Mikio Nishizawa

Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat

Rat VIP Rat PHI‐27 VIP precursor VIP precursor mRNA Human PHM‐27 Sequence homology

Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter.

We have previously shown that the VIP precursor contains a novel PHI-27-like peptide, PHM-27, and that the synthesis of the prepro-VIP/PHM-27 mRNA is induced with cAMP and TPA in human neuroblastoma cells. In this study, we have determined the complete nucleotide sequence of the human VIP/PHM-27 gene. The gene spans 8,837 bp and consists of seven exons and six introns. Exon I of 165 bp consists of the 5 untranslated region of the gene, exon II of 117 bp encodes the signal peptide of prepro-VIP/PHM-27, exon III of 123 bp encodes the amino-terminal region, exon IV of 105 bp encodes PHM-27, exon V of 132 bp encodes VIP, exon VI of 89 bp contains the termination codon of the prepro-VIP/PHM-27 mRNA, and exon VII of 724 bp consists of the 3 untranslated region of the gene. VIP and its structurally related peptide, PHM-27, were encoded in different exons V and IV, and the sequences around the splice junctions between these exons and their adjacent introns were highly conserved, suggesting that the VIP-encoding and PHM-27-encoding exons have been duplicated from an ancestral exon over a broad area containing its adjacent introns. We also determined the 1,929-bp sequence of the 5 flanking region of the human VIP/PHM-27 gene and found that four TATA-box sequences were present at 28 bp, 145 bp, 772 bp, and 900 bp upstream of the cap site. Primer extension, exon mapping, and mung bean nuclease mapping analyses revealed that only the TATA-box sequence 28 bp upstream of the cap site was the promoter that is inducible by cAMP and TPA in the human neuroblastoma cells. An 18-bp sequence 52 bp upstream from the TATA-box sequence was suggested to be a cAMP/phorbol esters-responsive element of the human VIP/PHM-27 gene.

Synergistic stimulation of VIPPHM-27 gene expression by cyclic AMP and phorbol esters in human neuroblastoma cells

Abstract The effects of dibutyryl cAMP (Bt2cAMP) and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate (PDD) on VIP PHM-27 gene expression in human neuroblastoma cells in culture were investigated. Bt2cAMP and phorbol esters increased the VIP PHM-27 mRNA level by about 9- and 4-fold, respectively. In the presence of both Bt2cAMP and phorbol esters, the VIP PHM-27 mRNA level increased by about 36-fold. The intracellular cAMP level was essentially unaffected by phorbol esters. The VIP PHM-27 gene dosage was unchanged by Bt2cAMP and phorbol esters. The results suggest that cAMP and phorbol esters synergistically induce the VIP PHM-27 gene expression through independent pathways.

Characterization of natural antisense transcripts expressed from interleukin 1β-inducible genes in rat hepatocytes

Abstract nBackground: Natural antisense transcripts (asRNAs) are transcribed from many genes in various species. Recently, we found that asRNAs were transcribed from the rat and mouse genes encoding inducible nitric

Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

BACKGROUNDnNobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes.nnnMETHODSnA C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed.nnnRESULTSnHigh-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei.nnnCONCLUSIONSnThe suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

Chlorogenic acid from the Japanese herbal medicine Kinginka (Flos Lonicerae japonicae) suppresses the expression of inducible nitric oxide synthase in rat hepatocytes

Abstract nBackground: nFlos Lonicerae japonicae (FLJ; Kinginka) is the dried flowers and buds of the Japanese honeysuckle Lonicera japonica Thunberg. FLJ has been used as a Japanese Kampo medicine to treat

The constituents of licorice (Glycyrrhiza uralensis) differentially suppress nitric oxide production in interleukin-1β-treated hepatocytes

