东方拟无枝酸菌原生质体激光诱变选育

为了选育去甲基万古霉素高产菌,以东方拟无枝酸菌07-8-32为出发菌株,制作原生质体,经氦氖激光处理后分离。经过21批次的筛选得到的突变株08-8-48效价比出发株高27.3%,且菌丝状态、传代稳定性好。     

抗药性基因突变筛选高产菌株

本文通过洁霉素对盐酸林可霉素产生菌 98-1菌株孢子的致死浓度测定 ,采用诱变剂EMS的四种不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含洁霉素 ( 2 0 0 0 0ug/ml)的高氏平板上 ,获得了大量的洁霉素抗性基因突变株 ,然后从 90 0株洁霉素抗性基因突变株中通过初筛获得高于诱变出发菌株产素能力的菌株 5 6株 ,再进一步通过摇瓶复筛 ,并结合菌丝生长及摇瓶代谢情况 ,获得优于出发菌株的诱变菌株 4株。将这 4个菌株连同出发菌株连续三批次进行摇瓶发酵 ,结果 4个突变株的产素能力 (产量 )及摇瓶代谢和菌丝生长情况均优于出发菌株 ,试验筛选出最优菌株 0 2 -0 3 -40 2。将 0 2 -0 3 -40 2进行罐上发酵生产试验 ,结果试验罐比对照罐平均效价提高 1 7.5 6% ,平均产量提高 1 9.3 4%。本文建立了林可霉素高产菌株的洁霉素抗性基因突变诱变快速高效的筛选方法     

复合诱变法筛选达托霉素高产菌株

以达托霉素产生菌HK402为出发菌株进行氦氖激光辐照-亚硝基胍复合诱变,最终获得一株达托霉素高产菌株14-10-98。该菌株遗传稳定性好,发酵单位比出发菌株高35%,比单用氦氖激光辐照诱变得到的高产菌株14-8-102高8%,比单用亚硝基胍诱变得到的高产菌株14-9-196高13%。     

紫外诱变复合重金属离子抗性突变选育螺旋霉素高产菌株

以螺旋霉素链霉菌(Streptomyces spiramyceticus)YL0113-01为出发菌株,采用紫外线诱变,并以重金属Co~(2+)为筛选因子,获得一株遗传稳定且螺旋霉素发酵效价比出发菌株提高6%的菌株。再对该菌株采用亚硝基胍(NTG)诱变,以缬氨酸结构类似物L-α-氨基丁酸作为筛选因子得到一株发酵效价较出发菌株提高14%的菌株。     

产黄青霉菌高能电子诱变选育

为了选育出青霉素高产菌株,以产黄青霉菌10-2-12为出发菌株,经高能电子诱变后分离。经过18批次筛选得到的突变株13-6-55效价比出发株高8.2%,且菌丝状态、传代稳定性好。在试验罐上的平均发酵单位比对照高3.6%。     

He-Ne激光与紫外线复合诱变选育西罗莫司高产菌株

以吸水链霉菌为出发菌株,采用He-Ne激光-紫外复合诱变对其单孢子悬液进行诱变筛选高单位突变株,经复筛选育出具有较好遗传稳定性的高产突变株11-1-6#,摇瓶效价达到401.9u/mL,比出发菌株提高了79%。     

头孢菌素C产生菌的诱变育种及发酵应用

以头孢菌素C产生菌顶头孢霉(Cephalosporium acremonium)B-1822-S-04为出发菌株,经过紫外线和丙二酸复合诱变,筛选得到了4株头孢菌素C高产菌株D-G-017、D-G-083、D-G-096和D-G-133,其中菌株D-G-096的效价高达6 816 mg·L~(-1),较出发菌株提高了50.80%,且高产特性稳定;50 L发酵罐小试结果显示,菌株D-G-096的效价达到了29 270 mg·L~(-1),较出发菌株提高了111.06%,可作为优良高产菌株保存使用。该实验筛选出的高产菌株及其在小试中的应用,可为后期工业化生产提供优质菌种资源,为发酵工艺提供指导。     

高产类金属硫蛋白假丝酵母菌株的筛选

目的筛选高产类金属硫蛋白(metallothionein,MT)假丝酵母菌株。方法以产朊假丝酵母菌为出发菌株,利用紫外照射诱变、亚硝基胍(nitrosoguanidine,NTG)化学诱变交替进行的方法进行诱变育种,检测总蛋白含量、巯基活性及类MT含量,筛选高产类MT的菌株。对筛选出的菌株传5代,分析其遗传稳定性。结果获得1株高产类MT的菌株N′′-6,其产类MT的能力由出发菌株的39.6 ng/L提高至165.2 ng/L,巯基活性由出发菌株的0.035μmo/L提高至0.147μmol/L。菌株N′′-6传5代,其总蛋白含量、巯基活性及类MT含量变异度较小。结论筛选出1株高产类MT的假丝酵母菌株,该菌株遗传稳定性较好,为类MT的工业化生产奠定了基础。     

