BaTiO3 doped Na0.5K0.5NbO3 thin films deposited by using eclipse shutter enhanced pulsed laser deposition method.

We have investigated structural, electrical, and electro-mechanical properties of lead-free piezoelectric BaTiO3 doped Na0.5K0.5NbO3 (BTO-NKN) thin films deposited by pulsed laser deposition (PLD) methods. BTO-NKN thin films have been deposited on La0.5Sr0.5CoO3 (LSCO) bottom electrodes with LaAlO3 (LAO) substrates. X-ray diffraction data have shown that all the BTO-NKN and bottom electrodes are highly oriented with their c-axes normal to the substrates. In order to improve the morphology of BTO-NKN thin films, we have located an eclipse shutter between a target and a substrate. Root-mean-square roughness was changed from 91 nm to 21 nm with eclipse shutter enhanced PLD (E-PLD) method. Furthermore, the enhanced surface morphology leads to the improvement in electrical or electro-mechanical properties mainly due to increased density. Typical capacitance and d33 values of a BTO-NKN film deposited by E-PLD method are 1000 pF and 30 pmN, respectively.

MDR-1 gene expression is a minor factor in determining the multidrug resistance phenotype of MCF7/ADR and KB-V1 cells.

The relevance of MDR-1 gene expression to the multidrug resistance phenotype was investigated. Drug-resistant cells, KB-V1 and MCF7/ADR, constantly expressed mRNA of the MDR-1 gene and were more resistant to vinblastine and adriamycin than drug-sensitive cells, KB-3-1 and MCF7. The drug efflux rate of KB-V1 was the same as KB-3-1 although the MDR-1 gene was expressed in only the resistant cell. The higher intracellular drug concentration of KB-3-1 than KB-V1 was due to the large drug influx. In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil. The amount of ATP, cofactor of drug pumping activity of P-glycoprotein, was not changed by the resistance. These observations suggested that drug efflux mediated by MDR-1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms.

Inhibition of cytokine-induced vascular cell adhesion molecule-1 expression; possible mechanism for anti-atherogenic effect of Agastache rugosa.

Adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) play an important role during the early stages of atherogenesis. Agastache rugosa has an anti-atherogenic effect in low density lipoprotein receptor -/- mice. Moreover, A. rugosa reduced macrophage infiltration and VCAM-1 expression has been localized in aortic endothelium that overlies early foam cell lesions. This study ascertained that tilianin (100 microM), a major component of A. rugosa, inhibits the tumor necrotic factor-alpha (TNF-alpha)-induced expression of VCAM-1 by 74% in cultured human umbilical vein endothelial cells (HUVECs). Also, tilianin (100 microM) reduced TNF-alpha-induced activation of nuclear factor-kappaB in HUVECs.

Dietary hematein ameliorates fatty streak lesions in the rabbit by the possible mechanism of reducing VCAM-1 and MCP-1 expression.

Hematein is a compound isolated from Caesalpinia sappan that has been used in oriental medicine as both an analgesic and an anti-inflammatory agent. In this study, we examined the anti-atherogenic potential of hematein using cholesterol-fed New Zealand White (NZW) rabbits. NZW rabbits were divided into a hematein-supplemented (0.05% in diet) group (n=6), a probucol-supplemented (0.25% in diet) group (n=6), and a control group (n=6). After 8 weeks of treatments, the extent of the atherosclerotic lesions was significantly reduced in the hematein-supplemented group and the probucol-supplemented group without changing plasma lipoprotein levels. Hematein and probucol prevented the up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) expression on the descending aorta induced by cholesterol diet. In culture, hematein also significantly inhibited the secretion of soluble VCAM-1 and of monocyte chemotactic protein-1 (MCP-1) respectively induced by tumor necrotic factor alpha (TNF-alpha) and mildly oxidized low density lipoprotein in human umbilical vein endothelial cell (HUVEC) culture. Also, hematein inhibited monocyte adhesion to endothelial cell and the activation of NF-kappaB in HUVECs stimulated with TNF-alpha. The results of the present study suggest that the anti-atherogenic effect of hematein is not related to control of the plasma lipid profile but probably related to the inhibition of VCAM-1 and MCP-1 expression resulting in an amelioration of lesion development in the rabbit.

