尿苷酸微生物转化菌株的选育

以产氨短杆菌K13为出发菌株，采用离子注入诱变，并以5-氟胞嘧啶筛选，最终获得了尿苷酸高转化突变株KU-F14-14。突变株生物转化生产尿苷酸能力约为900mg／L，比出发菌株提高了161．6％。     

苯乙酮酸的高特异性生物转化合成

从20株酵母菌中筛得一株解脂假丝酵母（Candida lipolytica）SIPI0201，可高特异性转化苯甘氨酸为苯乙酮酸。经优化转化条件后表明当温度30℃、pH9．0、Ca^2＋浓度0．75mmol／L，并于转化培养初始阶段加入底物，转化率可达47．9％，较优化前提高91％。该菌株具有较好的底物特异性和选择性。     

利用微生物转化法生产磷霉素的研究

本实验选择小棘青霉３１４９作为出发菌株，在合适的培养条件下，当顺式丙烯磷酸加入量为０．０２％～０．０４％时，小棘青霉３１４９对其转化率可达９０％，随着底物浓度的增加。转化率下降，当加入的底物浓度达到０．３％时，转化作用停止。在对小棘青霉３１４９进行ＵＶ－ＬｉＣｌ诱变处理后得到一株能利用较高浓度底物的变异株ＰＵ１１，在顺式丙烯磷酸加量为０．３％时，转化率为１３％，进一步将ＰＵ１１原生质体做ＮＴＧ处理获得ＰＵＮ１８变异株，该菌株在同样条件下对底物的转化率上升到３３％，与出发菌株相比，ＰＵＮ１８菌株对底物的耐受能力明显提高，高浓度顺式丙烯磷酸对转化的抑制作用基本被解除。     

赖氨酸6-氨基转移酶产生菌株的筛选鉴定

以赖氨酸为唯一氮源,从土壤中分离到36株能够利用赖氨酸的菌株,从中筛选到2株具有赖氨酸6-氨基转移酶活力的菌株。并对酶活较高的菌株进行形态以及ITS序列鉴定,菌株2-14鉴定为轮枝霉菌(Ver-ticilliumsp.)。该菌株能将L-赖氨酸代谢转化为L-哌可酸,并且其中几乎不含有D-哌可酸。该研究为国内L-哌可酸生物合成提供了有价值的资料。     

旱地小单孢菌FIM03-712生物转化雷帕霉素为14-去氧雷帕霉素

在生物转化研究中,获得对雷帕霉素具有转化作用的一株小单孢菌,经分类学研究鉴定为旱地小单孢菌(Micromonospora chersina)FIM03-712.该菌株转化雷帕霉素产生4个化合物,从转化发酵液中分离到一个分子量为900的转化产物,理化性质和波谱分析研究表明,该转化产物为14-去氧雷帕霉素.     

小单孢菌FIM10-2207生物转化卡那霉素B的初步研究

在卡那霉素B的微生物转化研究中,从台湾海峡海底沉积物中分离得到五株对卡那霉素B具有转化作用的菌株,选取其中转化率较高的一株菌,编号为FIM10-2207,经分类学研究鉴定为小单孢菌。初步考察了培养基的选择,底物卡那霉素B的初始浓度和添加时间这3个因素对转化率的影响,发现转化培养基C,添加时间32h和底物初始浓度1500μg/mL时,转化率较高。卡那霉素B经该菌株转化后,得到一个新化合物,波谱数据分析结果表明该产物为1,3″-二乙酰化卡那霉素B。     

微生物来源的乙酰胆碱酯酶抑制剂F99-909A的研究

研究利用丁酰硫代胆碱和二硫二硝基苯甲酸为底物和显色剂，建立一种精确、快速的高通量筛选微生物代谢产物来源的乙酰胆碱酯酶(AChE)抑制剂的体外筛选模型。利用此方法从2300株真菌中筛选到一株阳性菌株F99-909．该菌株的发酵液经有机溶剂提取、ODS柱层析及HPLC制备分离，得到一个活性化合物F99-909A，该化合物对乙酰胆碱酯酶的IC50为9．5μmol／L。通过对该化合物的紫外、红外、质谱、核磁等理化分析，得知该化合物与已知化合物radiclonic acid结构相同。但该化合物对Ache的抑制活性至今未见报道。     

