No abstract

Formation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-B. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-B. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.More than 300 publications dealing with pore-forming bacterial toxins have been published during the past decade, the majority focusing on structural aspects and mechanisms of pore formation (3, 6, 7). Biological consequences other than cell lysis have been described, e.g., secretory responses (4, 5), production of lipid mediators (11), interleukin-1␤ (IL-1␤) maturation (27), and programmed cell death (12). Killing of human keratinocytes by alpha-toxin is due to enhanced permeability for monovalent ions (27). Certain cells can repair a limited number of lesions (24). These findings all indicate that cells attacked by pore-forming toxins are not inevitably and instantly paralyzed and that they retain the capacity to mount active responses to membrane damage. In this investigation, we questioned whether cell damage by alpha-toxin might result in the activation and exploitation of transcriptional mechanisms. NF-B transcription factors are involved in the response to many types of stress. Proteins encoded by NF-B-regulated genes are devoted to intercellular signaling, cell adhesion, and other defense-related functions (9, 10). NF-B has also been implicated in the regulation of apoptosis (1,8). Biochemical features of NF-B include constitutive expression in the cytosol, rapid activation, and translocation into the nucleus.We report that alpha-toxin causes rapid activation of NF-B, which leads to the expression of IL-8 in monocytic THP-1 cells and in ECV304 cells. Transcriptional activation was also shown in 3T3 fibroblasts, where it was found that recovery to sublethal toxin attack was not dependent on activation of this transcription factor. MATERIALS AND METHODSWild type alpha-toxin and the nonlytic toxin mutant H35R were prepar...

The objective of this study was to report on the arsenic species present in tube-well water samples collected from West Bengal, India, especially dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA), whose existence has not been reported in the literature. The water samples were collected from Jalangi Gram Panchayet (Murshidabad district, West Bengal, India). The samples were speciated for arsenic 11 days after collection. The samples were collected in duplicate. One part was acidified with nitric acid (final concentration 0.1%), whereas the other part was left unacidified. A quick and highly sensitive high-performance liquid chromatography±inductively coupled plasma mass spectrometry (HPLC±ICPMS) technique was employed for the separation and detection of the arsenic species. Four arsenic species, namely arsenite [arsenic(III)], DMA, MMA and arsenate [arsenic(V)] were separated and analysed in less than 5 min. Total arsenic concentration was determined by flow injection (FI)-ICPMS. Most of the samples were found to contain low concentrations of DMA and MMA (<2.1 ppb) and high concentrations of inorganic arsenic (>300 ppb). The existence of DMA and MMA in both acidified and unacidified water samples and in similar concentrations suggests that their presence is natural and not due to acidification. The detection limit of the four arsenic species was 0.06±0.10 ppb. The method was validated by spike recovery and analysis of two water standard reference materials (SRMs). The percentage recoveries of added spikes of all four species were 97±112%. The total arsenic concentration obtained by FI-ICPMS and the sum of the four arsenic species obtained by HPLC±ICPMS for the two water SRMs agreed with the certified values. Moreover, the difference between the total arsenic and the sum of the four arsenic species for most of the water samples was less than 10%.

Even though fluoride toxicity is increasingly being considered to be important, very little information is available on the mechanism of action of fluoride. In the present study, the toxicity of fluoride on human leukemia (HL-60) cells was investigated and the involvement of caspase-3 was also studied. Fluoride induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Annexin staining and DNA ladder formation on agarose gel electrophoresis further revealed that HL-60 cells underwent apoptosis on exposure to 2-5 mM fluoride. Western blotting using polyclonal anti-caspase-3 antibody and mouse anti-human poly(ADP-ribose) polymerase (PARP) monoclonal antibody was performed to investigate caspase-3 and PARP activity. Fluoride led to the activation of caspase-3 which was evident by the loss of the 32 kDa precursor and appearance of the 17 kDa subunit. Furthermore, intact 116 kDa PARP was cleaved by fluoride treatment as shown by the appearance of a cleaved 89 kDa fragment. The results clearly suggest that fluoride causes cell death in HL-60 cells by causing the activation of caspase-3 which in turn cleaves PARP leading to DNA damage and ultimately cell death.

A low cost adsorbent prepared from material of biological origin Setaria verticillata (Grasses) has been utilized as an adsorbent for the removal of Reactive Yellow 15 (RY15) dye from an aqueous solution. The combined effect of the initial pH, adsorbent dose, and initial dye concentration was investigated using Response Surface Methodology (RSM). The most influential adsorption factor on each adsorption experimental design response was identified from the Analysis Of Variance (ANOVA). The experimental values of percentage removal were found to be in good agreement with the predicted values. pH value of 2, initial RY15 concentration of 50 mg L 21 and adsorbent dose of 50 mg are found to be the optimum conditions, for adsorption of RY15 from an aqueous solution. Thermodynamic parameters such as change in standard free energy change, enthalpy and entropy DG o , DH o , and DS o have been evaluated, and it has been found that the adsorption process is feasible, exothermic and spontaneous in nature. The experimental data were analyzed by the Langmuir, Freundlich, Sips and Temkin adsorption isotherms. Maximum monolayer adsorption capacity of the RY15 dye is found to be 138.6 mg g 21. The experimental data fitted well with the pseudo-second order kinetic model.