SPF-32629 A and B, novel human chymase inhibitors produced by Penicillium sp

Two new human chymase inhibitors, SPF-32629A and B, were isolated from the cultured broth of Penicillium sp. SPF-32629. These structures were determined by spectroscopic methods and identified as new pyridone compounds. SPF-32629B was the carboxylated derivative of SPF-32629A. SPF-32629A and B specifically inhibited human chymase among four serine proteases tested with the IC50 of 0.25 and 0.42 microg/ml, respectively.     

检测大肠杆菌菌体蛋白的夹心ELISA试剂盒的建立及应用研究

目的  建立特异、敏感的检测大肠杆菌菌体蛋白的夹心ELISA试剂盒。方法  采用大肠杆菌菌体蛋白免疫家兔,制备得到高效价抗菌体蛋白抗血清。将经饱和硫酸铵盐析、阴离子交换柱层析和亲和层析纯化的兔抗大肠杆菌菌体蛋白特异性多克隆抗体作为包被抗体和检测抗体,用生物素标记检测抗体,并加入辣根过氧化物酶（HRP）标记的亲和素。结果　建立的大肠杆菌菌体蛋白夹心ELISA检测方法的敏感性为0.32 μg/L,检测范围为1~100 μg/L,与国际同类商品化试剂盒相当。此法具有良好的稳定性,其批内变异系数小于7.7%,批间变异系数小于6.2%。结论  建立了特异、敏感、稳定的检测大肠杆菌菌体蛋白的夹心ELISA试剂盒,为生物制品中残留大肠杆菌菌体蛋白的质量控制提供了一种重要的检测方法。     

Development of a field test kit for coliphage detection in natural waters

Coliphages may be suitable alternative indicators of fecal pollution in natural waters. The relationship between coliphage and fecal coliform numbers may be expressed by log₁₀ transformed linear regression equations in surface waters (correlation coefficient, r² = 0.5872; p < 0.001) and in well waters (r² = 0.4767; p < 0.001). Using the American Public Health Association 919C single agar layer method, coliphages can be enumerated easily and at less cost and more rapidly than coliform enumeration methods. In adapting this test for use in a field test kit, however, further modifications and simplifications are required. The use of 2,3,5-triphenyl tetrazolium chloride (TPTZ) to enhance plaque visibility while helpful, was not essential. Matched-pair t-test analysis showed no significant difference between the plaque numbers enumerated with or without TPTZ to help plaque visibility. The Escherichia coli C host bacteria can be maintained either as a frozen preparation or dried on to paper disks with protective milk colloids. In the latter procedure, a resuscitation period of 18–24 h in broth at 30°C was found to be satisfactory in giving plaque numbers in the test that were not significantly different than when frozen host was used. A portable field kit has been designed to carry out a total of eight tests. The kit includes the media, the bacterial host, petri dishes, syringes, receptacles, and a camping gas cylinder. It measures 38 cm by 25.5 cm by 33.5 cm and weighs about 6.4 kg. Using the kit, the test procedure for a water sample can be carried out in under 15 min.     

抗感染新药重组人溶菌酶的临床前评价

重组人溶菌酶是经生物技术改构后产生的一类对G+和G-菌都有抗菌效应的一类新药。临床前数据表明它对1 040株致病菌具有抗菌活性,最低抑菌浓度(MIC)范围为0．125～>4000μg·mL-1;此外,它还有抑制冠状病毒、抗炎和镇痛作用。现综述其药效学、毒理学和药动学的研究情况。新型重组人溶菌酶在体内外均安全和有效,有望开发成为新型抗感染药物。     

Recombinant human basic-fibroblastic growth factor: different medical dressings for clinical application in wound healing.

