TSLC1 is a tumor-suppressor gene in human non-small-cell lung cancer.

The existence of tumor-suppressor genes was originally demonstrated by functional complementation through whole-cell and microcell fusion. Transfer of chromosome 11 into a human non-small-cell lung cancer (NSCLC) cell line, A549, suppresses tumorigenicity. Loss of heterozygosity (LOH) on the long arm of chromosome 11 has been reported in NSCLC and other cancers. Several independent studies indicate that multiple tumor-suppressor genes are found in this region, including the gene PPP2R1B at 11q23-24 (ref. 7). Linkage studies of NSCLC are precluded because no hereditary forms are known. We previously identified a region of 700 kb on 11q23.2 that completely suppresses tumorigenicity of A549 human NSCLC cells. Most of this tumor-suppressor activity localizes to a 100-kb segment by functional complementation. Here we report that this region contains a single confirmed gene, TSLC1, whose expression is reduced or absent in A549 and several other NSCLC, hepatocellular carcinoma (HCC) and pancreatic cancer (PaC) cell lines. TSLC1 expression or suppression is correlated with promoter methylation state in these cell lines. Restoration of TSLC1 expression to normal or higher levels suppresses tumor formation by A549 cells in nude mice. Only 2 inactivating mutations of TSLC1 were discovered in 161 tumors and tumor cell lines, both among the 20 primary tumors with LOH for 11q23.2. Promoter methylation was observed in 15 of the other 18 primary NSCLC, HCC and PaC tumors with LOH for 11q23.2. Thus, attenuation of TSLC1 expression occurred in 85% of primary tumors with LOH. Hypermethylation of the TSLC1 promoter would seem to represent the 'second hit' in NSCLC with LOH.

Juvenile hormone titre and related gene expression during the change of reproductive modes in the pea aphid.

Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.

The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function.

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

Effects of inflammatory cytokines on vascular tone.

OBJECTIVE: To assess the direct effects of the inflammatory cytokines interleukin-2 (IL-2), interleukin-6 (IL-6) and interleukin-8 (IL-8) on vascular smooth muscle contraction. METHODS: Smooth muscle contractility was studied in the thoracic aorta isolated from male Sprague-Dawley rats. Syntheses of cAMP and nitric oxide (NO) were investigated in cultured rat vascular smooth muscle cells (VSMC). RESULTS: Pretreatment of the rings with IL-6 (10 ng/ml) for 180 min caused a significant inhibition of their contraction in response to 10(-5) M phenylephrine, wile Il-2 (10 ng/ml) and IL-8 (100 ng/ml) showed no significant effect on the contraction. The inhibitory effect of IL-6 exhibited a dose-dependency (0.1 approximately 10 ng/ml). In cultured rat VSMC, synthesis of cAMP was increased time-dependently by IL-6, while IL-2 and IL-8 failed to show any significant effects. IL-2, IL-6 and IL-8 did not affect the production of nitrite, a stable metabolite of NO, by VSMC. CONCLUSIONS: IL-6, but not IL-2 and IL-8, is a potent inhibitor of vascular contraction, which effect is mediated through the increased cAMP synthesis.

Expression of intercellular adhesion molecule-1 on rat cardiac myocytes by monocyte chemoattractant protein-1.

