A functional promoter polymorphism of the human IL18 gene is associated with aspirin-induced urticaria.

BACKGROUND: Urticaria is the commonest cutaneous reaction caused by aspirin or other nonsteroidal anti-inflammatory drugs. The pathogenesis of aspirin-induced urticaria (AIU) is not fully understood, but appears to involve mast cell activation and neutrophil infiltration. OBJECTIVES: To investigate the genetic contribution of interleukin (IL)-18, which can amplify acute inflammation by promoting mast cell activation, neutrophil migration and cytokine production, to the pathogenesis of AIU. METHODS: A case-control association study was performed using 275 patients with AIU and 196 normal healthy controls in a Korean population. Two promoter polymorphisms of the IL18 gene (-607A/C and -137G/C) were genotyped using the primer extension method. The functional effect of the IL18 gene promoter polymorphism was investigated through in vitro studies including a luciferase reporter assay and electrophoretic mobility shift assays (EMSAs) and ex vivo studies involving neutrophil chemotaxis assays. RESULTS: A significant association was detected between both AIU in general and the aspirin-intolerant acute urticaria (AIAU) phenotype and the IL18 promoter polymorphism -607A/C. Patients with AIAU showed higher frequencies of the C(-607) G(-137) haplotype, ht1 [CG], compared with controls (P=0·02). Moreover, ht1 [CG] showed a high transcript haplotype by the luciferase activity assay, and EMSAs identified a -607C allele-specific DNA-binding protein as CREB2. Neutrophil chemotactic activity was highest in subjects with AIU exhibiting the high transcript haplotype, ht1 [CG] (P=0·019). CONCLUSIONS: The high transcript haplotype ht1 [CG] of the IL18 gene may contribute to the development of acute cutaneous inflammation sensitive to aspirin, leading to the clinical presentation of AIAU.

Hepsulfam induced DNA adducts and its excision repair by bacterial and mammalian 3-methyladenine DNA glycosylases.

A variety of alkylated base adducts are repaired by 3-methyladenine DNA glycosylases, one of the base excision repair enzymes. In this study, we examined the DNA adducts induced by hepsulfam and determined whether alkylated base adducts can be substrates for bacterial and mammalian 3-methyladenine DNA glycosylases by electrophoresis methods. Hepsulfam, a synthetic analogue of busulfan, is known to alkylate DNA and form interstrand cross-links. The extent of DNA interstrand cross-links induced by hepsulfam and busulfan was found to be similar but significantly lower than that induced by chlorambucil, as measured by an agarose gel assay. The major monofunctional alkylation site of hepsulfam was observed at the N7 position of guanine, and not at the N3 position of adenine. Both compounds did not exhibit any sequence selective DNA alkylation patterns. The excision of hepsulfam-induced DNA adducts has been determined by treatment with homogeneous recombinant bacterial, rat and human 3-methyladenine DNA glycosylases and successive treatments by formamidopyrimidine-DNA glycosylase. The Escherichia coli alkA protein was shown to completely excise N7 guanine adducts, whereas mammalian 3-methyladenine DNA glycosylase failed to excise them. In addition, the cytotoxicity assay showed that E. coli mutant strains defective in the alkA gene or the uvrA gene were more sensitive to killing by hepsulfam than the wild type.

Annomocherin, annonacin and annomontacin: a novel and two known bioactive mono-tetrahydrofuran annonaceous acetogenins from Annona cherimolia seeds.

A novel and two known bioactive mono-tetrahydrofuran (THF) annonaceous acetogenins, annomocherin (1), annonacin (2) and annomontacin (3), have been isolated from the fractionated ethanolic extracts of the seeds of Annona cherimolia, guided by the brine shrimp lethality test (BST). Their structures were elucidated on the basis of spectroscopic and chemical methods. All compounds have a relative stereochemistry of threo/trans/threo for the mono-THF ring with two flanking hydroxyls. Compound 1 has a double bond at C-23/24 of aliphatic chain. Compound 1 was isolated from natural sources for the first time, and was named annomocherin. Two known Compounds 2 and 3 which have never been isolated from this species before, were obtained. Compound 1 exhibited potent and selective cytotoxicities against the breast carcinoma (MCF-7) and kidney carcinoma (A-498) cell lines with 100 to 1,000 times the potency of adriamycin. In brine shrimp lethality test (BST), 1-3 exhibited cytotoxicity.