Recombinant human basic-Fibroblastic Growth Factor (rhb-FGF) is a basic single-chain protein showing high activity as mitogenetic and angiogenetic agent. The application of rhb-FGF in wound healing as stimulator of the tissue repair process is strictly connected with the covering of the wound by means of a proper dressing. A wide number of synthetic occlusive or non-occlusive wound dressings has been developed. Owing to the delicate proteic structure of rhb-FGF, and generally of all the Growth Factors, compatibility with the dressings has to be every time tested, to avoid its inactivation and consequent loss of tissue repair properties.     

Quantitation of nimesulide in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection and its application to a bioequivalence study

A rapid and simple high-performance liquid chromatography-reversed phase (HPLC-RV) method with ultraviolet detection (258 nm) was developed and validated for the quantitation of nimesulide (CAS 51803-78-2) in human plasma. After the plasma samples were extracted with 6.0 ml of dichloromethane containing the internal standard phenacetin 2 microg/ml in methanol, the analysis of the nimesulide level in the plasma samples was carried out using a reverse phase Supelcosil LC-18 (15 cm x 4.6 mm x 5 pm) column.The chromatographic separation was accomplished with an isocratic mobile phase consisting of a mixture of methanolphosphate buffer (pH 3.5; 0.01 mol/l) (55:45, v/v). The inter-assay accuracy ranged from 103.4 to 113.2%, while intra-day ranged from 105.6 to 117.5%. The inter-assay precision ranged from 11.7 to 14.6%, while intra-assay ranged from 3.2 to 9.5%. The recovery of nimesulide was determined as part of the assay validation process and was excellent. The linearity of the nimesulide curves ranged from 0.20 to 15.0 microg/ml (y = 0.3857-0.0081, r = 0.9975). Short-term stability showed that nimesulide is stable in plasma for at least 24 h at room temperature, while long-term stability studies showed that nimesulide is stable in plasma for at least 180 days when stored at -20 degrees C. This validated method was successfully applied to the bioequivalence study of nimesulide in tablets in healthy volunteers.     

一种ADR信号检测的改进算法及其在上海市ADR自发呈报数据库上的应用

目的 建立改进的信号检测算法并评估其实际检测效果.方法 建立基于新ADR报告(NAR)的信号检测算法;通过理论分析和实际演算比较基于报告数(REP)和基于药物-不良事件组合(DEC)两种算法的ADR检测效果,评估数据库中已知ADR报告对信号检测的影响;并将NAR算法应用于上海市ADR自发呈报数据库.结果 ①对上海市ADR自发呈报数据库部分数据(2006～2007年)进行了信号检测计算,结果发现基于DEC的算法会产生不合逻辑的虚假信号,而基于REP的算法能避免产生这类信号并能发现被DEC算法忽略的有价值信号;筛除已知ADR报告能提高信号检出率57%.②建立了包含2 650种药物,27 230种DEC的已知ADR数据库,可自动筛选NAR.③采用基于NAR的信号检测算法对上海市2004～2007年ADR自发呈报数据库进行检测,产生信号380条,包括加替沙星-低血糖反应、头孢他啶-过敏性休克,菌栀黄-荨麻疹等值得关注的信号.结论 基于NAR的算法能显著提高ADR信号的检测效率.     

The detection of cytotoxicity produced by short-lived reactive intermediates: a study with bromobenzene

1. A V79 cell incubation incorporating rat liver 9000 g supernatant (S9) fractions, used previously to detect the toxicity due to long-lived, stable metabolites of cyclophosphamide, has been used to study the toxicity of short-lived, reactive metabolites generated from bromobenzene. 2. Cytotoxicity was observed in the presence of S9 fractions from rats treated with phenobarbitone but not in the presence of S9 fractions from untreated or beta-naphthoflavone-treated animals. This toxicity was enhanced by depletion of the glutathione in the S9 fraction by prior treatment of the animals with diethyl maleate and was reduced by SKF 525 A, in agreement with results in vivo on the mechanism of bromobenzene-induced hepatotoxicity. 3. This study demonstrates that cytotoxicity due to the generation of short-lived, reactive metabolites can be detected in this system in vitro provided that procedures are used to modify the activating and detoxifying enzyme systems within the S9 fraction.     

