Stimulation of cell division by membrane-active agents

Agents that decrease membrane stability (e.g. dimethyl sulfoxide, lysolecithin, sodium oleate, and short-chain alcohols) stimulate multinucleoid, serpentine filaments of Agmenellum quadruplicatum strains SN12 and SN29 to divide into cellular equivalents within approximately one generation time. Agents that increase membrane stability (e.g. long-chain alcohols) antagonize this timulation. Thus, the physical properties of the cell membrane appear to be involved in the regulation of cell division. These observations suggest that the invagination of the cell wall may be regulated by agents that interact with the plasma membrane and with enzymes involved in peptidoglycan synthesis.     

Vascular endothelial cell killing by combinations of membrane-active agents and hydrogen peroxide

Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.     

Induction of petite yeast mutants by membrane-active agents

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Coarse-grained simulations of the membrane-active antimicrobial Peptide maculatin 1.1

Maculatin 1.1 (M1.1) is a membrane-active antimicrobial peptide (AMP) from an Australian tree frog that forms a kinked amphipathic α-helix in the presence of a lipid bilayer or bilayer-mimetic environment. To help elucidate its mechanism of membrane-lytic activity, we performed a total of ∼8 μs of coarse-grained molecular dynamics (CG-MD) simulations of M1.1 in the presence of zwitterionic phospholipid membranes. Several systems were simulated in which the peptide/lipid ratio was varied. At a low peptide/lipid ratio, M1.1 adopted a kinked, membrane-interfacial location, consistent with experiment. At higher peptide/lipid ratios, we observed spontaneous, cooperative membrane insertion of M1.1 peptide aggregates. The minimum size for formation of a transmembrane (TM) aggregate was just four peptides. The absence of a simple and well-defined central channel, along with the exclusion of lipid headgroups from the aggregates, suggests that a pore-like model is an unlikely explanation for the mechanism of membrane lysis by M1.1. We also performed an extended 1.25 μs simulation of the permeabilization of a complete liposome by multiple peptides. Consistent with the simpler bilayer simulations, formation of monomeric interfacial peptides and TM peptide clusters was observed. In contrast, major structural changes were observed in the vesicle membrane, implicating induced membrane curvature in the mechanism of active antimicrobial peptide lysis. This contrasted with the behavior of the nonpore-forming model peptide WALP23, which inserted into the vesicle to form extended clusters of TM α-helices with relatively little perturbation of bilayer properties.     

Azole antimicrobial pharmacophore-based tetrazoles: Synthesis and biological evaluation as potential antimicrobial and anticonvulsant agents

The azole pharmacophore is still considered a viable lead structure for the synthesis of more efficacious and broad spectrum antimicrobial agents. Potential antibacterial and antifungal activities are encountered with some tetrazoles. Therefore, this study presents the synthesis and antimicrobial evaluation of a new series of substituted tetrazoles that are structurally related to the famous antimicrobial azole pharmacophore. A detailed discussion of the structural elucidation of some of the newly synthesized compounds is also described. Antimicrobial evaluation revealed that twenty compounds were able to display variable growth inhibitory effects on the tested Gram positive and Gram negative bacteria with special efficacy against the Gram positive strains. Meanwhile, six compounds exhibited moderate antifungal activity against Candida albicans and Aspergillus fumigatus. Structurally, the antibacterial activity was encountered with tetrazoles containing a phenyl substituent, while the obtained antifungal activity was confined to the benzyl variants. Compounds 16, 18, 24 and 27 were proved to be the most active antibacterial members within this study with a considerable broad spectrum against all the Gram positive and negative strains tested. A distinctive anti-Gram positive activity was displayed by compound 18 against Staphylococcus aureus that was equipotent to ampicillin (MIC 6.25 μg/mL).On the other hand, twelve compounds were selected to be screened for their preliminary anticonvulsant activity against subcutaneous metrazole (ScMet) and maximal electroshock (MES) induced seizures in mice. The results revealed that five compounds namely; 3, 5, 13, 21, and 24 were able to display noticeable anticonvulsant activity in both tests at 100 mg/kg dose level. Compounds 5 and 21 were proved to be the most active anticonvulsant members in this study with special high activity in the ScMet assay (% protection: 100% and 80%, respectively).     

