Publications by authors named "Guang-Yuh Hwang"

31 Publications

Ovatodiolide isolated from Anisomeles indica induces cell cycle G2/M arrest and apoptosis via a ROS-dependent ATM/ATR signaling pathways.

Eur J Pharmacol 2018 Jan 3;819:16-29. Epub 2017 Oct 3.

Department of Life Science, National Taitung University, Taitung, Taiwan, ROC. Electronic address:

Ovatodiolide was isolated from the traditional Chinese medicinal herb Anisomeles indica, possesses anti-bacterial and anti-inflammatory properties; however, the anti-cancer activity and its mechanisms have been limitedly reported. This study aimed to examine the effect and molecular action of ovatodiolide in lung cancer cells. Cell cycle distribution and reactive oxygen species (ROS) generation were measured by flow cytometry. Apoptosis was detected by propidium iodide/annexin V staining and TUNEL assay. DNA damage was investigated by comet assay and γ-H2AX staining. Caspase activity was determined using caspase fluorometric kits. Moreover, protein levels were examined by western blot. Ovatodiolide provoked reactive oxygen species generation and DNA damage, as well as inhibited cell growth and induced apoptosis in human lung cancer A549 and H1299 cell lines. DNA damage-related molecules, ATM/ATR and CHK1/CHK2 were activated by ovatodiolide. Moreover, ovatodiolide-mediated G2/M arrest was associated with the decrease of Cyclin B1 and CDC25C levels, and increase of p21 expression. Additionally, ovatodiolide-triggered apoptosis was through both intrinsic and extrinsic pathways characterized by the elevating PUMA, Bax, and DR5 proteins, decreasing Bcl-2 and Mcl-1, and activating caspase-8, caspase-9 and caspase-3. Caffeine, an ATM/ATR inhibitor, rescued ovatodiolide-mediated cell cycle arrest and apoptosis, but not reactive oxygen species generation. Nevertheless, antioxidant N-acetyl-cysteine completely blocked ovatodiolide-mediated molecular events, G2/M arrest, and apoptosis. These observations suggest that ovatodiolide stimulates reactive oxygen species generation, causes oxidative stress and DNA damage; subsequently, provokes DNA damage signaling pathways, eventually leads to block cell cycle at G2/M phase and trigger apoptosis in lung cancer A549 and H1299 cells.
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http://dx.doi.org/10.1016/j.ejphar.2017.09.050DOI Listing
January 2018

Potent Antiarthritic Properties of Phloretin in Murine Collagen-Induced Arthritis.

Evid Based Complement Alternat Med 2016 4;2016:9831263. Epub 2016 Dec 4.

Institute of Biomedical Science, National Chung Hsing University, Taichung, Taiwan; Department of Biotechnology, Asia University, Taichung, Taiwan; Department of Medical Research, China Medical University Hospital, Taichung, Taiwan.

In the exploration of potential therapeutic agents for rheumatoid arthritis (RA), DBA/1J mice are used as the RA model of collagen-induced arthritis (CIA). Phloretin, a flavonoid compound extracted from , has been found to exhibit anti-inflammatory activity, making it a potential candidate for treatment of RA. The objective of this study was to evaluate the therapeutic effects of phloretin on CIA mice. CIA mice were dosed daily with phloretin at either 50 or 100 mg/kg among two treatment groups. CIA treated mice showed mitigation of clinical symptoms of RA in addition to reduced inflammation of hind-limbs compared to mice who did not receive phloretin. Histological analysis showed that phloretin suppressed the severity of RA and effectively mitigated joint inflammation and cartilage- and bone-destruction via reducing proinflammatory cytokine productions (TNF-, IL-6, IL-1, and IL-17). This was at least partially mediated by causing inadequate splenocyte activation and proliferation. Moreover, phloretin-treated CIA mice showed decreased oxidative stress and diminished levels of malondialdehyde (MDA) and hydrogen peroxide (HO) in paw tissues as well as reduced productivity of anti-collagen antibodies in serum. We have concluded that phloretin could be a potent and effective antiarthritis agent, demonstrating anti-inflammatory, antioxidative, and immunomodulatory effects in CIA mice.
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http://dx.doi.org/10.1155/2016/9831263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5164908PMC
December 2016

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2.

Allergy Asthma Immunol Res 2015 Jul 5;7(4):376-83. Epub 2015 Mar 5.

Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan.; Department of Life Science, Tunghai University, Taichung, Taiwan.; Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan.

Purpose: Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach.

Methods: The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry.

Results: Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces.

Conclusions: Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.
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http://dx.doi.org/10.4168/aair.2015.7.4.376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446636PMC
July 2015

Polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression.

PLoS One 2015 6;10(2):e0117602. Epub 2015 Feb 6.

School of Chinese Medicine, China Medical University, Taichung, Taiwan; Department of Biotechnology, Asia University, Taichung, Taiwan; Department of Gynecology, China Medical University Hospital, Taichung, Taiwan.

Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9-induced IFN-β production.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117602PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319845PMC
February 2016

Inhibition of reverse-mode sodium-calcium exchanger activity and apoptosis by levosimendan in human cardiomyocyte progenitor cell-derived cardiomyocytes after anoxia and reoxygenation.

PLoS One 2014 3;9(2):e85909. Epub 2014 Feb 3.

Clinical Immunology Center, China Medical University Hospital, Taichung, Taiwan ; College of Medicine, China Medical University, Taichung, Taiwan.

Levosimendan, a known calcium sensitizer with positive inotropic and vasodilating properties, might also be cardioprotective during ischemia-reperfusion (I/R) insult. Its effects on calcium homeostasis and apoptosis in I/R injury remain unclear. Na(+)/Ca(2+) exchanger (NCX) is a critical mediator of calcium homeostasis in cardiomyocytes, with reverse-mode NCX activity responsible for intracellular calcium overload and apoptosis of cardiomyocytes during I/R. We probed effects and underlying mechanisms of levosimendan on apoptosis and NCX activity in cultured human cardiomyocyte progenitor cells (CPC)-derived cardiomyocytes undergoing anoxia-reoxygenation (A/R), simulating I/R in vivo. Administration of levosimendan decreased apoptosis of CPC-derived cardiomyocytes induced by A/R. The increase in reverse-mode NCX activity after A/R was curtailed by levosimendan, and NCX1 was translocated away from the cell membrane. Concomitantly, endoplasmic reticulum (ER) stress response induced by A/R was attenuated in CPC-derived cardiomycytes treated with NCX-targeted siRNA or levosimendan, with no synergistic effect between treatments. Results indicated levosimendan inhibited reverse-mode NCX activity to protect CPC-derived cardiomyocytes from A/R-induced ER stress and cell death.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085909PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911900PMC
December 2014

Hepatitis B virus X protein disrupts stress fiber formation and triggers apoptosis.

Virus Res 2013 Jul 13;175(1):20-9. Epub 2013 Apr 13.

Graduate Institute of Natural Products, Chang Gung University, Taoyuan, Taiwan.

Cytoskeletal proteins are key participants in the cellular progression to apoptosis. In a previous study we injected nude mice with CCL13-HBx cells and identified in contrast to non-HBx transfected cells a differentially phosphorylated myosin light chain (p-MLC) by two-dimensional PAGE and mass spectrometry of the tumor material. To investigate the role of HBx in myosin light chain kinase (MLCK) signaling pathways, we analyzed the key molecules, p-MLC and MLCK, by western blotting. Immunofluorescence staining analysis showed that HBx disrupted stress fiber formation and that focal adhesion kinase (FAK) and integrin-linked kinase (ILK) were regulated by HBx-mediated phosphatase and tensin homolog (PTEN). We also used pharmacological inhibitors to explore the correlation between cytoskeletal rearrangements and HBx-mediated cell apoptosis via an MLCK and a PTEN-dependent pathway. The results showed that both ML9 and bvp restored the effects caused by HBx induction. Our findings suggest that HBx disrupts stress fiber formation and triggers apoptosis via an MLCK and a PTEN-dependent pathway.
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http://dx.doi.org/10.1016/j.virusres.2013.03.017DOI Listing
July 2013

Herbal Supplement Ameliorates Cardiac Hypertrophy in Rats with CCl(4)-Induced Liver Cirrhosis.

Evid Based Complement Alternat Med 2012 6;2012:139045. Epub 2012 Nov 6.

Division of Cardiovascular Surgery, China Medical University Hospital, Taichung 40402, Taiwan ; Department of Life Science, Tunghai University, Taichung 40704, Taiwan.

We used the carbon tetrachloride (CCl(4)) induced liver cirrhosis model to test the molecular mechanism of action involved in cirrhosis-associated cardiac hypertrophy and the effectiveness of Ocimum gratissimum extract (OGE) and silymarin against cardiac hypertrophy. We treated male wistar rats with CCl(4) and either OGE (0.02 g/kg B.W. or 0.04 g/kg B.W.) or silymarin (0.2 g/kg B.W.). Cardiac eccentric hypertrophy was induced by CCl(4) along with cirrhosis and increased expression of cardiac hypertrophy related genes NFAT, TAGA4, and NBP, and the interleukin-6 (IL-6) signaling pathway related genes MEK5, ERK5, JAK, and STAT3. OGE or silymarin co-treatment attenuated CCl(4)-induced cardiac abnormalities, and lowered expression of genes which were elevated by this hepatotoxin. Our results suggest that the IL-6 signaling pathway may be related to CCl(4)-induced cardiac hypertrophy. OGE and silymarin were able to lower liver fibrosis, which reduces the chance of cardiac hypertrophy perhaps by lowering the expressions of IL-6 signaling pathway related genes. We conclude that treatment of cirrhosis using herbal supplements is a viable option for protecting cardiac tissues against cirrhosis-related cardiac hypertrophy.
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http://dx.doi.org/10.1155/2012/139045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517219PMC
December 2012

Effector mechanisms of sunitinib-induced G1 cell cycle arrest, differentiation, and apoptosis in human acute myeloid leukaemia HL60 and KG-1 cells.

