Induction of beta-lactamase and penicillin-binding proteins in Escherichia coli by introduction of Streptomyces DNA

Introduction of hybrid plasmids, which were constructed by ligation of pCR1 or pMN1 vector plasmid and SalI restriction endonuclease cleaved segments of Streptomyces cacaoi chromosome, resulted in the production of new beta-lactamase and penicillin-binding protein in Escherichia coli. The beta-lactamase and penicillin-binding protein were not from S. cacaoi but rather induced by the plasmids. Close relationship was observed between plasmids and penicillin-binding proteins but not with beta-lactamase.     

Interaction of beta-lactamase of Streptomyces cacaoi. II. CP-45,899, izumenolide and cephamycins

Inhibition of a beta-lactamase of Streptomyces cacaoi by CP-45,899, izumenolide and cephamycins was investigated and compared with that of a beta-lactamase of Bacillus cereus. S. cacaoi enzyme could not hydrolyze CP-45,899. Instead, hydrolysis of benzylpenicillin by the enzyme was inhibited in the presence of CP-45,899. Although inhibition increased gradually with time, the inhibition line produced by CP-45,899 with time less curved than that produced by clavulanic acid and PS-5. Furthermore, preincubation of S. cacaoi beta-lactamase with CP-45,899 for up to 120 seconds did not obviously affect the degree of inhibition. When the concentration was lowered, it behaved as a competitive inhibitor, a Ki value being 6.2 X 10(-7) M. Izumenolide, on the other hand, did not inhibit the enzyme activity of S. cacaoi beta-lactamase at 1.28 X 10(-4) M, although it inhibited B. cereus enzyme slightly in a competitive manner. Oganomycins were inert to the both beta-lactamases.U     

Beta-lactams against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) have developed resistance to virtually all non-experimental antibiotics. They are intrinsically resistant to beta-lactams by virtue of newly acquired low-affinity penicillin-binding protein 2A (PBP2A). Because PBP2A can build the wall when other PBPs are blocked by beta-lactams, designing beta-lactams capable of blocking this additional target should help solve the issue. Older molecules including penicillin G, amoxicillin and ampicillin had relatively good PBP2A affinities, and successfully treated experimental endocarditis caused by MRSA, provided that the bacterial penicillinase could be inhibited. Newer anti-PBP2A beta-lactams with over 10-fold greater PBP2A affinities and low minimal inhibitory concentrations were developed, primarily in the cephem and carbapenem classes. They are also very resistant to penicillinase. Most have demonstrated anti-MRSA activity in animal models of infection, and two--the carbapenem CS-023 and the cephalosporin ceftopibrole medocaril--have proceeded to Phase II and Phase III clinical evaluation. Thus, clinically useful anti-MRSA beta-lactams are imminent.     

Combined antioxidant effects of rutin and vitamin C in Triton X-100 micelles

UV-vis spectra, fluorescence emission spectra and cyclic voltammetric measurements were used to study the influence of Vitamin C on the antioxidant of rutin in Triton X-100 micelles. Rutin can be located in Triton X-100 micelles spontaneously through hydrophobic force, and the binding constant K between rutin and Triton X-100 increases with the rutin concentration. The embedment of two hydroxyl groups on rutin into the more hydrophobic micellar microenvironment makes the oxidation of rutin harder and the radical scavenging activity decrease. With low concentration of Vitamin C, the antioxidant capacity of rutin against hydroxyl radical is enhanced, while that capacity is partly inhibited when the concentration of Vitamin C become higher.     

Isolation of two types of Pseudomonas aeruginosa mutants highly sensitive to a specific group of beta-lactam antibiotics and with defect in penicillin-binding proteins

Two types of mutants highly sensitive to beta-lactam antibiotics were obtained from Pseudomonas aeruginosa PAO 2142 by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. One type of mutant showed over 30 times higher sensitivity to mecillinam, carbenicillin and sulbenicillin than did the parent, but not to most other beta-lactam antibiotics tested. In contrast, the other type mutant was about 30 times more sensitive to ampicillin, cephaloridine, cefoxitin and cefmetazole, but resistant to mecillinam, carbenicillin and sulbenicillin at the same level as the parent. Beta-lactamase activity of these mutants ws not different from that of the parent. Defect in either of penicillin-binding proteins 1A/1B or 5 was observed in some mutants of P. aeruginosa highly sensitive to beta-lactam antibiotics.     

