YAKUGAKU ZASSHI
Online ISSN : 1347-5231
Print ISSN : 0031-6903
ISSN-L : 0031-6903
Volume 128 , Issue 11
Showing 1-24 articles out of 24 articles from the selected issue
Symposium Reviews
  • Takashi TSURUO, Gozoh TSUJIMOTO, Hiroshi YAMAMOTO
    Type: Foreword
    2008 Volume 128 Issue 11 Pages 1517
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
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  • Gozoh TSUJIMOTO
    Type: Review
    2008 Volume 128 Issue 11 Pages 1519-1523
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Following the completion of Human Genome Project, new discipline of drug discovery has emerged, namely Novel Drug Discovery beased on IT. Novel Drug Discovery beased on IT would be of improtance in performing “Peronalized Medicine” based on pharmacogenomics. Also, this must be critical for Japan to keep the leadershiop in drug discovery science.
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  • Satoru MIYAZAKI
    Type: Review
    2008 Volume 128 Issue 11 Pages 1525-1535
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      We have expected Bioinformatics as tools to extract new knowledge from whole genome sequences of various organisms. In the post-genome era, to find some knowledge of the gene regulation including locations of cis-regulatory elements, modules and those combinations became one of the big challenges on Bioinformatics field. Because, it is difficult and inefficient to determine all possible combinations of cis-regulatory elements by bio-chemical approach. However, computational ways might allow us to find out all cis-elements within a time frame. In this review, we introduce the current status of public available databases on Internet comparing our original database for the cis-modules. We also explain our new mathematical measurement to characterize sequence patterns for cis-elements of each transcription factors and its application to predict the gene expression regulation network.
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  • Jun-ichi SATOH
    Type: Review
    2008 Volume 128 Issue 11 Pages 1537-1545
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Following the completion of the Human Genome Project in 2003, we were able to clarify the comprehensive profile of the whole human genome on DNA microarray. KeyMolnet is a bioinformatics tool for analyzing molecular interactions on the curated knowledge database. It promotes genome-based drug discovery research aimed at mining the most relevant molecular target to personalized medicine. Multiple sclerosis (MS) is an inflammatory demyelinating disease with a relapsing-remitting clinical course, affecting exclusively the human central nervous system white matter. By DNA microarray, we identified a set of differentially expressed genes in T lymphocytes between MS and healthy subjects, and between acute relapse and complete remission. Hierarchical clustering of discriminator genes established the molecular classification of MS subgroups, associated with the therapeutic response to interferon-β. The molecular network of the genes involved in development of MS and induction of acute relapse of MS was related to NF-κB-regulated gene expression. Prion diseases are an intractable neurodegenerative disease, mediated by an abnormal prion protein PrPSc. The protein conformational conversion from the cellular prion protein PrPC to PrPSc requires an as yet unidentified species-specific chaperone named “Protein X”. By protein microarray, we identified a set of novel PrPC interactors, serving as the candidate for X. The molecular network of PrPC and interactors was closely associated with signaling pathways essential for cell survival, differentiation, proliferation and apoptosis. Thus, the molecular network analysis is highly valuable to extract biological implications from a huge amount of microarray data.
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  • Kei YURA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1547-1555
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      A vast amount of DNA sequence data, protein three-dimensional (3D) structure data, and RNA expression data have been produced by the efforts of genome sequencing, structural genomics, and omics projects, and we are at the stage where comprehensive views of cell activity and molecular mechanisms of life can be deduced. But in reality, we are inundated with massive amounts of data and are still in the process of finding ways to fully utilize the data. In this report, I would like to present our observations on the growth of protein 3D structure data and our effort to deduce the functions from the 3D structures. We found that the 3D structure of quite a high proportion of proteins derived from genome sequences can be now predicted and methods to predict the functions from 3D structures are in high demand. The methods we have developed can be used to predict some functions, namely RNA and ligand interfaces, based on those 3D structures and DNA sequences with relatively high accuracy. The methods enable predictions that are accurate enough to help with deducing the atomic structures of the complexes.