Ann Hematol 2013 Mar 20;92(3):301-13. Epub 2012 Nov 20.

Division of Hematology/Oncology, Department of Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China.

Acute myeloid leukaemia (AML) is a heterogeneous disease with dismal outcome. Sunitinib is an orally active inhibitor of multiple tyrosine kinase receptors approved for renal cell carcinoma and gastrointestinal stromal tumour that has also been studied for AML in several clinical trials. However, the precise mechanism of sunitinib action against AML remains unclear and requires further investigation. For this purpose, this study was conducted using human AML cell lines (HL60 and KG-1) and AML patients' mononucleated cells. Sunitinib induced G1 phase arrest associated with decreased cyclin D1, cyclin D3, and cyclin-dependent kinase (Cdk)2 and increased p27(Kip1), pRb1, and p130/Rb2 expression and phosphorylated activation of protein kinase C alpha and beta (PKCα/β). Selective PKCα/β inhibitor treatment abolished sunitinib-elicited AML differentiation, suggesting that PKCα/β may underlie sunitinib-induced monocytic differentiation. Furthermore, sunitinib increased pro-apoptotic molecule expression (Bax, Bak, PUMA, Fas, FasL, DR4, and DR5) and decreased anti-apoptotic molecule expression (Bcl-2 and Mcl-1), resulting in caspase-2, caspase-3, caspase-8, and caspase-9 activation and both death receptor and mitochondria-dependent apoptosis. Taken together, these findings provide evidence that sunitinib targets AML cells through both differentiation and apoptosis pathways. More clinical studies are urgently needed to demonstrate its optimal clinical applications in AML.
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http://dx.doi.org/10.1007/s00277-012-1627-7DOI Listing
March 2013

Apoptosis induced by hepatitis B virus X protein in a CCL13-HBx stable cell line.

Oncol Rep 2012 Jul 23;28(1):127-32. Epub 2012 Apr 23.

Department of Life Science, Tunghai University, Taichung, Taiwan, ROC.

The hepatitis B virus X protein (HBx) critically modulates cell growth by inducing apoptosis or proliferation. We sought to clarify whether HBx-mediated apoptosis in a CCL13 stable cell line (Chang-HBx) with inducible HBx expression proceeds through the extrinsic (death receptor-mediated) and/or intrinsic (mitochondrial-mediated) pathways of apoptosis. We used western blotting, cell viability assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, caspase activity assays, JC-1 staining and DNA fragmentation analysis to study the role of HBx in apoptosis. The expression of the pro-apoptotic proteins Bax and Bad and the release of cytochrome c also increased slightly upon HBx induction. JC-1 staining showed a loss of mitochondrial membrane potential upon HBx induction. Additionally, induction of HBx increased the levels of cleaved caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway) and the common effector caspase-3 as measured by western blotting. This elevation of cleaved caspase-8 or caspase-3 and caspase-9 or caspase-3 decreased in the presence of caspase-8 inhibitor Z-IETD-FMK or caspase-9 inhibitor Z-LEHD-FMK, respectively. Both inhibitors also rescued cell growth, and the caspase-8 inhibitor Z-IETD-FMK prevented apoptotic phenomena including the TUNEL signal. DNA fragmentation analysis showed that these phenomena were not detected in the presence of higher concentration of inhibitors. Our data suggest that HBx induces apoptosis through both extrinsic and intrinsic pathways.
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http://dx.doi.org/10.3892/or.2012.1775DOI Listing
July 2012

Sensitization to Per a 2 of the American cockroach correlates with more clinical severity among airway allergic patients in Taiwan.

Ann Allergy Asthma Immunol 2012 Apr 14;108(4):243-8. Epub 2012 Feb 14.

Department of Education and Research, Taichung Veterans General Hospital, and General Education Center, Overseas Chinese University, Taichung, Taiwan.

Background: In Taiwan, 57.5% of asthmatic patients are allergic to cockroaches, which are a major indoor allergen for immunoglobulin E (IgE)-mediated respiratory diseases.

Objective: To determine whether sensitization to different cockroach allergenic components correlates with different clinical manifestations and severities.

Methods: The complementary DNAs (cDNAs) encoding for Per a 1 through 7 and Per a 9 were generated by reverse transcription polymerase chain reaction and cloned into the Escherichia coli expression system. Sixty-four subjects were divided into 3 groups based on the clinical severity of their allergic reaction: those with persistent asthma and rhinitis (AS), those with allergic rhinitis only (AR), and the nonallergic controls (NA). Serum levels of interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), chemokine (C-C motif) ligand 20 (CCL-20), and granulocyte macrophage colony-stimulating factor (GM-CSF) were measured, and the binding frequencies to each recombinant allergen were examined.

