N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca²⁺ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca²⁺]ᵢ) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca²⁺]ᵢ in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca²⁺]ᵢ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca²⁺]ᵢ in human neutrophils was observed. In Ca²⁺-free buffer, NAC- and cysteine-induced [Ca²⁺]ᵢ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca²⁺]ᵢ in human neutrophils occur through Ca²⁺ influx. NAC- and cysteine-induced [Ca²⁺]ᵢ increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na⁺-free HEPES, both NAC and cysteine induced a marked increase in [Ca²⁺]ᵢ in human neutrophils, arguing against the possibility that Na⁺-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca²⁺]ᵢ increasing activity. Our results show that NAC and cysteine induce [Ca²⁺]ᵢ increase through Ca²⁺ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.
Acetylcysteine* ; Calcium Channels ; Calcium* ; Cysteine* ; HEPES ; Humans* ; Neutrophils* ; Ruthenium ; Ruthenium Red

Arterial allografts have known advantages over prosthetic vascular conduit for treatment of heart valvular disease, congenital heart disease and aortic disease. Cell viability may play a role in determining the longterm outcome of allografts. Endothelial cell is one important part in determining the allograft viability. To evaluate the viability of endothelial cells using current allograft preservation technique, porcine heart valve leaflets and arterial wall were subjected to collagenase digestion. Single endothelial cell suspension was labeled with GSA-FITC(Griffonia simplicifolia agglutinin- fluorescein isothiocyanate), a vascular endothelial cell specific marker. The cell suspension was washed and incubated with PI(Propidium Iodide), which does not bind with viable cells. Endothelial cell viability was evaluated by calculating the percentage of GSA-FITC(+) and PI(-) group using flowcytometric analysis. Allografts were treated with 4degrees C antibiotic solution for 24 hours for sterilization. After this, half of allografts were stored in 4degrees C RPMI 1640 with HEPES buffer culture medium with 10% fetal bovine serum for 1 to 14 days(Group I). Another half of allografts were cryopreserved with a currently used technique (Group II). During the procurement and sterilization of arterial allografts, 22.8% and 24.4% of endothelial cell viability declined, respectively. In Group I, 11.9% of endothelial cell viability declined further steadily during 14 days of storage. In Group II, 13.7% of endothelial cell viability declined. These results show that largest loss of endothelial cell viability occurs during the initial process. After 14 days of arterial allograft storage under 4degrees C nutrient medium or cryopreservation, about 40% of endothelial cell viability is maintained. There were no differences between the endothelial cell viability from aortic valve leaflet, pulmonic valve leaflets, aortic wall and pulmonic wall.
Allografts* ; Aortic Diseases ; Aortic Valve ; Cell Survival ; Collagenases ; Cryopreservation ; Digestion ; Endothelial Cells* ; Fluorescein ; Heart ; Heart Defects, Congenital ; Heart Valves ; HEPES ; Sterilization

Phenolic compounds affect intracellular free Ca2+ concentration ([Ca2+]i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca2+ signaling in PC12 cells using fura-2-based digital Ca2+ imaging and whole-cell patch clamping. Treatment with ATP (100 micrometer) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 micrometer) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84 micrometer). Treatment with octyl gallate (3 micrometer) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca2+ with nominally Ca2+-free HEPES HBSS or depletion of intracellular Ca2+ stores with thapsigargin (1 micrometer). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1 micrometer) significantly inhibited the ATP-induced [Ca2+]i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca2+]i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 micrometer) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca2+]i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca2+ from extracellular space and P2Y receptor-induced release of Ca2+ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca2+ responses by inhibiting the secondary activation of voltage-gated Ca2+ channels.
Adenosine Triphosphate ; Animals ; Calcium ; Constriction ; Extracellular Space ; Gallic Acid ; Genistein ; HEPES ; Indoles ; Maleimides ; Nimodipine ; PC12 Cells ; Phenol ; Protein Kinase C ; Protein-Tyrosine Kinases ; Thapsigargin