OBJECTIVE: Cytokine induction of intercellular adhesion molecule-1 (ICAM-1) on cardiac myocytes may be a critical step in cardiac inflammation associated with acute myocardial infarction and myocarditis. The aim of this study was to investigate the involvement of monocyte chemoattractant protein-1 (MCP-1), a homologue of mouse JE, in the neutrophil-myocyte adhesion in vitro. METHODS: MCP-1/JE and ICAM-1 mRNA expression in cultured neonatal rat cardiac myocytes was evaluated by northern blot analysis. ICAM-1 molecule content on myocytes was determined by ELISA. For adherence assay, myocytes and neutrophils were co-incubated and the number of bounded neutrophils was counted. RESULTS: MCP-1/JE transcripts were not clearly observed in cultured neonatal rat cardiac myocytes; however, its transcripts were clearly detected by exposure to interleukin 1 alpha (100 U.ml-1), lipopolysaccharide (1 microgram.ml-1), or hypoxia (95% N2 + 5% CO2). In ELISA analysis, the expression of ICAM-1 molecules on cardiac myocytes was significantly stimulated by MCP-1 in a dose dependent manner, and the effect of MCP-1 was observed as early as at 6 h. In northern blot analysis, ICAM-1 mRNA expression was constitutively observed in myocytes, and the expression was markedly stimulated by exposure to MCP-1 with a peak elevation at 2 h. In adherence assay, MCP-1 stimulated the adhesion of rat neutrophils to rat cardiac myocytes, and this effect of MCP-1 was inhibited by an anti-ICAM-1 MAb. CONCLUSIONS: These results suggest that cardiac myocytes produce MCP-1, which could in turn promote the adhesion of neutrophils to myocytes via ICAM-1 expression, suggesting the involvement of MCP-1 in cardiac inflammation associated with acute myocardial infarction and myocarditis.

Adrenomedullin increases inducible nitric oxide synthase in rat vascular smooth muscle cells stimulated with interleukin-1.

We investigated the effects of adrenomedullin on nitric oxide synthesis by measuring the production of nitrite, a stable metabolite of nitric oxide, in cultured rat vascular smooth muscle cells. Incubation of cultures with interleukin-1beta (10 ng/mL) for 24 hours caused a significant increase in nitrite generation. The interleukin-1beta-induced nitrite production by vascular smooth muscle cells was significantly increased by adrenomedullin in a dose-dependent manner (10(-10) to 10(-6) mol/L). This effect of adrenomedullin was significantly inhibited in the presence of Ng-monomethyl-L-arginine. The adrenomedullin-induced nitrite production by interleukin-1beta-stimulated cells was accompanied by increased inducible nitric oxide synthase mRNA accumulation. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, interleukin-1beta-induced nitrite accumulation was further increased, but the effect of adrenomedullin was not additive or synergistic. Adrenomedullin dose dependently increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that adrenomedullin augments nitric oxide synthesis in interleukin-1beta-stimulated vascular smooth muscle cells, at least partially through a cAMP-dependent pathway.

Studies of hypertension-induced vascular hypertrophy in cultured smooth muscle cells from spontaneously hypertensive rats.

Mechanisms of vascular hypertrophy induced by hypertension were studied in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) and compared with those from normotensive Wistar-Kyoto (WKY) rats. Fetal calf serum-stimulated ornithine decarboxylase (ODC) activity of cultured smooth muscle cells was greater in SHR and SHRSP than in WKY. Beta- but not alpha-adrenergic agonist stimulated ODC activity acutely in cultured smooth muscle cells from WKY, and isoprenaline-induced activation was blocked by the beta-blocker, propranolol, and enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. These results indicate that cultured vascular smooth muscle cells from SHR and SHRSP are more prone to increase the protein synthesis than those from WKY through the trophic induction of ODC activity and that the regulation of ODC activity by catecholamines is mediated through beta-agonistic effect in cultured smooth muscle cells.

Endothelin-1 inhibits nitric oxide synthesis in vascular smooth muscle cells.

We investigated the effects of endothelin-1 on nitric oxide synthesis in vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of nitric oxide, and the expression of inducible nitric oxide synthase mRNA and protein in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite production. Endothelin-1 significantly decreased the interleukin-1 beta-induced nitrite production by vascular smooth muscle cells in a dose-dependent manner (10(-11) to 10(-8) mol/L). Incubation with interleukin-1 beta for 24 hours induced expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells, whereas endothelin-1 showed a suppressive effect on their expressions. Addition of the endothelin type A receptor antagonist BQ-485, but not the endothelin type B receptor antagonist BQ-788, dose-dependently inhibited the effect of endothelin-1. After protein kinase C activity was functionally depleted by treatment of cells with phorbol 12-myristate 13-acetate for 24 hours, the effect of endothelin-1 was abolished. These results indicate that endothelin-1 acts on endothelin type A receptors and inhibits nitric oxide synthesis in interleukin-1 beta-stimulated vascular smooth muscle cells at least partially through a protein kinase C-dependent pathway.

Humoral trophic influence on cardiovascular structural changes in hypertension.

