OBJECTIVETo investigate the effect of hydrogen dioxide (H(2)O(2)) on the release and translocation of high mobility group box 1 release (HMGB1) from normal human bronchiolar epithelial cells (HBE).

METHODSMTT assay was used to assess the viability of HBE135-E6E7 cells exposed to different concentrations of H(2)O(2). The expression and location of HMGB1 in the cytoplasm, nuclei and culture medium of the exposed cells were determined using Western blotting and immunofluorescence assay.

RESULTSExposure to 125 µmmol/L H(2)O(2) did not obviously affect the cell viability. At the concentration of 250 µmmol/L, H(2)O(2) significantly decreased the cell viability (P<0.05), but significant cell death occurred only after exposure to 400 µmmol/L H(2)O(2) (P=0.000). Compared with the control cells, the cells exposed to 12.5, 125 and 250 µmmol/L H(2)O(2) for 24 h showed significantly increased levels of HMGB1 in the culture medium (P<0.05), and exposure to 125 µmmol/L H(2)O(2) for 12 and 24 h also caused significantly increased HMGB1 level (P<0.05). Exposure to 125 µmmol/L H(2)O(2) for 24 h significantly increased HMGB1 expression in the cytoplasm but decreased its expression in the nucleus. HMGB1 translocation from the nuclei to the cytoplasm and to the plasmalemma occurred after 125 µmmol/L H(2)O(2) exposure for 12 h and 24 h, respectively.

CONCLUSIONH(2)O(2) can induce HMGB1 translocation and release in human bronchial epithelial cells, suggesting the involvement of HMGB1 in airway oxidative stress in chronic inflammatory diseases such as asthma and COPD.

Bronchi ; cytology ; Cell Line ; Epithelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; drug effects ; metabolism ; Humans ; Hydrogen Peroxide ; pharmacology ; Protein Transport

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans- Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.
Animals ; Brefeldin A ; pharmacology ; Cell Line, Tumor ; Cytosol ; drug effects ; metabolism ; Humans ; Intracellular Space ; drug effects ; metabolism ; Membrane Proteins ; metabolism ; Protein Transport ; drug effects ; Rats ; Vesicular Transport Proteins ; metabolism ; trans-Golgi Network ; drug effects ; metabolism

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-kappaB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-kappaB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.
Active Transport, Cell Nucleus/drug effects ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Enzyme Activation/drug effects ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Humans ; Hyaluronic Acid/*pharmacology ; Interleukin-6/*pharmacology ; Keratinocytes/*metabolism ; MAP Kinase Signaling System/drug effects ; NF-kappa B/metabolism ; Phosphorylation/drug effects ; Protein Transport/drug effects ; Wound Healing ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo examine the expression and subcellular localization of transcription factor USF1 in odontoblasts and investigate whether nuclear translocation occurs under stimuli.

METHODSOdontoblasts MDPC-23 were cultured on coverslips and divided into 2 groups. Group 1 received no stimuli, and group 2 was stimulated by nicotine with various concentrations respectively for 1h. Then the mountings of odontoblasts were prepared and immunocytochemical staining was performed with specific USF1 antibody via SABC method. Hela cells were used as positive control.

RESULTSThe staining was positive in the cytoplasm of odontoblasts in group 1, but in the nuclei of Hela cells and in 100 mg/L nicotine-stimulated odontoblasts in group 2.

CONCLUSIONSThere exists USF1 protein in odontoblasts, which locates in the cytoplasm and could translocate into nuclei under the stimulation of nicotine.

Cells, Cultured ; HeLa Cells ; Humans ; Nicotine ; pharmacology ; Odontoblasts ; drug effects ; metabolism ; Protein Sorting Signals ; Protein Transport ; drug effects ; Upstream Stimulatory Factors ; metabolism

