Resistance system of Mycobacterium bovis B.C.B. to aminoglycoside- and peptide-antibiotics. Comparison of resistant phenotypes between Mycobacterium tuberculosis and Mycobacterium bovis

The resistance system of Mycobacterium bovis (B.C.G.) to aminoglycoside-and peptide-antibiotics has been studied. The phenotype of mutants isolated from the parent B.C.G. strain by a single-step selection with an antibiotic were classified into the following three types: (1) resistant only to a low concentration (200 μg/ml) of kanamycin in Ogawa egg medium (k1R); (2) resistant to a low concentration (200 μg/ml) of viomycin and of capreomycin (2R); and (3) resistant to a high concentration (1,000 μg/ml or more) of kanamycin and low concentrations (100 to 200 μg/ml) of lividomycin and of paromomycin (KR). The mutants showing these phenotypes, k1R, 2R, and KR, were isolated from the parent strain by inoculating the strain into media containing 100 μg/ml of kanamycin, and 100 μ/g/ml of viomycin or capreomycin, and 1,000 μg/ml of kanamycin, respectively, at rates of 10^?5-10^?6, 10^?5-10^?6, and 10^?6-10^?7, respectively, in a total viable population of the parent strain. Unlike in the case of M. tuberculosis, no mutant could be isolated from the parent strain by use of enviomycin, lividomycin, and/or paromomycin. In contrast to the fact that quadruply resistant mutants were isolated directly from the parent H37Rv strain of M. tuberculosis, such mutants could be isolated only by two-step selections. Furthermore, the phenotypes of the quadruply resistant mutants were those showing a higher resistance or a broader spectrum than expected by the addition of phenotypes of individual mutations. In addition, it was shown that, in contrast to the fact that hextuply resistant mutants could be isolated directly from the parent strain of M. tuberculosis, such mutants were not isolated directly from the parent B.C.G. strain, but could be isolated only after pre-incubation of the strain on a medium containing Tween 80.     

槲皮素对人脑胶质瘤干细胞生物学行为及miR-29s家族的影响分析

目的探讨分析槲皮素对人脑胶质瘤干细胞（BGSCs）生物学行为及miR-29s家族的影响。 方法使用干细胞培养液对U87人脑胶质瘤细胞进行培养,采用CCK-8法检测槲皮素对BGSCs细胞增殖抑制率,采用流式细胞技术检测槲皮素对BGSCs细胞凋亡影响,并采用real-time PCR鉴定槲皮素对BGSCs细胞中miR-29a、miR-29b以及miR- 29c表达的影响。采用t检验以及方差分析进行统计学分析。 结果随着槲皮素浓度的增加,BGSCs细胞增殖抑制率增加24 h 0 μg/ml（0.00±0.12）%、10 μg/ml（1.36±0.38）%、20 μg/ml（15.33±3.01）%、40 μg/ ml（29.50±4.57）%、80 μg/ml（40.21±6.42）%、160 μg/ml（61.21±7.48）,F = 76.273,P < 0.05;48 h 0 μg/ml （0.09±0.05）%、10 μg/ml（9.84±2.17）%、20 μg/ml（28.57±3.84）%、40 μg/ ml（43.59±5.21）%、80 μg/ml（59.50±3.28）%、160 μg/ ml（70.21±9.48）%,F = 85.392,P < 0.05,且同浓度槲皮素作用48 h对BGSCs细胞增殖抑制率高于作用24 h（P < 0.05）。随着槲皮素浓度的升高,BGSCs细胞凋亡率升高[0 μg/ml（13.42±1.21）%、20 μg/ml（38.47±9.28）%、40 μg/ml（59.34±7.20）%、80 μg/ml（71.42±9.47）%,F = 57.493,P < 0.05]。不同浓度槲皮素处理BGSCs细胞后,可促进BGSCs细胞miR-29s家族miR- 29a/ b/ c相对表达量,且随着槲皮素浓度的增加,BGSCs细胞miR-29s家族相对表达量增加[miR-29a 0 μg/ml（1.04±0.08）、20 μg/ml（1.16±0.05）、40 μg/ml（1.30±0.10）、80 μg/ ml（1.41±0.09）,F = 19.281,P < 0.05;miR-29b 0 μg/ml（1.06±0.09）、20 μg/ml（1.13±0.05）、40 μg/ml（1.25±0.07）、80 μg/ml（1.30±0.09）,F = 13.427,P < 0.05;miR-29c 0 μg/ml（1.03±0.07）、20 μg/ml（1.15±0.03）、40 μg/ml（1.22±0.06）、80 μg/ml（1.31±0.08）,F = 14.502,P < 0.05]。 结论槲皮素可有效抑制人脑BGSCs增殖,促进人脑BGSCs凋亡,并促进人脑BGSCs中miR- 29s家族表达。     

Simultaneous Preparation of a DNA Ligase and Restriction Endonucleases from Bacillus amyloliquefaciens N

An N-acetylgalactosamine (GalNAc)-specific Ca²⁺-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 μg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 μg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 μg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.     