Membrane-bound penicillin-binding proteins of Escherichia coli. Comparison of a strain carrying an R-factor and the parent strain.

Membrane-bound penicillin-binding proteins of an Escherichia coli carrying an R factor which mediated the resistance to penicillins were examined by slab gel electrophoresis and fluorography using beta-lactamase inhibitors such as methicillin, clavulanic acid and MC-696-SY2-A, and by affinity chomatography. By fluorography, it appeared that the penicillin-binding proteins of the strain carrying the R factor could not be distinguished from those of the parent strain. In both strains, methicillin had a preferential affinity for penicillin-binding proteins 2 and 3, clavulanic acid for 2 and 4, and MC-696-SY2-A for 1A at the concentration which was needed to inhibit about 75 approximately 80% of beta-lactamase activity of the membrane fraction from a strain carrying an R factor. This with other facts indicates that MC-696-SY2-A has a unique character in the binding to penicillin-binding proteins. By affinity chromatography using cephalexin-CH-Sepharose 4B column, two major cephalexin-binding proteins were detected. Their molecular weights were found to be 110,000 and 32,000, respectively. These two proteins correpsonded to penicillin-binding proteins 1 and 5/6. From these results it was suggested that the R factor had no influence on the penicillin-binding proteins in the E. coli strain examined.     

Characterization of a phosphatidic acid phosphatase from rat brain cell membranes

We have characterized a phosphatidic acid phosphatase (PAP, EC 3.1.3.4) that is associated with cell membranes from rat brain using [³²P] phosphatidic acid as substrate in a simple assay. The enzyme could be activated by Triton X-100, cholic acid and Chaps and inhibited by Lubrol PX and sodium dodecyl sulfate. The optimal pH was between 6.0 and 7.0. Mg²⁺ was not essential for enzyme activity. The enzyme activity was decreased by about 50% by Ca²⁺ at concentrations of 0.1 to 1 mmol/1. Zn²⁺ inhibited the enzyme by 50% at concentrations of about 10 mol/l in the absence of, and 100 nmol/1 in the presence (3 mmol/1) of, Triton X-100. NaF decreased the activity by about 50% at concentrations between 0.3 and 1 mmol/l when Triton X-100 was added, but did not inhibit the enzyme if the detergent was not present. N-Ethylmaleimide (NEM) did not affect the enzyme. In the absence of Triton X-100, propranolol and metoprolol enhanced the PAP activity. In the presence of 3 mmol/1 Triton X-100, the enzyme was inhibited by about 50% by propranolol at a concentration of 10 mmol/l, whereas metoprolol caused only a slight inhibition of PAP. The K _m for phosphatidic acid was 150 mol/1 and was changed to 20 mol/1 by 3 mmol/1 Triton X-100 without the V _max being changed. Enzyme activity could be solubilized by 1–5% (w/v) Triton X-100. Gel filtration chromatography showed a M _r of 320000. This membrane-associated PAP from neuronal tissue probably belongs among the NEM-insensitive forms of PAP enzymes which have been proposed to play a role in transmembrane signal transduction via phospholipase D. Correspondence to: Ariane Hoer at the above address     

Effects of Triton X-100 nanoaggregates on dimerization and antioxidant activity of morin

Dimerization and antioxidant activity of morin in the Triton X-100 micelles were studied by electronic absorption, ATR-FTIR spectra, cyclic voltammetric, DSC, freeze-fracture TEM, molecular modeling and ab initio quantum calculations. Morin can be solubilized in the Triton X-100 micelles and show selective dimerization in Triton X-100 micelles with different structures. In Triton X-100 spherical micelles, morin always exists in the form of dimer, and in Triton X-100 rodlike micelles, it is always in the form of monomer. The solubilization of morin dimer in Triton X-100 spherical micelles changes the micelle morphology from spherical to cubelike, and the size of the single micelle is also increased, while morin monomer links the Triton X-100 rodlike micelles and forms a kind of network micelle structure with the size of the "rod" unchanged. Solubilized and concentrated in Triton X-100 micelles, morin can protect human serum albumin from the damage induced by hydroxyl radicals effectively and even can form a kind of protein complex with human serum albumin showing more thermal stability.     

