头孢哌酮/他唑巴坦(CFP/TAZ)对β-内酰胺酶稳定性及抑酶增效作用研究

目的：研究头孢哌酮／他唑巴坦（cefoperazone/tazobactam CFP／TAZR:1)对β－内酰胺酶稳定性及抑酶增效作用。方法：以生物法测定酶对抗生素的相对水解率，及抑酶保护率，并与同类复方制剂头孢哌酮／舒巴坦（CFP／SBT）和哌拉西林／他唑巴坦（PIP／TAZ）进行比较，结果：头孢哌酮／他唑巴坦（4：1配比）使单头孢哌酮水解率由100％降至16％，抑梅保护率由0％增至84．0％。头孢哌酮／他唑巴坦4：1时相对水解率16％低于同类复方制砂孢哌酮／舒巴坦1：21和哌拉西林／他唑巴坦8：1，它们分别为19．5％、18．8％，但无统计学意义（P＞0．05）。头孢哌酮／他唑巴坦4：1时的抑酶保护率84％高于同类复方制头孢哌酮／舒巴坦1：1，后者为80．5％，但无统计学意义（P＞0．05）。     

还原型谷胱甘肽的金属螯合亲和色谱纯化

制备了以壳聚糖为载体的金属螯合亲和色谱分离纯化还原犁谷胱甘肽（GSH）。研究了pH、铵离子浓度等对GSH洗脱的影响。根据试验结果确定洗脱液为pH4．2、0．02mol／L磷酸盐缓冲液（含0．5mol／L NH4Cl），所得GSH纯度可达49．8％，平均提取率为67．9％。     

阻塞性睡眠呼吸暂停低通气综合征合并肺动脉高压者血浆肾上腺髓质素、内皮素-1水平及意义

目的测定阻塞性睡眠呼吸暂停低通气综合征（OSAHS）合并肺动脉高压（pH）患者血浆肾上腺髓质素（adrenomedullin，ADM）、内皮素-1（ET-1）水平，并探讨其临床意义。方法选2002年3月至2004年10月在郑州大学第一附属医院经多导睡眠仪监测诊断的58例OSAHS患者，分为单纯OSAHS组、OSAHS并pH组，选取30名健康体检者为正常对照组。检测各组血浆ADM、ET-1水平、RVPEP／AT、AHI、minSaO2及TSaO2水平，并进行相关性分析。结果单纯OSAHS组及OSAHS并pH组血浆ADM、ET-1水平[ADM：（54．19±11．60）pg／ml，ET-1：（52．2±8．96）pg／ml；ADM：（79．82±15．33）pg／ml，ET-1：（79．3±11．25）pg／ml]均高于正常对照组[ADM：（44．73±8．52）pg／ml，ET-1：（37．4±9．85）pg／ml，P〈0．01]；OSAHS并pH组又高于单纯OSAHS组（P〈0．01）。经nCPAP治疗后OSAHS并pH患者血浆ADM、ET-1水平明显降低[ADM：（79．82±15．33）pg／ml，ET-1：（79．3±11．25）pg／ml；ADM：（63．48±10．76）pg／ml，ET-1：（62．4±8．23）pg／ml，P〈0．01]，血浆ADM水平始终与ET-1、RVPEP／AT、AHI、TSaO2呈正相关，与minSaO2呈负相关。结论ADM、ET-1可能参与了OSAHS患者pH的发生发展过程。     

重组芽孢杆菌脂肪酶拆分制备(R)-氟比洛芬

利用大肠杆菌M15表达芽孢杆菌的脂肪酶基因，重组蛋白经亲和色谱纯化，得到比活力为30.25u／mg的纯化酶。以纯化酶催化水解50mmol／L的氟比洛芬乙酯，在55℃、pH9．0的条件下，所得产物（R）-氟比洛芬ee值为99.6％。     