利用组合抗药性突变选育秦岭霉素高产菌株

以秦岭链霉菌为出发菌株,采用链霉素和庆大霉素对其进行抗药性诱变.通过对致死率和活性最大提高率的测定,确定了链霉素和庆大霉素对出发菌株的最小抑制质量浓度(MIC),并在最小抑制质量浓度下筛选抗药性突变菌株.结合对诱变菌株的传代稳定性测定,获得了3株抗药性突变的高产菌株GS-4-04、GS-4-05和GS-4-06,其发酵液对5种细菌和4种病原真菌的抗菌活性比原始菌株显著提高.     

我国狂犬病流行毒株JX08-45适应细胞培养后的生物学特性

目的鉴定我国狂犬病流行毒株JX08-45适应细胞培养后(命名为JX08-45CC株)的生物学特性。方法通过全基因组序列分析比对、小鼠脑内和外周攻毒试验和免疫原性试验,分别对JX08-45CC株的基因组特点、毒力和免疫原性进行鉴定。结果 JX08-45CC株的基因组序列与JX08-45株比对,共出现16个核苷酸突变点和7个氨基酸突变点,其中6个氨基酸变异发生在G蛋白(333位精氨酸未改变)编码区,L蛋白仅有1个氨基酸突变;在G蛋白氨基酸变异中,4个突变序列在其他细胞适应毒株CVS-11、CTN-1、Nishigahara、SAG2、Flury-LEP和SRV9中也普遍存在。JX08-45CC株培养滴度≥107TCID50/ml时,脑内接种小鼠的致死率为100%,滴度为102～106TCID50/ml时,脑内接种小鼠的致死率为20%～80%;滴度为105～108TCID50/ml时,外周肌肉接种小鼠,致死率为10%～70%;脑内和外周攻毒小鼠的潜伏期均为7～9 d,并于发病后24～48 h死亡。JX08-45CC株与狂犬病病毒CVS-11、ERA、SRV9和Flury-LEP株的中和抗体滴度差异均无统计学意义(P>0.05)。结论已对JX08-45CC株的基因组序列、毒力和免疫原性等生物学特性进行了鉴定,为开发具有我国自主知识产权的兽用狂犬病灭活疫苗候选株奠定了基础。     

Physosmotic Induction of Chondrogenic Maturation Is TGF-β Dependent and Enhanced by Calcineurin Inhibitor FK506

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.     

A “Clickable” MTX Reagent as a Practical Tool for Profiling Small‐Molecule–Intracellular Target Interactions via MASPIT

We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ‐selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three‐hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration‐dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non‐regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full‐length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.     

An aldol approach to a building block corresponding to the C21–C26-part of FK506

Starting from D-tartrate 2 , the chiral aldehyde 10 was prepared in 8 steps. Key steps include the reductive opening of the p-methoxybenzyl acetal 4 and the elongation of the aldehyde 7 via Wittig and hydroboration reaction providing the alcohol 9 . Subsequent Evans aldol reaction provided compound 12 which corresponds to the C21–C26 part of the immunosuppressive FK506.     

FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolylisomerase that binds the macrolides FK506 and rapamycin. Wehave examined the role of the binding pocket residues of FKBP12in protein–ligand interactions by making conservativesubstitutions of 12 of these residues by site-directed mutagenesis.For each mutant FKBP12, we measured the affinity for FK506 andrapamycin and the catalytic efficiency in the cis–transpeptidyl-prolyl isomerase reaction. The mutation of Trp59 orPhe99 generates an FKBP12 with a significantly lower affinityfor FK506 than wild-type protein. Tyr26 and Tyr82 mutants areenzymatically active, demonstrating that hydrogen bonding bythese residues is not required for catalysis of the cis–transpeptidyl-prolyl isomerase reaction, although these mutationsalter the substrate specificity of the enzyme. We conclude thathydrophobic interactions in the active site dominate in thestabilization of FKBP12 binding to macrolide ligands and tothe twisted-amide peptidyl-prolyl substrate intermediate.     

