This experiment was designed to explore the pattern of K562 and HL60 leukemia cells death, the effects on their cell cycle and the cytoplasmic free calcium concentration ([Ca2+]i) induced by 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Under the transmission electron microscope (TEM), two kinds of leukemia cells' ultrastructure were observed. Flow cytometry combined with Annexin V-FITC/PI labeling was used to detect the pattern of K562 and HL60 cells' death induced by ALA-PDT. Flow cytometry combined with PI labeling was used to analyze the change in the cell cycle induced by ALA-PDT, and confocal laser scanning microscopy (CLSM) combining with calcium fluorescence probe was used to detect the change in the cytoplasmic free calcium concentration ([Ca2+]i). Immediately after irradiation, many typical apoptotic bodies were seen in the cells treated. Most of the cells treated were necrotic at 24 hours following irradiation. Flow cytometry analysis suggested that the main patterns of the cells' death were apoptosis immediately after irradiation and necrosis post-apoptosis at 24 hours post irradiation. Immediately and 24 hours after irradiation, the proportion of S phase of K562 was 57. 67% +/- 1.13% and 84.77% +/- 6.20% respectively, and the proportion of S phase of HL60 was 74.60% +/- 7.27% and 84.60% 1.74% respectively. Both [Ca+]i of the treated K562 and HL60 were increased obviously. In the best experiment condition, the initial pattern of the K562 and HL60 leukemia cells' death induced by PDT was apoptosis and the main pattern was necrosis post apoptosis. The two kinds of cells were arrested at S phase by ALA-PDT. During the death of the leukemia cells, the increase in intracellular free calcium concentration could be responsible for the ALA photodynamically induced damage to K562 and HL60 cells.
Aminolevulinic Acid ; pharmacology ; Apoptosis ; drug effects ; Calcium ; chemistry ; Cell Division ; drug effects ; Cytosol ; chemistry ; HL-60 Cells ; drug effects ; Humans ; K562 Cells ; drug effects ; Photochemotherapy

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

The effects of insulin on ATP-citrate lyase, its mRNA in cytosol, and the transcriptional activity in nuclei of diabetic rat liver were studied. Experimental diabetes was induced by an intraperitoneal injection of streptozotocin, and livers were removed from rats at 0, 1, 3, 6, 16, and 72 hours after the administration of insulin. ATP-citrate lyase began to increase at 16 hours, and continuously increased until 72 hours. The amount of mRNA encoding ATP-citrate lyase increased abruptly at 16 hours, then decreased to near basal level in 72 hours. No change in the transcription rate was observed until 3 hours after insulin administration. However, the activity increased 4-fold at 6 hours and 7-fold at 16 hours, 16-fold at 6 hours and 28-fold at 16 hours when pGACL1 and pGACL2 were used as probes, respectively, preceding the increase in the amounts of mRNA and the enzyme. It is suggested that the increase in the amount of ATP-citrate lyase by insulin is primarily due to the increase in the transcriptional activity of the gene in nuclei, which results in the subsequent increase in the amount of mRNA for the biosynthesis of ATP-citrate lyase in cytosol.
ATP Citrate (pro-S)-Lyase/*biosynthesis/genetics ; Animal ; Cell Nucleus/enzymology ; Cytosol/enzymology ; Diabetes Mellitus, Experimental/*enzymology ; Enzyme Induction/drug effects ; Insulin, Isophane/*pharmacology ; Liver/*enzymology ; Male ; RNA, Messenger/drug effects ; Rats ; Rats, Sprague-Dawley ; Transcription, Genetic/drug effects

OBJECTIVETo study the protective effect of recombinant adenovirus-mediated human cytosolic glutathione peroxidase (hCGPx) gene transfection on vascular endothelial cells ECV304 from oxidative damage.

METHODSpGEM-T Easy Vector containing hCGPx cDNA and recombinant adenovirus shuttle plasmid pACCMV-pLpA were used to construct the shuttle plasmid pACCMV-hCGPx for cotransfection of 293 cells with pJM17, thereby to obtain the recombinant adenovirus AdCMV-hCGPx. Cultured ECV304 cells were transfected with AdCMV-hCGPx for 24, 48 and 72 h, respectively, with the cells transfected with the empty vector serving as control, and hCGPx gene expression was then examined in the transfected cells. The transfected cell viability and apoptotic cell ratio were evaluated after treatment of the cells with H(2)O(2).

RESULTSThe expression ratio of hCGPx gene was significantly higher in the AdCMV-hCGPx-transfected cells than in those with empty vector transfection (P<0.01). The hCGPx gene-transfected cells showed significantly higher viability and significantly lower apoptotic ratio than the control cells following challenge with H(2)O(2)-induced oxidative damage.

CONCLUSIONhCGPx gene transfer mediated by recombinant adenovirus protects the vascular endothelial cells from oxidative damage in vitro, possibly due to the antioxidative and apoptosis-inhibiting effect of hCGPx.