Cytotoxic constituents from Solidago virga-aurea var. gigantea MIQ.

Activity-guided fractionation of the whole plant of Solidago virga-aurea var. gigantea M(IQ). (Compositae) has led to the isolation of three cytotoxic compounds, erythrodiol-3-acetate (1), alpha-tocopherol-quinone (2), and trans-phytol (3) from the hexane soluble fraction. It is the first report of those compounds from the genus.

Requirement of metabolic activation for estrogenic activity of Pueraria mirifica.

A wide range of chemicals derived from plant and human-made xenobiotics are reported to have hormonal activities. The present study was performed to examine the estrogenic effect of Kwao Keur, Pueraria mirifica (PM), that has been used as a rejuvenating folk medicine in Thailand, using recombinant yeast, MCF-7 cell proliferation and HepG2 cell transient transfection assay. In recombinant yeast assay, 0.025, 0.25, 2.5, 25, 2.5 x 10(2), 2.5 x 10(3), 2.5 x 10(4) ng/ml concentrations of PM did not show any estrogenic activities, while 10(-9) of 17 beta-estradiol (positive control) showed high estrogenic activity. Estrogenic activities were induced at 2.5 ng/ml to 25 microg/ml concentrations of PM in a dose-dependent manner on MCF-7 cells and the estrogenic effect of PM was blocked by tamoxifen treatment, a well-known anti-estrogen. PM also showed estrogenic effect on human hepatoma cell line, HepG2 cells, containing estrogen receptor and luciferase reporter gene. Taken together, PM in itself may have no estrogenicity in yeast system, but it has estrogenicity in MCF-7 & HepG2 cells that have human metabolic enzymes. The results indicated that PM may require metabolic activation for estrogenic activity.

Wool cortisol is a better indicator of stress than blood cortisol in ewes exposed to heat stress and water restriction.

This study investigated the effect of water restriction on wool and blood cortisol concentrations and water consumption patterns in heat-stressed sheep. Nine Corriedale female sheep (average BW=43±6.5 kg) were individually fed diets based on maintenance requirement in metabolic crates. They were assigned to three treatments according to a Latin square design (3×3) for three periods with a 21-day duration for each period (nine sheep per treatment). Treatments included free access to water (FAW), 2 h water restriction (2hWR) and 3 h water restriction (3hWR) after feeding. Average temperature-humidity index in the experimental room was 27.9 throughout the experiment that defines heat stress conditions. Wool samples were taken at the end of each period on day 21. No differences were found in cortisol concentration in each fragment (dried, washed and residual extract) of wool (P<0.05). Total wool cortisol concentration was higher in the 3hWR group than the other treatments (P<0.05). Blood cortisol was not different among the treatments (P>0.05) and resulted in higher variable data compared with wool cortisol. Blood neutrophils and neutrophil/lymphocyte ratio suppressed in FAW and 3hWR groups compared with the 2hWR group (P<0.05). The duration of water consumption recorded after feeding in the 3hWR group was higher than in the 2hWR group when recorded in the afternoon (P<0.01). Water consumption rate was higher in the 3hWR group than in the 2hWR group (P<0.01). However, total water consumed was lower in the 3hWR group compared with other treatments (P>0.05). It can be concluded that wool cortisol provides more precise and accurate data than blood cortisol during heat stress conditions. Water restriction for 3 h after feeding can act as a stressor and is critical for sheep during heat stress as the consumption of water decreases with restriction.

TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potentially useful anticancer agent with exquisite selectivity for cancer cells. Unfortunately, many cancers show or acquire resistance to TRAIL. In this study we report that TRAIL activates a TGF-beta-activated kinase 1 --> mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)/MKK6 --> p38 pathway in prostate cancer cells that transcriptionally upregulates expression of the antiapoptotic BCL-2 family member MCL-1. TRAIL alone triggered robust formation of the 'death-inducing signaling complex' (DISC), activation of the initiator caspase-8, and truncation of the BH3-only protein BID (tBID). Nevertheless, simultaneous disruption of the p38 MAPK pathway was required to suppress MCL-1 expression, thereby allowing tBID to activate the proapoptotic BCL-2 family member BAK and stimulate mitochondrial outer membrane permeabilization (MOMP). Release of the inhibitor-of-apoptosis (IAP) antagonist, Smac/DIABLO, from the intermembrane space was sufficient to promote TRAIL-induced apoptosis, whereas release of cytochrome c and activation of the apoptosome was dispensable. Even after MOMP, however, mitochondrial-generated reactive oxygen species (ROS) activated a secondary signaling pathway, involving c-Jun N-terminal kinases (JNKs), that similarly upregulated MCL-1 expression and partially rescued some cells from death. Thus, stress kinases activated at distinct steps, before and after mitochondrial injury, mediate TRAIL resistance through maintenance of MCL-1 expression.

DA-9601 inhibits activation of the human mast cell line HMC-1 through inhibition of NF-kappaB.

Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 with immune regulatory properties. The formulated ethanol extract of Artemisia asiatica Nakai (DA-9601) has been reported to have antioxidative and anti-inflammatory activities. In this report, we investigated the effect of DA-9601 on the expression of pro-inflammatory cytokines by the activated human mast cell line HMC-1 and studied its possible mechanisms of action. DA-9601 dose-dependently decreased the gene expression and production of TNF-alpha, IL-1beta, and IL-6 on phorbol 12-myristate 13-acetate (PMA)- and calcium ionophore A23187-stimulated HMC-1 cells. In addition, DA-9601 attenuated PMA- and A23187-induced activation of NF-kappaB as indicated by inhibition of degradation of IkappaBalpha, nuclear translocation of NF-kappaB, NF-kappaB/DNA binding, and NF-kappaB-dependent gene reporter assay. Our in vitro studies provide evidence that DA-9601 might contribute to the treatment of mast cell-derived allergic inflammatory diseases.

Drastic reduction of shot noise in semiconductor superlattices.

We have found experimentally that the shot noise of the tunneling current I through an undoped semiconductor superlattice is reduced with respect to the Poissonian noise value 2eI, and that the noise approaches 1/3 of that value in superlattices whose quantum wells are strongly coupled. On the other hand, when the coupling is weak or when a strong electric field is applied to the superlattice, the noise becomes Poissonian. Although our results are qualitatively consistent with existing theories for one-dimensional multibarrier structures, the theories cannot account for the dependence of the noise on superlattice parameters that we have observed.

Cyclic guanosine-3',5'-monophosphate and biopteridine biosynthesis in Nocardia sp.

Nocardia sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H(4)B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3',5'-monophosphate (cGMP) at 9.56 pmol of cGMP h(-1) mg of protein(-1). Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter(-1) to a maximum of 315 pmol liter(-1), in response to additions of GTP, L-arginine, H(4)B, and sodium nitroprusside to growing Nocardia cultures. On the other hand, the NOS inhibitor N(G)-nitro-L-arginine and the GC inhibitor 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H(4)B biosynthesis, were present in Nocardia culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h(-1) mg of protein(-1), respectively. In Nocardia spp., as in mammals, GTP is a key intermediate in H(4)B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.

Isolation and structure determination of new macrolide antibiotics.

Three new bafilomycin-like compounds PD 118,576-A1 [1], PD 118,576-A2 [2], and PD 118,576-A3 [3] were isolated from a new soil Streptomyces species (WP 3913). The structures of PD 118,576-A1, PD 118,576-A2, and PD 118,576-A3 were elucidated on the basis of spectroscopic studies including 2D nmr.

New naphthalenyl glycosides from the roots of Juglans mandshurica.