PURPOSE: To gain insight on the role of AP-1 in transcriptional regulation of vimentin gene during differentiation of HL-60 cells by 12-0-tetradecanoylphorbol-13-acetate (TPA), the levels of vimentin mRNA and AP-1 have been investigated with Northern blot hybridization and DNA mobility shift assay. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics in a humidified 5% CO2 at 37 degree C. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. EcoRI fragment of pVIM-GEM was used as probe for vimentin mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA probe(Upper strand, 5'-CGCTTGATGAGTCAGCCG- 3') for AP-1 binding activity was mixed with nuclear extracts in a 20 microliter reaction volume containing 300 mM KC1, 60 mM HEPES, pH 7.9, 25 mM MgCl2, 1 mM EDTA, 1 mM DTT, 60% glycerol, and 2 microgram of poly[dI-dC]. RESULTS: TPA increased vimentin mRNA levels, with maxima1 stimulation reached at 24 hr. The level of vimentin mRNA was induced in proportion to the concentration of TPA. TPA-induced vimentin mRNA was almost reduced by actinomycin-D pretreatment. TPA- induced stimulation of vimentin gene was completely reduced by staurosporin pretreatment. In DNA mobility shift assay, AP-I newly appeared at 24 hr during TPA- induced differentiation and was almost not detected after the pretreatment of staurosporin. CONCLUSIONS: These results suggest that the induction of vimentin mRNA during TPA- dependent differentiation in HL-60 cells may be mediated by protein kinases C signal transduction and AP-1 is important to transcriptional regulation.
Anti-Bacterial Agents ; Blotting, Northern ; DNA ; Edetic Acid ; Electrophoretic Mobility Shift Assay ; Glycerol ; HEPES ; HL-60 Cells* ; Humans ; Hydrogen-Ion Concentration ; Magnesium Chloride ; Protein Kinases ; RNA ; RNA, Messenger ; Signal Transduction ; Transcription Factor AP-1* ; Vimentin*

It has been suggested that increased number and activity of phagocytes in periodontitis lesion results in a high degree of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, nitric oxide and peroxynitrite. There are few reports on the relationship between ROS and MMPs expressions in gingival fibroblast. We studied to elucidate whether and how ROS, especially nitric oxide affects the MMP expression. Human gingival fibroblasts and HT1080 cells (human fibrosarcoma sell line as reference) were grown in DMEM supplemented with 10 mM HEPES, 50 mg/L gentamicin, and 10% heat inactivated fetal bovine serum with addition of various reactive oxygen species (ROS). Culture media conditioned by cells were examined by gelatin zymography. HT1080 cells expressed proMMP-2 and proMMP-9, but human gingival fibroblasts (HGF) produced only proMMP-2. Hydrogen peroxide upregulated MMP-9 expression in HT1080 cells, whereas in human gingival fibroblast SNP treatment showed marked increase in MMP-2 level compared to other ROS. These results suggest that the effects of ROS on MMPs expressions are cell-type specific. RT-PCR for MMP-2 and TIMP-2 m-RNA were performed using total RNA from cultured cells under the influence various kinase inhibitors. In HT1080 cells, treatment with FPTI III (Ras processing inhibitor) and LY294002 (PI3-kinase inhibitor) resulted in inhibition of MMP-2 and MMP-9 expressions, suggesting that Ras/PI3-kinase pathway is important for MMPs expression in HT1080 cells. In gingival fibroblasts, treatment with FPTI III and PDTC (NFkB inhibitor) showed marked decrease in MMP-2 regardless of the of SNP, suggesting that Ras/NF-kB could be the key pathway for NO-induced MMP-2 expression in gingival fibroblasts. This study showed that ROS, especially nitric oxide, could be the critical mediator of periodontal disease progression through control of MMP-2 expression in gingival fibroblasts possibly via Ras/NF-kB pathway.
Cells, Cultured ; Culture Media ; Fibroblasts* ; Fibrosarcoma ; Gelatin ; Gentamicins ; HEPES ; Hot Temperature ; Humans* ; Hydrogen Peroxide ; Matrix Metalloproteinases ; Nitric Oxide* ; Periodontal Diseases ; Periodontitis ; Peroxynitrous Acid ; Phagocytes ; Phosphotransferases ; Reactive Oxygen Species ; RNA ; Superoxides ; Tissue Inhibitor of Metalloproteinase-2

