Extreme light conditions repressed the levels of mRNAs accumulation of multiple genes encoding light-harvesting chlorophyll-(LHC) proteins of photosystem (PS)II in the unicellular green alga, genes encoding the major LHC (LHCII) proteins and two genes (and genes is usually coordinately repressed when the energy input through the antenna systems exceeds the requirement for CO2 assimilation. reaction centers and electron transport events. Photosynthesis is usually regulated at various levels in response to fluctuating light intensity under various ambient heat and nutrient conditions. The proper responses to the various environmental cues are necessary for photosynthetic plants to use light energy efficiently and to safeguard themselves from photoinhibitory damage caused by excessive irradiance (Aro et al., 1993; Long et al., 1994; Osmond, 1994). Excessive light energy absorbed by chlorophyll is usually dissipated by non-radiative processes (Crofts and Yerkes, 1994; Horton et al., 1996; Gilmore, 1997) and is usually properly distributed between two photosystems (PS) by state transition (Allen, 1995; Gal et al., 1997), whereas the energy input is usually regulated by changes in the size of the light-harvesting antenna systems to modulate the optical cross section. Light-harvesting chlorophyll (LHC)II proteins, which are major components of light-harvesting antennae of PSII in higher plants and green algae, typically switch their abundance in response to the intensity of irradiance (Anderson et al., 1988, 1995). Under stress and intense light, enhanced amounts of reactive oxygen species will react with proteins and lipids, not only in chloroplasts but also in the cytosol, and will induce various types of photodamage. Consequently, the quality and quantity control of the LHC proteins complex must prevent photodamage by alleviating excitation energy pressure. Even though LHC protein complicated could be managed by different mechanisms which includes pigment synthesis, the repression of the genes under demanding light conditions should be a significant antistress response of plant life. However, small is well known about the system of the way the extreme light intensity is certainly sensed and the way the transmission is certainly transduced to improve gene expression. One proposal is certainly that the redox condition of the photosynthetic electron transportation carrier(s) between your two PS in green algae monitors the energy stability because such carriers will be over-reduced if the energy input exceeds the requirement for the dark reaction. The abundance of Calcipotriol LHCII protein and/or mRNA decreases with the increase of the reduced QA populace probed by chlorophyll fluorescence in (Maxwell et al., 1995a) and (Maxwell et al., 1995b). Expression of the LHCII gene in is usually enhanced by interrupting electron transfer from QA to QB with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and it is repressed by inhibiting the oxidation of plastoquinol with 2,5-dibromo-3-methyl-6-isopropyl-genes to light intensity have mainly focused on the gene encoding the most abundant LHC (LHCII) protein. Whether each gene is usually regulated Calcipotriol independently or whether they are all coordinately regulated in response to Lum the light intensity remains unknown. To understand the light-dependent regulation of the entire antenna system, comprehensive studies on the light response of all genes are required. The unicellular green alga has been extensively applied as a model experimental system for studies of photosynthesis. The composition of LHC proteins in this alga has been best characterized in algal species (Bassi and Wollman, 1991; Bassi et al., 1992; Allen and Staehelin, 1994). We characterized the gene family encoding the LHC proteins of PSII using the expressed sequence tag (EST) databases (Teramoto et al., 2001). The results revealed that this alga has at least six genes encoding the major LHC (LHCII) proteins and two genes for the minor LHC proteins (CP29 and CP26). The highly homologous LHCII proteins in cannot be assigned to any of the three proposed types in higher Calcipotriol plants (Lhcb1-Lhcb3), but they can be classified into four unique types. Type I is usually encoded by the three genes: should provide a promising experimental system with which to study regulation of gene expression under various environmental conditions. The present study uses quantitative reverse transcriptase (RT)-PCR to examine the amounts of the multiple mRNAs that accumulate Calcipotriol in cells exposed to various intensities of light at various temperatures and under different CO2 conditions. The mRNA levels were.
Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis and it can stimulate the expression of Cyt387 a variety of inflammatory signals. mechanism we investigated the effect of different concentrations of Ox-LDL (50 100 150 μg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression. model of endothelial cell injury. In addition we investigated the underlying mechanisms involved in order to provide a new theoretical basis and targets for drug therapy for prevention and treatment of atherosclerosis. Material and Methods Cell lines and reagents Human umbilical vein endothelial cells (HUVECs) were obtained from Chongqing Medical University (Chongqing China) and cultured in RPMI-1640 (Gibco USA) supplemented with 10% fetal bovine serum (Gibco). For Ox-LDL injury HUVECs were treated with various concentrations of Ox-LDL (XieSheng Bio China) and Poly I (Santa Cruz USA) for 24 h. Cells were cultivated in a humidified atmosphere with 5% CO2 at 37°C. Cell proliferation assays The cell counting kit (CCK-8 Beyotime Institute of Biotechnology China) assay was used to determine cell proliferation. Increasing concentrations of Ox-LDL (50 100 and 150 μg/mL) were added to cell cultures which were then incubated for 24 h. Absorbance was detected with a microplate reader at a wavelength of 450 nm using a 96-well multichannel auto reader (Biotech Instruments USA). The percentage inhibition of cell proliferation was determined by comparing the absorbance of treated with untreated controls as follows: Inhibition (%) = [1-(A of the experimental sample/A of the control)]×100%]. Assessment of cell cycle and apoptosis Proliferating HUVECs were serum-starved overnight and treated with Ox-LDL (100 μg/mL) in complete media for 24 h. Following treatment the cells were harvested and fixed in 70% ice-cold ethanol. The percentages of cells in the G0/G1- S- Cyt387 G2- and M-phases were quantitated by flow cytometry. The extent of apoptosis was evaluated by Annexin-V staining. HUVECs were incubated in the presence of Ox-LDL (100 μg/mL) for 24 h and stained with Annexin-V-fluorescein isothyocyanate (FITC) and propidium iodide (PI). Samples were analyzed by flow cytometry. The data shown are representative of at least three impartial experimental sets. Immunoblotting HUVECs were plated in a culture flask 1 day before the experiment. The cells were then incubated Cyt387 for 24 h under the following conditions: a) no Ox-LDL b) 100 μg/mL Ox-LDL or c) 250 μg/μL Poly I plus 100 μg/mL Ox-LDL (14). The cells were washed three times with ice-cold PBS lysed with RIPA lysis buffer (Beyotime) and placed on ice for 30 min. Proteins were separated by SDS-PAGE (12% gels) and subsequently transferred to a PVDF membrane (Millipore USA). The membrane was blocked with 5% BSA in Tris-buffered saline and Tween-20 (10 mM Tris pH 7.5 140 mM NaCl 0.05% Tween-20) for 2 h at room temperature. A rabbit polyclonal Lum antibody against OX40L (1:1000 Santa Cruz) and a rabbit polyclonal antibody against LOX-1 (1:1000 Abcam Hong Kong) were used as the primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG was used as a secondary antibody. BeyoECL Plus (Beyotime) was used for antibody detection according to the manufacturer’s instructions. Immunocytochemistry Following the same stimulation conditions described above HUVECs were fixed in 4 paraformaldehyde for 20 min and washed three times in PBS. A rabbit polyclonal primary antibody against OX40L (1:1000 Santa Cruz) was added at 4°C overnight. Cells were washed three times in PBS and incubated with the blocking solution which included FITC-conjugated goat Cyt387 anti-rabbit IgG (CWBio China) at 37 for 2 h. Cells were washed three times in PBS and incubated with PI at 37°C for 5 min. Cells were observed with a laser confocal microscope and the average fluorescence value of eight cells from a random.