Results: Serum levels of IL-8, MCP-1, and CCL-20 were significantly higher in the AS group than in the AR and NA groups. The numbers of IgE-binding allergens did not correlate with the clinical severity of airway allergy to cockroaches. However, 81% in the AS group had IgE-binding activity to Per a 2, which was significantly higher than that of the AR group (45%, P < .05). In contrast, 80% of AR patients had IgE-binding activity to Per a 9 compared with only 28.5% of AS patients (P < .01).

Conclusion: Allergens from American cockroaches do not have equal importance in terms of pathogenicity. Sensitization to Per a 2 correlates with more severe airway allergy and elevated proinflammatory chemokines. This may help in selecting target allergens for component resolved diagnosis and immunotherapeutic agents.
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http://dx.doi.org/10.1016/j.anai.2012.01.014DOI Listing
April 2012

Specific IgE and IgG responses and cytokine profile in subjects with allergic reactions to biting midge Forcipomyia taiwana.

Int Arch Allergy Immunol 2009 2;150(1):66-74. Epub 2009 Apr 2.

Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan.

Background: Forcipomyia taiwana is a tiny blood-sucking midge whose habitat covers large parts of Taiwan and southern China. Female midges bite during the day, causing intense pruritus and swelling in allergic individuals. In this study, we investigated the immune responses of different allergic reactions to midge bites.

Methods: F. taiwana (midge)-specific IgE, -IgG and -IgG subclasses were examined by ELISA in 62 human subjects. Peripheral blood mononuclear cells (PBMC) from 6 subjects with solely delayed reactions (SDR) to midge bites and 6 nonallergic controls (NAC) were cultured with midge extract at various time points and assayed. Proliferation of PBMC was measured by MTT assay. Expression of cytokine mRNA was measured by real-time PCR and protein levels by cytometric bead immunoassay or ELISA. Protease activity in midge extract was determined by the Azocoll method.

Results: Midge-specific IgE among subjects with an immediate reaction were significantly elevated compared to SDR and NAC subjects. There were no differences in the level of midge-specific-IgG, -IgG(1), -IgG(2), -IgG(3) and -IgG(4) among subjects with different biting reactions. Midge extract elicited significantly more PBMC proliferation, higher expression of IFN-gamma, IL-10, IL-6 and TNF-alpha in SDR subjects than in NAC. Protease activity was detected in midge extract. Protease inhibitors E64 and pepstatin suppressed midge-extract-induced IL-8 production.

Conclusions: Our results suggest that an immediate reaction to midge bites is IgE-mediated. IFN-gamma, IL-6 and TNF-alpha are involved in delayed reactions to midge bites. A protease-activated pathway may also be involved in the intense, itchy reactions to midge bites.
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http://dx.doi.org/10.1159/000210382DOI Listing
September 2009

Pregnancy-induced hemophagocytic lymphohistiocytosis combined with autoimmune hemolytic anemia.

J Chin Med Assoc 2009 Mar;72(3):156-9

Department of Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China.

Hemophagocytic lymphohistiocytosis (HLH), presenting with fever, cytopenia, liver dysfunction, hepatosplenomegaly, hypertriglyceridemia, and hyperferritinemia, is associated with various etiologies, including infections, collagen vascular diseases, and malignancies. The present report describes a 28-year-old woman who developed HLH combined with autoimmune hemolytic anemia (AIHA) at 23 weeks of gestation. Without response to corticosteroid, the patient completely recovered from both HLH and AIHA after termination of the pregnancy. Pregnancy-induced immune dysregulation and cytokine overproduction in genetically susceptible women may play critical roles in HLH. The differential diagnosis of pregnant women with fever and cytopenia should include HLH. Pregnancy termination should be considered when pregnancy-induced HLH is refractory to medical treatment.
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http://dx.doi.org/10.1016/S1726-4901(09)70043-7DOI Listing
March 2009

Functional characterization of hepatitis B virus X protein based on the inhibition of tumorigenesis in nude mice injected with CCL13-HBx cells.

Intervirology 2008 30;51(4):253-60. Epub 2008 Sep 30.

Department of Life Science, Tunghai University, Taichung, Taiwan.

Objective: This study aimed to determine the effects of HBx on the inhibition of tumorigenesis in nude mice injected with CCL13-HBx cells. Therefore, the characteristics of the induced tumors and the phenomenon of apoptosis were assessed.

Methods: The induced tumors were identified using the specific marker of hepatocellular carcinoma (HCC), anti-alpha-fetoprotein (AFP), and their characteristics were pathologically examined. Apoptosis was detected by DNA fragmentation, and the expression of the proapoptotic proteins p53, Bax, Bad, caspase-3, and caspase-8 and the anti-apoptotic protein Bcl-2 was detected by Western blotting. To identify possible molecules involved in the inhibition of tumorigenesis, extracts of the induced tumors were separated by 2D-PAGE, and the proteins were identified by MS.