新分离到的嗜热拟青霉中不含纤维素酶的耐热木聚糖酶的分离纯化及特性研究

新分离得到的嗜热拟青霉（Paecilomyces themophila）J18在含4％（w／v）玉米芯的培养基上，50℃培养5d，能生产1470u／ml的木聚糖酶。木聚糖酶经2．4倍浓缩纯化后收率为38.3％，在SDS-PAGE凝胶电泳上呈现分子量为25．8k的单一条带。该木聚糖酶是中性糖含量为21．0％的糖蛋白，最适pH为7．0，在pH6．0～11．0稳定。最佳温度为75～80℃，在pH7．0、75℃时也稳定。     

重楼总皂苷的纯化工艺研究

目的：优选重楼总皂苷的纯化工艺。方法：综合运用醇溶液调节pH、醇提和大孔吸附树脂技术,以总皂苷含量、总皂苷收率为指标,优选重楼总皂苷的纯化工艺。结果：优化后的重楼总皂苷的纯化工艺条件为：70％乙醇提取液调节pH值至9,抽滤,将滤液调pH值至中性,浓缩至0．5g生药／mL,调pH值至8,离心（4000r／min,30min）,上清液部分上D101大孔树脂柱纯化（上样量为70％饱和吸附量,径高比为1：5,上样流速为4．5BV／h,纯化溶剂为20％乙醇,用量为6BV,洗脱溶剂为70％乙醇,用量为8BV,流速为7BV／h）。采用本工艺制备的中间体中总皂苷含量达71．6％,总皂苷收率为85．71％。结论：验证试验结果表明,优选出的工艺稳定、合理、可行。     

抑瘤肽注射液的研制及其抑瘤作用研究

目的研制抑瘤肽注射液。方法采用生化方法从乳猪肝中提取可溶性胶原，经胃蛋白酶水解后制成抑瘤肽注射液，同时拟订质量标准并进行检验；观察抑瘤肽注射液对荷瘤昆明小鼠HepA肝瘤和S180纤维肉瘤及小鼠体内肝癌H22移植瘤的影响。结果抑瘤肽注射液生产工艺路线合理，质量达到拟订标准。抑瘤肽注射液能明显抑制HepA肝瘤生长，25，50，100，200mg／（kg·d）的抑制率分别为43．26％，55．06％，74、71％，40．47％；对于荷瘤小鼠S180纤维肉瘤，25，50，100mg／（kg·d）的抑制率分别为53．87％，76．53％，58．20％，而阿霉素阳性对照组[2mg／（kg·d）]的抑制率为62．41％；对小鼠体内肝癌H22移植瘤生长的抑制呈剂量相关性，50，100，200mg／（kg·d）的抑制率分别为48．1％，60.0％，74、8％，而卡铂阳性对照组[20mg／（kg·d）]的抑制率为63．7％。结论制备出的抑瘤肽注射液合格，且适合于大规模生产，能明显地抑制荷瘤小鼠肿瘤的生长。     

细胞色素P450 2C19基因多态性对奥美拉唑胶囊抑酸效果的影响

目的研究细胞色素P4502C19（CYP2C19）基因多态性对奥美拉唑胶囊抑酸效果的影响。方法在15名幽门螺杆菌感染阴性的健康志愿者中，用聚合酶链反应-限制性片段长度多态性方法确定CYP2C19基因型，分为纯合子强代谢型（hom EM），杂合子强代谢型（het EM）和弱代谢型（PM）3组，每组5人。受试者口服奥美拉唑每天20mg，连续8天。分别在服药第1天和第8天，用24h胃内pH测定仪分析24h胃内pH中位值、pH〉4及pH〉3的总时间。结果24h胃内pH中位值、pH〉4和pH〉3的总时间，PM组显著高于hom EM组和het EM组（P〈0．05）。hom EM组和her EM组的24h胃内pH监测参数，在服药第8天显著高于第1天（P〈0．05）。夜间抑酸效果，PM组也优于hom EM组和het EM组（P〈0．05）。结论CYP2C19基因多态性对奥美拉唑的抑酸效果及夜间酸突破现象均有明显影响。     

