(AC), a highly valued polypore mushroom native only to Taiwan, has been traditionally used as a medicine for the treatment of food and drug intoxication, diarrhea, abdominal pain, hypertension, skin itching, and cancer. In this study, both of solid-state-cultured AC (S-AC) and wood-cultured AC (W-AC) were evaluated the anti-inflammatory effects on hyperoxia-induced lung injury in NF-κB-luciferase transgenic mice. The homozygous transgenic mice (NF-κB-luciferase ) were randomly assigned to four groups for treatment ( = 6) including Normoxia/DMSO group, Hyperoxia/DMSO group, Hyperoxia/S-AC group, and Hyperoxia/W-AC group. After 72 h of hyperoxia, we examined the bioluminescence images, reactive oxygen species (ROS), the mRNA and protein expression levels of inflammation factors, and histopathological analyses of the lung tissues. Hyperoxia-induced lung injury significantly increased the generation of ROS, the mRNA levels of , , and , and the protein expression levels of IKKα/β, iNOS and IL-6. Pulmonary edema and alveolar infiltration of neutrophils was also observed in the hyperoxia-induced lung tissue. However, treatment with either S-AC or W-AC obviously decreased hyperoxia-induced generation of ROS and the expression of IL-6, TNF-α, IL-1β, IL-8, IKKα/β and iNOS compared to hyperoxia treatment alone. Lung histopathology also showed that treatment with either S-AC or W-AC significantly reduced neutrophil infiltration and lung edema compared to treatment with hyperoxia treated alone. To find out their major compounds, eburicoic acid and dehydroeburicoic acid were both isolated and identified from S-AC and W-AC by using HPLC, MS, and NMR spectrometry. These results demonstrated that methanolic extracts both of S-AC and W-AC have excellent anti-inflammatory activities and thus have great potential as a source for natural health products.
Accumulating evidence shows that several cell types have the capacity to secrete membrane proteins by incorporating them into exosomes, which are small lipid vesicles derived from the intralumenal membranes of multivesicular bodies (MVBs) of the endocytic pathway. Exosomes are expelled in the extracellular space upon fusion of the MVB with the plasma membrane. Exosomal release is a way of secreting membrane proteins meant to be discarded, or to be passed on to other cells. Here, we demonstrate, using primary cortical cultures, that neurones and astrocytes can secrete exosomes. We find that exosomes released by cortical neurones contain the L1 cell adhesion molecule, the GPI-anchored prion protein, and the GluR2/3 but not the NR1 subunits of glutamate receptors. We also show that exosomal release is regulated by depolarisation. Our observation suggests that exosomes may have a regulatory function at synapses and could also allow intercellular exchange of membrane proteins within the brain.
Cells contain a large number of antioxidants to prevent or repair the damage caused by reactive oxygen species, as well as to regulate redox-sensitive signaling pathways. General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, whereas the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein required for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissues and cells. In general, these assays require 24-48 h to complete.
Modification of cellular functions by overexpression of genes is increasingly practised for research of signalling pathways, but restricted by limitations of low efficiency. We investigated whether the novel technique of magnetofection (MF) could enhance gene transfer to cultured primary endothelial cells. MF of human umbilical vein endothelial cells (HUVEC) increased transfection efficiency of a luciferase reporter gene up to 360-fold compared to various conventional transfection systems. In contrast, there was only an up to 1.6-fold increase in toxicity caused by MF suggesting that the advantages of MF outbalanced the increase in toxicity. MF efficiently increased transfection efficiency using several commercially available cationic lipid transfection reagents and polyethyleneimine (PEI). Using PEI, even confluent HUVEC could be efficiently transfected to express luciferase activity. Using a green fluorescent protein vector maximum percentages of transfected cells amounted up to 38.7% while PEI without MF resulted in only 1.3% transfected cells. Likewise, in porcine aortic endothelial cells MF increased expression of a luciferase or a β-galactosidase reporter, reaching an efficiency of 37.5% of cells. MF is an effective tool for pDNA transfection of endothelial cells allowing high efficiencies. It may be of great use for investigating protein function in cell culture experiments.
► Kisspeptin immunoreactive substance is expressed in chicken ovarian granulosa cells. ► Kisspeptin-10 (Kp-10) stimulates progesterone secretion in these cells from preovulatory (F –F ) follicles. ► Stimulated progesterone secretion parallels upregulation of StAR, P450scc and 3β-HSD mRNA expression. The effect of kisspeptin-10 (Kp-10) on the secretion of progesterone (P ) was investigated in cultured granulosa cells from F to F follicles of hens. The results showed that granulosa cells were stained with clear signals for kisspeptin using immunocytochemistry with the specific antibody against Kp-10. Among 10, 100 and 1000 nM concentrations tested, 100 nM Kp-10 treated for 24 h significantly increased P secretion in granulosa cells from F to F follicles. After 24 h and 48 h of treatment, 100 nM Kp-10 showed a significant increase in P secretion, while after 72 h of treatment P secretion was markedly decreased by Kp-10 compared to the control group ( 0.05). These results indicate that Kp-10 stimulates P secretion in cultured chicken granulosa cells, which was associated with an up-regulation in StAR, P450scc and 3β-HSD gene transcription.
The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after maturation (IVM) of cumulus–oocyte complexes (COCs) from antral follicles (>2–6 mm) grown (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC.
This study reports that the spontaneous 50‐fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13‐day chicken embryo, relies on a Ca2+‐dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant‐negative form of the cAMP‐responsive element‐binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca2+/calmodulin‐dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca2+ rather than cAMP. This was confirmed by the inhibitory effect of general and T‐type selective Ca2+ channel blockers. Oscillations in Ca2+ fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization‐activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage‐gated Ca2+‐channels, activation of Ca2+/calmodulin‐dependent kinase II and phosphorylation of cAMP‐responsive element‐binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+‐dependent mechanism whereby hyperpolarization‐activated cyclic nucleotide‐gated channels (HCN) activate T‐type voltage‐dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin‐dependent kinase II (CaMKII) to phosphorylate the cAMP‐responsive element‐binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+‐dependent mechanism whereby hyperpolarization‐activated cyclic nucleotide‐gated channels (HCN) activate T‐type voltage‐dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin‐dependent kinase II (CaMKII) to phosphorylate the cAMP‐responsive element‐binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo.
During human pregnancy, a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4%-8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) and, in some patients, fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously, we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, we cultured CTBs isolated from PE and control placentas for 48 hours, enabling differentiation and invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation, including upregulation of SEMA3B, which resolved in culture. The addition of SEMA3B to normal CTBs inhibited invasion and recreated aspects of the PE phenotype. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression; the autocrine actions of the upregulated molecules (including SEMA3B) impair CTB differentiation, invasion and signaling; and patient-specific factors determine the signs.