New findings in beta-lactam and metronidazole resistant Bacteroides fragilis group

Beta-lactam antibiotics and 5-nitroimidazoles have been extensively used against anaerobic bacteria. However, antibiotic resistance is increasingly common among anaerobic Gram-negative bacilli. The classical mechanisms of resistance to beta-lactams are, (1) production of beta-lactamases; (2) alteration of penicillin-binding proteins (PBPs); and (3) changes in outer membrane permeability to beta-lactams. The 5-nitroimidazole molecule is a prodrug whose activation depends upon reduction of the nitro group in the absence of oxygen. Decreased uptake and altered reduction are believed to be responsible for metronidazole resistance. Five nim genes (A, B, C, D and E) have been identified in Bacteroides fragilis group spp. that confer resistance to 5-nitroimidazole antibiotics. Knowledge of the status and the mechanisms of resistance is critical for both the selection of antimicrobial therapy and the design of new antimicrobial agents. The purpose of this article is to review the mechanisms for and the prevalence of beta-lactam and metronidazole resistance in strains belonging to the B. fragilis group.     

Use of enhancers in the HPTLC fluorescence analysis of thiols

Several thiols of biological and pharmacological interest, including glutathione,

Abstract

coenzyme A, acetylcysteine and captopril were derivatized with the fluorogenic reagents SBD-F and ABD-F and analysed by high-performance thin-layer chromatography (HPTLC)-fluorodensitometry on silica gel 60 plates, using isopropyl ether-methanol-water-acetic acid (9:8:2:1, v/v/v/v) as the developing solvent. The luminescence was considerably increased when several types of enhancers were applied as dipping reagents: Triton X-100, liquid paraffin and cyclodextrins; thus the delectability of the thiol fluorophores was improved. The influence of enhancer concentration, method of application, sample concentration, drying conditions and measuring time after plate dipping were investigated. The greatest enhancement was achieved using a 40% (v/v) solution of Triton X-100 in toluene as a dipping reagent for the determination of SBD-acetylcysteine; more than a 10-fold increase of the fluorescence signal was obtained, allowing low picogram detection limits.     

The affinity of imipenem (N-formimidoylthienamycin) for the penicillin-binding proteins of Staphylococcus aureus--binding and release

Penicillin-binding proteins 1, 2 and 3 in Staphylococcus aureus were found to possess common properties. All have very strong affinities for both benzylpenicillin and imipenem (N-formimidoylthienamycin), and all have an activity which releases bound imipenem, but not bound benzylpenicillin. Lower molecular weight penicillin-binding protein 4, which has a rather weak affinity for benzylpenicillin and also weak penicillinase activity showed an extraordinarily high affinity for imipenem but no antibiotic-releasing activity.     

Interactions of rat brain acetylcholinesterase with the detergent Triton X-100 and the organophosphate paraoxon.

Inhibition of the critical enzyme acetylcholinesterase (E.C. 3.1.1.7) with subsequent cholinergic crisis is the mechanism of acute toxicity of the organophosphorus insecticides (B. E. Mileson et al., 1998, Toxicol. Sci.41, 8-20). Consequently, measurement of acetylcholinesterase activity is important for evaluating the mammalian toxicity of this commonly used class of insecticides. While mammalian acetylcholinesterase activity has often been determined in tissue homogenates in the presence of the nondenaturing detergent Triton X-100 at a concentration of 1%, the potential actions of this detergent on the activity of this critical enzyme are not understood. In the current study, homogenization of rat brain in buffer containing 1% Triton X-100 slightly elevated the (app)V(max) for hydrolysis of acetylthiocholine, without affecting the (app)K(m) or the (app)K(ss). However, the presence of both 1% Triton X-100 and paraoxon (at concentrations of 5 nM-100 nM) resulted in complex kinetic interactions with acetylcholinesterase, as evidenced by a curvilinear secondary plot for determination of the (app)k(i). These results suggest that measurement of acetylcholinesterase activity in the presence of up to 1% Triton X-100, but in the absence of oxon, should pose no problems with regard to data interpretation, provided it is recognized that the detergent slightly elevates activity. However, measurement of acetylcholinesterase activity after enzyme was exposed simultaneously to Triton X-100 and oxon could be problematic. Caution is warranted when interpreting data where acetylcholinesterase activity was determined under such conditions since in the presence of 1% Triton X-100, the capacity of oxon to inhibit acetylcholinesterase might change as a function of oxon levels.     