肠膜样明串珠菌B-1149中蔗糖磷酸化酶基因的克隆及其在大肠杆菌中的表达

分离和鉴定了肠膜样明串珠菌NRRL B-1149的蔗糖磷酸化酶（SPase）基因1149sp。它由1479bp的核苷酸构成，编码了1149 SPase的492个氨基酸残基，分子量为56．1k。该基因具有独特的C端氨基酸序列（^439DVETPSDTHKITRKDKSGENVAVLVANA ADKTFTITANGEEILANTEADKQQL^492）。在大肠杆菌中表达1149sp，纯化的1149 SPase对蔗糖的比活力为1．49IU／mg。SPase在宽泛的反应温度和pH范围内均有活性，分别为20～50℃，pH6．0～7．5。最适温度和pH分别为37℃和6．7。1149 SPase对底物蔗糖的米氏常数Km为6．3mmol／L，催化速度常数放。是1．59s^-1。其具有宽泛的受体特异性，能将蔗糖的部分葡萄糖基或葡萄糖-1-磷酸转移至多种受体。     

应用来源于肺炎克雷伯杆菌的D-阿拉伯糖异构酶的新底物特异性以D-阿洛酮糖制备D-阿卓糖

从肺炎克雷伯杆菌40bXX中纯化了D-阿拉伯糖异构酶，经电泳得单一条带，收率为62．5％，比活增加了12倍。纯化的酶在最适条件下（50mmol／L glycine-NaOH，pH9．0，40℃）对D-可拉伯糖和L-岩藻糖有最强活性，但对不同的D／L醛糖有较宽泛的底物专一性，例如：D-阿拉伯糖、L-岩藻糖、D／L-木糖、D-甘露糖、     

Effects of product formulation on in vitro activity of pancreatic enzymes

The lipase activity of two enteric-coated and two uncoated pancreatic enzyme formulations was evaluated in vitro at different pH values and compared with postprandial duodenal pH data in cystic fibrosis patients. Lipase activity was measured over a pH range of 4-8 in four formulations: Viokase tablets and Viokase powder (Viobin), Cotazym-S (Organon), and Pancrease enteric-coated spherules (McNeil). A pH-stat technique was used in which the amount of hydroxyl ion that must be added to maintain a preset pH value is measured to determine the amount of lipase substrate (tributyrin) that is split into butyric acid. At least three determinations of activity were made at each pH. Six capsules of each enteric-coated formulation were subjected to disintegration testing at various pH values. These data were then compared with data available for postprandial duodenal pH in patients with cystic fibrosis. The lipase activity of all formulations studied decreased when pH decreased, especially when the pH was below 5.75. At a pH value between 5 and 5.5, which represents the postprandial duodenal pH in cystic fibrosis patients, activity was reduced 50% or more for all formulations tested. The two enteric-coated products displayed no activity at pH 5.5 and below because the coating did not dissolve in this pH range. Lipase activity in enteric-coated pancreatic enzyme preparations is limited because the enteric coating of these products dissolves slowly in the duodenal pH range (5-5.75) that is found in patients with cystic fibrosis.     