Results: The tumors of the nude mice injected with CCL13 and CCL13-HBx cells were identified as HCCs. Moreover, HBx was found to suppress tumor growth via apoptosis in the nude mice injected with CCL13-HBx cells. The MS findings revealed that phosphorylated myosin light chain was a candidate molecule involved in the inhibition of tumorigenesis.

Conclusion: HBx suppressed tumorigenesis in the nude mice injected with CCL13-HBx cells, which proved to be a good animal model for the in vivo study of the effects of HBx on tumorigenesis.
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http://dx.doi.org/10.1159/000158522DOI Listing
December 2008

HBx inhibits the growth of CCL13-HBX-stable cells via the GSK-3beta/beta-catenin cascade.

Intervirology 2008 16;51(2):130-6. Epub 2008 Jun 16.

Department of Life Science, Tunghai University, Taichung, Taiwan.

Objective: The hepatitis B virus X protein (HBx) plays critical roles in cell survival via modulation of signaling pathways. In our previous studies, we reported that HBx inhibited the growth of CCL13-HBx-stable cells (Chang-HBx cells) in vitro and tumor formation in vivo in CCL13-HBx-cell-injected nude mice; however, this inhibition mechanism is unclear.

Methods: To investigate the role of HBx in Wnt-3/beta-catenin signaling pathways, we focused on the key molecules GSK-3beta and beta-catenin, and analyzed by Western blotting and immunofluorescence staining.

Results: Results indicated that following HBx induction, GSK-3beta activity was up-regulated, the expression and accumulation of beta-catenin in the nucleus were decreased, and cell proliferation was suppressed. Inhibition of GSK-3beta activity by pharmacological inhibitors rescued the expression and accumulation of beta-catenin in the nucleus and facilitated cell proliferation and growth following HBx induction. The localization of beta-catenin, which is involved in cell proliferation, and mediated by GSK-3beta activation was also demonstrated.

Conclusion: Our findings suggest that HBx negatively regulated proliferation of CCL13-HBx-stable cells via the GSK-3beta/beta-catenin cascade.
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http://dx.doi.org/10.1159/000139131DOI Listing
September 2008

Primary splenic lymphoma associated with hemophagocytic lymphohistiocytosis complicated with splenic rupture.

J Chin Med Assoc 2008 Apr;71(4):210-3

Division of Hematology/Oncology, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, R.O.C.

Primary splenic lymphoma (PSL) is a rare disease with ambiguous definition, comprising less than 1% of non-Hodgkin's lymphoma. Even rarer is PSL combined with hemophagocytic lymphohistiocytosis (HLH), which has presentations of fever, cytopenia, hepatosplenomegaly, hyperferritinemia, and phagocytosis of hematopoietic cells in the reticuloendothelial system. We report the case of a 77-year-old man who presented with HLH initially. Refusing diagnostic splenectomy, he received chemotherapy. Spontaneous splenic rupture occurred after chemotherapy. In the following emergency operation, PSL was diagnosed. He received another 5 courses of chemotherapy with the R-CNOP regimen (rituximab, cyclophosphamide, mitoxantrone, vincristine, prednisolone). Now he has no residual or relapsed disease. Diagnostic splenectomy for adult HLH patients without definite etiologies may play an important role.
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http://dx.doi.org/10.1016/S1726-4901(08)70106-0DOI Listing
April 2008

Effects of hepatitis B virus X protein (HBx) on cell-growth inhibition in a CCL13-HBx stable cell line.

Intervirology 2008 29;51(1):26-32. Epub 2008 Feb 29.

Department of Life Science, Tunghai University, Taichung, Taiwan.

Objective: The known function of hepatitis B virus X protein (HBx) is to determine the fate of cells by modulating various signaling pathways. In our previous study, we demonstrated that HBx inhibits tumor formation in nude mice injected with CCL13-HBx stable cell lines; however, the mechanism underlying this inhibition is unclear.

Methods: To investigate the possible mechanisms underlying HBx involvement in CCL13-HBx cells, gene profiles were initially analyzed by DNA microarray technology and subsequently confirmed by performing semiquantitative RT-PCR and Western blotting. Furthermore, the phenomenon of cell death via apoptosis was detected via DNA fragmentation, TUNEL staining, caspase-3 activity assay, and propidium iodide (PI) staining.

Results: The results indicated that HBx induction downregulated Wnt-3 and beta-catenin that are involved in cell proliferation. Moreover, HBx induction repressed cell growth and downregulated the expressions of cyclin D1, CDK4, cyclin E, CDK2, and cyclin B1. Furthermore, HBx induction triggered cell death via apoptosis, as determined by DNA fragmentation, TUNEL staining, caspase-3 activity assay, and PI staining.

