Altered expression of dermokine in skin disorders.

BACKGROUND: Although dermokine-ß, a glycoprotein expressed in epithelial cells, does not have significant homology to other proteins, its carboxyl-terminal domain shares a high pI value with many cytokines, suggesting similar functions. OBJECTIVE: To better understand the biology of dermokine, we here determined its localization under pathological conditions and examined factors that regulate its expression. METHODS: We generated an anti-human dermokine-ß/γ monoclonal antibody cross-reacting with the mouse protein. Using this antibody, immunohistological staining and Western blotting of dermokine-ß/γ were performed with various tissue samples. RESULTS: Although human dermokine-ß/γ was expressed in almost all granular layers, upper spinous layers of the skin were also stained with anti-dermokine-ß/γ antibody in inflammatory skin disorders. Dermokine-ß/γ was expressed in keratoacanthoma and a part of well-differentiated squamous cell carcinoma (SCC). However, dermokine-ß/γ was not detected in poorly differentiated SCC or tumours derived from non-keratinocytes. In mice, dermokine-ß/γ-expressed keratinocytes were increased in models of contact hypersensitivity, ultraviolet-irradiated skin injury and wound healing. Consistent with expanded distribution in inflammatory skin diseases, proinflammatory cytokines such as interleukin-1ß, interleukin-12, and tumour necrosis factor-α augmented dermokine-ß/γ expression in cultured human keratinocytes. In contrast, growth factors including epidermal growth factor, insulin-like growth factor-I, keratinocyte growth factor and transforming growth factor-α significantly reduced dermokine expression. CONCLUSION: These results provide novel insights into the physiological and pathological significance of dermokine in the epidermis.

Enhanced in vitro and in vivo antioxidant activity and mobilization of free phenolic compounds of soybean flour fermented with different beta-glucosidase-producing fungi.

AIMS: To evaluate the soybean polyphenol glucosides bioconversion to aglycone forms by different beta-glucosidases-producing filamentous fungi to enhance their antioxidant activity. METHODS AND RESULTS: Soybean defatted flour was submitted to solid-state fermentation with Aspergillus niger, Aspergillus niveus and Aspergillus awamori. The fungi studied produced approximately the same beta-glucosidase activity units amount when p-nitrophenyl-beta-d-glucopyranoside was used as substrate for the assay. However, electrophoretic analysis, using 4-methylumbellipheryl-beta-d-glucopyranoside as substrate, showed that beta-glucosidase produced by A. niveus was more active. Fermented methanolic extracts showed an increase in polyphenol and genistein contents and antioxidant activities. The highest genistein content was found in soybean fermented by A. niveus. Methanolic extracts of the soybean fermented by the different fungi showed a similar capacity of scavenging H(2)O(2) generated in vivo by the tumour promoter 12-O-tetradecanoyl phorbol-13-acetate. CONCLUSIONS: A. niveus synthesized a beta-glucosidase with higher specificity to hydrolyse genistin beta-glycosidic bond than those produced by A. awamori and A. niger. SIGNIFICANCE AND IMPACT OF THE STUDY: The utilization of these beta-glucosidases-producing fungi in soybean fermentation processes resulted in the obtaining of methanolic extracts with different antioxidant potentials that could be used either therapeutically or as an antioxidant in nonphysiological oxidative stress conditions, as the one induced in skin by UV radiation.

Cloning and characterization of 5'-flanking region of mouse prostacyclin synthase gene.

To gain an insight into the mechanisms of prostacyclin expression, a genomic DNA clone harboring 2.0 kb of the 5'-flanking sequence of the mouse prostacyclin synthase (PGIS) gene was isolated. The 5'-flanking region did not possess a TATA box, but contained a GC-rich region and several consensus cis DNA elements. The major product of the primer extension analysis suggested that the transcription of the gene started from 72 bases upstream of the translational initiation codon. To analyze the PGIS promoter activity, the 2.0 kb fragment was fused to the luciferase gene and transient transfection assays were conducted with cultured rat vascular smooth muscle cells (VSMC). The fragment showed significant promoter activity in the cells. Analysis of a series of 5'-deletion constructs showed that the 5'-flanking regions spanning bases -371 to -285 and -229 to -119 were important for the basal transcriptional activity of the mouse PGIS gene. Gel mobility shift assays revealed that DNA-protein complexes were formed with the nuclear extracts from VSMC, and that the formation of these complexes was inhibited by excess consensus Sp1 oligonucleotide. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in supershift of the band for the DNA-protein complex. In addition, mutation of two Sp1 recognition motifs residing at bases -297 to -289 and -197 to -192 markedly reduced the basal PGIS promoter activity and retarded the band in a gel mobility shift assay. These results indicated that binding of one Sp1 to two Sp1 sites on the promoter region activated the basal transcription of the PGIS gene.