Effect of oxytocin on neuroblastoma cell viability and growth

Oxytocin, released in response to different physiological stimuli, could play a key role in reducing stress reaction. It was suggested that it has protective effect against inflammation and consequences of oxidative stress. Mechanisms how oxytocin effects mediated in the brain tissue are unclear. In this study, oxytocin effect on cell growth and neuronal viability was examined. Human neuroblastoma (SH-SY5Y and SK-N-SH) and glioblastoma (U87MG) cells were exposed to different concentrations of oxytocin for 12-96 h. Potential protective effect of oxytocin treatment was investigated after exposing cells to oxidative stress using hydrogen peroxide (50 mM, 2 h) or 6-hydroxydopamine (25 μM, 24 h). Cell proliferation was measured by cell counting and cell viability was examined by MTT assay. Protein expression of selected neurotrophic factors was measured as an additional parameter. Oxytocin (1 μM) significantly increased cell number in all three cell types. Viability of SH-SY5Y cells was increased in the presence of oxytocin without significant effect of dose (0.01-1 μM). Cell death induced by hydrogen peroxide was not prevented by incubation with oxytocin. Oxytocin pretreatment blunted neurotoxin 6-OHDA reduction of cell viability in SH-SY5Y cells. Oxytocin (1 μM, 12 h) elevated amount of total proteins without increasing levels of brain-derived neurotrophic factor and neurotrophic growth factor. In conclusion, oxytocin increases growth and viability of neuroblastoma and glioblastoma cells without activation of neurotrophic factors. Oxytocin does not have protective effect in oxidative stress; however, it might be important for neuroprotection to dopaminergic neurons. Its proliferative effect might be important in native cell life, euplastic processes, and tumor progression.     

Induction of petite yeast mutants by membrane-active agents.

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

Abnormal deposition of amyloid-β(Aβ) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer‘s disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of Aβ on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/Aβ and neurofilament was extended to neurites, whereas those of APP-transfected cells were still restricted within the cell body. Levels of both APP and Aβ were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of Aβ on the inhibition of cell differentiation,we added exogenously the similar level or about 10-times of the Aβ level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawalinduced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of Aβ into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of Aβ1-40 or Aβ1-42, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of Aβ could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability.     

Design,synthesis and evaluation of carbazole derivatives as potential antimicrobial agents

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5–2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure–activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.     

定位在线粒体的肝刺激因子对线粒体膜电势的影响

目的肝刺激因子（hepatic stimulator substance,HSS）可以保护肝细胞免受各种毒素的影响,但机制尚未清楚,研究探讨肝刺激因子保护肝细胞的可能机制。方法利用稳定转染FLAG-pcDNA3．0／hHss的肝癌细胞BEL-7402为模型,使用Alexa Flour 488、Hoechst 33342、MitoTracker 580分别将HSS、细胞核以及线粒体染色,观察HSS在细胞中的定位情况。当野生型7402细胞、转染空载体FLAG-pcDNA3．0的7402细胞以及转染FLAppcDNA3．0／hHSS的7402细胞受到线粒体膜孔道开放剂羰基氰化间氯苯腙（carbonyl cyanide m—chlorophenylhydrazone,CCCP）的损伤后,用电镜观察线粒体形态、荧光素酶检测ATP、流式细胞仪测定线粒体膜电位（mitoehondrial membrane potential,MMP）等,综合观察过表达HSS的肝细胞的抗损伤能力。结果在稳定转染hHSS基因的7402细胞中,大部分HSS与线粒体共定位;在CCCP作用下,对照组野生型7402细胞以及转染空载体的7402细胞MMP下降明显,线粒体肿胀,嵴断裂、消失,ATP下降显著;实验组稳定转染hHSS基因的7402细胞MMP下降幅度较小,线粒体肿胀与嵴形态的改变明显减轻,ATP的含量较对照组高。结论肝刺激因子HSS在细胞中主要定位于线粒体,可以稳定MMP,维持线粒体形态及细胞内ATP的水平,从而增强肝细胞抗损伤的能力。     

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10⁶ spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.     

