Nyeon-Hyoung An

The activation of PI 3-K and PKC ζ in PMA-induced differentiation of HL-60 cells

The human myelocytic leukemia cell line HL-60 is a useful model for the study of cellular differentiation. Phorbol 12-myristate 13-acetate (PMA) induces the monocyte/macrophage-like differentiation of HL-60 cells and results in growth arrest, increasing adherence. In PMA-induced differentiation of HL-60 cells, phosphoinositide 3-kinase (PI 3-K) activity was measured as phosphatidylinositol(3)P recovery from phosphatidylinositol by in vitro kinase assay. PI 3-K activity was increased in HL-60 cells that were stimulated by 20 nM PMA and the activity was inhibited by pretreatment with 20 μM LY294002, a specific inhibitor of PI 3-K. Members of the protein kinase C (PKC) family have been suggested to be one of the downstream targets of PI 3-K. PKC ζ is one of the atypical PKCs, non-diacylglycerol-responsive PKCs, and the activity was measured by the ability of phosphorylation onto myelin basic protein. PMA also induced the activation of PKC ζ during monocytic differentiation of HL-60 cells, and LY294002-pretreated cells failed to induce PKC ζ activation. The activity of PI 3-K is essential for PKC ζ activation, and LY294002 blocks both monocytic differentiation of HL-60 cells and activation of PKC ζ during PMA-induced cell differentiation. This implies that activated PI 3-K subsequently stimulates the PKC ζ in the process of PMA-induced monocytic differentiation.

Hypoxia-induced IL-6 production is associated with activation of MAP kinase, HIF-1, and NF-κB on HEI-OC1 cells

In the present study, we investigated the signal transduction pathways of expression of IL-6 in the desferrioxamine (DFX)-stimulated cochlear auditory cell line, HEI-OC1 cells. DFX increased the expression of HIF-1alpha and NF-kappaB in HEI-OC1 cells. DFX significantly increased the production of IL-6 (P<0.05) and expression of IL-6 mRNA but did not affect TNF-alpha production. DFX also induced the activation of mitogen-activated protein kinase (MAPK) including p38, ERK, and JNK on HEI-OC1. Increased IL-6 by DFX was significantly inhibited by p38 inhibitor, SB203580 (about 72% inhibition, P=0.027) but not ERK inhibitor, PD98059 or JNK inhibitor, SP600125. SB203580 inhibited the expression of IL-6 mRNA. Increased IL-6 production was partially inhibited by treatment of iron (HIF-1 inhibitor) or pyrriolidine-dithiocarbamate (PDTC, NF-kappaB inhibitor). DFX also induced IL-6 production and HIF-1alpha expression in the inner ear. We demonstrated the regulatory effects of MAPK, HIF-1alpha, and NF-kappaB on DFX-induced IL-6 production in a HEI-OC1 for the first time. In conclusion, these data indicate that regulation of inflammatory cytokine IL-6 by DFX, through mimicking hypoxic conditions, might explain its beneficial effect in the treatment of hypoxia-induced inner ear diseases.

Hypoxia induces apoptosis by caspase activation accompanying cytochrome C release from mitochondria in MC3T3E1 osteoblasts. p38 MAPK is related in hypoxia-induced apoptosis.

The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, western blot analysis was performed using anticaspase-3 or -6 antibody. The 10-kDa protein, corresponding to the active products of caspase-3 and the 10-kDA protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which processing of the full length form of caspase-3 and -6 was evident. With a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we have observed that pre-treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAP kinase -JNK/SAPK or ERK- was not or modestly activated. These results suggest that p38 MAPK positively regulates hypoxia-induced apoptosis in MC3T3E1 osteoblasts.

Inhibition of tumor necrosis factor-α-induced apoptosis by Asparagus cochinchinensis in Hep G2 cells

Abstract A human hepatoma cell line, Hep G2 cells, is a reliable system for the study of alcohol-induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Asparagus cochinchinensis MERRIL (Liliaceae) roots (ACAE) on ethanol (EtOH)-induced cytotoxicity in Hep G2 cells. ACAE (1–100 μg/ml) dose-dependently inhibited the EtOH-induced tumor necrosis factor-α (TNF-α) secretion. ACAE (1–100 μg/ml) also inhibited the EtOH and TNF-α-induced cytotoxicity. Furthermore, we found that ACAE inhibited the TNF-α-induced apoptosis of Hep G2 cells. These results suggest that ACAE may prevent the EtOH-induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells.

Effect of Vitex rotundifolia on immediate-type allergic reaction.

We investigated the effect of aqueous extract of Vitex rotundifolia (L.) (Verbenaceae) fruits (VRFE) on the immediate-type allergic reactions in vivo and in vitro. VRFE (10(-4)-1.0 g/kg) dose-dependently inhibited systemic allergic reaction induced by compound 48/80. When VRFE was employed in a systemic allergic reaction test, the plasma histamine levels were reduced in a dose-dependent manner. VRFE (5x10(-1) and 1.0 g/kg) inhibited passive cutaneous anaphylaxis activated by anti-dinitrophenyl (DNP) IgE. VRFE (10(-3)-1.0 mg/ml) also dose-dependently inhibited the histamine release from the rat peritoneal mast cells (RPMC) by compound 48/80 or anti-DNP IgE. Moreover, VRFE (10(-3) mg/ml) had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha production from RPMC. These results suggest that VRFE may be beneficial in the regulation of immediate-type allergic reaction.

