Dose-dependent Antiherpesvirus Activity of an Ethnomedicinal Phytophores: Search for Magic Bullet
CHATTOPADHYAY D1*, ARUNACHALAM G1, MANDAL AB2, MAHMUD TAREQ HASSAN KHAN3, AND CHAKRABARTY S1
1ICMR Virus Unit, I.D. & B. G. Hospital, GB 4, 1ST Floor, 57, Dr. Suresh C Banerjee Road, Beliaghata, Kolkata 700010, India;
2Department of Biotechnology, Central Agricultural Research Centre, Port Blair, Andaman and Nicobar Islands, India; and
3School of Molecular and Structural Biology, Department of Pharmacology, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway.
Background: Ophiorrhiza nicobarica Balkr., a wild herb popularly used as an antiinfective medicament by the Shompen and Nicobarese tribes of Grate Nicobar Islands, India, was investigated for its anti-herpesvirus and antibacterial activities.
Methods: The whole herb was extracted in water and ethanol, and fractioned in n-butanol for the isolation of bioactive compounds using TLC, HPLC, HPTLC and NMR. The in vitro and in vivo toxicity of the extract were determined in Vero cell and in mouse model. The antiviral activity and its mechanism was tested by Cytopathic effect (CPE), Plaque reduction assay, Yield reduction assay, and Dose response curve using the Herpes Simplex Virus type 1 (HSV-1) and HSV type 2 (HSV-2). The EC50 (50% protection against virus induced cytopathic effect) and Selective Index (SI; ratio of 50% cellular cytotoxicity to EC50) was determined. The antibacterial activity was tested by disc diffusion and agar dilution methods on 150 bacterial isolates of human origin, and the mechanism was tested by biochemical and transport studies.
Results: The extract was non-toxic up to 3.6 mg ml-1, while at 300 mg ml-1 it completely inhibits plaque formation in HSV-1 and HSV-2. The antibacterial activity was noticed at 128-2000 µg ml-1 against 25 Gram negative and 53 Gram positive isolates. These data are highly significant (p<0.05) compared to the control. The bioactive part of the extract contains ursolic acid (triterpene), quercetin (flavonoid), ?-sitosterol and harmine (?-carboline indole alkaloid). The isolated ursolic acid and quercetin in combination (100?g ml-1 each) showed highly significant (P>0.001) anti-HSV activity (EC50 = 37.2 for HSV-1 and 45.1 for HSV-2), while harmine showed moderate anti-HSV activity at 300?gml-1 (EC50 = 74.4 for HSV-1 and 82.2 for HSV-2). Interestingly the eclipse phase initiation of HSV-2 was delayed at 100 mg ml-1 of ursolic acid. The yield reduction inhibition was 85% with ursolic acid, 68% with harmine and 51% with quercetin respectively for HSV-1. Moreover, the ursolic acid in combination with acyclovir showed the highest activity (EC50 20.1 and 32.2, SI was 87.5 and 90.4 respectively). The results revealed that the early stage of multiplication was blocked by quercetin while ursolic acid and harmine block the late stage of multiplication. While membrane destabilization in susceptible bacteria is noticed with ursolic acid (50-100?g ml-1).
Conclusion: The O. nicobarica extract have good anti-HSV activity, along with moderate antibacterial activity possibly due to its ursolic acid, quercetin and or harmine content. Studies showed that the extract is acting on early and late stage of HSV multiplication and have SI value grater than 20. These results suggest that this herb have the potential in the management of HSV infections, particularly in primary health care.
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Blocking Effect of an Immuno-Suppressive Agent, Cynarin, on CD28 of T-Cell Receptor Found by a Novel Pharmaceutical Method
CHEN HM
National Nano Device Laboratories, HsinChu, Taiwan, R.O.C.
Background: Resting T-cells are stimulated to initiate immune activity in response to specific external stimuli. The “signal 2” pathway is in part initiated by a binding interaction between CD28 (T-cell receptor) and CD80 (B-cell receptor). Recent efforts to find new immuno-suppressive drugs have focused on cell membrane receptor’s antibodies. However, using antibodies as drugs has many drawbacks. Also, unlike small molecules, which mimic metabolites in the body, the large antibodies might induce other immune system problems and therefore reduce their therapeutic utility.