Involvement of the Golgi region in the intracellular trafficking of cholera toxin

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)-elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID₅₀ value similar to the LD₅₀ of BFA in Y1 adrenal cells. Binding and internalization of [¹²⁵I]-cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK₁ cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley-Liss, Inc.     

Technical Note: Bioluminescence Measurements of the Antilisterial Activity of Nisin: Comparison with Ampicillin and Streptomycin

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.     

Rapid and extensive vacuolation of the budding yeastsSaccharomyces cerevisiae andCandida albicans by amphotericin B

The effect of amphotericin B (AMPH) on vacuolation in the budding yeastsSaccharomyces cerevisiae andCandida albicans was studied. The minimum inhibitory concentration of AMPH for growth ofS. cerevisiae andC. albicans was 1 µg/ml. In untreated control cultures, mature cells had large central vacuoles in the exponential phase, which hampered the detection of vacuolation effect. Small buds in untreated exponential phase cells, however, only rarely showed vacuoles under the light microscope. Treatment with 0.2 µg/ml of AMPH for 20–30 min induced extensive vacuolation not only in mothers but also buds ofS. cerevisiae. Extensive vacuolation lasted 4 h or more, and growth rate of the cells was much reduced for 8 h or more. Vacuolation itself was not fatal: on removal of the drug most cells gradually recovered from vacuolation and eventually multiplied. A similar effect of AMPH was also observed inC. albicans but at a higher concentration (0.5 µg/ml).     

奥沙利铂对子宫内膜癌HEC-1A侵袭转移的影响

目的研究奥沙利铂对人子宫内膜癌细胞HEC-1A侵袭转移的影响。方法体外稳定培养HEC-1A细胞株系,用不同浓度奥沙利铂(40、80、160μg/ml)给药72h后,采用Transwell法测定奥沙利铂对HEC-1A侵袭能力的影响,重组基底膜试验测定奥沙利铂对HEC-1A粘附能力的影响,划痕试验检测奥沙利铂对HEC-1A迁移能力的影响。结果与未处理对照组比较,奥沙利铂(40、80、160μg/ml)明显抑制HEC-1A侵袭性,提高侵袭抑制率(P0.01),显著降低肿瘤细胞的迁移能力(P0.01),降低HEC-1A粘附程度(P0.01)。结论奥沙利铂有效抑制子宫内膜癌细胞继发性侵袭及转移,从而发挥抗癌作用。     

Simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, respectively.     

Effects of Lectins on the Hemolysis of Rabbit Erythrocytes by Staphylococcal Alpha Toxin

When concanavalin A (1 μg/ml) or wheat germ agglutinin (2 μg/ml) was preincubated with a suspension of 2% rabbit erythrocytes for 5 min at 20 C, the binding of [¹²⁵I]-labeled staphylococcal alpha toxin to these erythrocytes was greatly inhibited and the hemolytic action of alpha toxin was decreased. The inhibitory effect of concanavalin A on hemolysis by alpha toxin was completely reversed in the presence of 0.1 M α-methyl-D -glucoside or α-methyl-D -mannoside. Phytohemagglutinin-P from Phaseolus vulgaris and soybean agglutinin inhibited hemolysis by the toxin at concentrations exceeding 20 μg/ml. The effect of concanavalin A on alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Double reciprocal plots were made of hemolysis against alpha toxin concentrations, and the data suggested that inhibition of the initial rate of the hemolysis by concanavalin A is competitive in nature. This was probably due to an interaction with the alpha toxin binding sites on the cell membrane surface.     

Effect of Cytochalasin B on the Cytokinesis of Thuricola folliculata (Ciliophora,Peritrichida)1

ABSTRACT. Binary fission of the sessiline loricate peritrich Thuricola folliculata was examined by light microscopy. Cytokinesis, which occurs in the oral-aboral median plane, is 35-40 min in duration. Cytochalasin B (CB) was used at final concentrations of 50–100 μg/ml in dimethyl sulfoxide (DMSO). A transition point occurs at about 20 min after the beginning of binary fission and about 10 min before cytokinesis. In most cases, 50 μg/ml and 100 μg/ml CB added to cells before the transition point resulted in delays of cytokinesis which were significant compared to DMSO controls; sometimes, cytokinesis was blocked completely. If added after the transition point, CB had no effect on cells.     