The detection of cytotoxicity produced by short-lived reactive intermediates: a study with bromobenzene

1. A V79 cell incubation incorporating rat liver 9000 g supernatant (S9) fractions, used previously to detect the toxicity due to long-lived, stable metabolites of cyclophosphamide, has been used to study the toxicity of short-lived, reactive metabolites generated from bromobenzene.

2. Cytotoxicity was observed in the presence of S9 fractions from rats treated with phenobarbitone but not in the presence of S9 fractions from untreated or β-naphthoflavone-treated animals. This toxicity was enhanced by depletion of the glutathione in the S9 fraction by prior treatment of the animals with diethyl maleate and was reduced by SKF 525 A, in agreement with results in vivo on the mechanism of bromobenzene-induced hepatotoxicity.

3. This study demonstrates that cytotoxicity due to the generation of short-lived, reactive metabolites can be detected in this system in vitro provided that procedures are used to modify the activating and detoxifying enzyme systems within the S9 fraction.     

Recombinant human follicle-stimulating hormone: a controversial issue--without controversy

Recent developments in recombinant DNA technology have resulted in a rapidly expanding range of new diagnostic and therapeutic opportunities. This technology paved the way for identification, isolation, cloning and production of specific proteins. Recently, recombinant human gonadotropins became available for clinical use. Pharmacokinetics, receptor availability, pharmacodynamics and safety were extensively studied and the drugs were found to be identical, if not superior, to urinary gonadotropins that have been used in reproductive medicine for the last 40 years. It is currently apparent that the use of recombinant human gonadotropins is expected to provide improved batch-to-batch consistency, steady supply and most importantly, a purified compound with highly specific activity, which accounts for > 99% of the protein content of the preparation, making it possible to administer subcutaneously. The purpose of this report is to describe and evaluate recent developments in the use of recombinant human follicle-stimulating hormone.     

Characterization of angiotensin II formation in human isolated bladder by selective inhibitors of ACE and human chymase: a functional and biochemical study

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle.
2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO₂Me) and a recently developed human chymase substrate Pro¹¹-,D-Ala¹²)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used.
3. Angiotensin I (AI) (0.3 μM) induced a contractile response amounting to 58±5% (n=12) of the initial K⁺ (124 mM)-induced contractions. This response was reduced to 36±3% (n=8) by the ACE-inhibitor enalaprilat (10 μM), while pretreatment with soybean trypsin inhibitor (STI 200 μg ml⁻¹) or CH 5450 (10 μM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19±5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1±1% (n=6).
4. The substrate (Pro¹¹-,D-Ala¹²)-AI (3 μM) produced contractions which amounted to 57±4% (n=13) of the initial K⁺ (124 mM) contractions. These contractions were not affected by enalaprilat (10 μM). On the other hand, STI (200 μg ml⁻¹) and CH 5450 (10 μM) added separately, depressed the (Pro¹¹-,D-Ala¹²)-AI-induced contractions to 34±5% (n=6) and 24±4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition.
5. Experiments with detrusor membrane fractions incubated with AI (50 μM) were performed. In the presence of enalaprilat (100 μM), carboxypeptidase inhibitor CPI (10 μg ml⁻¹) and aprotinin (15 μM), CH 5450 (10 nM–1 μM) caused a concentration-dependent inhibition of AII formation.
6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

     