R-（-）扁桃酸及其衍生物的手性合成

以苯乙酮酸和苯乙酮酸甲酯为底物，从10株不同种类的菌株中筛选出具有不对称合成R(-)-扁桃酸及其甲酯活性的酵母菌S.c1。以此菌为出发菌株，进一步采用紫外和微波复合诱变技术。分别筛选获得突变菌S.cl-MA16和Scl-ME10，其对应的扁桃酸和扁桃酸甲酯得率分别达99.0%和93．7%，R构型对映体过量值分别为99.8％和99.2％；其最优转化条件均为：转化培养基pH6．5和温度38℃。     

胞外青霉素酰化酶产生菌的筛选及产酶条件

采用青霉素梯度琼脂平皿法，从土壤中快速筛选出３５株产胞外青霉素酰化酶的菌株，经复筛有５株酶活力较高，经鉴定均为芽胞杆菌，Ｂ３５－１７菌株酶活力最高．研究了Ｂ３５－１７菌株的胞外青霉素酰化酶产生条件，该菌株在最适产酶条件下，酶活力达２．６７ｕ／ｍＬ．在发酵培养基中添加０．２％的玉米浆能降低Ｆｅ２＋对酶合成的抑制作用．     

一株新的肺炎克雷伯氏菌将甘油快速转化为1，3-丙二醇

从39株样品中筛得5株能在厌氧条件下将甘油转化为1，3-丙二醇的细菌，本研究鉴定了其中一株菌株XJ—Li，并进行了包括形态学、生理学、生化特征及16SrDNA序列的研究。     

Effects of d-pseudoephedrine on tracheo-bronchial muscle and the cardiovascular system (author's transl)

The action of d-psuedoephedrine on bronchial smooth muscle, respiratory resistance and blood pressure was compared with that of 1-ephedrine. The following results were obtained. 1) Administration of dl-isoproterenol on isolated guinea pig tracheal muscle previously constricted with acetylcholine (ACh) or histamine (his) was the most effective and d-pseudoephedrine had the same effect as 1-ephedrine. The relaxing effect of dl-isoproterenol, 1-ephedrine or d-pseudoephedrine on ACh or His induced tracheal constriction was competitively antagonized by the pretreatment of propranolol. 2) The intravenous or sublingual administration of d-pseudoephedrine on the increased respiratory resistance induced by His produced the same effect as 1-ephedrine. 3) The increase in blood pressure with d-pseudoephedrine was weaker than that of 1-ephedrine. 4) When 1-ephedrine of d-pseudoephedrine was repeatedly injected into the same animal, signs of tachyphylaxis were observed. It was found that d-pseudoephedrine, the main alkaloid of Ephedrine Helba, exhibited the relaxing effect on bronchial smooth muscles as did 1-ephedrine. The respiratory resistance increased by His was inhibited by the sublingual administration of d-pseudoephedrine and 1-ephedrine.     

Cloning and expression of a gene encoding N-glycosyltransferase (ngt) from Saccarothrix aerocolonigenes ATCC39243

In the course of our bioconversion studies on the derivatives of an indolocarbazole, J-104303, Saccharothrix aerocolonigenes ATCC39243 was found to convert J-104303, which was added into the culture medium, to its glycosylated derivative, J-109384. In order to clone the gene having the ability to convert J-104303 to J-109384, a library of Saccharothrix aerocolonigenes ATCC39243 DNA fragments was constructed using Streptomyces lividans TK21 and pIJ702 as host strain and vector, respectively. By examining more than 5,000 transformants, one was found to convert J-104303 to J-109384. Sequence analysis of the inserted DNA fragment revealed an open reading frame with 1,245 base pairs, named ngt. The transformant containing this ngt gene was also found to introduce a D-glucose moiety into 6-N-methylarcyriaflavin C. Furthermore, when ngt was introduced into Streptomyces mobaraensis BA13793, a producer of J-104303, the resulting transformant produced J-109384 directly.     