Conclusion: Most importantly, our results indicated that HBx induction in the CCL13-HBx stable cell line downregulated Wnt-3/beta-catenin expression and suppressed cell growth by repressing cell proliferation or triggering cell apoptosis.
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http://dx.doi.org/10.1159/000118793DOI Listing
April 2008

Characterization of porcine arterial endothelial cells cultured on amniotic membrane, a potential matrix for vascular tissue engineering.

Biochem Biophys Res Commun 2007 Jun 18;357(4):984-90. Epub 2007 Apr 18.

Department of Life Science, Tunghai University, Taichung, Taiwan.

The existing of basement membrane improves the development of endothelium while constructing blood vessel equivalent. The amniotic membrane (AM) provides a natural basement membrane and has been used in ocular surface reconstruction. This study evaluated the molecular and cellular characteristics of porcine vascular endothelial cells (ECs) cultured on AM. ECs cultured on AM expressed the endothelial marker vWF and exhibited normal endothelial morphology. Here, we demonstrated that AM enhanced the expression of intercellular molecules, platelet-endothelial cell adhesion molecule-1 (PECAM-1), and adhesion molecule VE-cadherin at the intercellular junctions. The expression level of integrin was markedly higher in ECs cultured on AM than on plastic dish. Furthermore, the AM downregulated the expression of E-selectin and P-selectin in both LPS-activated and non-activated ECs. Consistently, adhesion of leukocytes to both activated and non-activated cells was decreased in ECs cultured on AM. Our results suggest that AM is an ideal matrix to develop a functional endothelium in blood vessel equivalent construction.
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http://dx.doi.org/10.1016/j.bbrc.2007.04.047DOI Listing
June 2007

Lactate dehydrogenase, not vascular endothelial growth factor or basic fibroblast growth factor, positively correlates to bone marrow vascularity in acute myeloid leukemia.

J Chin Med Assoc 2006 Nov;69(11):534-7

Division of Hematology/Oncology, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan.

Background: Angiogenesis has been extensively studied in acute myeloid leukemia (AML). Lactate dehydrogenase (LDH), a common biochemical marker for tumor burden and anaerobic glycolysis, is a poor prognostic factor for AML. Regulated by hypoxia-induced factor, both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are responsive to cancer-related angiogenesis. To study the roles of serum LDH, VEGF and bFGF in AML angiogenesis, we investigated bone marrow vascularity in untreated AML patients, and analyzed its relationship to serum LDH, VEGF and bFGF levels.

Methods: Eighteen (11 males, 7 females; mean age, 57.7 years) de novo, untreated AML patients were enrolled. Bone marrow vascularity was evaluated by staining bone marrow core biopsy tissue with endothelial cell marker CD31 or CD34. Serum LDH was determined with the Wroblewski-La Due method. Serum VEGF and bFGF were determined with enzyme-linked immunoassay. The relationship of LDH, VEGF and bFGF level to bone marrow vessel numbers was examined by linear regression.

Results: Log LDH significantly correlated to AML bone marrow vascularity (r = 0.61; p = 0.007). VEGF and bFGF concentrations did not correlate with AML angiogenesis.

Conclusion: These results suggest that serum LDH, but not VEGF and bFGF concentrations, can be used as a simple parameter for predicting vessel formation in AML bone marrow.
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http://dx.doi.org/10.1016/S1726-4901(09)70324-7DOI Listing
November 2006

Increased prevalence of interleukin-1 receptor antagonist gene polymorphism in patients with chronic rhinosinusitis.

Arch Otolaryngol Head Neck Surg 2006 Mar;132(3):285-90

Department of Otolaryngology, China Medical University Hospital, Taichung, Taiwan.

Objective: To assess the association of the interleukin (IL)-1beta and the IL-1 receptor antagonist (IL-1Ra) gene polymorphisms with chronic rhinosinusitis (CRS).

Design: Genotyping of the 2 IL-1beta gene (IL1B) polymorphisms (promoter and exon) and the IL-1Ra gene (IL1RN) polymorphism (intron 2) was performed using polymerase chain reaction and restriction length fragment polymorphism analyses.

Setting: Prospective study, tertiary medical center.

Patients: The study population comprised 88 consecutive adult Taiwan-Chinese patients who met stringent criteria for CRS and received endoscopic sinus surgery and 103 healthy volunteers of the same ethnicity and similar age range. Of the 88 patients, 61 had CRS with nasal polyps, while the other 27 had CRS without nasal polyps.

Results: There were significant differences in the distribution of the IL1RN polymorphism between the control subjects and patients with CRS (P<.05). The II allele of IL1RN occurred more frequently in the CRS patient group, and the odds ratio for subjects with I/II genotype was 3.39 (95% confidence interval, 1.25-9.18). In the case of CRS without nasal polyps, the odds ratio for subjects with I/II genotype was further increased to 4.75 (1.39-16.25). There was no association between the other 2 polymorphisms of IL1B and CRS.