Three new glycosides, 1,4,8-trihydroxynaphthalene-1-O-D-glucopyranosyl-(1-->6)-beta-D- xylopyranoside (1), 1,4,8-trihydroxynaphthalene-1-O-beta-D-glucopyranosyl-(1-->6)-alph a-L- arabinopyranoside (2), 1-hydroxy-4-methoxynaphthalene-1-O-beta-D-glucopyranosyl-(1-->6)- alpha-L-rhamnopyranoside (3), were isolated from the roots of Juglans mandshurica and their structures were elucidated on the basis of spectroscopic studies.

Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-beta signaling.

Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-beta (TGF-beta) signaling pathway. We infected a TGF-beta-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-beta. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-beta-induced promoter (p15), it up-regulates a TGF-beta-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-beta. Our results suggest that BF-1 is a general inhibitor of TGF-beta signaling and as such may play a key role during brain development.

Annomolin and annocherimolin, new cytotoxic annonaceous acetogenins from Annona cherimolia seeds.

Two new cytotoxic annonaceous acetogenins, annomolin (1) and annocherimolin (2), were isolated from an ethanolic extract of the seeds of Annona cherimolia. Annomolin has a mono-THF ring with one flanking hydroxyl and possesses a 1,2-diol at C-7/8 of the aliphatic chain. Annocherimolin has a mono-THF ring with two flanking hydroxyls and possesses a double bond at C-21/22. Their structures were elucidated by spectral data and chemical derivatization. Compound 1 showed cytotoxic selectivity for the human prostate tumor cell line (PC-3), with a potency of over 10,000 times that of adriamycin. Compound 2 showed cytotoxic potencies about 10,000 times those of adriamycin in the breast (MCF-7) and colon (HT-29) cancer cell lines.

Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase.

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M(-1) min(-1). The pH-inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.

Quinoline alkaloids from the fruits of Evodia officinalis.

A new quinoline derivative, 2-hydroxy-4-methoxy-3-(3'-methyl-2'-butenyl)-quinoline and five known quinoline alkaloids were isolated from the fruits of Evodia officinalis. The structure of 1 was determined by spectroscopic methods.

Cytotoxic compounds from the roots of Juglans mandshurica.

Three new compounds, a diarylheptanone glucoside (1), 4,5, 8-trihydroxy-alpha-tetralone 5-O-beta-d-[6'-O-(3", 5"-dimethoxy-4"-hydroxybenzoyl)]glucopyranoside (2), and 1,4, 8-trihydroxy-3-naphthalenecarboxylic acid 1-O-beta-d-glucopyranoside methyl ester (3), were isolated from the roots of Juglans mandshurica, and their structures were elucidated on the basis of spectroscopic studies including 2D-NMR.

Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein.

BACKGROUND/AIMS: Chronic infection with hepatitis B virus is a high-risk factor for hepatocellular carcinoma in humans. The HBV X-protein, a multi-functional viral regulator, has been suspected to play a positive role in hepatocarcinogenesis, as demonstrated by the high incidence of hepatocellular carcinoma in HBx-expressing transgenic mice, although it is still controversial. The aim of this study was to generate transgenic mice expressing the HBV X-gene under authentic promoter control and to test whether the gene products can cause hepatic tumors. METHODS: Three transgenic mouse lines were generated by microinjecting the X-gene construct into hybrid (C57BL/6 x DBA) eggs. Gene expression was tested by protein and mRNA analyses. During an observation period of 18 months, mice were sacrificed and organs subjected to histologic examinations. RESULTS: Grossly defined hepatocellular carcinomas reproducibly were observed in mice expressing the X-protein, which were investigated through six generations from the age of 11 to 18 months. Among 14 transgenic mice investigated from the age of 11 to 18 months, 12 were found to have hepatocellular carcinoma, grossly or microscopically. The lesion of the hepatocellular carcinoma disclosed a significant increase in the proliferating cell nuclear antigen in the nuclei. CONCLUSION: The incidence of hepatocellular carcinoma (86%) in our HBV X transgenic mice may be highly significant, since, except for one case, HBV X-gene transgenic mice produced in other laboratories did not develop liver tumor or any other pathologic phenomena.