Methods: In this study, cynarin was purified from Echinacea purpurea extracts on a silica gel open column with serial elution buffers. Based on HPLC analysis, the active fraction including cynarin was collected at a retention time of 32.612 min. The UV absorption peak shifted from 338 nm (crude extract) to 330 nm (cynarin pure fraction). The cytotoxicity of cynarin treatment of T-cells was measured by MTT colorimetric assay. Molecular modeling and docking among cynarin and CD28/CD80 were done by PRODRG2 based on AutoDockTools.
Results: Cynarin, a potential immunosuppressant that blocks the interaction between the CD28 of T-cell receptor and CD80 of antigen presenting cells, was found in Echinacea purpurea extract by a new pharmaceutical screening method: After Flowing Through Immobilized Receptor, (AFTIR; G.-C. Dong et al., J. Med. Chem., 2006, 249: 1845-1854). This Echinacea component is a small molecule that is able to specifically block “signal 2” of T-cell activation. In this study, we further confirm that cynarin effectively blocked the binding between CD80 of B-cells and CD28 of T-cells, and provide details of its mechanism of action.
Conclusions: The results above confirm both that AFTIR is a promising method for screening selective active compounds from herbal medicine and that cynarin has great potential as an immuno-suppressive agent.
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Synergistic inhibition of taxol- reisistance primary ovarian cancer cells by oridonin and wogonin
CHEN S1, WADES J1, JONES M1, BUTLER-MANUEL S2
1 Ovarian and Prostate Cancer Research Trust Laboratory, Guildford, Surrey, UK,
2 St. Luke Cancer Centre, Guildford, Surrey, UK
Abstract: In researching the molecular principle of Chinese herbal medicine for cancer therapy, we use the composition of the original US phyto product “PC SPES“ as a study model. Now no longer available, PC SPES was reported by several research groups to be active in suppressing hormone refractory prostate cancer. Among the isolated phytoactive chemicals, baicalein, oridonin isoliquiritigenin and wogonin were found to individually or combinatorially inhibit the ovarian cancer cell lines sensitive (A2780) or resistant (PTX10) to taxol. The activity was confirmed by MTS cell viability assay, SRB antiproliferation assay and colony formation assay. Although the four agents share some common molecular targets including the inactivation and down regulation of transcription factors NF-kB and androgen receptor, oridonin seems to display the most potent activity in modulating cancer cell apotosis and stabilization of p53 protein. To fully understand the action of botanical medicine, it is essential to investigate the combinatory effect of active agents. Our investigation has led to the discovery that differential synergism is dependant on the cancer cell type. While the strongest synergistic inhibition was observed in prostate cancer cell line DU-145 (isolated from hormone therapy refractory patients and androgen receptor insensitive) induced by the combination of oridonin and baicalein, the combined pair of oridonin and wogonin exhibited the most potent antiproliferative activity in ovarian cancer cells A2780 and PTX10. Since taxol is the last treatment option in ovarian cancer therapy, we further tested our finding on the primary ovarian cancer cells directly isolated from the ascitic fluid of patients. Preliminary results reveal that the individual and combined agents markedly suppress the proliferation of primary cancer cells. In contrast, taxol failed to inhibit both primary cancer cell cultures up to a concentration of 100 nM. In view of the different chemical structure and biological activity of terpenoids and flavonoids, we are interested to understand the scientific rationale of the combination. On this ground, we presently are studying the underlying molecular mechanism and the systems biology network affected by phytochemicals. This report discusses our initial findings. We thank Dr. Marianne Poruchynsky of NIH (Rockville, Maryland) for the gift of PTX10 cell line.