具有生物碱转化活力的4株喜树内生真菌的鉴定

从喜树（Camptotheca acuminata Decne）的根、枝条、叶和果实中分离纯化了48株内生真菌，其中菌株J-3、J-4、J-5、J-8对盐酸罂粟碱、硫酸长春碱等天然活性产物具有转化能力。采用PCR技术对这4株真菌rD-NA的ITS区进行扩增、测序，得到4株真菌的ITS序列，用Blast调出与菌株ITS同源的序列，用DANMAN6．0软件中的Multiple Sequence Alighment进行同源性分析，构建系统进化树，并辅以生理形态指标进行种属鉴定。菌株J-3、J-4分别鉴定为Phomopsis acuminata J-3和Phomopsis acuminata J-4；J-5鉴定为Phomopsis acuminata J-5；J-8鉴定为Phomopsis acuminata J-8。上述工作为充分利用药用植物内生真菌对天然活性产物进行微生物转化，丰富天然化合物类型并进行构效关系的分析提供了基础。     

复方盐酸伪麻黄碱分散片的研制

目的:研制复方盐酸伪麻黄碱分散片。方法:采用正交设计法筛选处方,导数光谱法测定两主药含量,并对制备的分散片样品进行稳定性影响因素考察。结果:该分散片崩解迅速,主药含量在光、湿、热的条件下均可保持稳定。结论:该制剂制备工艺简便,服用方便,选用的分析方法简便快速,精密度和稳定性均符合标准,有临床应用价值。     

Inhalation toxicity of methyl n-amyl ketone (2-heptanone) in rats and monkeys

A subchronic inhalation study was conducted in which male rats and monkeys were exposed at 0 (controls), 131, or 1025 ppm methyl n-amyl ketone (MAK) for 10 months (6 hr/day, 5 days/week). Comprehensive cardiopulmonary, clinical chemistry, metabolism, and tissue distribution studies were performed. No statistically significant alterations of pulmonary function, electrocardiographic, or clinical chemistry parameters were observed. Methyl n-amyl ketone and methyl n-amyl alcohol were detected in urine and serum from monkeys exposed to MAK for 10 months. Inhaled MAK did not induce rat liver microsomal enzymes. Results of tissue distribution studies of [¹⁴C]MAK in rats comparing ip and inhalation routes of exposure were similar, and preexposure to unlabeled MAK did not alter the tissue distribution compared to animals without prior exposure to MAK. Liver tissue had the highest level of radioactivity regardless of the route of administration; however, no liver pathology was observed. Urinary excretion accounted for 25% of the administered dose after 12 hr. In general, a lack of toxicity was noted in both species exposed to MAK.     

A methyl jasmonate derivative,J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro

The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J-7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway.     

Hepatobiliary Transport of a Nonpeptidic Endothelin Antagonist, (+)-(5S,6R, 7R)-2-Butyl-7-[2((2S)-2-Carboxypropyl)-4-Methoxyphenyl]-5-(3,4-Methylenedioxyphenyl) Cyclopentenol[1,2-b]Pyridine-6-Carboxylic Acid: Uptake by Isolated Rat Hepatocytes and Canalicular Membrane Vesicles

Purpose. Hepatobiliary excretions of drugs from the blood to the bile include two essential transmembrane processes: uptake into hepatocytes and secretion from hepatocytes. The purpose of this study was to clarify the transport mechanisms underlying these processes for a new non-peptide endothelin antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxy- phenyl)cyclopentenol[1,2-b]pyridine-6-carboxylic acid (J-104132). Methods. Biliary excretion of J-104132 was assessed in rats after intravenous injection. To evaluate the hepatic uptake process, J-104132 was incubated with freshly isolated rat hepatocytes and the uptake of J-104132 was calculated. To evaluate the biliary secretion process, the uptake of J-104132 into rat canalicular membrane vesicles that were isolated from normal Sprague-Dawley rats or Eisai hyperbilirubinemic rats was measured. Results. After intravenous injection, J-104132 was recovered from the bile quantitatively (99.7 ± 1.3%) as its intact form. J-104132 was taken up by isolated rat hepatocytes in a time- and temperature-dependent manner. The uptake was saturable with K _m and V _max of 5.7 M and 564 pmol/min/10⁶ cells, respectively. The uptake was Na⁺ independent and was reduced in the presence of ATP depleters (rotenone and carbonyl cyanide-p-(trifluoromethoxy)-phenylhydra- zone), organic anions (dibromosulfophthalein, indocyanine green, BQ-123, and pravastatin), and bile acids (taurecholate and cholate). In Sprague-Dawley rats, J-104132 was taken up by canalicular membrane vesicle ATP-dependently with K_m and V_max values of 6.1 M and 552 pmol/min/mg protein, respectively. However, ATP-dependent uptake disappeared in Eisai hyperbilirubinemic rats. Conclusions. These data suggest that energy-dependent and carrier-mediated transport systems play important roles in hepatobiliary excretion of J-104132 (both uptake and secretion processes), which is the main excretion route in rats. As for the secretion process of J-104132, an involvement of mrp2 was demonstrated.     