Up-Regulation of ninjurin expression in human hepatocellular carcinoma associated with cirrhosis and chronic viral hepatitis.

The cDNA clones, which were highly expressed in liver tissues of hepatocellular carcinoma (HCC) patients, were identified using dot hybridization and reconfirmed by Northern blot analysis. One of the clones, ninjurin (nerve injury induced protein), showed a much higher expression level in the liver tissue of HCC patients than in normal liver tissue. Interestingly, the presence of ninjurin mRNA transcripts was detected with high intensity in HCC tissues when combined with viral infection and cirrhosis, but not with a normal liver or HCC tissue unrelated with viral infection and cirrhosis. We produced a N-terminal part of recombinant ninjurin protein, as well as a monoclonal antibody specific to this polypeptide. The intensity of immunohistochemical staining of the liver tumor tissue, and regenerating tissue for the ninjurin protein, was stronger than that of normal liver tissue. These results suggest that ninjurin may play an important role in the development of HCC combined with cirrhosis and viral infection.

Limitation of Hu-PBL-scid mouse model in direct application to immunotoxicity assessment.

Hu-PBL-scid mice were directly introduced to the methods of immunotoxicity assessments. Human IgG and IgM was detected 1 week after transplantation. Cyclosporin A (CsA) and cyclophosphamide (CP), which were injected i.p. 4 weeks after transplantation, decreased the serum concentration of IgM after 2-4 days of treatment but not that of IgG. Lymphocyte proliferation induced by various mitogens and primary T-dependent antibody responses to sheep red blood cells could not be measured by using splenocytes of hu-PBL-scid mice. These results were correlated with the fact that human cells were not detected in the spleen, thymus, or blood of hu-PBL-scid mouse but were detected in lymph nodes of the intestine, which were observed by flow cytometric and immunohistochemical examinations. The present results suggest using hu-PBL-scid mice in routine immunotoxicity investigations: lymph nodes of intestines could be used as the lymphocyte source. In addition, the determination of serum Ig concentration might be used as a experimental item.

Efficient fixation procedure of human leukemia cells in sulforhodamine B cytotoxicity assay.

The fixation procedures in sulforhodamine B (SRB) assay for human leukemia cells were modified to produce more reliable results. It was found that the concentration of the fixative agent, trichloroacetic acid (TCA), was critical in the selective fixation of cellular protein. While a TCA solution of 80% fixed both cells and serum proteins, a 50% solution fixed only cells with a very low interference of the serum proteins. Accordingly, we selected 50% TCA as a fixative agent which made the final absorbance of the SRB assay to be exactly matched to the cell density with a small deviation and a low background. Besides the change of TCA concentration, a precentifugation of microplate just before fixation also improved the previous assay procedures in the two points of view. The 2-h standing step was simply substituted for only 1 min of centrifugation. Both the rapid and slow application of TCA solution in fixation produced the same extents of fixation. In an actual application, these two kinds of modifications in the previous SRB assay procedure were also proved to be effective in the determination of cytotoxicities of doxorubicin by using human leukemias.

Rapid determination of in vivo and in vitro antibody responses by suspension hemolytic assay.

A suspension hemolytic (SH) assay newly modified in this laboratory was applied to the measurement of primary IgM antibody responses, such as T-dependent, T-independent, and polyclonal B cell responses in in vivo and in vitro assays. All results of the SH assay were equal to those of the conventional plaque-forming cell (PFC) assay. With the SH assay in in vitro assays, we could separately measure the amount of the IgM that had been produced during the whole immunization period and measure the IgM newly produced during the shorter time of the assay. The immunomodulations by dioxin and glycopeptide were accurately evaluated by the SH and the PFC assay. Our study suggested that the SH assay modified in this laboratory was more advantageous because of the simplicity of the assay and better analysis of data than the PFC assay, and could be used as the primary method in studying the effects of pharmaceutical and toxic agents on humoral immune functions.