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  • Hidefumi MUKAI, Yusuke ETO
    Type: Review
    2008 Volume 128 Issue 11 Pages 1557-1558
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
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  • Shinsaku NAKAGAWA
    2008 Volume 128 Issue 11 Pages 1559-1565
    Published: 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Recently, nanoscopic systems that incorporate therapeutic agents, and molecular targeting and diagnostic imaging capabilities are emerging as the next generation of functional nanomedicines to improve the outcome of drug therapeutics. Among the many nanoparticulate systems, micelle-like aggregates or nanoparticles formed with amphiphilic block- or graft- copolymers are currently being studied for possible application as protein carriers. We recently developed a technique to prepare uniform nanoparticles (γ-PGA NPs) using amphiphilic γ-PGA (γ-PGA-L-PAE), in which L-phenylalanine ethyl ester (L-PAE) is introduced as a hydrophobic residue into the α-position group carboxyl of poly(γ-glutamic acid) (γ-PGA) which is a biodegradable polymer derived from a natto mucilage. γ-PGA NPs are excellent vaccine carriers capable of delivering antigenic proteins to antigen-presenting cells (APCs) and eliciting potent immune responses based on antigen-specific cytotoxic T lymphocytes. In mice, subcutaneous immunization with γ-PGA NPs entrapping ovalbumin (OVA) more effectively inhibited the growth of OVA-transfected tumors than immunization with OVA emulsified using Freund's complete adjuvant. In addition, γ-PGA NPs did not induce histopathologic changes after subcutaneous injection or acute toxicity through intravenous injection. Importantly, γ-PGA NPs efficiently delivered entrapped antigenic proteins into APCs through cytosolic translocation from the endosomes, which is a key process of γ-PGA NP-mediated anti-tumor immune responses. These antigen-capturing APCs migrated to regional lymph nodes. Our results demonstrate that a γ-PGA NP system for antigen delivery will advance the clinical utility of vaccines against cancer.
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  • Kentaro HATANAKA, Kosuke SHIMIZU, Tomohiro ASAI, Naoto OKU
    Type: Review
    2008 Volume 128 Issue 11 Pages 1567-1575
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Small interfering RNA (siRNA), which induces sequence-dependent gene silencing, has been widely studied. We previously developed polycation liposomes (PCL) as carriers of plasmid DNA and succeeded in showing their potent gene expression efficiency. In the present study, we optimized PCL for siRNA transfection and used it to determine the role of Argonaute2 (Ago2), a main constitution protein of RNA-induced silencing complex (RISC), on angiogenesis. We determined the biological effect of Ago2 knockdown on the angiogenic potential of endothelial cells. Human umbilical vein endothelial cells (HUVECs) stimulated with cytokines including vascular endothelial growth factor (VEGF) were used as an in vitro angiogenesis model. Our data showed that Ago2 knockdown using polycation liposomal Ago2-siRNA (siAgo2) suppressed indispensable processes of angiogenesis, namely endothelial cell proliferation and tube formation. Furthermore, TUNEL staining indicated that the treatment with siAgo2 increased apoptotic cells in comparison to that with control siRNA. These results might be caused by disorder of microRNA system. Next, we attempted to construct systemic siRNA delivery system targeting to angiogenic vessels. Synthetic siRNA was incorporated in polycation liposomes modified with polyethyleneglycol (PEG) and the functional peptide on their surface. Peptide-modified liposomes enhanced cellular uptake of siRNA in comparison with non-modified liposomes. Thus APRPG-modified liposomal siAgo2 may be useful for tumor treatment.