Conclusion: Increased prevalence of IL1RN polymorphism in patients with CRS suggests that this polymorphism, or a polymorphism in linkage disequilibrium with it, may be involved in the development of CRS.
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http://dx.doi.org/10.1001/archotol.132.3.285DOI Listing
March 2006

Altered expression profile of superoxide dismutase isoforms in nasal polyps from nonallergic patients.

Laryngoscope 2006 Mar;116(3):417-22

Department of Otolaryngology, China Medical University Hospital, Taichung, Taiwan.

Objective/hypothesis: Nasal polyposis (NP) is a chronic inflammatory disease of the upper respiratory tract. The pathophysiology is unknown but has been shown to be multifactorial. Free radical-mediated damage has been implicated in the pathogenesis of NP. Superoxide dismutases (SODs) are the first and the most important line of antioxidant enzyme defense against reactive oxygen species. Moreover, isozymes of the SOD family are critical for modulating the activity of nitric oxide, a gaseous free radical that is believed to play roles in the physiology and pathology of respiratory tracts. However, the expression profile of SOD isoforms in NP remains unclear. We aimed to investigate the expression profile of the SOD isoforms in nasal polyps from nonallergic patients.

Study Design: Prospective study.

Methods: Nasal polyp tissues were obtained from eight nonallergic patients who underwent elective polypectomy; mucosal specimens from the middle turbinates were acquired from eight subjects without NP as control tissues. The expression profile of SOD isoenzymes, SOD1, SOD2, and SOD3, in the nasal tissues was determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB).

Results: NP in all eight of the NP patients manifested as severe or recurrent sinonasal polyposis clinically. The expression pattern of SOD isoenzymes evaluated by RT-PCR analysis indicated that the mean levels of SOD1 mRNA and, to a greater extent, SOD3 mRNA were higher in polyp tissues than in control tissues. There was no significant difference in the expression levels of SOD2 mRNA between the two groups. The data from ELISA and WB analysis showed that there were increased expressions of SOD1 and SOD3 protein in polyp tissues compared with the control tissues, but there was no difference in the expression of SOD2 protein between the two groups. The results from RT-PCR, ELISA, and WB were paralleled and revealed that the expressions of SOD1 and, to a greater extent, SOD3 were higher in polyp tissues than in the control group.

Conclusions: The expressions of SOD3 and SOD1 were higher in polyp tissues. These results are consistent with previously reported data and support the hypothesis that there is increased oxidative stress in NP. Our data also suggest that the SODs might be important in the pathogenesis of NP; however, the roles these SOD isoforms, especially SOD3, play in both normal nasal mucosa and NP require further clarification.
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http://dx.doi.org/10.1097/01.MLG.0000199738.37455.55DOI Listing
March 2006

Identification of hevamine and hev B 1 as major latex allergens in Taiwan.

Int Arch Allergy Immunol 2006 4;139(1):38-44. Epub 2005 Nov 4.

Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, ROC.

Background: Proteins from latex gloves have been documented to trigger occupational latex allergy among health care workers. Allergen characterization of latex glove extract has never been studied in Taiwan. This study aimed to identify allergenic proteins from latex gloves.

Methods: Crude extracts of latex gloves were prepared with phosphate-buffered saline and 20 medical workers with a history of latex allergy were enrolled in this study. The specific IgE antibody was determined by the Pharmacia CAP system and in-house enzyme-linked immunoassay and immunoblotting. The target proteins were excised from two-dimensional PAGE and analyzed by electrospray ionization tandem mass spectrometry.

Results: Immunoblotting of glove extracts revealed three IgE-binding proteins at a molecular mass of 45, 30 and 14 kDa. Peptide mass fingerprinting revealed that the protein at 45 kDa, which was recognized by 10% (2/20) of atopic sera tested, was an allergenic lipolytic esterase from Hevea brasiliensis (Hev b 13). The 30- and 14-kDa proteins, which were recognized by 55% (11/20) and 85% (17/20) of patients' sera, were found to be hevamine and rubber elongation factor (Hev b 1), respectively.

Conclusions: Our results indicated that hevamine and Hev b 1 are the major allergens from latex gloves in Taiwan, which differs from the reports in Western countries.
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http://dx.doi.org/10.1159/000089521DOI Listing
February 2006

Molecular cloning of Indian jujube (Zizyphus mauritiana) allergen Ziz m 1 with sequence similarity to plant class III chitinases.

Mol Immunol 2006 Mar 24;43(8):1144-51. Epub 2005 Aug 24.

Taichung Veterans General Hospital, Department of Education and Research, 160 Chang Kang Road, Section 3, Taichung 407, Taiwan.

