80%以上。     

拟无枝酸菌B37与游动放线菌E92—70属间隔合

以去甲基万古霉素产生菌Ｂ３７和替考拉宁类抗生素产生菌Ｅ９２－７０为出发菌株，将Ｂ３７原生质体热灭活，进行属间原生质体融合，融合率１．４×１０＾－６－４．６×１０＾－６，融合子５０％以上抗菌活性增强。     

通过双亲株灭活原生质体融合方法提高链霉素产量

<正> 关于带遗传标记的链霉菌原生质体融合已有报道。近年来,国内外研究人员为了节约杂交育种时间,避免进行遗传标记可带来的不利于生产的突变株,故在摆脱遗传标记方面做了一定的工作。我们在进行链霉素产生菌灰色链霉菌的细胞融合工作中,采用高产菌株两株和野生菌株两株均不带遗传标记,将4株菌的原生质体混合在一起,然后分成两份,分别用热和UV灭活,使原生质体的存活率接近于零,融合得到重组子。其重组率低于文献报道。通过筛选,得到一株生长快、效价与对照相当的菌株,并得到10株摇瓶效价提高10～35%的菌株,其中前者已通过中试。这就为缩短微生物杂交育种时间提供了可行途径。     

生米卡链霉菌原生质体电融合重组研究

采用电融合新技术可提高生米卡链霉菌原生质体融合频率,并且在原生质体稳定液中加入2.5mol/L的MgCl_2,比使用PEG助融方法的融合频率高10倍。经过UV灭活高产菌株和另一脯氨酸缺陷型菌株的原生质体的电融合,其融合子的抗生素产量变异幅度大,发酵产物的各组份比例改变,经过选择获得了一新的高产菌株,麦迪霉素产量提高77％,其有效组份A_1比例增加。     

麦角菌与雀稗麦角菌种间灭活原生质体融合

探索了在麦角菌中进行省却营养缺陷型标记的种间灭活原生质体融合。亲株Ⅰ（Ｃ．ｐｕｒｐｕｒｅａ,麦角隐亭产生菌）用紫外线灭活（１５Ｗ,照射距离３０ ｃｍ ）７５ ｍ ｉｎ,存活率约６×１０－ ５;亲株Ⅱ（Ｃ．ｐａｓｐａｌｉ,麦角酰胺产生菌）用热灭活（５０～５２℃）６０ ｍ ｉｎ,存活率为零。融合后的再生菌落均为亲株Ⅰ的形态,其再生频率比灭活的亲株Ⅰ提高约２０ 倍,推测绝大多数为融合子。后者经５ 次传代,大多数的产碱能力与亲株Ⅰ相当或略有提高。     

微生物原生质体灭活及其在育种中的应用

灭活原生质体融合技术的依据是,用适当的方法处理单一亲株或双亲株的原生质体,使之失去再生的能力,经细胞融合后,由于致死损伤部位的互补可以形成能再生的融合体,除有诱变作用的灭活剂外,一般的灭活处理对细胞DNA的遗传功能和重组力未发现有明显的影响。该技术在动、植物细胞融合中应用较多,近几年来在微生物研究中的应用亦有较大发展。一、原生质体灭活实验技术主要采用热、紫外线(UV)、电离辐射(X-射线、γ-射线)以及某些生化试剂(包括抗生素)等作为灭活剂。 (一)热灭活通常用50-52℃处理2h。在处理过程中,可将原生质体悬液用移液管移至预热过的高渗琼脂培养基表面     

新型常压室温等离子体-紫外复合诱变选育埃莎霉素I高产菌株#br#

目的 通过对埃莎霉素Ⅰ产生菌WSJ-IA进行诱变选育研究,以期获得埃莎霉素Ⅰ高产菌株。方法 使用多功能等离子体诱变系统(multifunctional plasma mutagenesis system, MPMS)对出发菌株的孢子进行等离子体和紫外复合诱变,设定不同的诱变时间处理孢子悬液,通过致死率确定合适的诱变条件,利用突变株摇瓶发酵效价筛选出正突变菌株。结果 在MPMS射频功率为100W,处理距离5mm,气体流量12.5SLM,等离子体-紫外辐射时间为50s时,菌株致死率为96.08%。在此诱变条件下,以突变株的初筛效价为指标的突变率、正突变率分别达到63.96%和22.52%,复筛效价是出发菌株1.5倍以上的有5株,占复筛菌株的9%。最终筛选出一株发酵单位比出发菌株提高221%、埃莎霉素Ⅰ组分含量提高192%的正突变株IA-425。42L自动发酵罐发酵结果表明,该菌株埃莎霉素Ⅰ产量达到(2000±200)μg/mL左右。结论 新型等离子体复合紫外诱变方式,可有效提高菌株的埃莎霉素Ⅰ发酵产量和组分含量。这为埃莎霉素Ⅰ的大规模发酵和临床前研究奠定了良好基础。     