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  • Hidefumi MUKAI, Shigeru KAWAKAMI, Mitsuru HASHIDA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1577-1586
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      The kidney is one of the most important organs that play a crucial role in homeostasis and, therefore, congenital or acquired renal dysfunction causes refractory diseases, i.e., Alport's syndrome, Fabry's disease, diabetic nephropathy, IgA nephropathy, kidney cancer, transplant glomerulopathy. Nucleic acid transfection technology to the kidney is indispensable for the progress of biomedical research and the realization of gene therapy and nucleic acid drug for renal diseases. Control of renal nucleic acid transfection was difficult because of the structural complexity; however, the study of recombinant virus, synthetic carrier and physical force-mediated nucleic acid transfection to the kidney has advanced. Recombinant virus and synthetic carrier-mediated methods require long-term block of the blood or urinary flow for efficient transfection of nucleic acid because of the rich blood flow of the kidney. In contrast, physical force-mediated methods that transfect with nucleic acid via transient membrane permeability do not apprehend ischemia-reperfusion injury and, therefore, may be beneficial for nucleic acid transfection to the kidney. In this article, we collect the information of therapeutic gene, target molecule of the nucleic acid drug and target cells for renal diseases and structural property of the kidney from the point of view of nucleic acid transfection. Additively, current status of nucleic acid transfection technology to the kidney is reviewed.
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  • Yutaka SADAKANE, Makoto TSUNODA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1587-1588
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
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  • Makoto TSUNODA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1589-1594
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Catecholamines, namely, dopamine, norepinephrine and epinephrine, play important roles in higher animals as neurotransmitters or hormones, and are metabolized by catechol-O-methyltransferase (COMT). To elucidate the role of COMT in blood pressure regulation, we have developed simultaneous determination methods for catecholamines and their 3-O-methyl metabolites using high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence reaction detection. Using the developed method, we have found that inactivation of catecholamines by COMT is attenuated in hypertensive rats compared to normotensive rats. Furthermore, both the activities and the amounts of membrane-bound (MB-)COMT in the liver were found to be lower in hypertensive rats than in normotensive rats, which indicated that liver MB-COMT may be a relevant factor in blood pressure regulation.
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  • Yoshihide TANAKA, Nahoko NARUISHI
    Type: Review
    2008 Volume 128 Issue 11 Pages 1595-1604
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Psychological stress is of major importance to all age groups in recent years, and may lead to mental disorder and various diseases. An objective and quantitative method for measuring salivary stress-related substances is highly desired because saliva collection is easy, stress free and noninvasive. We have developed a rapid and easy-to-use analytical tool for the measurement of cortisol and secretory immunoglobulin A (sIgA) based on microchip technology, immunoselectivity and electrophoretic separation technique. Performing immunoreaction and capillary electrophoresis (CE) separation on microchips is a promising technique for on-site determination of biogenic substances, and has a few advantages over conventional immunoassay methods: reduced sample size, shortening analysis times, high separation efficiency, reduced cost, and downsizing of analytical system. At this stage of our research, some preliminary prototypes of a high-sensitive microchip CE instrument were constructed to determine the stress-related substances in real saliva samples. However, there is not enough detection sensitivity for cortisol analysis. On the other hand, sIgA was successfully analyzed using a laboratory-built microchip CE system and optimal analytical conditions. The sIgA determination is rapid compared with a conventional immunoassay method, and provides an acceptable degree of repeatability and recovery. In the future, microchip technologies will enable total automation and integration of sample preparation. This research has widespread future potential for monitoring multiple stress-related markers within minutes from a trace of saliva, and can contribute to disease prevention and overall good health.