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From Mono- to Dimeric-IRFs: The Heart of the Matter in Activation of the Interferon Regulatory Factors
CHEN W, LAM SS, SRINATH H, SCHIFFER CA, LIN K, ROYER WE
Dept. of Biochemistry and Mol. Pharm., Univ. of Massachusetts Med. School, Worcester, MA 01605
Background: Members of the Interferon Regulatory Factor (IRF) family of proteins play important roles in development of the immune system, host defense, inflammation, apoptosis and tumorigenesis. Activation of these proteins in the cytoplasm is triggered by phosphorylation of Ser/Thr residues in a C-terminal autoinhibitory region. Phosphorylation stimulates dimerization, transport into the nucleus and assembly with the coactivator cAMP response element-binding binding protein (CBP)/p300 to activate transcription of type I interferons and other target genes. However, it is unknown how the phosphorylation of IRF proteins activates dimerization and why dimerization favors binding of CBP/p300.
Methods: To that end, x-ray crystallography, size-exclusion chromatography, isothermal titration calorimetry, and mutational studies are used in this study.
Results: We present here that the 2.0Å resolution crystal structure of a dimeric form of the IRF-5 transactivation domain (residues 215-477) in which Ser 440 has been mutated to the phosphomimetic Asp. The structure reveals a striking mechanism of dimerization in which the C-terminal autoinhibitory domain attains a highly extended conformation permitting extensive contacts to a second subunit. Based on comparison with crystal structures of IRF-3, these results provide a structural basis for the coupling between dimerization and CBP binding in IRF family members, in which the C-terminal autoinhibitory domain plays a dual role. In the unphosphorylated form, the C-terminal autoinhibitory domain binds to and masks the hydrophobic CBP/p300 binding surface.
Conclusion: Phosphorylation stimulates the unfolding of the C-terminal autoinhibitory domain which then forms extensive contacts with a second IRF-5 subunit, leaving a hydrophobic surface free for binding CBP/p300.
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iTRAQ-coupled 2D LC-MS/MS Analysis of Proteomics Profile in Cells Incubated with S- or R-enantiomers of Chiral Drugs: from Metabolic Pathways to Biomarker Discovery
CHING CB, SUI JJ, ZHANG JH, CHEN WN
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore
Background: Protein profile in cells incubated with individual enantiomers of chiral drugs is important to understand their differential mechanisms of action. Using propranolol, a β-blocker used for controling hypertension and myocardial infarction, the main aims are: 1) To establish cellular protein profile by LC-MS/MS apparoach. 2) To identify specific proteins secreted from cells incubated with propranolol.
Methods: The vascular smooth muscle A7r5 cells were cultured and incubated with the S- or R-propranolol at a concentration which showed no significant effect on the cell viability. A 4-plex multiplex strategy was used to simultaneously detect and quantitate differences in intracellular as well as extracellular proteins in untreated vascular smooth muscle cells and those incubated with S- or R-propranolol. The analysis was performed on an Agilent 1200 nanoflow LC system interfaced with a QSTAR XL mass spectrometer (Applied Biosystems). Relative abundance quantitation and protein identification were performed using ProteinPilotTM Software 2.0 (Applied Biosystems).
Results: A total of 40 and 13 unique proteins were identified in three independent experiments in cells and culture medium of cells incubated with S- or R-propranolol, respectively. For intracellular proteins, the higher level of enzymes involved in cellular anabolism and antioxidant activity in cells incubated with the S-propranolol was supported by Real-Time PCR and Western blot analyses. The increase in the anabolic activity was also supported by the higher intracellular concentration of the metabolic cofactor NAD+. For extracellular protein, only T-kininogen was found to be significantly increased in cells incubated with S-propranolol by Western blot analysis.
Conclusions: 1) Metabolic effect associated with propranolol treatment was revealed. The relevant metabolic enzymes may be useful targets for future pharmaceutical interventions to reduce clinical side effects following propranolol treatment. 2) The higher level of secreted T-kininogen from cells incubated with S-propranolol may provide a link of this vasoactive peptide to treatment of cardiovascular disease. 3) Our approach may be a platform for drug-target cells analysis and biomarker discovery.
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New targets for new drug discovery opportunities.
CHENE P
Oncology Research, Druggability-Enzymology-Profiling Unit, Novartis, Basel, Switzerland.