The relationship of protein synthesis to cell division and oral development in synchronized Tetrahymena pyriformis GL-C: An analysis employing cycloheximide

The effects of cycloheximide on synchronized Tetrahymena pyriformis strain GL-C were investigated at concentrations ranging from 0.01 to 10 μg/ml. The initial inhibition of protein synthesis was nearly total (>85%) at 1 μg/ml and above, partial (50–80%) at 0.2 to 0.05 μg/ml, and slight (<30%) at 0.02 μg/ml. Eventual recovery of protein synthesis to a rate approaching that of the controls took place at concentrations of 1 μg/ml and less. When the drug was added before a “transition point” at 55 minutes after the end of the synchronizing treatment (EST), cell division was blocked by 10 μg/ml, and delayed at concentrations of 1 μg/ml or less. The duration of delay was related to the degree of initial inhibition, and to the time required for recovery of protein synthesis; it also depended on the time after EST at which the drug was added. At a given concentration, maximum division delay was observed just prior to the “transition point;” this maximum delay was correlated with resorption of differentiating oral primordia, followed by the appearance of new primordia. The lesser delays observed at earlier times were correlated with temporary blockage of development of primordia in the “stomatogenic field” stage. Resumption of oral primordium development was, in both cases, temporally correlated with a substantial recovery of protein synthesis. After the “transition point,” cell division, and completion of oral development, was delayed slightly at the lower concentrations, and more substantially at 1 and 10 μg/ml, with some division-arrest at the latter concentration. Except for the recovery phenomenon, the developmental responses elicited by cycloheximide were similar to those observed earlier with puromycin. The bearing of these findings on the mechanism of synchronization in Tetrahymena is considered in the Discussion.     

Exploring the Therapeutic Properties of Alga-Based Silver Nanoparticles: Anticancer,Antibacterial, and Free Radical Scavenging Capabilities

The current study was designed to evaluate the antioxidant, anticancer and antimicrobial activities of silver nanoparticles (AgNPs) biosynthesized by Spirulina platensis extract. The biosynthesized silver nanoparticles were characterized using Fourier transform infrared (FT-IR) analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) analysis. The antioxidant activity of the biosynthesized AgNPs were determined via DPPH radical scavenging assay while its anticancer activity was determined using the MTT assay. The antimicrobial activity of the biosynthesized AgNPs were analyzed by disc diffusion method. Spirulina platensis acts as a reducing and capping agent. The efficacy of silver nanoparticles (AgNPs) in inhibiting the growth of Gram-negative bacteria, specifically Acetobacter, Klebsiella, Proteus vulgaris, and Pseudomonas aeruginosa, was assessed by the utilisation of the diffusion method. The study aimed to evaluate the efficacy of biosynthesized silver nanoparticles (AgNPs) against many strains of Pseudomonas aeruginosa bacteria. The findings of the study revealed that when administered in doses of 50 μl, 75 μl, and 100 μl, the largest observed zone of inhibition corresponded to measurements of 10.5 mm, 14 mm, and 16 mm, respectively. A zone of inhibition with dimensions of 8 mm, 10.5 mm, and 12 mm was detected during testing against Acetobacter at concentrations of 50 μl, 75 μl, and 100 μl, respectively. The findings also indicate that there is a positive correlation between the concentration of AgNP and the DPPH scavenging ability of silver nanoparticles. The percentage of inhibition observed at concentrations of 500 μg/ml, 400 μg/ml, 300 μg/ml, 200 μg/ml, and 100 μg/ml were recorded as 80±1.98, 61±1.98, 52±1.5, 42±1.99, and 36±1.97, respectively. In addition, it was observed that the silver nanoparticles exhibited the greatest antioxidant activity at a concentration of 500 g/ml, with a measured value of 80.89±1.99. The IC-50 values, representing the inhibitory concentration required to achieve 50 % inhibition, were found to be 8.16, 19.15, 30.14, 41.13, and 63.11 at inhibition levels of 36±1.97, 42±1.99, 52±1.5, 61±1.98, and 80±1.98, respectively.     