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  • Kazuhisa FUJIMOTO, Masahiko INOUYE
    2008 Volume 128 Issue 11 Pages 1605-1613
    Published: 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      In this review, we report DNA duplex-based fluorescence probes/sensors using pyrene monomer-excimer switching. The review mainly comprises two topics: 1) excimer-monomer switching molecular beacons (EMS-MB) and 2) monomer-excimer switching sensors based on the structural motif of antibodies. The EMS-MBs have two pyrene fluorophores connected both at 3′ and 5′ ends of a single-stranded oligonucleotide. Emission switching occurs from excimer to monomer accompanying isoemissive points when the probes hybridized with target DNAs. The isoemissive points indicate the presence of only two fluorescent species, nonhybridized and hybridized probes in the mixtures, and thereby unambiguous detection of the targets is available. The probes can detect target 19-mer DNAs and can discriminate the targets from their single-nucleotide mismatches at 1 nM concentration. Furthermore, the EMS-MBs have been recently applied to kinetic study for RNase H activity by Tan et al. The structures and emission-switching properties of the EMS-MBs encourage us to develop a new class of fluorescent sensors based on the structural motif of antibodies. The sensors consist of three functional regions, benzo-15-crown-5 ether (or per-O-methylated β-cyclodextrin), DNA, and pyrene as guest-binding, dimerizing, and sensing sites, respectively. The crown- and CD-modified sensors can detect potassium cation and porphyrin derivatives, respectively, by monomer-excimer emission switching.
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  • Yutaka SADAKANE, Yasumaru HATANAKA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1615-1622
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Formation of a covalent bond using photoreactive groups allows one to maintain the complex between the ligand and its binding protein even under denaturing conditions. Thus, the photoreactive groups functioned as the powerful hook in the fishing of specific binding proteins. This is a very useful feature for developing the efficient and sensitive methods in molecular biology. Among the photoreactive groups, carbene-generating phenyldiazirine is a first choice for the application to the methods because the use of phenyldiazirine seems to be promising for achieving efficient crosslinking. This review describes improvements of phenyldiazirine usability and an application of phenyldiazirine to displayed phage screening. We synthesized the photoreactive diazirine units for a solution to preparing photoreactive ligands. Since the photoreactive units can be easily integrated into physiological ligands such as peptides, proteins, DNAs, and sugars by chemoselective reaction, the biochemists, who are not familiar with organic synthesis, can prepare the photoaffinity ligands using their interested ligands. We applied the phenyldiazirine to screening of displayed phages, and investigated the screening efficiency. The phages displayed specific-binding protein were extremely concentrated by using photoreactive peptide bearing diazirine. High efficiency in the screening is due to carry out intensive washing which removes almost all the unspecific binding phages. Moreover, such application overcomes the unavoidable drawback of photoreactive groups, the low efficiency of crosslinking because the isolated genes can be amplified.
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  • Yuichi HIRATSUKA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1623-1630
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Living system use many types of micro or nano-mechanical systems, which are called “motor protein”. Those biological motors have unique features, such as nano-meter scaled molecular motor, high efficiently energy transduction from chemical energy or having a capacity of self-assembly. The realization of bio-hybrid micro-machines to integrate such motor proteins and micro-or nano-structures fabricated of inorganic materials, would have some potential values that are not achieved by traditional electronic, magnetic or optical devices. In this paper, we discuss a possibility of motor proteins to use as driving unit for micro analysis systems, such as Lab on a chip or μTAS (micro Total Analysis System) devices.
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Reviews
  • Hitoshi NAKAYAMA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1631-1643
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Introduction to bioorganic chemistry by Prof. Kanaoka at the entrance of my research works affects greatly throughout the life afterward. Chemical modification studies of enzyme proteins taught me quality of chemical reactions. For example, triethyloxonium fluoroborate (Et3O+ BF4), a Meerwein reagent, selectively reacted with a particular carboxyl group (Asp-177) in the substrate binding site of trypsin, even though the reaction was performed in aqueous solution. A series of ion channel studies intoxicate me how exciting the science works are. Purification of sodium channel protein from electric eels initiated the collaboration work to reveal total primary structure of the molecule, as an inaugurating work of ion channel molecules. Photoaffinity labeling proved to be an efficient method to elucidate ligand binding sites, such as TTX binding site within the sodium channel and the sites for calcium anatagonists in L-type calcium channels. Encounter with CD36 molecule expands our works to more pathobiochemical field. We revealed CD36, a class B scavenger receptor, is related to development of atherosclerosis by phagocytosis of ox-LDL in macrophages and even matured adipocytes. In microglia, however, CD36 plays clearance role of oligomeric β-amyloid peptides in IL-4 activated type-2 microglia, suggesting the activation of type-2 microglia may be useful for developing a new method to treat or prevent from Alzheimer's disease.