PURPOSE: Nocodazole, a microtubule disrupting reagent, is known to arrest cells in the M phase, To gain insight on the regulatory mechanism of H2B histone gene expression by nocodazole in HL-60 cell, the binding pattern of nuclear proteins to cis element in the human H2B histone gene promoter has been investigated with DNase I footprinting and DNA mobility shift assay. MATERIALS AND METHODS: Northern blot hybridization was performed by the method of Virca et al. A Hinc II-Sac I fragment of pSPH28 was used as probe for Northern blot analysis of H2B histone mRNA. DNase I footprinting and DNA mobility shift assay were performed by the method of Lim et al. End labeled DNA oligomer (upper strand, 5'-CTTCACCTTATTTGCATAA GCGATTC-3') for octamer binding activity was mixed with nuclear extracts in a 20 ul reaction volume containing 60 mM KC1, 12 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 12% glycerol, and 2 ug of poly [dI-dC]. RESULTS: The level of H2B histone mRNA rapidly was reduced at 24 hours in nocodazole-treated HL-60 cells and the mRNA was repressed in proportion to the concentration of nocodazole. Nocodazole-dependent repression of H2B histone gene was restored by replacement with nocodazole-free media. In DNase I footprinting analysis, one nuclear factor bound at 42 bp site (octamer motif) in the absence of nocodazole. In the presence of nocodazole, the binding of nuclear factor on octamer motif partially vanished. In DNA mobility shift assay, one DNA-protein complex (Octl) was formed when octamer motif was incubated with nuclear extract of HL-60 cell. After nocodazole treatment, Octl binding activity was reduced by time dependent manner. CONCLUSION: These results suggest that nocodazole-dependent repression of H2B histone gene is correlated with reduction of Octl binding activity in HL-60 cell.
Blotting, Northern ; Cell Division ; Deoxyribonuclease I ; DNA ; Edetic Acid ; Electrophoretic Mobility Shift Assay ; Gene Expression* ; Glycerol ; HEPES ; Histones* ; HL-60 Cells* ; Humans ; Hydrogen-Ion Concentration ; Magnesium Chloride ; Microtubules ; Nocodazole* ; Nuclear Proteins ; Repression, Psychology ; RNA, Messenger

BACKGROUND: Vimentin is the major intermediate-size filament in the cytoplasm of cells from mesenchymal origin. The HL-60 cell is a unique human leukemic cell line capable of terminal differentiation with several chemical inducers, and then the cell line becomes a fre#quently described model system for cell differentiation in vitro. Vimentin mRNA is reduced during all-trans retinoic acid (retinoic acid) -dependent differentication but increased by 12-0-tetradecanoylphorbol-13-acetate (TPA). In this paper, we have investigated on the mechanism of transcriptional repression of vimentin gene during retinoic acid-dependent differentication of HL-60 cell. METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics in a humidified 5% CO at 37C. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. EcoRI fragment of pVIM-GEM was used as probe for vimentin mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA probe (Upper strand, 5-CGCITGATGAGTCAGCCG-3) for AP-1 binding activity was mixed with nuclear extracts in a 20 pL reaction volume containing 300 mM KCI, 60 mM HEPES, pH 7.9, 25mM MgC1, 1mM EDTA, 1mM DTT, 60% glycerol, and 2 pg of poly[dI-dC]. RESULTS: The level of vimentin mRNA was decreased at 12 hours after retinoic acid treatment, and not detected at 48 hours. The level of vimentin mRNA was reduced in proportion to concentration of retinoic acid, Retinoic acid-reduced vimentin mRNA was no change in cells treated with cycloheximide. Retinoic acid-dependent decrease of vimentin mRNA was partially recovered by staurosporin pretreatment. In DNA mobility shift assay, AP-1 binding activity was reduced at 48 hr during retinoic acid-induced differentiation. CONCLUSION: These results suggest that the transcriptional repression of vimentin gene during retinoic acid-induced differentiation in HL-60 cells is correlated with reduction of DNA binding activity of AP-1.
Anti-Bacterial Agents ; Blotting, Northern ; Cell Differentiation ; Cell Line ; Cycloheximide ; Cytoplasm ; DNA ; Edetic Acid ; Electrophoretic Mobility Shift Assay ; Glycerol ; HEPES ; HL-60 Cells* ; Humans ; Hydrogen-Ion Concentration ; Repression, Psychology* ; RNA ; RNA, Messenger ; Transcription Factor AP-1 ; Tretinoin ; Vimentin*