Partial purification and characterization of arachidonate 12-lipoxygenase from rat lung.

Incubation of rat lung cytosol with arachidonic acid produced 12-hydroxy-5,8,10,14-eicosatetraenoic acid as a major product, which was identified by gas chromatography-mass spectrometry. By ammonium sulfate fractionation and DEAE-cellulose chromatography the arachidonate 12-lipoxygenase was purified about 30-fold from the rat lung cytosol. The partially purified enzyme was mostly free of the glutathione peroxidase activity and transformed arachidonic acid to its 12-hydroperoxide. 5,8,11,14,17-Eicosapentaenoic acid was also an active substrate, and the oxygenation at C-12 was confirmed by mass spectrometry. A significant amount of 12-lipoxygenase activity was also found in the microsomes and other particulate fractions.

2,3,5-Trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of the 5-lipoxygenase reaction and the biosynthesis of slow-reacting substance of anaphylaxis.

2,3,5-Trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861) inhibited 5-lipoxygenase of guinea pig peritoneal polymorphonuclear leukocytes (ID50, 0.8 microM). The inhibition was of competitive type. 12-Lipoxygenases and fatty acid cyclooxygenase were not affected below 10 microM. The formation of slow-reacting substance of anaphylaxis by the sensitized guinea pig lung was almost fully suppressed by the compound at 10 microM.

Decreased messenger RNA of arachidonate 12-lipoxygenase in platelets of patients with myeloproliferative disorders.

On the basis of the previous work by Okuma and Uchino [Blood 54, 1258-1271, 1979], three patients with myeloproliferative disorders were investigated with a special reference to arachidonate 12-lipoxygenase in their platelets. The cytosol of the patients' platelets showed a markedly reduced activity of arachidonic acid oxygenation to 12-hydroperoxy acid. A peroxidase-linked immunoassay for the 12-lipoxygenase demonstrated only 7-12% of the normal level of the enzyme protein in the cytosol fraction of platelets. Furthermore, 12-lipoxygenase mRNA level was determined quantitatively by a reverse transcriptase-polymerase chain reaction with an internal standard cRNA which was synthesized by in vitro transcription of human platelet 12-lipoxygenase cDNA with a 105-bp deletion. The 12-lipoxygenase mRNA content was 4.7 +/- 2.0 (mean +/- S.D.) ng/10(11) platelets in 13 normal subjects. In contrast, the mRNA content was as low as 0.15, 0.11 and 0.10 ng/10(11) platelets in the three patients. Taken together, the 12-lipoxygenase deficiency in these patients was attributable to the decreased 12-lipoxygenase mRNA level and thus the impaired synthesis of the enzyme protein in their platelets.

Gene transfer of human prostacyclin synthase ameliorates monocrotaline-induced pulmonary hypertension in rats.

BACKGROUND: Prostacyclin is a potent vasodilator that also inhibits platelet adhesion and cell growth. We investigated whether in vivo gene transfer of human prostacyclin synthase (PGIS) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. METHODS AND RESULTS: The cDNA encoding PGIS was intratracheally transfected into the lungs of rats by the hemagglutinating virus of Japan-liposome method. Rats transfected with control vector lacking the PGIS gene served as controls. Three weeks after MCT injection, mean pulmonary arterial pressure and total pulmonary resistance had increased significantly; the increases were significantly attenuated in PGIS gene-transfected rats compared with controls [mean pulmonary arterial pressure, 31+/-1 versus 35+/-1 mm Hg (-12%); total pulmonary resistance, 0.087+/-0.01 versus 0.113+/-0.01 mm Hg x mL x min(-1) x kg(-1) (-23%), both P:<0.05]. Systemic arterial pressure and heart rate were unaffected. Histologically, PGIS gene transfer inhibited the increase in medial wall thickness of peripheral pulmonary arteries that resulted from MCT injection. PGIS immunoreactivity was intense predominantly in the bronchial epithelium and alveolar cells. Lung tissue levels of 6-keto-PGF(1alpha), a stable metabolite of prostacyclin, were significantly increased for >/=1 week after transfer of PGIS gene. The Kaplan-Meier survival curves demonstrated that repeated transfer of PGIS gene every 2 weeks increased survival rate in MCT rats (log-rank test, P:<0.01). CONCLUSIONS: Intratracheal transfer of the human PGIS gene augmented pulmonary prostacyclin synthesis, ameliorated MCT-induced pulmonary hypertension, and thereby improved survival in MCT rats.