Adenoviridae ; genetics ; Apoptosis ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Cytosol ; enzymology ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Genetic Vectors ; Glutathione Peroxidase ; biosynthesis ; genetics ; Humans ; Hydrogen Peroxide ; pharmacology ; Oxidative Stress ; Plasmids ; genetics ; Time Factors ; Transfection

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

Mammalian acetyl-CoA carboxylase (ACC) is present in two isoforms, alpha and beta, both of which catalyze formation of malonyl-CoA by fixing CO2 into acetyl-CoA. ACC-alpha is highly expressed in lipogenic tissues whereas ACC-beta is a predominant form in heart and skeletal muscle tissues. Even though the tissue-specific expression pattern of two ACC isoforms suggests that each form may have a distinct function, existence of two isoforms catalyzing the identical reaction in a same cell has been a puzzling question. As a first step to answer this question and to identify the possible role of ACC isoforms in myogenic differentiation, we have investigated in the present study whether the expression and the subcellular distribution of ACC isoforms in H9c2 cardiac myocyte change so that malonyl-CoA produced by each form may modulate fatty acid oxidation. We have observed that the expression levels of both ACC forms were correlated to the extent of myogenic differentiation and that they were present not only in cytoplasm but also in other subcellular compartment. Among the various tested compounds, short-term treatment of H9c2 myotubes with insulin or okadaic acid rapidly increased the cytosolic content of both ACC isoforms up to 2 folds without affecting the total cellular ACC content. Taken together, these observations suggest that both ACC isoforms may play a pivotal role in muscle differentiation and that they may translocate between cytoplasm and other subcellular compartment to achieve its specific goal under the various physiological conditions.
Acetyl-CoA Carboxylase/metabolism* ; Acetyl-CoA Carboxylase/drug effects ; Animal ; Cell Differentiation/drug effects ; Cell Line ; Cell Membrane Permeability ; Chromones/pharmacology ; Cytosol/enzymology* ; Cytosol/drug effects ; Digitonin/pharmacology ; Immunoblotting ; Insulin/pharmacology* ; Isoenzymes ; Morpholines/pharmacology ; Myocardium/cytology ; Okadaic Acid/pharmacology* ; Phosphorylation ; Rats

AIMTo study the relaxation mechanisms of tetrandrine (Tet) on the corpus cavernosum smooth muscle.

METHODSThe corpus cavernosum smooth muscle cells from New Zealand white rabbits were cultured in vitro. [Ca(2+)](i) was measured by Fluorescence Ion Digital Imaging System, using Fluo-2/AM as a Ca(2+)-sensitive fluorescent indicator.

RESULTSTet (1, 10 and 100 micromol/L) had no effect on the resting [Ca(2+)](i) (P>0.05). In the presence of extracellular Ca(2+) (2.5 mmol/L), Tet (1, 10 and 100 micromol/L) inhibited [Ca(2+)](i) elevation induced by high K(+) and phenylephrine (PE) in a concentration-dependent manner (P>0.05). In calcium free solution containing egtaic acid, Tet (1 and 10 micromol/L) had no inhibitory effects on [Ca(2+)](i) elevation induced by PE (P>0.05). However, Tet (100 micromol/L) inhibited [Ca(2+)](i) elevation induced by PE (P>0.05).

CONCLUSIONTet inhibited the Ca(2+) influx from the extracellular site via voltage-activated Ca(2+) channel and alpha(2)-adrenoceptor-operated Ca(2+) channel. At a high concentration, Tet might inhibit the cytosolic calcium pool release in cultured corpus cavernosum smooth muscle cells. This inhibitory action on [Ca(2+)](i) might be one of the relaxation mechanisms of Tet on the corpus cavernosum smooth muscle.

Alkaloids ; pharmacology ; Animals ; Benzylisoquinolines ; pharmacology ; Calcium ; metabolism ; Cytosol ; drug effects ; metabolism ; Male ; Muscle Relaxation ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Penis ; cytology ; metabolism ; Phenylephrine ; pharmacology ; Potassium Chloride ; pharmacology ; Rabbits