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  • Yasushi OKUNO
    Type: Review
    2008 Volume 128 Issue 11 Pages 1645-1651
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      With the near completion of the human genome sequencing, bioinformatics and chemoinformatics are expected as promising tools in genome-based drug discovery. The emerging field of chemical genomics is accumulating large-scale assay data on compound-protein interactions. We are now developing new mining methods for the chemical genomics data based on the integration of bioinformatics and chemoinformatics. Here we present a GPCR-ligand database (GLIDA) and a novel in silico screening method, which we have developed. GLIDA is a novel public GPCR-related chemical genomics database that is primarily focused on the correlation of information between GPCRs and their ligands. Our in silico screening method is based on statistical machine learning of the conserved patterns of molecular recognition extracted from comprehensive compound-protein interaction data. These are promising approaches to accelerating drug discovery processes.
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  • Hirotatsu KOJIMA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1653-1663
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      The number of reports on new techniques in molecular imaging has been recently increasing because of their usefulness in biological, medical, and clinical research. Fluorescence imaging methods are generally superior in terms of sensitivity, selectivity and ease of use. Cyanine dyes have been employed as fluorescent labels in fluorescence imaging studies of biological mechanisms. In particular, tricarbocyanines have the advantage that light at their emission and absorption maxima in the near-infrared (NIR) region around 650-900 nm is relatively poorly absorbed by biomolecules, and so can penetrate deeply into tissues. There is also less autofluorescence in this region. In addition to cyanine dyes for straightforward fluorescence labeling, we successfully developed cyanine dyes whose fluorescence intensity changes upon specific reaction with nitric oxide, which is an important signaling molecule involved in the regulation of a wide range of physiological and pathophysiological mechanisms, and many disorders. Then, we synthesized dipicolylcyanine (DIPCY), consisting of tricarbocyanine as a fluorophore and dipicolylethylenediamine as a heavy metal chelator, and investigated its response to various heavy metal ions. Upon addition of zinc ion, a red shift of the absorbance maximum was observed. Namely, DIPCY can work as a ratiometric fluorescent sensor for zinc ion in the NIR region. Moreover, we have recently developed several pH probes based on the amine-substituted tricarbocyanine fluorophore. We could measure pH with these fluorescent probes by a ratiometric monitoring method.
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  • Yorishige IMAMURA
    Type: Review
    2008 Volume 128 Issue 11 Pages 1665-1672
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      In this paper, the structure and function of a new tetrameric carbonyl reductase (TCR) is reviewed. TCRs were purified from rabbit and pig heart, using 4-benzoylpyridine as a substrate. Partial peptide sequencing and cDNA cloning of rabbit and pig TCRs revealed that both enzymes belonged to the short-chain dehydrogenase/reductase family and that their subunits consisted of 260 amino acid residues. Rabbit and pig TCRs catalyzed the reduction of alkyl phenyl ketones, α-dicarbonyl compounds, quinones and retinals. Both enzymes were potently inhibited by flavonoids and fatty acids. 9,10-Phenanthrenequinone, which is efficiently reduced by rabbit and pig TCRs, mediated the formation of superoxide radical through its redox cycling in pig heart. The C-terminal sequences of rabbit and pig TCRs comprised a type 1 peroxisomal targeting signal (PTS1) Ser-Arg-Leu, suggesting that the enzymes are localized in the peroxisome. In fact, pig TCR was targeted into the peroxisomal matrix, in the case of transfection of HeLa cells with vectors expressing the enzyme. However, when the recombinant pig TCR was directly introduced into HeLa cells, the enzyme was not targeted into the peroxisomal matrix. The crystal structure of recombinant pig TCR demonstrated that the C-terminal PTS1 of each subunit of the enzyme was buried in the interior of the tetrameric molecule. These findings indicate that pig TCR is imported into the peroxisome as a monomer and then forms an active tetramer within this organelle.