The regional distribution and cellular localization of mRNA encoding rat prostacyclin synthase.

The cloned cDNA for rat prostacyclin synthase was found to contain a 1503-bp open reading frame which encoded a 501-amino acid protein sharing 84.0% identity with the human enzyme. RNA blot analysis revealed that the rat prostacyclin synthase mRNA, as a single species of 2.1 kb, is expressed abundantly in the aorta and uterus. High levels of expression were also observed in the stomach, lung, heart, testis, liver, and skeletal muscle. Low but significant expression was also seen in the brain and kidney. Furthermore, the regional distribution and cellular localization of prostacyclin synthase mRNA were examined by in situ hybridization analysis of rat tissue sections. The definitive signals for the mRNA were localized in smooth muscle cells of the arteries, bronchi and uterus, and in the cells of the fibrous tunic surrounding the seminiferous tubules, which are characterized as smooth muscle cells. Besides smooth muscle cells, signals were also detected in the fibroblasts of the heart myocardium, lung parenchyma cells and kidney inner medulla tubules and interstitial cells.

Eicosanoid biosynthetic enzymes in placental and decidual tissues from preeclamptic pregnancies: increased expression of thromboxane-A2 synthase gene.

Preeclampsia is a disease of late pregnancy characterized by hypertension, edema, and proteinuria, in which vasoconstriction, platelet aggregation, and reduced uteroplacental blood flow contribute to preterm delivery, perinatal morbidity, and mortality. Increased thromboxane-A2 (TXA2) and/or decreased prostacyclin (PGI2) have been implicated as causative factors of this disease. The present studies investigated the expression of TXA2 synthase gene along with those of TXA2 receptors, PGI2 synthase, cyclooxygenase-1 (COX-1), and COX-2 in placental and decidual tissue from preeclamptic and normal pregnancies. In situ hybridization and immunocytochemistry showed that primarily trophoblast layer and decidual cells express TXA2 synthase, COX-1, and COX-2 enzymes. Immunocytochemistry for PGI2 synthase and in situ hybridization for TXA2 receptors showed similar results. Trophoblast layer and decidua from preeclamptic pregnancies contained a greater abundance of mRNA and protein of TXA2 synthase than the matched normal pregnancies. In summary, our findings suggest that an increased local expression of TXA2 synthase could be responsible for local and/or peripheral vascular changes in preeclampsia.

Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence.

The complete amino acid sequence of prostaglandin endoperoxide synthase from sheep vesicular gland has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by digestion of the enzyme with carboxypeptidase Y and by automated Edman degradation of the intact enzyme polypeptide and peptide fragments obtained by limited proteolysis of the enzyme with Achromobacter proteinase I. Mature sheep prostaglandin endoperoxide synthase is shown to be composed of 576 amino acids with an Mr of 66,175. The precursor peptide is predicted to contain a 24-residue signal peptide. The serine residue susceptible to acetylation by aspirin is found to be located near the C-terminus of the enzyme polypeptide.

The cyclic AMP response element plays an essential role in the expression of the human prostaglandin-endoperoxide synthase 2 gene in differentiated U937 monocytic cells.

The promoter activity of 1432 bp upstream of the human prostaglandin-endoperoxide synthase 2 gene (PTGS2) was examined in differentiated U937 monocytic cells expressing prostaglandin-endoperoxide synthase 2 mRNA. Transient transfection experiments were performed using these cells and reporter vectors containing the upstream region of the gene with deletions or site-specific mutations and the luciferase gene. The deletion or destruction of the cyclic AMP response element (nucleotides -59 to -53) markedly reduced the promoter activity of this gene. Electrophoretic mobility shift assays showed that a nuclear protein(s) binding to the cyclic AMP response element was induced during monocytic differentiation of U937 cells. These results indicate that expression of the human prostaglandin-endoperoxide synthase 2 gene in differentiated U937 monocytic cells is regulated by the cyclic AMP response element.

Induction of thromboxane synthase and prostaglandin endoperoxide synthase mRNAs in human erythroleukemia cells by phorbol ester.

The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the mRNA levels of two enzymes, thromboxane synthase (TXS) and prostaglandin endoperoxide synthase (PES), responsible for the synthesis of thromboxane A2 from arachidonic acid, were studied in human erythroleukemia (HEL) cells by RNA blot analysis. TPA induced both TXS and PES mRNAs in HEL cells in a dose-dependent manner at 36 h. The half-maximal and maximal effects for the induction of both mRNAs were at approximately 3 x 10(-9) M and at 10(-8) M, respectively. TXS and PES mRNA levels increased in a time-dependent fashion by TPA, and reached to 7- and 3.5-fold of the control, respectively after 48 h of TPA treatment. These results suggest that expression of TXS and PES genes in HEL cells were simultaneously stimulated by TPA.