β-内酰胺类抗生素作用机制探讨——青霉素结合蛋白研究方法介绍

本文详细介绍了十二烷基硫酸钠聚丙烯酰胺电泳及荧光放射自显影研究青霉素结合蛋白的方法及研究过程中的注意事项.研究方法主要包括:(1)经超声破碎和超速离心制备细菌细胞膜;(2)利用~(14)C-青霉素G作为放射性示踪剂与待测抗生素竞争性结合位于细胞膜中的青霉素结合蛋白;(3)经含十二烷基硫酸钠(SDS)的聚丙烯酰胺不连续板状凝胶电泳分离蛋白质;(4)将闪烁剂PPO掺入凝胶并将凝胶干燥,感光材料与凝胶板在超低温环境中曝光,显定影处理后用显微光密度计对感光材料进行黑度扫描;(5)利用线性插值法处理实验结果.主要注意事项:(1)方法的标准化;(2)细胞膜液的蛋白含量;(3)关于标记抗生素;(4)关于荧光放射自显影.本方法是一简便、灵敏、准确的β-内酰胺类抗生素靶位蛋白研究方法,已被各国许多开展分子药理的实验室接受.在我国介绍、推广青霉素结合蛋白研究方法将促进我国抗生素药理深入发展及在分子水平进行药物评价.     

Verapamil Potentiates 4′-Epidoxorubicin Cytotoxicity in a Rat Hepatoma Cell Line and in a Substrain Resistant to 4′-Epidoxorubicin

We have examined the influence of verapamil (VP) on the in vitro effect of 4′-epidoxorubicin (Epi-A) in a rat hepatocarcinoma cell line (MH1C1) and in an Epi-A resistant substrain. A VP concentration of 500 ng/ml (1.1 μmol/l) markedly potentiated the cytotoxic effect of Epi-A in the parent line. The resistant cells grow at an Epi-A concentration of 7500 ng/ml (12.9 μmol/l). This is approximately 15-fold higher than the concentration tolerated by parental cells. In these cells VP reversed the acquired resistance to Epi-A in a concentration dependent manner; thus, a concentration in the range of 500–750 ng/ml (1.1–1.7 μmol/l) of VP restored the sensitivity to Epi-A in the resistant cells. Our results demonstrate that VP increases the sensitivity to Epi-A in hepatocarcinoma cells never exposed to this drug, as well as in hepatocarcinoma cells with acquired Epi-A resistance.     

Triton-X-100-modified polymer and microspheres for reversal of multidrug resistance.

Triton X-100 is a non-ionic detergent capable of reversing multidrug resistance (MDR) due to its interaction with cell membranes. However, it interacts with cells in a non-specific way, causing cytotoxicity. This work aimed to develop polymeric chemosensitizers that possess the ability to reverse MDR and lower toxic side effects. When being delivered to tumours, the polymeric chemosensitizers may also have longer retention times in tumours than the free detergent. Triton-X-100-immobilized dextran microspheres (T-MS) and inulin (T-IN) were prepared and characterized. Their cytotoxicity against multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was compared with that of free Triton X-100 solutions. The in-vitro effect of the products on 3H-vinblastine accumulation by CH(R)C5 cells was determined. Both T-MS and T-IN showed a marked decrease in the cytotoxicity, as compared with free Triton solutions at equivalent concentrations. Drug accumulation by CH(R)C5 cells was increased over two fold in the presence of T-MS or T-IN. These results suggest that polymeric drug carriers with MDR-reversing capability and lower cytotoxicity may be prepared by immobilization of chemosensitizers.     