Impaired NK Response of Cancer Patients to IFN-α but Not to IL-2: Correlation with Serum Immunosuppressive Acidic Protein (IAP) and Role of Suppressor Macrophage

In vitro NK responses of cancer patients (N=21) to rIFN-αA and rIL-2 were examined. The serum concentration of IAP (immunosuppressive acidic protein) was determined in parallel. Five out of seven patients whose serum IAP contents were within the normal range (270 μg/ml to 470 μg/ml), had their NK activities significantly augmented by rIFN-αA and rIL-2. On the other hand, NK cells from ten out of fifteen patients whose serum IAP concentrations were 650 μg/ml or more, were not activated by rIFN-αA. NK cells of these fifteen patients yet were capable of responding to rIL-2. NK cells from cancer patients, however, became responsive to rIFN-αA by either removal of adherent cells or treatment with indomethacin. Therefore, macrophages in PBMC of cancer patients with high serum IAP levels seem to selectively suppress NK response to rIFN-αA by an indomethacin-sensitive mechanisms. It was further shown that PGE₂ was not the mediator of this suppression.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

中药大蒜素体外抗弓形虫效应及其机制的研究

目的 观察大蒜提取物体外抗弓形虫的作用效果及其机制.方法 将弓形虫RH株速殖子与兔肾细胞共同培养,加入不同浓度的大蒜素(实验组)和磺胺嘧啶(阳性对照组),培养不同时间后取出细胞,固定染色后观察细胞感染率及每个纳虫泡中弓形虫速殖子的数量;采用MTT比色法观察大蒜素对弓形虫速殖子侵袭细胞及其正常细胞增殖的影响;采用台盼兰着色法观察大蒜素对弓形虫速殖子活性的影响;采用TUNEL末端标记法检测弓形虫速殖子凋亡率,对药物的效应和机制进行探讨.结果 (1)10～80 μg/ml的大蒜素能抗弓形虫的感染,呈现剂量依赖性,与时间无明显的相关性.(2)40 μg/ml、80 μg/ml的大蒜素不能抑制细胞的增殖,对细胞无明显的毒副作用;160 μg/ml的大蒜素对细胞有明显的毒副作用.(3)大蒜素80 μg/ml时,台盼兰着色率最高,弓形虫的活力最低.(4)大蒜素80 μg/ml的浓度时,弓形虫速殖子凋亡率最高.结论 大蒜素可以抑制弓形虫速殖子的活力、对宿主细胞的感染力、在细胞内的增殖,无明显的毒副作用,最适宜浓度为80 μg/ml.大蒜素是一种良好的抗弓形虫药物,诱导弓形虫速殖子凋亡是其抗弓形虫机制之一.     

Cephalexin-Induced Morphological Alterations in the Surface Structures of Staphylococcus aureus and Escherichia coli Demonstrated by Scanning Electron Microscopy

The scanning electron microscope was used to investigate the alterations in surface morphology of Staphylococcus aureus 209P and Escherichia coli NIH induced by the action of cephalexin known to interfere with cell-wall synthesis. Exposure to cephalexin produced a series of changes on the surface morphology in proportion to the concentrations of cephalexin added. Untreated S. aureus cells had smooth contours. Exposure to 1 μg/ml of cephalexin during the logarithmic phase of growth in S. aureus did not produce any detectable changes. Upon exposure of S. aureus to 5 μ/ml or 10 μg/ml, some cells were larger than normal and showed abnormal cell division-like structures in part. When S. aureus was exposed to 50 μg/ml, cell division was completely inhibited, and no formation of grape-like clusters was observed. Untreated E. coli cells appeared to have smooth and regular contours. E. coli propagated almost normally upon exposure of the organisms to 1 μg/ml of cephalexin. Filamentous structures were observed with the exposure of E. coli to 12.5 μg/ml or 25 μg/ml, but spheroplast-like structures were not observed. Exposure to 100 μg/ml of cephalexin resulted in the formation of marked filamentous cells and spheroplast-like structures having multiple small saccular outpouchings. Scanning electron microscope demonstrated more completely the morphological abnormalities induced by cephalexin.     

Inhibitory Effects of Apple Polyphenol on Induced Histamine Release from RBL-2H3 Cells and Rat Mast Cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC₅₀ values for histamine release were 30 μg/ml and 25 μg/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

Cytolytic differences among lepidopteran cell lines exposed to toxins ofBacillus thuringiensis subst.Kurstaki. (HD-263) andAizawai (HD-112): Effect of aminosugars and N-glycosylation