Since the early development of structural cardiovascular change in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) indicated the involvement of non-pressure-dependent factors in this process in hypertension, smooth muscle cells (SMC) from the aorta of SHR, SHRSP, and normotensive Wistar-Kyoto rats (WKY) were investigated under tissue culture conditions free from blood pressure and humoral factors in vivo. By the observation of such factors as growth rate and DNA or protein synthesis vascular SMC from these rats with genetic hypertension were proved to have intrinsically greater growth activity independently of blood pressure. Although serum from SHR and SHRSP had no specific stimulative effect on SMC growth, circulating epinephrine may accelerate cardiovascular structural changes because isoproterenol added to the culture media enhanced ornithine decarboxylase (ODC) activity. Moreover, SMC from SHR and SHRSP showed greater thymidine incorporation than those from WKY even in response to lower extracellular Na+ concentration. Local nutritional conditions of SMC, which were proved to have a great effect on the morphology and structure of cultured SMC, may be a basic determinant of the development of hypertension-induced structural vascular changes or lesions.

Phylogenetic relationship and mode of evolution of yeast DNA topoisomerase II gene in the pathogenic Candida species.

We have determined the nucleotide sequences of about 55% of the region of the DNA topoisomerase II gene (approximately 2.3 kb) isolated from the pathogenic Candida species, C. dubliniensis, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, C. guilliermondii and C. lusitaniae. Evolutionary relationships among nine Candida species including those of C. albicans and C. glabrata were studied based on the DNA topoisomerase II gene. The nucleotide sequences of 2192 bp, which covered two catalytic domains, ATPase and cutting/resealing, were subjected to phylogenetic analysis. Sequence comparison and evolutionary analysis have revealed that the Candida species tested here are not monophyletic, and the two strains within the species C. tropicalis and C. parapsilosis are too diverse to be in a single species. A wide variety of divergence was observed among the functional domains of DNA topoisomerase II, suggesting that Candida species were in different evolutionary paths at least as regarding the DNA topoisomerase II gene. Sequence information and the observation on the species-specific manner of molecular evolution of DNA topoisomerase II in Candida will be applied to develop a method of identification and characterization of the Candida species in both natural and clinical isolates.

Folding and aggregation of DNA chains induced by complexation with lipospermine: formation of a nucleosome-like structure and network assembly.

Dioctadecylamidoglycylspermine (DOGS) is a cationic lipid vector capable of efficiently introducing DNA into various eukaryotic cells. We investigated the higher-order structure of the DNA/DOGS complex using fluorescence and electron microscopy. Our results show that the DNA/DOGS complex exhibits a nucleosome-like structure in which DNA wraps around an aggregate of DOGS molecules. In addition, DNA/DOGS complexes tend to associate with each other to form network structures. The resulting network assembly may play a role in effective gene transfection.

Improvements and important considerations of an ex vivo assay to study Candida albicans-splenic tissue interactions.

Improvements were made on an ex vivo assay to study adherence properties of Candida albicans to host internal organs. The assay is applicable to understanding mechanisms of C. albicans dissemination following a fungemia. Binding patterns of yeast forms to splenic tissue are intriguing and we found the following modifications to be especially relevant. Mice serving as spleen donors for the assay should be injected with 0.1 ml i.v. of a 10% concentration of luconyl blue, 5 min before killing. After collecting splenic sections on a glass slide, 100 microliters of a 1-2 x 10(8)/ml suspension of stationary yeast cells should be applied to the sections. The assay does not require a carbonate buffering system or serum supplements. Attachment of yeasts to host tissue occurs best if the interaction is allowed to proceed without agitation by rotation. Assessment of binding is facilitated by staining the slides with crystal violet and computer image analysis can be used for quantification of binding.

Antigenic components of Malassezia species for immunoglobulin E antibodies in sera of patients with atopic dermatitis.