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Regular Articles
  • Makoto TANABE, Machiko WATANABE, Mashiho YANAGI, Satoru NISHIZAWA, Yos ...
    Type: Regular Article
    2008 Volume 128 Issue 11 Pages 1673-1679
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      To develop a film formulation allowing controlled release for long-term analgesia, we selected ethyl cellulose (EC) as a novel additive, prepared a film formulation using indomethacin (IM film), and evaluated it in vitro and clinically. In the in vitro experiments, the effects of the EC concentration on the release rate of IM and on the adhesion force to the mucous membrane were investigated. The addition of 10% EC resulted in more sustained slow release compared with no EC, and the adhesion of the film with 10% EC added was similar to that of films containing carboxyvinyl polymer, which we reported previously showed significantly increased adhesion. A two-layered film consisting of an adhesive layer with 2% or 1% IM and 10% EC and a nonadhesive layer with 2% polyethylene glycol as a softening agent, was investigated for clinical use. Film consisting of an adhesive layer with 2% IM and 10% EC exhibited rapid onset of potent analgesia and was expected to prolong the duration of analgesia. These results suggest that IM film with EC added may be useful clinically, since it shows both immediate analgesic effects and prolonged duration of release.
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  • Hyo-Jung LEE, Hyo-Jeong LEE, Venkataraman MAGESH, Dongwoo NAM, Eun-Ok ...
    Type: Regular Article
    2008 Volume 128 Issue 11 Pages 1681-1688
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Lithospermum erythrorhizon has been used for treatment of inflammatory diseases and cancer as a folk remedy. Based on the evidences that anti-inflammatory agents frequently exert antiangiogenic activity, thus we examined comparatively the antiangiogenic activities of three naphthoquinone derivatives (shikonin, acetylshikonin, and isobutyroylshikonin) isolated from the plant. Three derivatives exhibited weak cytotoxicity against human umbilical vein endothelial cells (HUVECs) with IC50 of over 20 μM. Shikonin had more specific inhibitory effects on proliferation and vascular endothelial growth factor (VEGF) production by VEGF compared with different derivatives. All of derivatives significantly suppressed the migration of VEGF treated HUVECs at different optimal concentrations. Also, shikonin and acetylshikonin significantly disrupted VEGF-induced tube formation. Furthermore, three derivatives effectively downregulated the expression of urokinase-type plasminogen activator (uPA), but not its receptor uPAR. Additionally, shikonin significantly inhibited tumor growth in LLC-bearing mice, whereas its derivatives had relatively mild effects. Taken together, our findings suggest that shikonin and its derivatives exhibit the antiangiogenic and antitumorigenic effects by suppressing proliferation and angiogenic factors.