Effects of the ethanol extract of Cichorium intybus on the immunotoxicity by ethanol in mice

Effects of the ethanol extract of Cichorium intybus (CIEE) on the immunotoxicity of ethanol (EtOH) were investigated in ICR mice. Mice were divided into four groups, and CIEE at dose of 300 mg/kg was orally administered to mice daily for 28 consecutive days, and normal mice were given vehicle. Mice treated with EtOH were given freely with 20% w/v EtOH solution. The results of this study are summarized as follows: The combination of CIEE and EtOH showed significant increases in the circulating leukocytes and the relative weights of liver, spleen and thymus, as compared with those in mice treated with EtOH alone. However, the body weight gain was not affected. Splenic plaque forming cells (PFC) and hemagglutination (HA) titers to sheep red blood cells (SRBC), and the secondary IgG antibody response to bovine serum albumin (BSA) were markedly enhanced by CIEE plus EtOH treatment as compared with the treatment of EtOH alone. In mice receiving the combination of CIEE and EtOH when compared with EtOH alone-treated mice, there were also significant increases in delayed-type hypersensitivity (DTH) reaction, phagocytic activity, natural killer (NK) cell activity and cell proliferation as well as interferony (IFN-gamma) secretion. In the case of interleukin-4 (IL-4) content, however, an insignificant induction observed by CIEE plus EtOH treatment. These findings indicate that the immunotoxicity induced by EtOH is significantly restored or prevented by CIEE treatment.

High molecular weight water-soluble chitosan protects against apoptosis induced by serum starvation in human astrocytes ☆

The effect of high molecular weight water-soluble chitosan (WSC) on serum starvation-induced apoptosis in human astrocytes (CCF-STTG1 Cells) was investigated. WSC, having an average molecular weight of 300 kDa and a degree of deacetylation over 90%, can be produced using a simple multi-step membrane separation process. Serum starvation led to growth arrest, rounding up of cells and appearance of p53 bands. Prolonged (48 h) incubation in serum starved medium led to cell detachment and death. WSC significantly protected the serum starvation-induced cellular rounding up and protected the serum starvation-induced cell death as tested by flow cytometry. WSC also protected serum starvation-induced p53 activation as determined by Western blot. These results suggest that WSC may prevent serum starvation-induced apoptosis of CCF-STTG1 cells via p53 inactivation.

Polymorphism of the angiotensin-converting enzyme gene in patients with cerebral infarction in koreans

The relationship between cerebrovascular disease and an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene is still being debated. The frequency of the DD genotype of the ACE gene was significantly higher in subjects with than those without cerebral infarction in Japan. The aim of the present study was to assess the relationship between ACE gene polymorphism and the development of cerebral infarction in a population from Korea. We examined its possible role as a risk factor in patients with cerebral infarction. The association between ACE gene polymorphism and cerebral infarction was examined in 106 patients with cerebral infarction and 498 controls without cerebral infarction. Frequencies of the genotypes and alleles of the ACE gene were investigated. The ACE genotype was analyzed by the polymerase chain reaction (PCR). The frequency of D allele was 37.7% in patients and 39.1% in controls (X2=0.128, p=0.720). The frequencies of the genotypes of the ACE gene were II:39.6%, ID:45.3%, and DD:15.1% in patients, and II:37.1%, ID:47.6%, and DD:15.3% in controls (X2=0.127, p=0.721). There was no significant difference in the frequency of the DD genotype of the ACE gene, and we did not find any association between ACE polymorphism and cerebral infarction. These results indicate that ACE polymorphism is not a risk factor for the development of cerebral infarction in a Korean population.

The nitric oxide-producing properties of Solanum lyratum

We examined the effect of Solanum lyratum Thunb. (Solanaceae) (SL) on the production of nitric oxide (NO). Stimulation of mouse peritoneal macrophages with SL after the treatment of recombinant interferon-gamma (rIFN-gamma) resulted in increased NO synthesis. SL had no effect on NO synthesis by itself. When SL was used in combination with rIFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of SL on NO synthesis was shown 6 h after treatment with rIFN-gamma. The increased production of NO from rIFN-gamma plus SL-stimulated cells was decreased by the treatment with staurosporin. In addition, synergy between rIFN-gamma and SL was mainly dependent on SL-induced tumor necrosis factor-alpha (TNF-alpha) secretion. All the preparations of SL were endotoxin free. The present results indicate that the capacity of SL to increase NO production from rIFN-gamma-primed mouse peritoneal macrophages is the result of SL-induced TNF-alpha secretion via the signal transduction pathway of PKC activation.

Auto-ADP-ribosylation of NAD glycohydrolase from Neurospora crassa

NAD glycohydrolase (NADase; EC 3.2.2.5) is an enzyme that catalyzes hydrolysis of NAD to produce ADP-ribose and nicotinamide. We recently demonstrated that self-inactivation of NADase from rabbit erythrocytes was due to an auto-ADP-ribosylation. In the present study, a mechanism of self-inactivation of NADase from Neurospora crassa by its substrate was investigated by using intact mycelia of N. crassa and purified NADase, which had molecular characteristics different from mammalian NADases. The results suggested that inactivation of NADase from N. crassa was also due to an auto-ADP-ribosylation. These findings indicate that the auto-modification of NADase is one of the universal phenomena to regulate enzyme functions.