患者服FK506的剂量、血药浓度、服药天数的三维图像解析

研究分析老、中、青年肾移植患者服用普乐可复剂量、血药浓度、服药天数的三维图像,使患者临床用药形象化显现,为个体用药提供信息。将60岁以上老年患者、40-59岁中年患者与小于40岁青年患者分三组,采用MATLAB7.0做三维图像,分析图像特征并且对老年、中年、青年组图像进行比较。三维图像显示出各项指标间存在内在关系与个体差异。体内指标与服药天数相关,体内指标对临床个体用药具有重要意义。     

A Practical Synthesis of the Cyclohexyl Part of the Immunosuppressant FK506

Starting from the benzylidene lactone 3 of D-(−)-quinic acid the cyclohexyl fragment 15 (C-28-C-34 part) of the immunosuppressant FK506 was synthesized. Key steps include homolytic deoxygenation reactions on compounds 4 and 6 as well as a regioselective opening of the benzylidene acetal 5 . Opening of the lactone 7 to provide the methyl ester 8 was followed by methylation of the hydroxy group to give 9 . Further steps provided the aldehyde 12 which was elongated to the alkyne 15 . This sequence provides 15 in gram quantities.     

Exosomes Secreted by Adipose-Derived Stem Cells Following FK506 Stimulation Reduce Autophagy of Macrophages in Spine after Nerve Crush Injury

Macrophages emerge in the milieu around innervated neurons after nerve injuries. Following nerve injury, autophagy is induced in macrophages and affects the regulation of inflammatory responses. It is closely linked to neuroinflammation, while the immunosuppressive drug tacrolimus (FK506) enhances nerve regeneration following nerve crush injury and nerve allotransplantation with additional neuroprotective and neurotrophic functions. The combined use of FK506 and adipose-derived stem cells (ADSCs) was employed in cell therapy for organ transplantation and vascularized composite allotransplantation. This study aimed to investigate the topical application of exosomes secreted by ADSCs following FK506 treatment (ADSC-F-exo) to the injured nerve in a mouse model of sciatic nerve crush injury. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile the potential exosomal proteins involved in autophagy. Immunohistochemical analysis revealed that nerve crush injuries significantly induced autophagy in the dorsal root ganglia and dorsal horn of the spinal segments. Locally applied ADSC-F-exo significantly reduced autophagy of macrophages in the spinal segments after nerve crush injury. Proteomic analysis showed that of the 22 abundant exosomal proteins detected in ADSC-F-exo, heat shock protein family A member 8 (HSPA8) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) are involved in exosome-mediated autophagy reduction.     

鼬獾狂犬病病毒分离株的培养及其免疫原性

目的对分离的鼬獾狂犬病病毒BHK-21细胞适应株进行毒力和免疫原性检测,为兽用狂犬病疫苗的生产奠定基础。方法将分离获得的鼬獾狂犬病病毒野毒株JX08-45在BHK-21细胞上连续传代,采用直接免疫荧光法测定病毒滴度(TCID50);PCR法检测外源病毒和支原体。以β-丙内酯灭活病毒液,将灭活的病毒液免疫犬,采用FAVN法检测狂犬病病毒中和抗体水平,分析其免疫原性。结果鼬獾狂犬病病毒野毒株JX08-45在体外培养至130代时,病毒滴度可达1.0×107.75 TCID50/ml;未从狂犬病病毒JX08-45株中扩增出犬瘟热病毒、细小病毒、冠状病毒、腺病毒和支原体的特异性核酸;灭活的病毒液接种犬后产生的中和抗体可持续1年以上,且均在0.5 IU/ml以上。结论鼬獾狂犬病病毒野毒株JX08-45经BHK-21细胞传代适应性较好,病毒滴度较高,且具有较好的免疫原性,已具备制备疫苗的基本条件。     

The prospect of FKBP51 as a drug target

The FK506 binding protein 51 (FKBP51) is best known as an Hsp90-associated co-chaperone that regulates the responsiveness of steroid hormone receptors. In human genetic association studies, FKBP51 has repeatedly been associated with emotion processing and numerous stress-related affective disorders. It has also been implicated in contributing to the glucocorticoid hyposensitivity observed in New World primates. More recently, several research groups have consistently shown a protective effect of FKBP51 knockout or knockdown on stress endocrinology and stress-coping behavior in animal models of depression and anxiety. The principal druggability of FKBP51 is exemplified by the prototypic FKBP ligands FK506 and rapamycin. Moreover, FKBP51 is highly suited for X-ray co-crystallography, which should facilitate the rational drug design of improved FKBP51 ligands. In summary, FKBP51 has emerged as a promising new drug target for stress-related disorders that should be amenable to drug discovery.