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  • Hong-ping PAN, Gao LI
    Type: Regular Article
    2008 Volume 128 Issue 11 Pages 1689-1698
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      The present study was designed to investigate the possible properties of the injured brain neurocytes, the expression of heat shock protein70 (HSP70) and Fas protein after acute local ischemia brain injury and local cerebral ischemia-reperfusion injury in rats and to investigate the protecting mechanism of puerarin on the brain neurocytes of rats in acute local ischemia brain injury and local cerebral ischemia-reperfusion injury. A rat model of acute local cerebral ischemia was made by ligatting the middle cerebral artery. The rat model of local cerebral ischemia and reperfusion injury was made by ligatting the middle cerebral artery for 30 min then opened for 30 min. Rats of puerarin treating group were injected with puerarin in dose of 30 mg/kg-1 by intraperitoneal injection 30 min before ischemia. HSP70 and Fas protein expressions in brain tissue were detected by SP method of histochemistry. In addition, dead brain neurocytes were counted and their morphology was observed. The results indicated that puerarin can limit the tissue injury caused by local cerebral ischemia injury through improving expression of HSP70, and limit the tissue injury caused by local cerebral ischemia-reperfusion through decreasing the Fas expression and improving expression of HSP70. On the basis of these results, it may be concluded that puerarin can protect the brain neurocytes of rats in acute local ischemia brain injury and local cerebral ischemia-reperfusion injury, which may be different according to the different injury mechanism.
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  • Nadeem KHAN, Nirmal SINGH, Amteshwar Singh JAGGI
    2008 Volume 128 Issue 11 Pages 1699-1705
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      The present study was designed to investigate a possible role of vanilloid receptors, CGRP and spleen in the induction of diabetes induced hyperalgesia in mice. Tail flick latency, an index of hyperalgesia, was assessed using analgesiometer. Serum nitrite levels and an index of nitric oxide were analyzed using Griess reaction. Mice were rendered diabetic with streptozotocin (200 mg/kg-1, i.p.) and kept for 30 days for development of diabetic pain. To explore the involvement of spleen in diabetic pain, spleen homogenate supernatant (SHS) was prepared from spleen of 30th day diabetic mice and administered in normal mice for 14 days. Both in diabetic and SHS treated mice, significant degree of hyperalgesia was developed, suggesting the possible role of spleen derived factor in induction of diabetic pain. Moreover, the levels of nitric oxide were also elevated in 30 day diabetic mice and SHS treated mice. Administration of ruthenium red (1 mg/kg-1, i.p.), vanilloid receptor antagonist, and sumatriptan (50 mg/kg-1, i.p.), a CGRP release inhibitor, attenuated diabetes and SHS induced decrease in nociceptive threshold and increase in serum nitrite oxide levels. These results suggest that spleen derived factor induced activation of vanilloid receptors and CGRP release may be contributing in the development of hyperalgesia in diabetic mice.
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Notes
  • Jun OGATA, Ruri KIKURA-HANAJIRI, Kayo YOSHIMATSU, Fumiyuki KIUCHI, Yuk ...
    2008 Volume 128 Issue 11 Pages 1707-1711
    Published: November 01, 2008
    Released: November 01, 2008
    JOURNALS FREE ACCESS
      Cannabis plants show a high Δ9-tetrahydrocannabinol content and are used as a psychoactive drug. Therefore the cultivation of hemp and its possession are prohibited by law in Japan. Meanwhile, Cannabis seeds have been used as a component of shichimi-togarashi (a Japanese spice), bird feed, or a crude drug (mashinin). To exclude the possibility of germination, it is officially noticed that hemp seeds must be killed. However, the number of violators has increased in recent years. To judge the ability of seed germination, a germination test is performed. However, the test requires several days and thus has not been used for on-site inspection. In this study, we developed a rapid detection method to determine the ability of Cannabis seeds to germinate using 2,3,5-triphenyl-2H-tetrazolium chloride (TTC). The principle of the assay is as follows. The endogenous respiratory enzymes in hemp seeds convert added colorless TTC into red 1,3,5-triphenylformazan. Consequently, a living embryo is stained red, while red does not appear in the dead seeds. The reaction was active over a pH range of 8.0-9.0, and the optimum activity was found from 40 to 50°C. Under the optimum conditions, we were able to determine the ability of seeds to germinate based on the presence of color within 20 min. Since this method is rapid and simple, it is applicable to on-site inspections. In addition, it could be used as an alternative technique to the germination test, because erroneous decisions is cannot occur under the assay principle.
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