高效液相色谱法测定质粒pUDKH中曲拉通X-100的含量

目的检测质粒pUDKH最终产品中去污剂曲拉通X 10 0 (TritonX 10 0 )的残留含量。方法用高效液相色谱法测定TritonX 10 0残留含量 ,色谱柱为C18柱 ,流动相为乙腈 水 (70∶30 ) ,流速为 1.0ml/min ,进样量 10 μl,检测波长 2 2 3nm。结果平均回收率为 10 0 .19% ,日内精密度为 4 .36 % ,日间精密度为 4 .17% ,TritonX 10 0在 1.2 5～2 0 μg/ml浓度范围内呈线性关系。pUDKH产品中的TritonX 10 0含量在 2 .2 7～ 3.0 0 μg/ml之间。TritonX 10 0的最低检测限可达 1.0 μg/ml。 结论此法有良好的准确度与精密度 ,供试品不需预处理 ,不受其它成分的干扰。     

Acute toxicity of polyethylene glycol p-isooctylphenol ether in Syrian hamsters exposed by inhalation or bronchopulmonary lavage

Dose-response studies were conducted with Syrian hamsters exposed to polyethylene glycol p-isooctylphenyl ether (Triton X-100) via inhalation or bronchopulmonary lavage. Syrian hamsters were exposed to an aerosol of Triton X-100 with a mass median aerodynamic diameter of 1.5 μm and a concentration of 3.0 mg/liter. Estimated initial lung burdens of Triton X-100 ranged from 800 to 3100 μg. Hamsters were lavaged with concentrations of Triton X-100 ranging from 0.01 to 0.10% in isotonic saline resulting in initial lung burdens of Triton X-100 that ranged from 300 to 3200 μg. The $\text{LD}\text{50}\text{7}$ values were 1700 μg (1300–2100 μg, 95% confidence limits) for the inhalation study and 2100 (1900–2700) μg for the lavage study. The difference between the $\text{LD}\text{50}\text{7}$ values for the two methods of exposure was not significant. However histopathological examination revealed differences in the nature and distribution of pathologic changes observed in animals exposed by the two routes of administration. Animals exposed by inhalation died as a result of ulcerative laryngitis and laryngeal edema with only minimal pulmonary pathologic alterations. Animals exposed by lavage, where the larynx was not exposed to Triton X-100, died from pulmonary edema and acute exudative pneumonia, these results demonstrate the need for careful selection of exposure methods to meet the specific objectives of a toxicology study.     

Metal-induced alteration of the cell membrane/cytoplasm complex studied by flow cytometry and detergent lysis

Flow cytometric analysis of the cell cycle is most effectively accomplished with membrane-/cytoplasm-free ("clean") nuclei. Non-ionic detergents (e.g. NP40 or Triton X-100) commonly are employed to solubilize cell membranes/cytoplasm to produce "clean" nuclei. Treatment of murine erythroleukemic cells (MELC) with tri-n-butyltin methoxide, cadmium acetate, zinc sulfate, or lead acetate alters the properties of the cell membrane/cytoplasm complex making it resistant to NP40 dissolution. On a molar basis, the organotin compound was more effective in inducing resistance to detergent-mediated dissolution than the inorganic metal compounds. Resistance to NP40-mediated dissolution was manifested as an increase in the flow cytometric parameters 90 degrees scatter and fluorescein isothiocyanate (FITC) fluorescence and was confirmed by light microscopy.     

耐万古霉素微生物筛选模型

为获得耐万古霉素的肠球菌和金葡球菌，利用紫外线对Enterococcus faecalis458和Staphylococcus aureus9918进行诱变，确定在不同处理中以紫外线照射30s为最佳。最后获得对万古霉素中敏的E．faecalis458 V20和S．aureus9918 V16，药物敏感性试验表明前者对青霉素G钾和红霉素耐药，而后者对青霉素G钾、红霉素、苯唑青霉素钠和氨苄青霉素钠耐药。两者对万古霉素的耐药性在无药培养基上传10代后略微下降。     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.