Today most pharmaceutical companies use similar strategies to target the same proteins. As a consequence there is a very high competition between the different pharma, a reduction of the potential markets, a lower innovation and a limited freedom to operate. Overall the productivity is decreasing. The identification of new drug targets is then very important for the future of the pharmaceutical industry.
During this presentation we shall examine the ATPases as a new family of drug targets. From a brief structural analysis of the ATP-binding site of these enzymes, we shall see that some these enzymes are very good drug targets for a strategy aiming for the identification of ATP competitive inhibitors.
Finally, we will show that it is possible to identify low molecular weight inhibitors of these enzymes and to develop them up to the clinic.
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Association between Alpha-2a-Adrenergic Receptor Gene and Methylphenidate Response in Korean Children and Adolescents with ADHD
CHEON K-A1*, CHO D-Y2, SHIM J-O1, KOO M-S1
1Univ. Kwandong, Koyang City, South Korea; 2Labgenomics, Seoul, South Korea.
Background: Methylphenidate (MPH), known to be effective for the attention deficit problems, blocks norepinephrine transporters and low oral doses of MPH have more effect on norepinephrine than on dopamine in subcortical areas. Alpha-2a-adrenergic receptor (ADRA2A) is a key component of the noradrenergic system. The aim of this study was to evaluate the association between the ADRA2A polymorphism and the clinical improvement of symptoms with MPH treatment in Korean subjects with ADHD.
Methods: This study included 114 ADHD children (mean age= 9.08±1.94 years) who were recruited from the child psychiatric clinic at university hospital in South Korea. The subjects who had an greater than or equal to 50% compared with the baseline ADHD rating scale (ARS) scores and who had 1 or 2 point of Clinical Global Impression–improvement (CGI-I) score after 8 weeks of treatment were considered as the ‘good response’ group. After performing genotyping for ADRA2A, we examined the correlation the ADRA2A polymorphism with MPH response and also compared the change of total ARS scores between genotypes at ADRA2A.
Results: e found that while 76.9% of the subjects with G/G genotype showed a good response, 46.0% and 41.7% of the subjects with C/G and C/C genotype showed a good response to MPH treatment according to ARS assessed by parent (Pearson χ2value=11.929, df=2, p=.003). We also found a significant difference of the change at total ARS scores between the subjects with and without G/G. (t=2.21, df=1, p=.029). In terms of treatment response according to the CGI-I, significant correlation was found between genotypes at ADRA2A (Pearson χ2value=7.250, df=2, p= .027).
Conclusions: Our findings provide evidence of an association between the ADRA2A genotype and response to MPH treatment assessed by both parents and clinician in ADHD subjects.
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From Gene Expression Profile to Identification of Molecular Target Granulin-Epithelin Precursor for Liver Cancer
CHEUNG ST, FAN ST
Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong.
Background: Primary liver cancer, hepatocellular carcinoma (HCC), is the third cancer killer worldwide. The majority of HCC patients have no effective therapeutic option. Aims: 1) To identify novel therapeutic target from the HCC microarray genome-wide expression profiles. 2) To develop neutralizing monoclonal antibody (mAb) against the therapeutic target and examine its functional effect.
Methods: The expression profiles of more than 200 HCCs and adjacent liver tissues were examined. Genes that demonstrated differential overexpression in significant portions of tumors, revealed clinically relevant biological functions, and preferably a secretory factor (diagnostic therapeutic concern) were short listed for further evaluation. The granulin-epithelin precursor (GEP, also named progranulin, acrogranin, or PC-derived growth factor) was identified as potential therapeutic target. The GEP expression on mRNA level was subsequently validated by real-time quantitative RT-PCR, on protein level by western blot and immunohistochemistry. The GEP mAb was raised against the peptide designed at the carboxyl-terminal of GEP. Neutralization effect was examined on hepatoma cells and normal liver cells in vitro and in vivo.