Pathogenic potential of vibriophages against an experimental infection with Vibrio cholerae O1 in the RITARD model

Cholera continues to be an important public health problem in developing countries, including India. This study concerns the feasibility of possible exploitation of bacteriophages as a biocontrol agent to eliminate the pathogen Vibrio cholerae from the gut using the removable intestinal tie–adult rabbit diarrhoea (RITARD) model. A control rabbit challenged with 10⁹ colony-forming units (CFU)/mL of V. cholerae MAK 757 developed Grade II to IV diarrhoea, but the phage-treated rabbit that received 10⁹ CFU/mL MAK 757 and 10⁸ plaque-forming units (PFU)/mL cocktail phages produced only Grade II diarrhoea. Histological results revealed that in the control rabbit (MAK 757-treated), villi lost their normal shape and showed more inflammatory cellular infiltration in the lamina propria compared with the experimental rabbit. Our data suggest that phages could be valuable as prophylaxis against V. cholerae infection.     

Activity and stability of alpha- and beta-mannosyltransferases in Candida albicans cells cultured at high temperature and at low pH

We measured the activity and stability of three mannosyltransferases to ascertain the mechanisms of changes in the antigenicity and the mannan structure of Candida albicans cells cultured at high temperature (37 degrees C) under acidic (pH 2.0) conditions in a liquid medium. The alpha-1,6-mannosyltransferase (alpha-1,6-MT) activity of the particulate-insoluble enzyme fractions prepared from C. albicans J-1012 (J-1012) cells cultured at 37 degrees C was retained compared to those at 27 degrees C, whereas beta-1,2-mannosyltransferase II (beta-1,2-MT II) activity was detected in the 27 degrees C fraction but not in the 37 degrees C fraction. Similar results were obtained in the fraction prepared from J-1012 cells cultured at pH 2.0. The alpha-1,6-MT activities of fractions prepared from C. albicans NIH B-792 (B-792) strain cells cultured at 37 degrees C were retained compared to those at 27 degrees C, whereas beta-1,2-mannosyltransferase VI-6 (beta-1,2-MT VI-6) activity was detected in the fraction of B-792 cells cultured at 27 degrees C but not detected in the 37 degrees C fraction. We also found that the beta-1,2-MT II and beta-1,2-MT VI-6 activity of C. albicans cells was more sensitive to both high temperature and low pH compared with alpha-1,6-MT activity.     

Sugar Ester J-1216 Enhances Percutaneous Permeation of Ionized Lidocaine

Percutaneous absorption enhancers affect not only the permeability of skin but also the thermodynamic properties of active ingredients in the vehicle. The present study examined the effect of J-1216, a sucrose laurate with hydrophilic–lipophilic balance = 16, on the percutaneous permeation of lidocaine (LC) from this point of view. The percutaneous permeation of LC from aqueous vehicles (pH 6.0, 7.0, 8.0, and 10.0) with or without 1.5% J-1216 was examined with excised hairless mouse skin mounted on flow-through-type diffusion cell. The permeation of LC without J-1216 increased with an increase in the vehicle pH and could be basically explained by pH-partition theory. J-1216 increased the LC permeation at pH 6.0 and 7.0 but decreased it at pH 8.0 and 10.0. The interaction between LC and J-1216 was examined using an ultrafiltration technique. J-1216 micelles interacted predominantly with unionized LC. A theoretical calculation suggested that J-1216 enhances the permeability coefficient of ionized LC, whereas it has almost no effect on that of unionized free LC. J-1216 directly affects the skin to increase the permeation of ionized LC, whereas J-1216 micelles interact with unionized LC to decrease the permeation. The effect of J-1216 is therefore a function of vehicle pH and LC concentration. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4482–4490, 2011