BACKGROUND: Volatile general anesthetics have been widely used to produce reversible unconsciousness and analgesia in clinical practice over the last one hundred years, but the basic mechanism of anesthetic action is not yet completely understood. In addition to the well known mechanism of GABAA and glycine channels, accumulating evidence indicates that neuronal baseline K+ channels are also activated by volatile anesthetics. The goal of this study was to test the hypothesis that sevoflurane, one of the newly developed volatile anesthetics, activates baseline potassium channnels in the cerebellar granule neurons of rats. METHODS: Whole cell measurement techniques were performed from cultured cerebellar granule neurons of seven day old male Sprague-Dawley rats using patch clamp techniques to see the effects of two MACs of sevoflurane on baseline K+ channels. Holding potentials were set to 20 mV and collect pulses from 90 to 90 in 10 mV increments of 300 ms duration. The electrode filling solution contained (in mM) 150 KOH, 105 aspartic acid, 3 NaCl, 10 HEPES, 86 glucose, 1 EGTA, 5 MgCl2 (pH 7.4) and standard saline were used as bath solution. The bath contained 150 NaCl, 3 KCl, 10 HEPES, 14 glucose, 1 EGTA, 5 MgCl2 (pH 7.4). RESULTS: Analysis of multiple patch clamp experiments showed the presence of outward-rectifying K+ selective ion channels with a conductance of 1.064 +/- 0.32 nS (n = 10) at depolarized potentials. Cerebellar granule neurons exhibit rapid rising, noninactivating, outward-rectifying currents. These channels are insensitive to conventional K+ channel blockers. Clinically relevant concentrations of sevoflurane (518nM) increased the baseline K+ channel outward currents from the control value by 225% in a standard saline perfusate (n = 10, P < 0.05, paired t-test). Channel activity enhanced during the duration of the exposure period to sevoflurane returned to the baseline activity level quickly upon wash. CONCLUSIONS: These outward-rectifying whole cell I V curves are consistent with the properties of tandem pore K+ channels. Activation of baseline K+ channels in central neurons by two MACs of sevoflurane causes membrane hyperpolarization and increases neuronal input conductances providing an additional inhibitory mechanism that could contribute to the overall central depressant effects of this compound.
Analgesia ; Anesthetics ; Anesthetics, General ; Animals ; Aspartic Acid ; Baths ; Egtazic Acid ; Electrodes ; Glucose ; Glycine ; HEPES ; Humans ; Ion Channels ; Magnesium Chloride ; Male ; Membranes ; Neurons* ; Patch-Clamp Techniques ; Potassium ; Potassium Channels ; Potassium Channels, Tandem Pore Domain* ; Rats ; Rats, Sprague-Dawley* ; Unconsciousness