斑蝥素对草地贪夜蛾Sf9细胞膜完整性和膜电位的影响

为明确斑蝥素对昆虫细胞膜的作用及其机理, 本研究利用草地贪夜蛾Spodoptera frugiperda的卵巢细胞系Sf9细胞作为实验材料, 采用透射电子显微技术(transmission electron microscope , TEM)、 激光共聚焦显微镜(laser scanning confocal microscope, LSCM)结合荧光探针FDA/PI及DiBAC4(3)技术研究斑蝥素(cantharidin, CTD)对Sf9细胞膜完整性及膜电位（membrane potential, MP）的影响。结果表明： 32 μmol/L CTD处理6 h和12 h后, 电镜观察均未发现细胞膜结构破损; FDA/PI染色后, 32 μmol/L CTD处理0.5 h后细胞FDA荧光强度比对照显著降低(P<0.05), 碘化丙啶（propidium iodide, PI）染色的细胞比例与对照无显著性差异(P≥0.05)。32 μmol/L CTD处理140 s后即引起MP发生显著性去极化(P<0.05); 64 μmol/L CTD处理瞬时MP发生显著性去极化(P<0.05); 32 μmol/L CTD处理3 h内及64 μmol/L CTD处理2 h 内MP仍保持显著性去极化(P<0.05), 之后去极化程度降低; 32 μmol/L CTD处理6 h及64 μmol/L CTD处理3 h时MP去极化与对照组相比已无显著性差异(P≥0.05)。结果说明, CTD处理短时间内可引起Sf9细胞膜电位去极化并维持一段时间, 同时导致细胞活性发生不可逆下降, 但未对细胞膜结构完整性产生破坏。     

The influence of various storage conditions on cell viability in amniotic membrane

Up to now freeze-dried, gamma-sterilised or glycerol-preserved amniotic membranes (AMs) have widely been used in the field of ophthalmology and wound care (e.g. leg ulcers, burns). After some preservation processes in use, like freeze-drying or glycerol-preserving, the cells in the AM are no longer viable. Within this study we evaluated the influence of different short-term and long-term storage conditions on cell viability in AM. Therefore AMs from cesarean section placentae were washed and biopsied to evaluate the microbiological status and to determine the viability of the tissue. Additionally, viability under various storage conditions was examined by assessment of mitochondrial activity. Preservation included temperatures above and below 0°C as well as various media compositions. As expected, cell viability in amnion decreases during storage, in fact the effect was more pronounced when stored frozen, but the higher viability of amnion obtained by storage above 0°C with medium is associated with the limitation to a short period of storage of about 28 days. The evaluated preservation methods are the basis for future non-clinical in-vivo studies in which the possible benefit of amnion as a viable biomaterial in wound healing will be investigated. A part of this work was presented at the World Congress on Tissue Banking in Rio in May 2005 and was honoured by the Poster Award Commission.     

Effect of trisodium citrate treatment on hybridoma cell viability

Trisodium citrate has been widely used to dissolve calcium alginate beads in order to estimate cell concentration in the beads. To obtain an accurate measurement of viable cell concentration in calcium alginate beads, the effect of trisodium citrate solutions on hybridoma cell viability was studied with regard to stage of growth and trisodium citrate concentration. The cells in the decline phase of growth were more sensitive to 30 minutes of trisodium citrate treatment than the cells in the exponential phase of growth. The cell viability did not decrease rapidly during citrate treatment regardless of cell growth phase and trisodium citrate concentration in the range of 1–1.5%. By using the commercially available sodium alginate, Keltone LV, dissolution time can be kept short enough (below 5 minutes) to keep the effect of trisodium citrate negligible to cell viability.     

Mitochondrial membrane potential selects hybridomas yielding high viability in fed-batch cultures

Prior research (Follstad, B. D.; Wang, D. I. C.; Stephanopoulos, G. Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures. Eur. J. Biochem. 2000, 267, 6534-6540.) identified mitochondrial membrane potential (MMP) as a marker of hybridoma subpopulations resistant to apoptosis caused by a variety of apoptosis inducers. In this study, we investigated the viability of hybridoma cell cultures inoculated with cells of varying MMP in regular fed-batch operation. A hybridoma cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123 (Rh123). These subpopulations showed dramatic differences in their apoptotic death kinetics. Fed-batches inoculated with a high MMP subpopulation reached higher viable cell concentrations and viabilities that were maintained for prolonged periods of time relative to fed-batches inoculated with low MMP subpopulations. These results underline the heterogeneous nature of hybridoma cell cultures and suggest that mitochondrial physiology is a critical parameter determining culture performance.     

Design, synthesis and characterization of antimicrobial pseudopeptides corresponding to membrane-active peptide.

To obtain active and metabolically stable analogues, peptide backbone modifications have been incorporated into many biologically active peptides. In this study, we designed and synthesized pseudopeptides corresponding to the antimicrobial peptide that acted on the lipid membrane of the pathogen. Most pseudopeptides exhibited a longer half-life than the peptide in the presence of serum as well as a considerable activity against test bacteria and fungi. Circular dichroism spectra and retention times of the pseudopeptides helped us to elucidate the effect of the incorporation of backbone modifications on the structural parameters necessary for the activity, indicating that alpha-helical structure was the most important factor for the activity and hydrophobicity had a considerable effect on the activity. Backbone modifications employed in this study can be a useful tool for structure-activity relationship studies and the development of therapeutic agents from membrane-active antimicrobial peptides.