Role of calcium in the efflux system of Escherichia coli

Efflux of antibiotics by Escherichia coli AG100 is performed by a variety of efflux pumps, ensuring survival of the bacterium in widely diverse media. At pH 5, efflux is independent of metabolic energy during the period of time the assay is conducted; at pH 8 it is totally dependent upon metabolic energy. Because calcium ions (Ca(2+)) are important for membrane transport channels and the activity of ATPases that provide energy functions, the role of Ca(2+) in the extrusion of an efflux pump substrate under conditions that challenge the bacterium was investigated. Real-time accumulation and efflux of ethidium bromide (EtBr) by E. coli K-12 AG100 strain [argE3 thi-1 rpsL xyl mtl Δ(gal-uvrB) supE44] was determined by a semi-automated fluorometric method in the presence and absence of Ca(2+) and agents that are known to inhibit access of calcium to enzymes that provide energy. Chlorpromazine (CPZ), an inhibitor of calcium binding to proteins (calcium-dependent enzymes), and ethylene diamine tetra-acetic acid (EDTA), a chelator of Ca(2+), increased accumulation and efflux of EtBr at pH 8 but not at pH 5. Ca(2+) reverses these effects when the assay is conducted at pH 8. In conclusion, the activity of the efflux pump system of E. coli is dependent upon metabolic energy at pH 8. Because at pH 8 hydrolysis of ATP is favoured and contributes protons for activation of the AcrAB-TolC efflux pump, CPZ is suspected of having its effects on accumulation/efflux of EtBr by indirectly affecting ATPase activity that is dependent upon Ca(2+).     

Synthesis, stability studies, anti-inflammatory activity and ulcerogenicity of morpholinoalkyl ester prodrugs of niflumic acid

In search for potential prodrugs for anti-inflammatory drug candidates in the niflumate series, novel morpholinoalkyl ester prodrugs of niflumic acid (CAS 4394-00-7) 5a-b were prepared by esterification of appropriate morpholinylalkyl alcohols 3a-b with niflumic acid 4 in the presence of dicyclohexyl carbodiimide (DCC) and catalyst dimethylamino pyridine (DMAP) at 0-5 degrees C. The structures were confirmed by elemental and spectral data (UV, IR, 1H-NMR, 13C-NMR, and EI-MS). The ester prodrugs 5a-b showed better solubility than the parent drug niflumic acid 4 in simulated gastric fluid (SGF) and phosphate buffer (pH 7.4). The in vitro hydrolysis studies were conducted at pH 1.3 (SGF), phosphate buffer (pH 7.4) and in human plasma diluted with phosphate buffer (pH 7.4) at 37+/-0.5 degrees C using HPLC with UV detection. The ester prodrugs 5a-b were quantitatively hydrolyzed to the parent drug niflumic acid 4 by enzymatic and/or chemical means. It is observed that an increase in the carbon chain length rendered the prodrugs 5a-b more stable in phosphate buffer (pH 7.4) than in pH 1.3 (SGF), but they were rapidly hydrolyzed in human plasma at 37+/-0.5 degrees C. They exhibited longer hydrolytic half-lives of 16.11-53.30 h in aqueous buffer solutions (pH 1.3 and 7.4) and 1.63-2.73 min in human plasma, respectively. The title compounds were evaluated in vivo for anti-inflammatory activity in carrageenan induced rat paw oedema model in rats at the doses 45, 90, 150 mg/kg b.w. The test compounds exhibited good anti-inflammatory activity (46.6-53.2 % at the dose of 150 mg/kg b. w.) with respect to niflumic acid (78.7 % at the dose of 90 mg/kg b.w.). The compounds were also screened for in vivo ulcerogenicity, it was observed that the prodrug 5b was significantly less irritating to gastric mucosa than compound 5a and the parent drug niflumic acid 4 following single and chronic oral administration in rats.     

IL-2基因工程菌的发酵及其包含体制备工艺的初探

本文对IL-2基因工程菌的发酵及其包含体制备工艺进行了研究.结果表明在发酵过程中pH的稳定(7.0)、糖的补充(3%)、O_2的及时供给(<10PSIG持续泵入)和发酵密度的控制(OD_(550)=1.9),对提高发酵产量至关重要(12g/L).在包含体制备及溶解工艺中,以4mol/L脲去除脂质及杂蛋白,1%SDS溶解包含体,可获最大IL_2活性单位(2.35×10~7u/ml,即相当于5L发酵可获3.53×10~9u).     