Results: The GEP overexpression was demonstrated in more than 70% of HCC tissues. GEP controls HCC cells proliferation, invasion and tumorigenicity. These in vitro and in vivo data corroborated the clinical findings that GEP overexpression is associated with aggressive cancer features including large tumors, venous infiltration (micrometastasis) and early recurrence after curative surgery. The GEP mAb inhibited the growth of human hepatoma cells Hep3B and HepG2, but revealed no significant effect on normal liver cells MIHA. In the nude mice model transplanted with human HCC cells Hep3B, GEP mAb decreased the serum GEP level and inhibited the growth of established tumors in a dose-dependent manner. The GEP mAb reduced tumor cell proliferation via the p44/42 MAPK and Akt pathways, and reduced tumor angiogenesis with reduced microvessel density and vascular endothelial growth factor (VEGF) level.
Conclusions: 1) GEP is a novel therapeutic target for liver cancer treatment. 2) GEP mAb inhibit tumor growth in a dosage dependent manner with anti-proliferative and anti-angiogenic functions.
Acknowledgement: The research work was supported in part by the Sun C.Y. Research Foundation for Hepatobiliary and Pancreatic Surgery, the Seed Funding Program of the University of Hong Kong, and the Hong Kong Research Grants Council (HKU 7392/03M, HKU 7560/06M, HKU 1/06C).
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Application of Superparamagnetic Nanoparticles in Purification of Plasmid DNA and Recombinant Protein from Bacterial Cells
CHIANG CL, CHEN CY
Department of Chemical and Materials Engineering,Southern Taiwan University, 710 Yung-Kang City, Tainan Hsien, Taiwan
Background: The purification of plasmid DNA or recombinant protein is fundamental to life science research, but some isolation methods can be physically and chemically damaging. Magnetic separation offers a gentle alternative. Targets are captured on magnetic particles coated with a target-specific surface, and separated from the sample using a magnetic field.
Methods: Nanosized superparamagnetic nanoparticles (Fe3O4) were prepared by chemical coprecipitation method using Fe2+, Fe3+ salt, and ammonium hydroxide under a nitrogen atmosphere. A quick and reliable method for the isolation and purification of transfection-grade plasmid DNA has been developed, using PEI-modified magnetic nanobeads as a solid-phase adsorbent. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of E. coli. A new immobilized metal ion affinity (IMA) adsorbent containing superparamagnetic nanoparticles and coated with hydrophilic resins are also proposed to improve the purification of His-tagged proteins.The GMA-IDA-coated magnetic Fe3O4 were employed for the direct extraction of recombinant protein, EGFP-(His)6, from E. coli lysates as a model system.
Results: Up to approximately 819 μg of high-purity (A260/A280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success.The Cu2+-charged GMA-IDA-coated adsorbent had the highest yield and purification factor at 70.4% and 12.3, respectively.
Conclusions: 1) PEI-modified magnetic nanobeads deliver significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. 2) GMA-IDA-coated magnetic adsorbent could be used as a suitable adsorbent for recombinant His-tagged protein from aqueous solution. Results proved that this new protein purification adsorbent provides a fast and efficient method for purifying His-tagged proteins with high yield and low background.
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PATZ1 gene has a critical role in the spermatogenesis and testicular tumors
CHIEFFI P
Department of Experimental Medicine, II University of Naples, Naples, Italy.
Background: PATZ1, also named MAZR or ZSG, is a recently discovered ubiquitously expressed transcriptional regulatory factor gene whose product binds to the RING finger protein RNF4 that in turn associates with a variety of transcription regulators. Due to the presence of the POZ domain, PATZ1 acts as a transcriptional repressor affecting the basal activity of different promoters. Recently, it has been shown that PATZ1 is an androgen receptor (AR) coregulator that acts by modulating the effect of the AR coactivator RNF4.
Methods: To gain insights into its biological role, we generated mice lacking the PATZ1 gene (PATZ1-/-)and utilized different technical approaches (Northern blot, Western blot and immunohistochemistry analyses) to characterize the expression PATZ1 gene.
Results: Male PATZ1-/- mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only spermatogonia and few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by Northern blot, Western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumor suppressor gene, we also looked at its expression in tumors deriving from testicular germ cells (TGCTs, carcinoma in situ, seminoma, teratoma, and embryonal carcinoma). Although expression of PATZ1 protein was increased in these tumors, it was delocalized in the cytoplasm, suggesting an impaired function.