Site-directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys-pocket, and Glu347 and Arg350 of the EXXR motif.

The possible active site Cys441 in the Cys-pocket and Glu347 and Arg350 of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441 Ala, Cys441 Ser, Cys441 His, Glu347 Ala and Arg350 Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1alpha/min per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys441 in the Cys-pocket, and Glu347 and Arg350 of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

Regulation of the activity of caspases by L-carnitine and palmitoylcarnitine.

L-Carnitine facilitates the transport of fatty acids into the mitochondrial matrix where they are used for energy production. Recent studies have shown that L-carnitine is capable of protecting the heart against ischemia/reperfusion injury and has beneficial effects against Alzheimer's disease and AIDS. The mechanism of action, however, is not yet understood. In the present study, we found that in Jurkat cells, L-carnitine inhibited apoptosis induced by Fas ligation. In addition, 5 mM carnitine potently inhibited the activity of recombinant caspases 3, 7 and 8, whereas its long-chain fatty acid derivative palmitoylcarnitine stimulated the activity of all the caspases. Palmitoylcarnitine reversed the inhibition mediated by carnitine. Levels of carnitine and palmitoyl-CoA decreased significantly during Fas-mediated apoptosis, while palmitoylcarnitine formation increased. These alterations may be due to inactivation of beta-oxidation or to an increase in the activity of the enzyme that converts carnitine to palmitoylcarnitine, carnitine palmitoyltransferase I (CPT I). In support of the latter possibility, fibroblasts deficient in CPT I activity were relatively resistant to staurosporine-induced apoptosis. These observations suggest that caspase activity may be regulated in part by the balance of carnitine and palmitoylcarnitine.

Expression of human thromboxane synthase using a baculovirus system.

Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system using cDNA encoding human TX synthase [(1991) Biochem. Biophys. Res. Commun. 78, 1479-1484]. A recombinant baculovirus TXS7 was expressed in Spodoptera frugiperda Sf9 insect cells. The expressed protein was recognized by monoclonal antibody, Kon 7 raised against human TX synthase [(1990) Blood 76, 80-85]. The recombinant TX synthase catalyzed the conversion of prostaglandin (PG) H2 to TXA2 and 12-hydroxy-heptadecatrienoic acid (HHT). Both conversions of PGH2 to TXA2 and HHT by the expressed TX synthase were completely inhibited by a specific TX synthase inhibitor, OKY-046 (5 microM).

Autoradiographic distribution of [3H]YM-09151-2, a high-affinity and selective antagonist ligand for the dopamine D2 receptor group, in the rat brain and spinal cord.

We determined the regional distribution of the dopamine D2 receptor group in the rat central nervous system by quantitative receptor autoradiography with a high-affinity and selective antagonist, [3H]YM-09151-2. Saturation and competition experiments demonstrated that the binding of [3H]YM-09151-2 to striatal sections was saturable (Bmax = 37.3 fmol/section), of high affinity (Kd = 0.315 nM), and was inhibited selectively by prototypic D2 ligands. The anatomical localization of binding sites was determined by comparison of autoradiograms and the original 3H-ligand-exposed sections stained with cresyl violet. Very high levels of [3H]YM-09151-2 binding were found in the caudate-putamen, nucleus accumbens, tuberculum olfactorium and the insula of Calleja, to each of which midbrain dopaminergic neurons project densely. High levels of binding were also observed in other regions rich in dopaminergic neurons and fibers including the glomerular layer of the olfactory bulb, the intermediate lobe of the pituitary, lateral septum, substantia nigra pars compacta, interfascicular nucleus, dorsal raphe nucleus, locus coeruleus, and nucleus of the solitary tract. Some regions poor in dopaminergic innervation, however, had high levels of [3H]YM-09151-2 binding including the molecular layer of gyrus dentatus, all layers of CA1 and the nonpyramidal layer of CA4 of hippocampus, and the deeper layer of medial entorhinal cortex. Motor neurons present in brainstem motor nuclei and spinal ventral horn were also strongly labeled. Neocortical, cerebellar, and thalamic regions had low levels of binding, except lobules 9-10 of the cerebellum, the olivary pretectal nucleus, zona incerta and lateral mammillary nucleus, in which moderate to high levels of binding were detected. Our findings concerning the widespread but region-specific localization of [3H]YM-09151-2 binding sites in the brain and spinal cord may prove useful for analyzing various dopaminergic functions in the central nervous system.