Stability of the Thrombolytic Protein Fibrolase: Effect of Temperature and pH on Activity and Conformation

The effect of temperature and pH on the activity and conformation of the thrombolytic protein fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (22°C). At 37°C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alphahelical structure was lost in a cooperative transition (T _m of 50°C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the T _m determined by NMR was 46°C at pD 8. The effect of pH on the proteolytic activity and structure of fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of fibrolase decreased to 43°C at pH 5. Protein concentrations determined over the pH range 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.     

Separation of plasma kallikrein and a kallikrein-like plasminogen activator generated by acetone in rat plasma

Plasminogen activator (PGA), kininogenase (Kase) and benzoyl arginine ethyl ester (BAEe) activities generated in plasminogen-free rat plasma by incubation with acetone (23% v/v) at 22 degrees were purified. The activities passed unadsorbed through columns of DEAE-Sephadex A-50 (pH 7.8) and arginine methylester-Sepharose 4B (pH 8.5). Part of the activities (rat plasma kallikrein) was adsorbed onto a soybean trypsin inhibitor (SBTI)-Sepharose 4B column at pH 8.5. At pH 7.0 a fraction with higher ratios PGA/BAEe esterase and Kase/BAEe esterase was also adsorbed. Both fractions could be eluted with 5 mM sodium hydroxide. The fraction not adsorbed at pH 8.5, but adsorbed at pH 7.0 was designated low molecular weight plasminogen activator (LMr-PGA), a plasminogen activator fraction with higher molecular weight, but without esterase activity being also present (Berstad & Briseid 1982). LMr-PGA was strongly inhibited by tranexamic acid (AMCA) 0.10 mM, whereas the fraction designated rat plasma kallikrein was not. By polyacrylamide gel electrophoresis Mr-values in the range 120,000 to 130,000 were established for native samples of both rat plasma kallikrein and LMr-PGA, whereas Mr-values of 78,000 to 80,000 were established after treatment with SDS.     

New renin inhibitors with hydrophilic C-terminus

Four new peptide-based renin inhibitors, Boc-Phe(4-OMe)-MePhe-AHPPA-epsilon Ahx-EA (11), Boc-Phe(4-OMe)-MeLeu-AHP-PA-epsilon Ahx-EA (15), Boc-Phe(4-OMe)-MePhe-Sta-epsilon Ahx-EA (20) and Boc-Phe(4-OMe)-MeLeu-Sta-epsilon Ahx-EA (21) have been synthesized in search of structures with improved biological properties. They were designed as compounds with moderate hydrophobicity (5.28, 4.79, 4.79 and 4.30), respectively. All synthesized inhibitors were resistant to chymotrypsin activity, all were poorly soluble in buffers pH 2.0 and pH 7.4. The inhibitory potency of renin activity in vitro of 11, 15, 20 and 21 expressed as IC50 was 7.0 x 10(-4), 7.5 x 10(-5), 6.0 x 10(-4) and 2.5 x 10(-4) M/l, respectively.     

Metabolism of platelet 5-hydroxytryptamine in acidified platelet concentrates

The present study was designed to understand the metabolism of 5-hydroxytryptamine (serotonin, 5HT), a dense granule constituent of platelets, in acidified platelet concentrates (PC) during in vitro storage in polyvinylchloride plastic containers at 22 degrees C. High-performance liquid chromatographic analysis showed that significant amounts of 5HT and its metabolites, 5-hydroxytryptophol and 5-hydroxyindole-3-acetic acid (5HIAA), were detected in the plasma of all PC stored for 3 days with a pH below 6.0, measured at 37 degrees C. PC with a platelet number over 7 x 10(10) cells maintained low PO2 values. Rapid fall in pH occurred in these PC with low PO2 values. The rate of lactate production of platelets increased several fold because of an insufficient oxygen supply to them. In PC with a sufficient supply of oxygen, the adenosine triphosphate (ATP) production rates were estimated to be 8 nmol/min/10(9) platelets. In PC acidified below pH 6.0, the oxidative phosphorylation nearly ceased, and glycolysis was also inhibited: ATP production rates averaged 1 nmol/min/10(9) platelets at 3-day storage. Numerous clusters of platelet aggregates were found in acidified PC, and platelets changed into spherical shapes with accompanying swelling. These platelets did not respond to adenosine triphosphate and failed to produce shrinking when suspended in hypotonic solutions. 5HT was not metabolized in acidified platelet-poor plasma. These data suggest that the metabolic activity of platelets was impaired when the pH of the PC fell below 6.0, leading to the release of platelet 5HT into the plasma. A substantial amount of 5HT might be metabolized by platelet monoamine oxidase into 5HIAA and 5-hydroxytryptophol.     