Conclusions:These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mis-localization could be associated to the development of TGCTs.
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Pathological Studies on Thymic Lymphoma in Medaka Fish
CHIEN1 C-H, CHIEN EJ1, LIN T-L2, AND LIAO CF1,2
1University, National Yang-Ming, Taipei, ROC; 2Institution Academia Sinica, Taipei, ROC
Background: Five orange color strain of Japanese medaka (Oryzias latipes) fish, age from 8 to 24 months old, were diagnosed with thymic lymphoma. Grossly, the tumors protruded unilaterally out of the side of the head from the thymus.
Methods: Specimens were anesthetized with MS-222. Peripheral blood smears and tumor cells imprints were stained by Giemsa–Wright stain. The bodies of victims were fixed in Bouin’s fluid ready for histopathological study. Obtained tumor cells and tissues were fixed by 2.5% glutaraldehyde in 0.1 M cacodylate buffer for SEM and TEM studies.
Results: Metastatic lesions had infiltrated via both direct extension and vascular system in all fish by histopathological examination. The neoplastic mononuclear cells with transverse splitting were observed in blood smears. Mitotic neoplastic cells commonly appeared two or three nuclei in tumor tissue imprint. No virus particles was found after the investigation by TEM examination. The results obtained from SEM studies, the thymic neoplastic cells obtained from victims were identified to neoplastic lymphocytoid and lymphoblastoid. The surface of lymphoblastoid was smooth with some lamellate and pits. In addition, lymphocytoid cells had microvilli in appearence. The results obtained from TEM studies, lymphoblastoid possessed nuclear pockets and swollen mitochodria. Besides, lymphocytoid possessed few cytoplasm and vesicles inside the cells.
Conclusions: The thymic lymphoma presented in medaka at least origined from two different kinds of cells. The lymphoblast lymphoma/leukemia type showed aberrance in nuclear and edema in mitochondria. The lymphocytic lymphoma/leukemia showed aberrance in few cytoplasm, and nuclear cleft. The victims must suffer severely from anemia and neoplastic cell proliferation.
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Mifepristone Acts as Progesterone Antagonist of Non-genomic Responses but Agonist of Immunosuppression in Human T Cells
CHIEN EJ 1, LAI JN2, CHIEN C-H1, SHIE MC 1, LEE WF 1
1University, National Yang-Ming,Taipei, 2Hospital, Taipei Municipal Yang-Ming, Taipei, Republic of China.
Background: Progesterone is an endogenous immunomodulator to suppress T cell activation during pregnancy. The stimulation of membrane progesterone receptors might be the cause for rapid non-genomic responses in elevating intracellular calcium ([Ca2+]i) and decreasing intracellular pH (pHi) in human T cells. Mifepristone (RU486) exhibits mixed agonist/antagonist effects of progesterone on immune cells. It is necessary to explore whether these complicated effects come from RU486 being antagonist of rapid non-genomic response by progesterone.
Methods: The responses in pHi and [Ca2+]i changes were measured using the fluorescent dyes, BCECF and fura-2, respectively, in T cells. Proliferation was determined by [3H]-thymidine incorporation into phytohemagglutinin (PHA)-stimulated T cells.
Results: Equimolar amounts of the progesterone antagonist RU486 blocked the progesterone-mediated non-genomic responses on [Ca2+]i increases and pHi decreases. RU486 did not block the inhibitory effects of progesterone on PHA-stimulated T cell proliferation. Rather, RU486 was inhibitory. This inhibitory effects on proliferation caused by progesterone and RU486 were additive.
Conclusions: These results demonstrate that RU486 has dose-dependent mixed progesterone antagonist/agonist effects in T cells. RU486 is antagonistic to progesterone-stimulated non-genomic responses, but agonistic and synergistic with progesterone to suppress PHA-stimulated T cell proliferation. Our findings suggest a new light on the clinical application of RU486.
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