新型酰基转移酶AT-EF080951的重组表达及底物结合分析

目的 研究新型聚酮合酶(PKS)基因簇EF568935(Gen Bank登录号)中酰基转移酶(AT)EF080951(GenBank登录号)底物特异性,为PKS的功能研究及组合生物合成研究提供新组件.方法 从酰基转移酶AT-EF080951结构域两端的连接肽区设计引物,通过PCR克隆其结构域基因at-EF080951;连接入原核表达载体pMAL-c2X,与麦芽糖结合蛋白(MBP)融合表达;直链淀粉树脂柱亲和层析纯化MBP-AT-EF08095l;融合蛋白用Xa因子切除MBP,得到AT-EF080951单体;以MBP、小牛血清白蛋白(BSA)为对照蛋白,用紫外分光光度法测定MBP-AT-EF080951与AT-EF080951对底物的结合能力,计算结合常数(Ka,μmol/L),分析其结合底物的特异性.结果 (1)MBP-AT-EF080951与底物的结合常数为甲基丙二酰辅酶A,0.0049±0.001;丙二酰辅酶A,0.0049±0.003;乙酰辅酶A,0.107±0.002;辅酶A,0.005±0.003.(2)AT-EF080951与底物的结合常数为甲基丙二酰辅酶A,0.005±0.002;丙二酰辅酶A,0.0041±0.002;乙酰辅酶A,0.120±0.001;辅酶A,0.0042±0.003.结论 MBP-AT-EF080951和AT-EF080951都特异性结合乙酰辅酶A,AT-EF080951可能是乙酰转移酶.     

Recombinant cell bioassays for endocrine disruptors: development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals

The ability of a variety of compounds to disrupt normal endocrine homeostasis, and potentially, the physiological and reproductive capacity of an organism, has gained worldwide attention in recent years. In an attempt to identify such compounds, and to examine the mechanism(s) by which they may exert their actions, we have constructed reporter plasmid vectors that contain the firefly luciferase gene under hormone-inducible control of estrogen-, androgen-, or retinoic acid-responsive DNA enhancer elements. Transient transfection of these vectors into appropriate receptor-containing cell lines revealed their ability to respond to their respective hormones with the induction of luciferase. Here, we describe development and optimization of a recombinant human ovarian carcinoma (BG-1) line, which has been stably transfected with the estrogen responsive luciferase reporter plasmid. The resulting recombinant cell line (BG1Luc4E(2)) responds to 17beta-estradiol at concentrations as low as 1 pM. The utility of BG1Luc4E(2) cells as a bioassay screening system for environmental estrogens was demonstrated by their response to known xenoestrogens, and also by the putative identification of two polychlorinated biphenyls (2,3',4, 4,'-tetrachlorobiphenyl and 2,2',3,5',6-pentachlorobiphenyl) as novel estrogenic chemicals. These cell bioassay systems have applications for rapid screening, identification, and characterization of endocrine disrupting chemicals.     

Total analysis system for tumor promoter microcystins produced by cyanobacteria

Microcystins, produced by freshwater cyanobacteria, are cyclic peptide hepatotoxins and tumor promoters. An outbreak of human poisoning attributed to microcystins has been reported in Caruaru, Brazil in 1996, where exposure through renal dialysis led to the death of 50 patients. Although such severe acute effects on human health seem to be rare, microcystins poses problems to human health which could result from low-level, chronic exposure to microcystins in drinking water. It is therefore important to monitor the levels of microcystins in water reservoirs where cyanobacterial blooms occur. We have developed a total analysis system for microcystins using GC-MS and LC-MS. This comprises initial screening of samples to check for the presence of microcystins by detecting 2-methyl-3-methoxy-4-phenylbutyric acid as an ozonolysis product using thermospray interface LC-MS and electron ionization/GC-MS. If a sample is positive in a screening test, it will be necessary to follow through with identification and quantification. Frit-FAB interface LC-MS allowed the rapid identification of microcystins in cyanobacteria and lake water, and also enabled us to identify microcystins and their metabolites formed in vivo in mouse liver. Finally, Frit-FAB/LC-MS using selected ion monitoring could be used for quantitative analysis of microcystins in lake water in the low nanogram range. The total analysis system proposed in the present study should be applicable to studies of the metabolism of microcystins, of their detoxification, and those of the mechanism(s) of the accumulation in the food chain.     