Selective suppression of in vitro T-dependent humoral immunity by synthetic food additive antioxidants.

Effect of antioxidants on humoral immune responses, such as butylated hydroxytoluene (BHT), n-propyl gallate (PG) and dimethyl sulfoxide (DMSO) is suppression in vitro antibody production. These antioxidants all inhibited T-dependent B cell response, not T-independent and polyclonal B cell response. These data suggest that antioxidants suppress humoral immunity by suppression of regulation of T cells or action of macrophages on B cells, not by direct suppression of B cells. The other possible explanation for antioxidant action is the lack of T-B cell contact required for the triggering of the B cell response with T-dependent antigens.

Functional human hepatocytes: isolation from small liver biopsy samples and primary cultivation with liver-specific functions.

Morphologically and functionally intact human hepatocytes were isolated from small liver biopsy samples weighing about 1-2 g by initial digestion with collagenase followed by repeated digestions with trypsin. The usual yield of hepatocytes was greater than 1 x 10(7) cells per g of liver sample and cell viability, as judged by dye exclusion test, was routinely over 90%. The isolated human hepatocytes showed intact morphology under scanning electron microscope. Formation of membrane protrusions upon phalloidin addition demonstrated that the actin in isolated hepatocytes was maintained with its structural integrity. The cultured human hepatocytes retained a variety of liver-specific functions which were similarly exhibited by rat hepatocytes isolated using the same procedure. The cultured human hepatocytes exhibited a specific cytochrome P-450 related enzyme activity, and active amino acid uptake that increased upon addition of hormones like glucagon and dexamethasone. Additionally, the cultured human hepatocytes synthesized DNA actively and, human serum albumin, and was found to be responsive to modulation by growth modulating hormones, cytokines and hepatotoxic agents. Based on the profile of activity described above, the presently established conditions for isolation and culturing of human hepatocytes demonstrate that functional liver cells can be obtained from small biopsied liver samples.

Genomic DNA sequence and transcription factor binding sites of mouse ninjurin.

The complete genomic DNA sequence of mouse ninjurin gene has been cloned and sequenced by screening a bacterial artificial chromosome (BAC) library of mouse 129/SvJ genomic DNA. The mouse ninjurin gene comprises four exons and the translatable sequences are included in the first three exons. The putative promoter region of the mouse ninjurin gene lacks the consensus "CAAT" or "TATA" sequence. Nonetheless, it has demonstrated the promoter activity in transient transfection experiment using the construct containing putative promoter sequence of mouse ninjurin and reporter gene. The nucleotide sequence of the putative promoter region shows 83% homology with the corresponding DNA sequence of human ninjurin gene that had been previously reported, and reveals a high degree of conservation between the two species. Analysis of the DNA sequence identified the putative promoters and the binding sites for a variety of transcription factors of mouse ninjurin.

Comparative studies of adriamycin and 28-deacetyl sendanin on in vitro growth inhibition of human cancer cell lines.

The limonoid compound (28-deacetyl sendanin) isolated from the fruit of Melia toosendan SIEB. et ZUCC. was evaluated on anticancer activity. According to a standard in vitro cytotoxicity assay, eight human cancer cell lines and SRB assay were introduced for present evaluation. As a positive standard, adriamycin was tested in parallel. The cell lines were originated from six different organs. In view of dose-response profiles to 28-deacetyl sendanin, the most sensitive cells were SF-539 and PC-3 which were derived from CNS and prostate, respectively. In contrast, all the cell lines responded similarly to adriamycin to give rise to nearly identical dose-response profiles. By comparison of Gl50 between 28-deacetyl sendanin and adriamycin, six cell lines were more sensitive to 28-deacetyl sendanin and two were more resistant. As a result, 28-deacetyl sendanin had more sensitive and selective inhibitory effects on in vitro growth of human cancer cell lines in a comparison with adriamycin.