Antigenic components of Malassezia furfur, M. globosa, M. restricta, M. slooffiae, and M. sympodialis were studied for immunoglobulin E antibodies in sera of patients with atopic dermatitis (AD). Antigenic components were extracted from Malassezia cells by treatment with beta-mercaptoethanol, referred to as 2-ME extract. CBB staining and lectin blots using Con A, LCA, PHA-E4, PNA or RCA120 showed that the 2-ME extracts contained several species-dependent components that differed quantitatively and qualitatively among the Malassezia species at the protein level. In the Western blot with the 2-ME extracts, of 54 sera of the patients with AD (54 patients), the patients' IgE antibodies most frequently recognized components showing molecular weights of 43-46 kDa for M. slooffiae, 12-22 kDa for M. sympodialis, 35-40 kDa for M. restricta, 45-50 kDa for M. globosa, and of 67-72 kDa for M. furfur, respectively. In the correlative study, in which the total band intensities generated for each extract in Western blot were compared among the Malassezia species, the intensity for M. globosa was well correlated with that for M. sympodialis (r=0.756). In the Western blot inhibition test, the 2-ME extract of M. globosa partially inhibited the reaction of the antigenic components of other Malassezia species with the patient's IgE antibodies. These results indicated that Malassezia species contained both species-specific and common antigenic components at the IgE antibody level.

Effects of methylxanthines and imidazole on the contractions of guinea-pig ileum induced by transmural stimulation.

Methylxanthines (10(-5) to 10(-3)M) were found to increase the amplitude of contractions of guinea-pig ileum induced by transmural stimulation but to inhibit those induced by acetylcholine or histamine. The order of the abilities of methylxanthines to augment the contractile responses was theobromine greater than caffeine greater than theophylline. When the contractions were completely suppressed by reduction of the calcium content in the medium or by addition of cyclic AMP, methylxanthines restored the responses effectively, just as does addition of calcium. Methylxanthines also accelerated the release of acetylcholine from the ileum associated with stimulation. Imidazole (3 X 10(-5) to 10(-3) M) had an essentially similar effect to methylxanthines in potentiating the contractile responses and in augmenting the release of acetylcholine. The present results indicate that the potentiating effects of methylxanthines and imidazole are due to an action on the nerve terminals, not on the postsynaptic membranes or contractile elements. Therefore, it si concluded that theit potentiating actions are due to facilitation of the movement of calcium in the nerve terminals on excitation, resulting in increased release of acetylcholine, and are not due to the effect of cyclic AMP formed as a result of their inhibitory actions on phosphodiesterase.

Ouabain enhances nitric oxide synthesis in rat vascular smooth muscle cells induced by interleukin-1 beta.

Incubation of cultured rat vascular smooth muscle cells with interleukin-1 beta caused a significant increase in the production of nitrite, a stable metabolite of nitric oxide (NO), in time- and dose-dependent manners. Addition of ouabain to the culture further enhanced interleukin-1 beta-induced nitrite production. Similarly, interleukin-1 beta produced a significant increase in the cellular level of guanosine 3',5'-cyclic monophosphate, and the increase was significantly enhanced by coincubation with ouabain. The calcium ionophore ionomycin also significantly enhanced interleukin-1 beta-induced nitrite generation. These findings indicate that ouabain enhances NO synthesis in vascular smooth muscle cells induced by interleukin-1 beta, presumably through an increase in intracellular calcium ion concentrations.

Gastric mucosal protection by OPC-12759, a novel antiulcer compound, in the rat.

OPC-12759, 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid, was studied for its efficacy to prevent the gastric mucosal damage induced by several necrotizing agents. Experiments were also performed to elucidate the mechanism of this mucosal protective activity. OPC-12759 dose dependently prevented the formation of mucosal necrosis induced by absolute ethanol, 0.2 N NaOH or 0.6 N HCl. PGE2 was also shown to prevent the gastric mucosal erosion induced by necrotizing agents. The mucosal protective effect of OPC-12759 was completely counteracted by pretreatment with indomethacin while that of PGE2 was not. In addition, OPC-12759 given alone increased the generation of gastric mucosal PGE2-like activity. OPC-12759 dose dependently reduced the volume, acid output and pepsin output of the gastric juice in pylorus-ligated rats. The inhibitory effect of OPC-12759 but not of cimetidine or atropine on gastric secretion was also abolished by concurrent administration of indomethacin. These findings suggest that the mucosal protective effect and antisecretory effect of OPC-12759 presumably result from enhancement of the generation of endogenous PGs.