Studies on the mechanism of action of omeprazole

The effects of omeprazole on preparations of pig gastric (H+ + K+)-ATPase have been studied. Omeprazole was found to inhibit the (H+ + K+)-ATPase activity in a time-dependent manner. Inhibition was more pronounced at pH 6.1 compared with pH 7.4 and decreased as the concentration of (H+ + K+)-ATPase preparation increased. The potency of omeprazole was therefore highly dependent upon the conditions used. When pre- incubated with (H+ + K+)-ATPase preparation (30 micrograms protein/ml) for 30 min at 37 degrees and pH 6.1, omeprazole inhibited the (H+ + K+)-ATPase activity with an IC50 of 3.9 microM. This inhibition was shown to be irreversible in nature. Whilst omeprazole itself was not very potent as an inhibitor of the (H+ + K+)-ATPase activity at pH 7.4 (IC50 = 36 microM), transient acidification of omeprazole resulted in the formation of a compound(s) which produced marked inhibition at this pH (IC50 = 5.2 microM). The effects of omeprazole in the absence of acidification may have resulted from the rate-limiting formation of this compound. Radiolabelled omeprazole was shown to incorporate into the (H+ + K+)-ATPase preparation in a time-dependent and pH-dependent manner. Omeprazole, radiolabelled in three separate positions (the sulphur atom and the two adjacent carbon atoms), incorporated with equivalent time courses suggesting that the incorporation did not involve a fragmentation of the omeprazole molecule. Under conditions shown to produce a 50% inhibition of (H+ + K+)-ATPase activity, [14C] omeprazole had incorporated to a level of 4-5 nmoles/mg protein. Incorporation continued beyond the point required to produce 100% inhibition of (H+ + K+)-ATPase activity and reached 30 nmoles/mg protein after 5 hr. Prior acidification of the omeprazole resulted in a more rapid initial rate of incorporation although the final level of incorporation was lower than for omeprazole. Omeprazole was also shown to interact with the (Na+ + K+)-ATPase from dog kidney. Omeprazole inhibited the (Na+ + K+)-ATPase activity (IC50 = 186 microM). Acid-degraded omeprazole inhibited the (Na+ + K+)-ATPase activity with greater potency (IC50 = 19 microM) and was also shown to incorporate into this enzyme preparation.     

The in vitro properties of a new hydroxypyridone antimycotic rilopirox, with special reference to its anti-Candida activity

In vitro properties of a new hydroxypyridone antimycotic rilopirox (RIL) with special reference to its anti-Candida activities were studied in comparison with the three reference drugs, ciclopirox olamine (CPO), oxiconazole nitrate (OCZ) and isoconazole nitrate (ICZ), using several strains of Candida albicans and Candida glabrata as the test organisms. RIL was potently fungicidal for growing cultures of these Candida strains, whereas all the three reference drugs were slightly fungicidal or fungistatic. Unlike OCZ and ICZ whose anti-Candida activity was decreased by lowering pH or adding serum to culture media, the activity of RIL was scarecely affected by change in pH or serum addition. However, RIL became less potent in the presence of Fe3+ at concentrations of 10(-5) mmol/ml or above. These findings suggest that RIL will be useful as a topical anti-Candida agent.