Recombinant adeno-associated virus: clinical application and development as a gene-therapy vector

Gene therapy is gaining momentum as a method of treating human disease. Initially conceived as a strategy to complement defective genes in monogenic disorders, the scope of gene therapy has expanded to encompass a variety of applications. Likewise, the molecular tools for gene delivery have evolved and diversified to meet these various therapeutic needs. Recombinant adeno-associated virus (rAAV) has made significant strides toward clinical application with an excellent safety profile and successes in several clinical trials. This review covers the basic biology of rAAV as a gene therapy vector as well as its advantages compared with other methods of gene delivery. The status of clinical trials utilizing rAAV is also discussed in detail. In conclusion, methods of engineering the vector to overcome challenges identified from these trials are covered, with emphasis on modification of the viral capsid to increase the tissue/cell-specific targeting and transduction efficiency.     

Application of baculovirus-insect cell expression system for human therapy

A major advantage of recombinant DNA technology is its flexibility allowing for "on demand" production of specific proteins with theurapeutic value in heterologous expression systems. Gene expression vectors based on baculovirus, insect virus attacking mostly lepidopteran species, are frequently used for relatively inexpensive and fast production of such proteins. This expression system is recognized as one of the most powerful technologies for commercial synthesis of glycoproteins originating from vertebrate themselves or from vertebrate viruses. Glycosylation pathways utilized by insects are not identical, though they are similar to vertebrate glycosylation pathways. In the review special attention is given to the development of new virus-like particles (VLPs) potential vaccines which represent a novel class of subunit vaccines that are able to stimulate efficiently cellular and humoral immune responses against viral agents. Apart from production of vertebrate proteins or VLPs "on demand " in insect cells, a new exciting field of using baculovirus as gene delivery system to vertebrate cells was recently open which has a great potential for future uses of baculovirus as effective gene therapy vector.     

Cytotoxicity assays for mycotoxins produced by Fusarium strains: a review

Mycotoxins are naturally occurring toxic secondary metabolites of fungi that may be present in food and feed. Several of these mycotoxins have been associated with human and animal diseases. Fusarium species, found worldwide in cereals and other food types for human and animal consumption, are the most important toxigenic fungi in northern temperate regions. The overall economical loss and the detrimental health effects in humans and animals of mycotoxin contamination are enormous and therefore, rapid screening methods will form an important tool in the protection of humans and animals as well as to minimize economical losses by early detection. An overview of methods for the determination of cytotoxicity and the application of such bioassays to screen solid fungal cultures, cereals, respectively, food/feedstuffs for the presence and toxic potential of Fusarium mycotoxins is presented. Various cell lines including different endpoints of toxicity using vertebrate cells and the predictive value of the in vitro assays are reviewed. Bioassays are compared with existing chemical analytical methods and the possibilities and limitations of such systems are discussed. The review is based on 149 references.     

LC-ESI-MS法测定人血浆中紫杉醇的浓度及其在药代动力学中的应用(英文)

建立了一种灵敏度高、特异性好的高效液相色谱-电喷雾质谱法 (LC-ESI-MS) 测定人血浆中紫杉醇浓度的方法。采用一步液液萃取法进行血浆样品预处理, 提取液为甲基叔丁基醚, 内标选用炔诺酮。色谱柱为Zorbax SB-C18 柱 (100 mm×2.1 mm, 3.5 μm, Agilent), 流动相为甲醇-0.2 mmol/L甲酸铵缓冲盐溶液 (包含0.1%甲酸), 采用梯度洗脱。选择离子监测 (SIM) 的目标离子为紫杉醇的[M+Na]+ m/z 876.5和内标的[M+H]+ m/z 299.4。方法学验证表明线性范围是1.0-400 ng/mL (r>0.998), 最低定量限为1.0 ng/mL, 方法的批内和批间精密度都小于9.0%, 准确度在6.8%以内。此方法已成功应用于紫杉醇脂质体注射液在患者体内的药动学研究。