Licorice (Glycyrrhizae radix) is the roots and stolons of Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra Linnaeus in the Japanese Pharmacopoeia. Glycyrrhizae radix has been widely used as a sweetener and a traditional medicine. A Glycyrrhizae radix extract contains many constituents and has antispasmodic, antitussive, anti-ulcer, and anti-inflammatory effects. However, reports comparing the anti-inflammatory effects of these constituents are very few. Here, we purified several constituents from the roots and stolons of G. uralensis and examined and compared their anti-inflammatory effects by monitoring the levels of the inflammatory mediator, nitric oxide (NO), in interleukin (IL)-1β-treated rat hepatocytes. From the G. uralensis extract, we purified the main constituent glycyrrhizin and the constituents that are characteristic of G. uralensis (chalcones and flavanones). These constituents suppressed NO production in IL-1β-treated rat hepatocytes, and isoliquiritigenin showed the greatest suppression activity. Isoliquiritigenin, isoliquiritin, and liquiritigenin significantly decreased both protein and mRNA for the inducible nitric oxide synthase. These constituents reduced the levels of mRNAs encoding tumor necrosis factor α and IL-6. In contrast, although glycyrrhizin is abundant, it showed a 100-fold lower potency in NO suppression. Therefore, both glycyrrhizin and the minor constituents (isoliquiritigenin, isoliquiritin, and liquiritigenin) may be responsible for the anti-inflammatory effects of G. uralensis. It is also implied that these constituents may have a therapeutic potential for inflammatory hepatic disorders.

Japanese Kampo Medicine, Ninjinyoeito, Inhibits the Induction of iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes

This work was carried out in collaboration between all authors. Authors YT, HM, MO and RN managed the analyses of the study. Author YT performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors MK, KT, MN, TO and AHK managed the coordination of the study. Authors MK, TO and MN conceived the study and participated in its design. This work was carried out in collaboration between all authors. All authors read and approved the final manuscript.

Inducible Nitric Oxide Synthase Expression in Liver Injury: Liver Protective Effects on Primary Rat Hepatocytes

BACKGROUND/AIMSnFollowing injury to the liver, liver cells, including Kupffer cells and hepatocytes express inducible nitric oxide synthase (iNOS), followed by the production of excess levels of nitric oxide (NO). NO produced by iNOS has been found to contribute to liver injury. Treatment of primary cultures of rat hepatocytes with the proinflammatory cytokine interleukin (IL)-1β stimulated iNOS expression and NO production. Experiments with this in vitro hepatocyte model of liver injury and with in vivo animal models of liver injury have demonstrated that drugs showing a liver-protective effect in vivo also inhibited the induction of iNOS expression and NO production both in vivo and in vitro. Thus, in this in vitro hepatocyte model, the prevention of iNOS expression and NO production are considered indicators of liver protection.nnnRESULTS/CONCLUSIONnThis review describes a simple in vitro liver injury model, consisting of IL-1β-stimulated cultured hepatocytes, and methods used to analyze the mechanisms of action of drugs that inhibit iNOS expression. This in vitro hepatocyte model may be used to assess the liver-protective effects of pharmaceutical agents, herbal medicines, and certain types of foods.

Systematic analyses of Japanese Kampo drugs

Herbal drugs from medicinal plants consist of many constituents which is difficult to isolate and identify the pharmacologically active constituents in the herbal drugs. We established a standard protocol to fractionate the herbal extracts of medicinal plants by hydrophobicity into crude fractions to estimate the pharmacological activity. We have been analyzed anti-inflammatory effects of herbal extracts and their constituents on the induction of nitric oxide, which is an inflammatory mediator-induced by interleukin 1β in the rat hepatocytes. When chemical and spectral analyses are performed, active constituents in herbal extracts can be identified. Our systematic approachment using the hepatocytes have been successfully applied to investigate many traditional Japanese herbal drugs (Kampo drugs). Traditional Indonesian herbal drugs (Jamu) may be also analyzed. This approachment may elucidate the mechanisms of action of these drugs and lead to understanding of the pathophysiology of various diseases.Herbal drugs from medicinal plants consist of many constituents which is difficult to isolate and identify the pharmacologically active constituents in the herbal drugs. We established a standard protocol to fractionate the herbal extracts of medicinal plants by hydrophobicity into crude fractions to estimate the pharmacological activity. We have been analyzed anti-inflammatory effects of herbal extracts and their constituents on the induction of nitric oxide, which is an inflammatory mediator-induced by interleukin 1β in the rat hepatocytes. When chemical and spectral analyses are performed, active constituents in herbal extracts can be identified. Our systematic approachment using the hepatocytes have been successfully applied to investigate many traditional Japanese herbal drugs (Kampo drugs). Traditional Indonesian herbal drugs (Jamu) may be also analyzed. This approachment may elucidate the mechanisms of action of these drugs and lead to understanding of the pathophysiology of various diseases.