Syntheses of 5,6,7- and 5,7,8-trioxygenated 3',4'-dihydroxyflavones having alkoxy groups and their inhibitory activities against arachidonate 5-lipoxygenase.

Arachidonate 5-lipoxygenase plays a pivotal role in the biosynthesis of leukotrienes. Cirsiliol (3',4',5-trihydroxy-6,7-dimethoxyflavone), a selective inhibitor of the enzyme, was derivatized by introducing alkyl groups of various chain lengths at positions 5, 6, 7, and 8 of the A ring of the flavone skeleton. Modification of the positions 5 and 6 with an alkyl group of 5-10 carbons markedly decreased the IC50 values for 5-lipoxygenase inhibition to the order of 10 nM. As tested with 5- or 6-hexyloxy derivatives, a relatively selective inhibition of 5-lipoxygenase was shown. Inhibition of 12-lipoxygenase required much higher concentrations of these compounds, and cyclooxygenase was not inhibited. Modification of positions 7 and 8 did not increase the inhibitory effect of most flavone compounds.

N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine reduces intracellular calcium response to noradrenaline in rat visual cortex.

Using the fluorescent indicator Fura-2, we investigated the effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), a noradrenergic neurotoxin, on intracellular calcium responses to noradrenaline, N-methyl-D-aspartate, and carbamylcholine chloride in brain slices of the rat visual cortex. Noradrenergic depletion in the visual cortex of young rats was induced by DSP-4, and its selectivity was confirmed by two different methods, i.e., immunostaining with anti-dopamine-beta-hydroxylase antibody and biochemical analysis by high-performance liquid chromatography. The treatment with DSP-4 (25 mg/kg i.p., x2) caused disruption of noradrenergic fibers throughout all cortical layers, and reduced the content of noradrenaline to 6.4% of that in the normal control. In the normal cortex, bath-applied noradrenaline (100 microM) increased the intracellular calcium to 123% of the control in terms of the F(340)/F(380) ratio of Fura-2 fluorescence. Quantitative analysis of the F(340)/F(380) ratio was performed in layers II to IV, since the increase was mainly observed in these layers. The intracellular calcium response to noradrenaline was significantly (P<0.0001) reduced in the DSP-4-treated animals to 63.2% of that in the normal control. The response to N-methyl-D-aspartate (100 microM) was also reduced, whereas the response to carbamylcholine chloride, a muscarinic cholinergic agonist (100 microM), was not affected by the DSP-4 treatment. From these findings we suggest that noradrenergic denervation by DSP-4 reduces the intracellular calcium response to noradrenaline through changes in the intracellular signal transduction.

Cyclooxygenase expression in bovine aortic endothelial cells exposed to cyclic strain.

The aim of this study was to investigate the effect of cyclic strain on cyclooxygenase (COX)-1 and 2 expression in bovine aortic endothelial cells (EC). EC, subjected to 10% average strain at 60 cycle/min, were analyzed for induction of COX by Northern blot analysis and confirmed by analysis of promoter activity in transient transfection experiments. Exposure of EC to cyclic strain induced promoter activity and expression of COX-2 but not of COX-1. The extent of induction, however, was lower than that seen with stimulation of 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or lipopolysaccharide (LPS). These results demonstrate that, unlike shear stress, cyclic strain does not affect COX-1 expression and is a weak inducer of COX-2 promoter activity in bovine aortic EC with minimal effect on mRNA expression.

Regional expressions of Fos-like immunoreactivity in rat cerebral cortex after stress; restraint and intraperitoneal lipopolysaccharide.

To demonstrate regional activation in the rat cerebral cortex related to stress-evoked neuroendocrine response, Fos expression in both the cerebral cortex and hypothalamic paraventricular nucleus (PVN) was immunohistochemically examined in two experimental groups; a lipopolysaccharide (LPS) intraperitoneally injected group for inflammatory stress and a restraint group for emotional stress. The LPS injection (100 microg/100 g b.w.) and restraint (for 30 min) had similar effect on Fos-like immunoreactivity (Fos-LI) in PVN with regard to the number of immunoreactive nuclei and their distribution pattern, while the times to maximize Fos-LI were different. Numerical analysis of cortical Fos-LI in untreated rats showed a distinct region-specific pattern. Statistical analysis revealed no significant increase in Fos-LI density in any cortical regions in the LPS group, but restraint resulted in a dramatic and region-specific increase. A significant increase was detected in the prefrontal cortex (the cingulate, orbital and agranular insular cortex), the frontal area 2, the agranular retrosplenial cortex, the parietal cortex, and the medial and lateral occipital area 2. These results indicate that cortical activation relevant to specific functions may be involved in stress-specific neural circuitry.