During ischemia-reperfusion injury, brief pre-exposure to oxidative stress renders organs resistant to subsequent severe damage. NF-kappaB is a transcription factor that is involved in reperfusion-induced inflammatory and immune responses. The activity of NF-kappaB has been shown to be modulated by oxidative stress in various cell types through different pathways. We studied the effect of pre-exposure to oxidative stress on subsequent NF-kappaB activation in TNFalpha-stimulated HEK293 cells. The cells were transiently exposed to 0.5 mM H2O2 for 20 min, prior to stimulation with TNFalpha, and the subsequent expression of NF-kappaB-dependent genes and the levels of NF-kappaB signaling molecules were measured. Pre-exposure to H2O2 significantly delayed the TNFalpha-induced expression of an NF-kappaB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1beta). The degradation of inhibitor of NF-kappaB alpha (IkappaBalpha) and the nuclear translocation of NF-kappaB were also delayed by H2O2 treatment, whereas IkappaBalpha phosphorylation and IkappaB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H2O2-treated cells, we could not detect significant changes in proteosomal peptidase activities, but we were able to detect a delay of IkappaBalpha poly-ubiquitination. Our results suggest that transient exposure to oxidative stress temporally inhibits NF-kappaB-dependent gene expression by suppressing the poly-ubiquitination of phosphorylated IkappaBalpha in HEK293 cells.
Active Transport, Cell Nucleus ; Cell Nucleus/metabolism ; Enzyme Activation/drug effects ; HEK293 Cells ; Humans ; Hydrogen Peroxide/*pharmacology ; I-kappa B Kinase/antagonists & inhibitors/*metabolism ; Phosphorylation/drug effects ; Protein Transport ; Tumor Necrosis Factor-alpha/*pharmacology ; Ubiquitination/*drug effects

OBJECTIVETo study the effect of total glucosides of paeony (TGP) on lipopolysaccharides (LPS)-induced nuclear factor-kappaB (NF-kappaB) activation in macrophages.

METHODRat peritoneal macrophages were pre-treated with TGP for 2 h and stimulated with LPS for 20 min or 0.5 h. Inhibitory kappaBalpha (IkappaBalpha) protein in the cytoplasm and NF-kappaB p65 protein in the nuclear were analyzed by western blot. Further, DNA binding activity of NF-kappaB complex was detected.

RESULTTGP enhanced the amounts of IkappaBalpha protein in the cytoplasm and decreased the amounts of NF-kappaB p65 protein in the nuclear of LPS-induced macrophages. TGP also inhibited the LPS-mediated DNA binding activity of NF-kappaB complex in macrophages.

CONCLUSIONTGP can inhibit LPS-induced NF-kappaB activation in macrophages through arresting IKBalpha protein degradation, NF-kappaB p65 protein nuclear translocation and DNA binding activity of NF-kappaB complex.

Animals ; Cell Nucleus ; drug effects ; metabolism ; Cytoplasm ; drug effects ; metabolism ; DNA ; metabolism ; Dose-Response Relationship, Drug ; Glucosides ; pharmacology ; Macrophages, Peritoneal ; cytology ; drug effects ; metabolism ; NF-kappa B ; metabolism ; Paeonia ; chemistry ; Protein Transport ; drug effects ; Rats ; Transcription Factor RelA ; metabolism

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the premature loss of motor neurons. While the underlying cellular mechanisms of neuron degeneration are unknown, the cytoplasmic aggregation of several proteins is associated with sporadic and familial forms of the disease. Both wild-type and mutant forms of the RNA-binding proteins FUS and TDP-43 accumulate in cytoplasmic inclusions in the neurons of ALS patients. It is not known if these so-called proteinopathies are due to a loss of function or a gain of toxicity resulting from the formation of cytoplasmic aggregates. Here we present a model of FUS toxicity using the yeast Saccharomyces cerevisiae in which toxicity is associated with greater expression and accumulation of FUS in cytoplasmic aggregates. We find that FUS and TDP-43 have a high propensity for co-aggregation, unlike the aggregation patterns of several other aggregation-prone proteins. Moreover, the biophysical properties of FUS aggregates in yeast are distinctly different from many amyloidogenic proteins, suggesting they are not composed of amyloid.
Amyotrophic Lateral Sclerosis ; metabolism ; pathology ; Cell Proliferation ; drug effects ; Cytoplasm ; drug effects ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Detergents ; pharmacology ; Humans ; Kinetics ; Peptides ; metabolism ; Prions ; chemistry ; metabolism ; Protein Binding ; drug effects ; Protein Multimerization ; drug effects ; Protein Structure, Quaternary ; Protein Transport ; RNA-Binding Protein FUS ; chemistry ; genetics ; metabolism ; Saccharomyces cerevisiae ; cytology ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; metabolism