Overexpression of cyclin D1 and cyclin E in 1,2-dimethylhydrazine dihydrochloride-induced rat colon carcinogenesis.

Deregulation of G1 cyclins has been reported in several human and rodent tumors including colon cancer. To investigate the expression pattern of G1 cyclins in 1,2- dimethyl-hydrazine dihydrochloride (DMH)-induced rat colon carcinogenesis, we studied the expression of cyclin D1 and cyclin E by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry (IHC). The mRNA level of cyclin D1 was increased 1.2-fold in adenocarcinomas but not significantly in adenomas, when compared with normal rat colonic mucosa (p<0.05). The cyclin E mRNA level was increased 2.7-fold in adenomas and 3.3-fold in adenocarcinomas (p<0.05). The PCNA mRNA level was also increased 1.9-fold in adenomas and 1.8-fold in adenocarcinomas (p<0.05). Immunohistochemical staining revealed exclusive nuclear staining of the neoplastic cells for cyclin D1, cyclin E and PCNA. Cyclin D1 expression was detected in 56.3% of the adenomas and in 61.5% of the adenocarcinomas examined, whereas cyclin E expression was detected in 87.5% of the adenomas and in 92.3% of the adenocarcinomas. Overall, cyclin D1, cyclin E and PCNA expression was significantly increased at both the mRNA and protein levels in normal colonic mucosa, adenomas and adenocarcinomas, but there was no significant difference in the degree of expression of these genes in adenomas and adenocarcinomas. Our results indicate that the overexpression of cyclin D1 and cyclin E may play an important role during the multistage process of rat colon carcinogenesis, at a relatively early stage, and may disturb cell-cycle control in benign adenomas, and thereafter, participate in tumor progression.

Arrest defective 1 regulates the oxidative stress response in human cells and mice by acetylating methionine sulfoxide reductase A.

Methionine sulfoxide reductase A (MSRA) protects proteins from oxidation, and also helps remove reactive oxygen species (ROS) by recovering antioxidant enzymes inactivated by oxidation. Although its functions have been investigated extensively, little is known about the mechanism by which MSRA is regulated. Arrest defective 1 (ARD1) is an enzyme that catalyzes not only N-terminal acetylation as a cotranslational modification but also lysine acetylation as a posttranslational modification. ARD1, which is expressed in most cell types, is believed to participate in diverse biological processes, but its roles are poorly understood. Given that MSRA was hunted in a yeast two-hybrid screen with ARD1 as the bait, we here investigated whether ARD1 is a novel regulator of MSRA. ARD1 was shown to interact with and acetylate MSRA in both cells and test tubes. It specifically acetylated the K49 residue of MSRA, and by doing so repressed the enzymatic function of MSRA. ARD1 increased cellular levels of ROS, carbonylated proteins and DNA breaks under oxidative stress. Moreover, it promoted cell death induced by pro-oxidants, which was attenuated in MSRA-deficient cells. When mice were exposed to hyperoxic conditions for 2 days, their livers and kidneys were injured and protein carbonylation was increased. The oxidative tissue injury was more severe in ARD1 transgenic mice than in their wild-type littermates. In conclusion, ARD1 has a crucial role in the cellular response to oxidative stress as a bona fide regulator of MSRA. ARD1 is a potential target for ameliorating oxidative injury or for potentiating ROS-producing anticancer agents.

Trichostatin A attenuates airway inflammation in mouse asthma model.