Summary Comparison of lytic-dose response behavior of seven lepidopteran cell lines to the activated delta-endotoxin polypeptides ofBacillus thuringiensis subspecieskurstaki (HD-263) andaizawai (HD-112) indicated distinct differences among the lines. The lines derived fromSpodoptera speciesS. exigua (URC-SE-1A) andS. littoralis (UIV-SL-575) were more susceptible to lysis byaizawai towin (Bta) thankurstaki toxin (Btk) as were cells from theLymantria dispar line (IPLB-LD652Y). However, the concentrations of Bta required for lysis of 50% of URC-SE-1A and IPLB-LD652Y cells (LC₅₀) were 0.2 to 0.8 μg/ml compared to 5 to 9 μg/ml for UIV-SL-575 cells. In comparison, Btk LC₅₀ concentrations for the three lines were similar (14 to 19 μg/ml). Cells fromS. frugiperda (IPLB-SF-21AE) andTrichoplusia ni (TN368) were similar in their response to Bta (LC₅₀=2.5 to 3.7 μg/ml) and Btk (LC₅₀=1.0 to 2.8 μg/ml) whereas the lines derived fromHeliothis spp. were the least susceptible to both toxins. The LC₅₀ concentrations for Bta with theH. zea line (IPLB-HA-1075) andH. virescens line (BCIRL-HV-AM1) were >50 μg/ml and for Btk were >50 μg/ml and 42 to 50 μg/ml, respectively, yet for both lines Btk was the more cytolytic. Cytolysis of TN368 cells could be inhibited to varying extents by preincubation of the toxins with the aminosugars of galactose, mannose, and glucose and theirN-acetyl derivatives. The unsubstituted hexoses were not inhibitory. The order of decreasing inhibitory effectiveness was the same for both toxins regardless of the derivative species and followed the order galactose, mannose, and glucose. Also, inhibition of cytolysis could be achieved to varying extents by assaying cells grown in medium with tunicamycin. Lysis with Btk was inhibited 68 and 37% using treated cells of TN368 and IPLB-LD652Y, respectively; however, no inhibition was observed with URC-SE-1A cells. Further, no inhibition of Bta-mediated lysis was obtained with tunicamycin-grown cells of the three lines.     

白花蛇舌草对人胃癌细胞MNK-45线粒体膜电位及凋亡相关基因表达的影响*

目的: 探讨白花蛇舌草(注射液)对人胃癌细胞MNK-45线粒体膜电位及凋亡相关基因表达的影响。方法: 将人胃癌细胞MNK-45分成4组,每组设置3个复孔,对照组为未加入白花蛇舌草的MNK-45细胞,3组实验组分别加入终浓度为20、30、40 μg/ml白花蛇舌草的MNK-45细胞,各组在5％的CO₂培养箱中孵育48 h后,利用激光共聚焦显微镜下观察细胞形态变化,流式细胞术检测线粒体膜电位,qRT-PCR检测Cytochrome C (Cyt c)、caspase3和caspase9基因的表达变化,Western blot检测Cytochrome C (Cyt c)、caspase3和caspase9蛋白的表达变化。结果: 与对照组相比,终浓度为20、30、40 μg/ml的各白花蛇舌草处理组,其MNK-45细胞的线粒体膜电位均明显降低(P＜0.01),Cyt c、caspase3和caspase9的基因表达均明显上调(P＜0.01)、蛋白表达也均显著升高(P＜0.05或 P＜0.01),40 μg/ml的白花蛇舌草处理组的表现最佳。结论: 在终浓度为20~40 μg/ml的范围内,白花蛇舌草能够降低人胃癌MNK-45细胞线粒体膜电位, 诱导细胞凋亡,并可上调Cyt c、caspase3和caspase9基因表达。     

Sensitive semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681, the active parent drug of a new oral carbapenem antibiotic,DZ-2640, in human plasma and urine using a column-switching system as sample clean-up procedure

DZ-2640 is a new oral carbapenem antibiotic having a dihydro-pyrroloimidazole ring as a side chain and a pivaloyloxymethyl (POM) ester prodrug of DU-6681, the active parent compound. A simple and sensitive column-switching semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681 in human plasma and urine has been developed. Human plasma was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was filtered through an Ultrafree C3GV. The resulting filtrate was injected without further cleanup onto the HPLC system. Human urine was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was directly injected onto the HPLC system. The analyte was detected by monitoring the column effluent with UV light at a wavelength of 300 nm, which resulted in the limit of quantitation of 0.008 μg/ml of plasma and 0.32 μg/ml of urine. Calibration curves were linear in the range of 0.008 to 5.85 μg/ml in plasma and 0.32 to 104.4 μg/ml in urine. The present methods showed greatly increased sensitivity for DU-6681 compared to conventional HPLC methods and also showed satisfactory recovery, selectivity, precision, and accuracy. Stability studies showed that 1 M MOPS buffer (pH 7.0) acted as a stabilizer. In plasma and urine diluted with equal volume of the buffer, DU-6681 showed good stability at −80°C for up to 4 weeks with no significant loss of the drug.