PURPOSE: To gain insight on transcriptional repression of Topo II a in HL-60 cells arrested to G2/M and M phase, the levels of Topo IIa mRNA and the binding activity of ATF have been investigated with Northern blot hybridization and DNA mobility shift assay, respectively. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-mactivated fetal bovine serum and antibiotics in a humidified 5% CO2 at 37C degree. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. A Xho I-Mlu I fragment of phTOP2 was used as probe for Northern blot analysis of Topo II a mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA oligomer (upper strand, 5-TCTCCGCTATGACGCCGAGTGGTG-3) for ATF binding activity was mixed with nuclear extracts in a 20 pl reaction volume containing 60 mM KC1, 12 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 12% glycerol, and 2 ug of poly [dI-dC]. RESULTS: HL-60 cells were arrested at G2/M phase and M phase after taxol or nocodazole treatment. The levels of Topo II a mRNA were reduced at 24 hours after exposure with nocodazole or taxol but the unknotting activities were not changed. DNA mobility shift assay using oligonucleotide containing the ATF binding site showed that ATF binding activity was reduced after pretreatment of nododazole or taxol. CONCLUSIONS: These results suggest that the reduction of ATF binding activity may be important to transcriptional repression of Topo II a gene by nocodazole and taxol in HL- 60 cells.
Anti-Bacterial Agents ; Binding Sites ; Blotting, Northern ; Cell Division* ; DNA Topoisomerases, Type I* ; DNA Topoisomerases, Type II* ; DNA* ; Edetic Acid ; Electrophoretic Mobility Shift Assay ; Genes, vif ; Glycerol ; HEPES ; HL-60 Cells ; Humans ; Hydrogen-Ion Concentration ; Magnesium Chloride ; Nocodazole ; Paclitaxel ; Repression, Psychology ; RNA ; RNA, Messenger

PURPOSE: Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidyl choline to phosphatidic acid (PA) and choline. Recently, PLD has been drawing much attentions and considered to be associated with cancer process since it is involved in cellular signal transduction. In this experiment, oleate-PLD activities were measured in various tissues of the living rats after whole body irradiation. MATERIAL AND METHODS: The reaction mixture for the PLD assay contained 0.1microCi 1,2-di[1-14C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50mM HEPES buffer (pH 6.5), 10mM CaCl2, and 25mM KF. phosphatidic acid, the reaction product, was separated by TLC and its radioactivity was measured with a scintillation counter. The whole body irradiation was given to the female Wistar rats via Cobalt 60 Teletherapy with field size of 10cm x 10cm and an exposure of 2.7Gy per minute to the total doses of 10Gy and 25Gy. RESULTS: Among the tissues examined, PLD activity in lung was the highest one and was followed by kidney, skeletal muscle, brain, spleen, bone marrow, thymus, and liver. Upon irradiation, alteration of PLD activity was observed in thymus, spleen, lung, and bone marrow. Especially PLD activities of the spleen and thymus revealed the highest sensitivity toward gamma-ray with more than two times amplification in their activities. In contrast, the PLD activity of bone marrow appears to be reduced to nearly 30%. Irradiation effect was hardly detected in liver which showed the lowest PLD activity. CONCLUSION: The PLD activities affected most sensitively by the whole-body irradiation seem to be associated with organs involved in immunity and hematopoiesis. This observation strongly indicates that the PLD is closely related to the physiological function of these organs. Furthermore, radiation stress could offer an important means to explore the phenomena covering from cell proliferation to cell death on these organs.
Animals ; Attention ; Bone Marrow ; Brain ; Cell Death ; Cell Proliferation ; Choline ; Cobalt ; Female ; Hematopoiesis ; HEPES ; Humans ; Hydrolysis ; Kidney ; Liver ; Lung ; Muscle, Skeletal ; Oleic Acid ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D* ; Phospholipases* ; Radiation Effects ; Radioactivity ; Rats* ; Rats, Wistar ; Scintillation Counting ; Signal Transduction ; Sodium ; Spleen ; Taurodeoxycholic Acid ; Thymus Gland ; Whole-Body Irradiation