Effect of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide production by vascular smooth muscle cells.

We investigated the effects of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in the accumulation of nitrite, a stable metabolite of NO. Although cilostazol itself showed no effect on nitrite accumulation, it stimulated interleukin-1 beta-induced nitrite accumulation in a concentration-dependent manner (10(-8)-10(-5) M). This effect of cilostazol was completely abolished in the presence of NG-monomethyl-L-arginine, actinomycin D or dexamethasone. The cilostazol-induced nitrite production was accompanied by increased inducible NO synthase protein expression. In the presence of dibutyryl-cAMP, interleukin-1 beta-induced nitrite accumulation was further increased, but the stimulatory effect of cilostazol on nitrite accumulation was blunted. The effect of cilostazol was also abolished in the presence of Rp-8-bromoadenosine-3',5-cyclic monophosphorothioate, a competitive inhibitor of protein kinase A. Addition of cilostazol to the cultures significantly increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that cilostazol increases NO synthesis in interleukin-1 beta-stimulated smooth muscle cells, at least partially through a cAMP-dependent pathway.

Effects of a bradykinin receptor antagonist (HOE140) on taurocholate-induced acute pancreatitis in rats.

The effect of a potent and long-acting bradykinin B2-receptor antagonist (HOE140) on acute pancreatitis induced by retrograde infusion of trypsin and taurocholate into the pancreatic duct was studied in rats. HOE140 was administered subcutaneously immediately before and 3 h after the induction of pancreatitis and the systemic blood pressure, ascites volume, serum amylase, 24-h survival rate, and pathology of the pancreas were evaluated. Plasma concentrations of bradykinin increased significantly 15 min after the induction of pancreatitis and decreased to basal levels at 90 min. HOE140 (0.1 mg/kg) alleviated hypotension developing immediately after the induction of pancreatitis and reduced the ascites volume. The 24-h survival rate in rats treated with 0.1 mg/kg HOE140 (70.3%) was significantly higher than that in controls (35.6%). Treatment with 0.01, 0.3, 1.0, and 3.0 mg/kg of HOE140, however, had no beneficial effect on the survival rate. Ascites volume, serum amylase, and pathology of the pancreas at 24 h were not improved by treatment with HOE140. These data suggest that HOE140 may improve the survival rate by maintaining hemodynamics in the early stage of experimental acute pancreatitis.

Aldosterone inhibits nitric oxide synthesis in rat vascular smooth muscle cells induced by interleukin-1 beta.

We investigated the effects of aldosterone on nitric oxide (NO) synthesis in vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase mRNA and protein in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite generation. The interleukin-1 beta-induced nitrite production by vascular smooth muscle cells was significantly inhibited by aldosterone in a dose (10(-9) approximately 10(-6) M)-dependent manner. Incubation with interleukin-1 beta for 12 approximately 24 h caused inducible NO synthase mRNA expression in vascular smooth muscle cells, whereas aldosterone had a suppressive effect on its expression. Aldosterone also decreased interleukin-1 beta-induced NO synthase protein accumulation. These results indicate that aldosterone inhibits NO synthesis under interleukin-1 beta-stimulated conditions in vascular smooth muscle cells.

Daunomycin unfolds compactly packed DNA.

Daunomycin is an antitumor antibiotic known to inhibit DNA replication and transcription. Although the inhibition is assumed to be caused by a direct interaction of the drug with DNA, the exact effect of daunomycin on the higher order DNA structure remains uncertain. We studied the effect of daunomycin on DNA compacted states using fluorescence and electron microscopies. Structural changes in individual DNA molecules were examined under the following conditions. T4 phage DNA (166 kbp) was first compacted by spermidine followed by the addition of daunomycin to the compacted DNA. A direct observation of individual single duplex DNAs by fluorescence microscopy indicated that daunomycin induced unfolding of the compacted DNA. Electron microscopic observation of the morphological changes of the higher order DNA structure supported the results obtained by fluorescence microscopy. We discuss here the mechanisms of the unfolding of the compacted structure following intercalation of daunomycin into DNA particularly in terms of the free energy.