To investigate Bax translocation from cytosol into mitochondria induced by staurosporine (STS) in GFP-Bax-tagged MCF7 stable cell line, the viability was measured by MTT method. Bax translocation from cytosol into mitochondria was investigated under the fluorescence microscope. The dose-effect and time-course relationships were also observed and the percentage of GFP-Bax punctuate cells were calculated. Immunofluoresence method was used to observe Bax translocation to mitochondria, Cyt-c release from mitochondria and Annexin V label. The TMRE assay was used to investigate membrane pertential (Deltapsim) and function of mitochondria. Western blotting was used to observe the mechanism of apoptosis induced by STS. The results showed that STS can induce Bax translocation from cytoplasm to mitochondria, Cyt-c release from mitochondria and Annexin V label. The Western blotting analysis presented the inhibitory effect on apoptosis induced by STS of SP600125 which is a specific JNK inhibitor. The study revealed the mechanism of STS induced apoptosis associated with JNK activated pathway.
Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Cytosol ; metabolism ; Humans ; MAP Kinase Kinase 4 ; antagonists & inhibitors ; Membrane Potentials ; drug effects ; Mitochondria ; metabolism ; Protein Transport ; drug effects ; Staurosporine ; pharmacology ; bcl-2-Associated X Protein ; metabolism

AIMTo investigate the effect of ciliary neurotrophic factor (CNTF) on the nuclear translocation of protein kinase C (PKC) following NMDA administration in the primary cultured hippocampal neurons.

METHODS(1) PKCGAMMA or PKCepsilon- immunocytochemistry staining method was used after treating neurons with NMDA or CNTF. (2) The gray of the nucleus of the PKC-positive neurons were measured under the image pattern analysis system.

RESULTS(1) After NMDA administration of different concentration and time, Nucleus appear PKCgamma and PKCepsilon activities, especially the 100 micromol/L NMDA 30 min group. (2) The gray of nucleus in CNTF + 500 micromol/L NMDA group is similar to control group.

CONCLUSIONNMDA can induce nuclear translocation of PKC in the primary cultured hippocampal neurons, and CNTF can inhibit the translocation. It suggests that the inhibition of PKC translocation induced by NMDA is one of the important reasons for the neuro-protective effects of CNTF.

Animals ; Cells, Cultured ; Ciliary Neurotrophic Factor ; pharmacology ; Hippocampus ; cytology ; N-Methylaspartate ; pharmacology ; Neurons ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Protein Transport ; Rats ; Rats, Sprague-Dawley

The peptide angiotensin IV (Ang IV) is a derivative of angiotensin II. While insulin regulated amino peptidase (IRAP) has been proposed as a potential receptor for Ang IV, the signalling pathways of Ang IV through IRAP remain elusive. We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A (N2A) cells treated with and without Ang IV using sta ble-isotope labeling by amino acids in cell culture (SILAC), and identified a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1 (PP1) upon Ang IV treatment. In addition, spinophilin (spn), a PP1 regulatory protein that plays important functions in the neural system, was expressed at higher levels. Immunoblotting revealed decreased phosphorylation of p70S6 kinase (p70(S6K)) and the major cell cycle modulator retinoblastoma protein (pRB). These changes are consistent with an observed decrease in cell proliferation. Taken together, our study suggests that Ang IV functions via regulating the activity of PP1.
Angiotensin II ; analogs & derivatives ; pharmacology ; Animals ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Membrane ; drug effects ; metabolism ; Cell Nucleus ; drug effects ; metabolism ; Cell Proliferation ; drug effects ; Humans ; Mice ; Microfilament Proteins ; metabolism ; Nerve Tissue Proteins ; metabolism ; Neurons ; cytology ; Phosphorylation ; drug effects ; Protein Phosphatase 1 ; chemistry ; metabolism ; Protein Transport ; drug effects ; Proteome ; metabolism ; Rats ; Threonine ; metabolism ; Up-Regulation ; drug effects