BACKGROUND: Histone deacetylase (HDAC) inhibition has been demonstrated to change the expression of a restricted set of cellular genes. T cells are essential in the pathogenesis of allergen-induced airway inflammation. It was recently reported that treatment with HDAC inhibitors induces a T cell-suppressive effect. OBJECTIVE: The purpose of this study was to determine whether treatment with trichostatin A (TSA), a representative HDAC inhibitor, would reduce allergen-induced airway inflammation in a mouse asthma model. METHODS: BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and challenged with an aerosol of OVA. TSA (1 mg/kg body weight) was injected intraperitoneally every 2 days beginning on day 1. Mouse lungs were assayed immunohistochemically for HDAC1, a major HDAC subtype, and for infiltration of CD4+ cells. The effect of TSA on airway hyper-responsiveness (AHR) was determined, and the bronchoalveolar lavage fluid (BALF) of these mice was assayed for the number and types of inflammatory cells, and for the concentrations of IL-4, IL-5, and IgE. RESULTS: HDAC1 was localized within most airway cells and infiltrating inflammatory cells of asthmatic lungs. Treatment with TSA significantly attenuated AHR, as well as the numbers of eosinophils and lymphocytes in BALF. TSA also reduced infiltration of CD4+ and inflammatory cells and mucus occlusions in lung tissue, and decreased the concentrations of IL-4, IL-5, and IgE in BALF. CONCLUSION: TSA attenuated the development of allergic airway inflammation by decreasing expression of the Th2 cytokines, IL-4 and IL-5, and IgE, which resulted from reduced T cell infiltration. Our results suggest that HDAC inhibition may attenuate the development of asthma by a T cell suppressive effect.

Genetic factors associated with endothelial dysfunction affect the early onset of coronary artery disease in Korean males.

The maintenance of balance between nitric oxide (NO) and the superoxide anion is required for proper functioning of the endothelium. To investigate the relationship between genetic factors associated with endothelial function and the development of coronary artery disease (CAD), endothelial nitric oxide synthase (ecNOS) gene a/b polymorphism and NADH/NADPH oxidase p22 phox gene C242T polymorphism were examined in 305 Korean male CAD patients and 215 healthy male control subjects. The beta-fibrinogen gene H1/H2 polymorphism was also analyzed. Both ecNOS a/b and p22 phox C242T polymorphisms were found to be associated with the development of CAD in the study population (p=0.020 and 0.011, respectively). When the association was analyzed by age, statistical significance was retained only in those <51 years (p=0.021 and 0.025 for the a/b and the C242T polymorphism, respectively) and not in those >51 years of age (p=0.155 and 0.278 respectively). However, the distribution of the beta-fibrinogen H1/H2 genotypes was not found to be associated with the development of CAD in either the < or =50 (p = 0.611) or >50 groups (p = 0.188). The ecNOS gene a/b polymorphism and the NADH/NADPH oxidase p22 phox gene C242T polymorphism were found to be significantly associated with the development of CAD in Korean male patients less than 51 years old.

Tumor necrosis factor receptor superfamily 12 may destabilize atherosclerotic plaques by inducing matrix metalloproteinases.

Immunohistochemical staining of human atherosclerotic plaques revealed expression of the tumor necrosis factor receptor superfamily (TNFRSF) 12 in regions rich in macrophage/foam cells. The role of TNFRSF12 in the functioning of monocytes in relation to atherogenesis was investigated by analysis of cellular events after stimulation of TNFRSF12 in a human macrophage-like cell line, THP-1. Activation of the THP-1 cells on plates coated with monoclonal antibody against TNFRSF12 induced the expression of matrix metalloproteinases (MMPs) -1, -9, and -13. Furthermore, the expression patterns of TNFRSF12 and the MMPs overlapped in atherosclerotic plaques. Signaling of TNFRSF12 may thus contribute to the induction of extracellular matrix degrading enzymes in macrophages.

