Authors:Hagen Frickmann, Elmar Schröpfer, and Gerhard Dobler
Rickettsiae are able to spread within infected cell mono-layers by modifying intra-cellular actin formations. The study analyzes whether a visualization of actin modifications in addition to specific immuno-fluorescence staining of rickettsiae might facilitate the proof of rickettsial growth in cell culture.
Cell mono-layers of Vero E6 und BGM cells were infected with Rickettsia honei. Intra-cellular actin was fluorescence stained with TRITC-(tetra-methyl-5,6-isothiocyanate)-labeled phalloidin in addition to specific immuno-fluorescence staining of rickettsiae with FITC-(fluorescein-isothiocyanate)-labeled antibodies. DNA of bacteria and cells was counter-stained with DAPI (4′,6- diamino-2-phenyl-indole). Cell cultures infected with Vaccinia virus were used as positive controls, cell cultures infected with Coxiella burnetii as negative controls.
High concentrations of R. honei are necessary to demonstrate characteristic modifications of the intra-cellular actin. This effect is more pronounced in Vero E6 cells than in BGM cells.
Actin staining with phalloidin is not suited for an early proof of rickettsial growth in cell culture but may confirm unclear findings in specific immuno-fluorescence staining in case of sufficient bacterial density.
selection of expression system is based on its productivity with acceptable product quality attributes [ 14 ].
The cellculture processes for therapeutic protein production can follow three different types. The simplest mammalian cellculture process
Authors:Z. Balla, Éva Szőke, G. Czéh, and János Szolcsányi
Effects of capsaicin on voltage-gated currents were examinedin vitro by whole-cell patch-clamp recordings from small neurones of rat trigeminal ganglia either in slice preparations or in different cell cultures. Cells were classified as sensitive to capsaicin if they responded with inward current and/or conductance change to the agent in nanomolar concentration. Capsaicin (150 to 330 nM) in sensitive cells reduced the mixed inward current evoked by depolarizing step or ramp commands in all preparations. In cultured cells, the inward current was depressed to 32.78±26.42% (n = 27) of the control. Both the tetrodotoxin-sensitive and -resistant inward currents were affected. The data support the concept that capsaicin besides acting on VR-1 receptors inhibits also some voltage gated channels. In 34 cultured cells, capsaicin increased the slope conductance to 170.5±68%. Percentage of capsaicin sensitive cells observed in nerve growth factor-treated cultured cell populations was higher (77.8%) than in the two other preparations (14.3 or 38.8%). It is concluded that 1) depression of the voltage-gated currents may play an important role in the functional desensitization of the sensory receptors and in the analgesic effect induced by the agent and 2) cell body of sensory neurones under native condition seems less sensitive to capsaicin then that of cells cultured in the presence of nerve growth factor.
Authors:Virág Somogyi, Tamás L. Horváth, István Tóth, Tibor Bartha, László Vilmos Frenyó, Dávid Sándor Kiss, Gergely Jócsák, Annamária Kerti, Frederick Naftolin, and Attila Zsarnovszky
Thyroid hormones (THs) and oestrogens are crucial in the regulation of cerebellar development. TH receptors (TRs) mediate these hormone effects and are regulated by both hormone families. We reported earlier that THs and oestradiol (E2) determine TR levels in cerebellar cell culture. Here we demonstrate the effects of low concentrations (10–10 M) of the endocrine disruptor (ED) bisphenol A (BPA) on the hormonal (THs, E2) regulation of TRα,β in rat cerebellar cell culture. Primary cerebellar cell cultures, glia-containing and glia-destroyed, were treated with BPA or a combination of BPA and E2 and/or THs. Oestrogen receptor and TH receptor mRNA and protein levels were determined by real-time qPCR and Western blot techniques. The results show that BPA alone decreases, while BPA in combination with THs and/or E2 increases TR mRNA expression. In contrast, BPA alone increased receptor protein expressions, but did not further increase them in combination with THs and/or E2. The modulatory effects of BPA were mediated by the glia; however, the degree of changes also depended on the specific hormone ligand used. The results signify the importance of the regulatory mechanisms interposed between transcription and translation and raise the possibility that BPA could act to influence nuclear hormone receptor levels independently of ligand–receptor interaction.
resistance, the aim of this study was to design antisense and intrusive peptide sequences to inhibit cag A virulence gene in the laboratory environment and cellculture in H. pylori bacteria.
Materials and Methods
Authors:P. Vidhyasekaran, A. Ramanathan, Luke Rathinakumar, and R. Samiyappan
Gowri, G., Paiva, N. L. and Dixon, R. A. (1991): Stress responses in alfalfa ( Medicago sativa L.). 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cellcultures and developing
Rickettsiae tend to have a rapid decrease of viability outside living cells. Therefore, the transport of samples containing viable rickettsiae for culturing in cell culture for diagnostic purposes is challenging.
The viability of rickettsiae in different transport media (commercially available transport medium COPAN “UTM-RT transport medium for viruses, chlamydia, mycoplasma, and ureaplasma,” minimal essential medium (MEM) with and without 10% foetal calf serum) at various time points at 4 °C and at ambient temperature (22 °C) was compared. Rickettsia honei was used as model organism.
After 2 weeks of storage at room temperature, no viable rickettsiae were detectable any more while storage at 4 °C kept rickettsiae viable for up to 4 weeks. The commercially available COPAN medium showed similarly good or slightly better stabilizing effects on rickettsiae compared with MEM + 10% foetal calf serum, pure MEM demonstrated the poorest results.
It is important to transport and store media with potentially rickettsiae-containing samples at 4 °C to prevent inactivation. MEM + 10% foetal calf serum can be used if no commercial medium is available with similarly good results.
Authors:Esma Kaya, Hatice Ozbilge, Mustafa Ustundag, and Yasemin Torun
The aim of this study was to investigate the effects on the immune response of levamisole alone and in conjunction with Candida albicans stimulation in human macrophage cell culture by determining the alterations in the levels of cytokine release.Levamisole treatment was performed before, during and after infecting U-937 human macrophage cells with C. albicans. In cell supernatants, interleukin (IL)-1b, IL-12, IL-18, tumour necrosis factor alpha (TNF-α) levels were measured by ELISA.In vitro levamisole treatment accompanied by C. albicans stimulation significantly increased IL-12, IL-1β and IL-18 production in macrophage cells (p < 0.05). It was observed that when administered before C. albicans infection, levamisole significantly increased IL-12 and IL-1β production in macrophage cells (p < 0.05). Another finding was that when applied to macrophage cells simultaneously with C. albicans infection, or before infection with C. albicans, levamisole suppressed the TNF-β production stimulating effect of C. albicans (p < 0.05).These results indicated that levamisole could be useful in treating patients infected with C. albicans or in protecting individuals under the risk of being infected with this pathogen. There is a need for further experimental and clinical studies on this hypothesis.