To investigate the existence of nongenomic action of 17β-estradiol (E(2)) in the delayed implantation mouse endometrial stromal cells, the changes in intracellular calcium concentration ([Ca(2+)](i)) and the upstream of calcium signal in vitro were detected. The experiment was composed of two parts. Firstly, the change in [Ca(2+)](i) in endometrial stromal cells induced by E(2) under different conditions was detected. The mice were divided into 6 groups as follows: 0.1% dimethylsulfoxide (DMSO) control group, 1×10(-8) mol/L bovine serum albumin (BSA) control group, 1×10(-8) mol/L E(2) group, 1×10(-8) mol/L E(2) conjugated with BSA (E(2)-BSA) group, 1×10(-8) mol/L E(2) + calcium-free medium group, 1×10(-8) mol/L E(2) + 5 mg/mL tamoxifen group, with 4 mice in each group. The endometrial tissue was obtained from delayed implantation mice at pregnant day 7, and digested by incubation of tissue minces in Hankos balanced salts (HBSS, pH 7.2), which contained glucose (1 g/L), and collagenase I (0.125%), for 1 h at 37 degrees C. The stromal cells were preloaded with 2.5 mmol/L Fluo-3/AM, a fluorescent probe of calcium, for 30 min. A confocal laser scanning microscope, which fixed the wave length of excitation and emission at 488 nm and 526 nm, respectively, was used to detect the change in [Ca(2+)](i). Secondly, the mechanism of E(2) effects in endometrial stromal cells was investigated. Immunofluorescent method was used to detect the change in phosphorylation of phospholipase C (PLC) before and after the stromal cells were treated with E(2) for 5 min, 15 min, and 30 min. Seven delayed implantation mice were used. The results were as follows. [Ca(2+)](i) increased immediately and reached the maximum at 15 min after the stromal cells were treated with 1×10(-8) mol/L E(2) and returned to the normal level at 30 min. In E(2)-BSA group and E(2) + calcium-free medium group the same results were obtained as that in E(2) group, but there was no increase of [Ca(2+)](i) in DMSO and BSA groups. Tamoxifen, a traditional antagonist of estrogen receptor, did not inhibit the increase in [Ca(2+)](i) induced by E(2). Immunofluorescent results showed that the change in phosphorylated-PLC level had the same trend as [Ca(2+)](i) after the cells were treated with E(2). Compared with that in the control group, the immunofluorescent intensity increased at the beginning and achieved the maximum at 15 min (P<0.001), then declined to the normal level at 30 min. These results suggest that the rapid response of [Ca(2+)](i) induced by E(2) in the endometrial stromal cells in delayed implantation mice is possibly carried out through a nongenomic pathway, and the transmembrane signal transduction is related to the phosphorylation of PLC in this process.
Animals ; Calcium ; chemistry ; Culture Media ; Cytosol ; chemistry ; Endometrium ; cytology ; Estradiol ; pharmacology ; Female ; Mice ; Phosphorylation ; Pregnancy ; Receptors, Estrogen ; Signal Transduction ; Stromal Cells ; cytology ; drug effects ; Tamoxifen

AIM: To study the effect of Buyang Huanwu Decoction (BYHWD) on the antioxidant enzymes and drug-metabolizing enzymes in rat liver. METHOD: Following treatment of rats with BYHWD at 6.42, 12.83, or 25.66 g·kg(-1) per day for 15 days, microsomes and cytosols isolated from the liver tissues were prepared by differential centrifugation according to standard procedures. The activities of the antioxidant enzymes and cytochrome b5, NADPH-cytochrome P450 reductase, CYP3A, CYP2E1, UGT, and GST of the rat livers were determined by UV-Vis spectrophotometer. RESULTS: The activities of ALT, AST, antioxidant enzymes, and the Hepatosomatic Index in serum were not significantly affected. In cytosols, the activity of CAT was significantly increased at the dosage of 12.83 g·kg(-1), and all the other antioxidant activities and MDA levels were not affected by this treatment. BYHWD had no effect on cytochrome b5, NADPH-cytochrome P450 reductase, CYP3A, and UGT. At the highest dose (25.66 g·kg(-1)), the activity of CYP2E1 was significantly inhibited, and the activities of GST and the level of GSH were increased. CONCLUSION BYHWD is safe for the liver, and has the functions of detoxification and antioxidant. Patients should be cautioned about the herb-drug interaction of BYHWD and CYP2E1 substrates.
Animals ; Antioxidants ; metabolism ; pharmacology ; Catalase ; metabolism ; Cytochrome P-450 CYP2E1 ; metabolism ; Cytosol ; Drugs, Chinese Herbal ; pharmacology ; Glutathione ; metabolism ; Glutathione Transferase ; metabolism ; Herb-Drug Interactions ; Inactivation, Metabolic ; drug effects ; Liver ; drug effects ; enzymology ; Male ; Microsomes ; Rats, Sprague-Dawley

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H2O2 leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)-induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF-kappaB activation, HeLa cells were incubated with H2O2 or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H2O2 and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H2O2 alone, nuclear NF-kappaB was induced after 2 h without detectible degradation of cytosolic IkBa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappaB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.
Cell Nucleus/drug effects/metabolism ; Cytosol/drug effects/metabolism ; Diamide/pharmacology ; Hela Cells/drug effects/metabolism ; Human ; Hydrogen Peroxide/pharmacology ; I-kappa B/drug effects/metabolism ; NF-kappa B/drug effects/genetics/*metabolism ; Oxidants/pharmacology ; *Oxidative Stress ; Protein-Serine-Threonine Kinases/metabolism ; Signal Transduction/drug effects ; Time Factors ; Transcription, Genetic ; Tumor Necrosis Factor/pharmacology