Catabolism of vasoactive polypeptides by perfused rat liver

Summary Exsanguinated rat liver preparations perfused in situ with oxygenated saline solutions inactivated recirculating bradykinin (BK) at rates of 2.3 to 9.1 and isoleucyl⁵ angiotensin II (AII) at rates of 2.8 to 15.0 nmolex x min^–1 x g^–1 of liver, depending on the initial concentration of the peptides in the perfusion fluid (3.1 to 18.9×10^–6 M for BK and 8.5 to 17.0×10^–6M for AII). On the other hand, at similar concentrations, recirculation of isoleucyl⁵ Angiotensin I (AI) for 8 min did not lead to decrease of its biological activity when assayed on the isolated rat uterus. Following a single passage through liver, picomole amounts of both BK and AII were inactivated by about 90% as revealed by assays on a superfused rat uterus.The potency ratio AI:AII, assayed on a superfused rat uterus was 1:22 and changed to 1:5 following a single passage of both peptides through liver. This finding and the separation of 4.9% of AII on carboxymethylcellulose columns following recirculation of AI through rat liver indicate a conversion of AI into AII. The dipeptides Phe-Arg, Ser-Pro and Gly-Phe were identified among the hydrolysis products of perfused BK. A peptidyldipeptide hydrolase (EC 3.4.15) may be responsible for both the BK inactivation and AI conversion. The inactivation of AII cannot be attributed to the same enzyme.Abbreviations BK bradykinin - AII isoleucyl⁵ angiotensin II - AI the corresponding angiotensin I - BPP 5a bradykinin-potentiating peptide PCA-Lys-Trp-Ala-Pro Work supported by grants from Project BIOQ/FAPESP (São Paulo) and FINEP (Rio de Janeiro)From Department of Medicine, EPMFrom Department of Physiology and Biophysics, EPM     

The sequence of the early steps in the metabolism of prostaglandin E1

The pathway in the metabolism of prostaglandins E₁ in kidney, spleen and liver from swine is 1) oxidation of the 15-hydroxyl group to a ketone, 2) reduction of the Δ¹³-double bond and partly 3) stereospecific reduction of the 15-keto group to dihydro-prostaglandin E₁ (15 S).     

改良碳青霉烯灭活试验在检测肺炎克雷伯菌碳青霉烯酶中的应用评价

目的评估改良碳青霉烯灭活试验(mCIM)和EDTA改良碳青霉烯灭活试验(eCIM)在中国产碳青霉烯酶肺炎克雷伯菌(CPKP)中的应用价值。方法分别用mCIM和eCIM筛查中国271株耐碳青霉烯肺炎克雷伯菌(CRKP)中产酶阳性菌株,并以碳青霉烯酶基因聚合酶链反应(PCR)及测序结果为标准计算灵敏度和特异度。结果 271株非重复CRKP菌株中碳青霉烯酶基因阳性菌有209株,未检测出耐药基因的菌株有62株。mCIM检测到209株基因阳性菌株中208株为阳性菌株,62株基因阴性菌均为阴性; eCIM检测出的阳性菌株有58株,阴性菌株有150株。mCIM检测敏感度为99. 5%,特异度为100. 0%; eCIM检测敏感度为100. 0%,特异度为99. 3%。其中有1株产IMP-4型碳青霉烯酶菌株的mCIM结果为阴性,1株产KPC-2型碳青霉烯酶菌株的mCIM和eCIM都为阳性。结论 mCIM和eCIM在肺炎克雷伯菌中检测碳青霉烯酶有很好的敏感度和特异度,值得推广使用。     

Kinetics of inhibition of aminoacylase activity by dithiothreitol or 2-mercaptoethanol

The previously described kinetic method of the substrate reaction during irreversible inhibition of enzyme activity [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61 . 381–436] has been used to study the inactivation kinetics of aminoacylase by dithiothreitol (DTT) and 2-mercaptoethanol (MET). The results show that the inactivation of aminoacylase by DTT or MET is competitive slow-reversible inhibition. The microscopic rate constants for the inactivation reaction were determined. Removal of these inhibitors by dialysis can lead to complete recovery of enzymatic activity. The present results also show that the presence of equimolar Zn²⁺ to DTT gives complete protection of the enzyme against the inactivation by DTT. Moreover, addition of equimolar amounts of Zn²⁺ to DTT can induce recovery of the enzymatic activity of DTT-inactivated enzyme. It is known that aminoacylase from pig kidney contains no disulfide bonds. Therefore, it may be suggested that inactivation of aminoacylase by dithiothreitol or 2-mercaptoethanol is not due to the reduction of disulfide bonds, and is a competitive slow-reversible inhibition.     

