ABSTRACT
Recent evidence established a crucial role for mammalian oxygen sensing transcription factor hypoxia inducible factor-1 (HIF-1) in innate immunity against intracellular pathogens. In response to most of these pathogens host phagocytes increase transcription of HIF-1α, the regulatory component of HIF-1 to express various effector molecules against invaders. Leishmania donovani (LD), a protozoan parasite and the causative agent of fatal visceral leishmaniasis resides in macrophages within mammalian host. The mechanism of HIF-1 activation or its role in determining the fate of LD in infected macrophages is still not known. To determine that J774 macrophages were infected with LD and about four-fold increase in HIF-1 activity and HIF-1α expression were detected. A strong increase in HIF-1α expression and nuclear localization was also detected in LD-infected J774 cells, peritoneal macrophages and spleen derived macrophages of LD-infected BALB/c mice. A two-fold increase in HIF-1α mRNA was detected in LD-infected macrophages suggesting involvement of a transcriptional mechanism that was confirmed by promoter activity. We further revealed that LD also induced HIF-1α expression by depleting host cellular iron pool to affect prolyl hydroxylase activity resulting in to stabilization of HIF-1α. To determine the role of HIF-1 on intracellular LD, cells were transfected with HIF-1α siRNA to attenuate its expression and then infected with LD. Although, initial infection rate of LD in HIF-1α attenuated cells was not affected but intracellular growth of LD was significantly inhibited; while, over-expression of stabilized form of HIF-1α promoted intracellular growth of LD in host macrophage. Our results strongly suggest that LD activates HIF-1 by dual mechanism for its survival advantage within macrophage.
Subject(s)
Gene Expression Regulation/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunity, Innate/immunology , Leishmania donovani/immunology , Phagocytes/parasitology , Animals , Blotting, Northern , Blotting, Western , Cell Line , DNA Primers/genetics , Female , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Phagocytes/metabolism , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Solid-phase extraction (SPE) of phenol and chlorophenols, their derivatization to methyl ethers, headspace single-drop microextraction (HS-SDME) of methyl ethers using 1-butanol as extraction solvent, and direct transfer of the drop into the injector for high performance liquid chromatography with diode array detection (HPLC-DAD) have been reported. A flanged-end polytetrafluoroethylene sleeve, 3 mm × 0.5mm, placed at the tip of the syringe needle, allowed the use of 10 µL solvent drop for extraction. The procedure has been optimized for variables involved in SPE and HS-SDME. A rectilinear relationship was obtained between the amount of chlorophenols and peak area ratio of their methyl ethers/internal standard (4-methoxyacetophenone) in the range 0.01-10 mg L(-1), correlation coefficient in the range 0.9956-0.9996, and limit of detection in the range 1.5-3.9 µg L(-1) when HS-SDME alone was used for sample preparation. When using coupled SPE and HS-SDME, the linear range obtained was 0.1-500 µg L(-1), correlation coefficient in the range 0.9974-0.9998, and the limit of detection in the range 0.04-0.08 µg L(-1). Spiked real samples have been analyzed with adequate accuracy, and application of the method has been demonstrated for the analysis of chlorophenols formed upon bamboo pulp bleaching.