Glucocorticoid-mediated repression of inflammatory cytokine production in fibroblast-like rheumatoid synoviocytes is independent of nuclear factor-kappaB activation induced by tumour necrosis factor alpha.

OBJECTIVE: To determine whether steroids inhibit the production of inflammatory cytokines by the inhibition of nuclear factor kappaB (NF-kappaB) activation in fibroblast-like rheumatoid synoviocytes (FLSs) under inflammatory conditions, and to determine whether steroids stimulate the induction of synthesis of the inhibitory protein IkappaB-alpha in the anti-inflammatory immune response of these cells. METHODS: Expression of the interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) genes was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the secreted IL-6 was measured with the enzyme-linked immunosorbent assay. Inhibition of the NF-kappaB activation was examined with the electrophoretic mobility shift assay (EMSA). In order to study dexamethasone (DEX)-dependent regulation of IkappaB-alpha expression, we performed Western blotting before and after stimulation with tumour necrosis factor alpha (TNF-alpha). RESULTS: The inflammatory cytokine study showed that DEX suppressed gene expression and the production of protein in FLSs. EMSA demonstrated that identical amounts of NF-kappaB were present in the nucleus of the FLSs stimulated by TNF-alpha, with or without pretreatment with DEX. Treatment of FLSs with DEX did not induce an increase in IkappaB-alpha sufficient to prevent nuclear translocation of NF-kappaB on stimulation with TNF-alpha. CONCLUSION: DEX may suppress the production of inflammatory cytokines, such as IL-6 and IL-1beta, but it neither prevents the translocation of NF-kappaB to the nucleus nor induces the synthesis of IkappaB-alpha protein in FLSs stimulated by TNF-alpha.

Homeostasis in mice with genetically decreased angiotensinogen is primarily by an increased number of renin-producing cells.

Here we investigate the biochemical, molecular, and cellular changes directed toward blood pressure homeostasis that occur in the endocrine branch of the renin-angiotensin system of mice having one angiotensinogen gene inactivated. No compensatory up-regulation of the remaining normal allele occurs in the liver, the main tissue of angiotensinogen synthesis. No significant changes occur in expression of the genes coding for the angiotensin converting enzyme or the major pressor-mediating receptor for angiotensin, but plasma renin concentration in the mice having only one copy of the angiotensinogen gene is greater than twice wild-type. This increase is mediated primarily by a modest increase in the proportion of renal glomeruli producing renin in their juxtaglomerular apparatus and by four times wild-type numbers of renin-producing cells along afferent arterioles of the glomeruli rather than by up-regulating renin production in cells already committed to its synthesis.

Hematein inhibits tumor necrotic factor-alpha-induced vascular cell adhesion molecule-1 and NF-kappaB-dependent gene expression in human vascular endothelial cells.

Monocyte adhesion to the endothelium via adhesion molecules is one of the earliest events in atherogenesis. It has been suggested that vascular cell adhesion molecule-1 (VCAM-1) plays a very important role in the recruitment of monocytes in atherosclerosis. The aim of our study was to evaluate whether hematein can influence the expression of VCAM-1 and the transcription of nuclear factor-kappaB (NF-kappaB)-dependent genes. Immunohistochemistry revealed that mouse aortic artery endothelial cells express VCAM-1 after feeding a high cholesterol diet for 8 weeks. Hematein dose dependently suppressed TNF-alpha-induced VCAM-1 in both surface (30.8%) and soluble protein (65%) production in HUVECs. The transcription level of VCAM-1 was measured by Northern blot analysis, and decreased VCAM-1 protein expression was associated with a reduction of VCAM-1 mRNA expression. Transient transfection study of NF-kappaB promoter construct and electrophoretic mobility shift assay suggested that hematein inhibited both NF-kappaB-dependent gene expression and NF-kappaB activation induced by TNF-alpha. Our results suggest that the down-regulation of VCAM-1 expression by hematein may in part be due to the inhibition of NF-kappaB-dependent gene expression.