尿胰蛋白酶抑制剂生产中病毒灭活工艺的效果观察

目的验证尿胰蛋白酶抑制剂(UTI)生产工艺中采用的60℃水浴10 h和乙醇处理3 h的病毒灭活效果。方法将Sindbis病毒、伪狂犬病毒(PRV)和脊髓灰质炎病毒(PV1)3种指示病毒分别加入不同的UTI原料样品中,进行60℃水浴10 h和乙醇处理3 h,处理不同时间后分别取样,用微量细胞病变法检测不同时间段样品中的病毒残留滴度。结果60℃水浴10 h可灭活(6.503±0.102)LgTCID50/mL的Sindbis病毒,灭活(6.42±0.158)LgTCID50/mL的PRV以及(6.587±0.061)LgTCID50/mL的PV1。乙醇处理3 h后可灭活(5.88±0.204)LgTCID50/mL的Sindbis病毒,灭活(6.378±0.268)LgTCID50/mL的PRV以及(5.963±0.118)LgTCID50/mL的PV1。经两种方法处理后的样品加入细胞经3代盲传均未见细胞病变(CPE)出现。结论UTI生产工艺中采用的60℃水浴10 h和乙醇处理3 h均可灭活所验证的3种指示病毒,灭活效果>6.0 LgTCID50/mL。     

吩噻嗪类化合物的合成及其光化学病毒灭活活性研究

光化学病毒灭活法目前被用于血液产品病毒灭活处理, 其中亚甲蓝 (MB) 光化学法能够有效地进行血细胞制品病毒灭活。以MB为先导物, 设计合成了12个新结构的吩噻嗪类化合物, 并进行了初步病毒灭活、红细胞损伤等实验, 结果显示测试样品中化合物YWW-7在活性和可能的副作用等方面的表现均优于MB, 有希望开发成为全新的血液产品病毒灭活剂。     

Biologically stable analogues of trh with increased neuropharmacological potency

Two simple analogues of TRH containing a 3-methyl or 3,3-dimethyl substituted prolineamide residue have been studied. In CNS tests both analogues showed increased activity, the dimethyl analogue being the most potent. The potency changes observed correlated with the increased biological stability of the analogues against enzymes located in the brain and various peripheral tissues. It is concluded that the increased resistance to inactivation shown by the analogues is a major contributor to their enhanced potency.     

Effects of ozone exposure on inactivation of intra- and extracellular enterovirus 71

In this study, the potential of ozone in inactivating enterovirus 71 (EV71) free particles was investigated using either various ozone flow rates of 100, 80 or 60 mg/h or a constant flow rate of 80 mg/h, given to culture medium or various pH culture media containing EV71, respectively. Results demonstrated that EV71 inactivation by ozone was related to the kinetics of ozone solubility, 99% inactivation being obtained in the exponential phase of ozone solubility. However, the inactivation rate was dependent on the ozone input flow rate and positively enhanced at acidic pH. Inactivation of intracellular EV71 was also studied. At a constant ozone supply of 60 mg/h, a significant reduction of intracellular virus titer (≥99%, p < 0.01) was obtained after 45 or 60 min exposure but with low cell viability. Upon 30 min exposure, however, 45% cell viability was retained. The results indicate that the inactivating effect of ozone on intracellular EV71 virus is dependent on exposure duration.     

Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels

BACKGROUND AND PURPOSE: L-type calcium channels (Ca((V))1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca((V))1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca((V))1.2 channels. EXPERIMENTAL APPROACH: Ca((V))1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. KEY RESULTS: Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca((V))1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC(50)=29.5+/-12 microM, which is close to that for slow activation and inhibition. CONCLUSIONS AND IMPLICATIONS: We conclude that roscovitine binds to an extracellular site on Ca((V))1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension.     

人血丙种球蛋白制备的新工艺

本文报道了利凡诺-低温乙醇-DEAE离子交换层析结合法制备人血丙种球蛋白的新工艺,按此工艺,IgG收率约0.5％（g/ml）,纯度>99％,制剂各项指标均符合规程要求。以VSV为病毒模型,制备过程至少可失活或排除10~(11)ID_(50)/ml病毒,工艺对灭活脂质壳病毒是有效的。