Indian jujube (Zizyphus mauritiana) is a sweet fruit that is abundantly cultivated in Taiwan. We have previously identified 42 and 30 kDa allergens that are cross-reactive with latex allergen from crude Indian jujube extract. This study aimed to clone the 30 kDa Ziz m 1 Z. mauritiana allergen. The Ziz m 1 encoding cDNA was isolated from a ZAPII cDNA library constructed from Z. mauritiana mRNA, sequenced and expressed in Pichia pastoris. The protein predicted from the cDNA sequence has 330 amino acids, the first 25 of which constituted a putative signal peptide. The deduced molecular mass of the mature protein is 33.86 kDa, while its isoelectric point is estimated at 4.36. The recombinant Ziz m 1 showed chitinase activity, possessed IgE binding capacity, and had IgE cross-reactivity with the latex allergen. Moreover, anti-recombinant Ziz m 1 antibody-based ELISA was able to detect commercial skin testing latex reagent, laboratory prepared latex and Indian jujube extracts. Recombinant Ziz m 1 showed 87.5% skin reactivities on eight latex- and Indian jujube-sensitive subjects. Although no sequence similarity was found to other known allergens, Ziz m 1 was found to have amino acid sequence identity (39-45.3%) to many plant chitinases including chitinase (45.2%) of Hevea brasiliensis (hevamine), class III chitinases of Vigna angularis (45.3%), Capsicum annuum (44.7%) and Oryza sativa (41.2%). A conserved domain search revealed that Ziz m 1 belongs to the family 18 glycosyl hydrolases. The recombinant allergen may therefore be of value for diagnosis and therapeutic purposes, and the further characterization of Indian jujube allergen may help to elucidate the mechanism underlying latex-fruit syndrome.
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http://dx.doi.org/10.1016/j.molimm.2005.07.021DOI Listing
March 2006

Histidines 345 and 378 of Bacillus stearothermophilus leucine aminopeptidase II are essential for the catalytic activity of the enzyme.

Antonie Van Leeuwenhoek 2005 May;87(4):355-9

Department of Biology, Tung-Hai University, 181 Taichung-Kan Road, Taichung, Taiwan.

The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the Km value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in Kcat/Km for H227L was partly caused by a 3.9-fold increase in Km value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII.
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http://dx.doi.org/10.1007/s10482-004-5777-zDOI Listing
May 2005

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan.

J Clin Virol 2005 Jan;32(1):33-7

Department of Life Science, Tunghai University, 181, Sec. 3, Chungkang Rd., Taichung, Taiwan, ROC.

Background: Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown.

Objectives: To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients.

Study Design: 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing.

Results: The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region.

Conclusions: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.
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http://dx.doi.org/10.1016/j.jcv.2004.06.011DOI Listing
January 2005

Inter-specific sequence conservation and intra-individual sequence variation in a spider silk gene.

Int J Biol Macromol 2004 Oct;34(5):295-301

Department of Life Science, Tunghai University, Taichung 407, Taiwan.

Currently, studies on major ampullate spidroin 1 (MaSp1) genes of non-orb weaving spiders are few, and it is not clear whether genes of these organisms exhibit the same characteristics as those of orb-weavers. In addition, many studies have proposed that MaSp1 might be a single gene with allelic variants, but supporting evidence is still lacking. In this study, we compared partial DNA and amino acid sequences of MaSp1 cloned from different spider guilds. We also cloned partial MaSp1 sequences from genomic DNA and cDNA of the same individuals of spiders using the same primer combination to see if different molecular forms existed. In the repetitive region of partial MaSp1 sequences obtained, GGX, GA and poly-A motifs were present in all Araneomorphae and Mygalomorpae species examined. An extreme similarity in MaSp1 non-repetitive portions was found in sequences of ecribellate, cribellate and Mygalomorphae web-builders and such a result suggested that this sequence might exhibit an important function. A comparison of sequences amplified from the same individual showed that substitutions in amino acids occurred in both repetitive and non-repetitive regions, with a much higher variation in the former. These results suggest that the MaSp1 of Araneomorphae spiders exhibits several forms in an individual spider and it might be either a multiple gene or a single gene with a multiple exon/intron organization.
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http://dx.doi.org/10.1016/j.ijbiomac.2004.08.002DOI Listing
October 2004

Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme.

Protein J 2004 May;23(4):295-302

Department of Biology, Tung-Hai University, 181 Talchung-Kan Road, Taichung, Taiwan.

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.
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http://dx.doi.org/10.1023/b:jopc.0000027854.56051.e4DOI Listing
May 2004

Inhibition of tumorigenicity of the hepatitis B virus X gene in Chang liver cell line.

Virus Res 2004 Jun;102(2):133-9

Department of Biology, Tunghai University, 181, Sec. 3, Chungkang Road, Taichung, Taiwan, ROC.

The hepatitis B virus X gene, which encodes the HBx protein, has multiple functions and is involved in hepatocarcinogenesis. However, the exact role of HBx in hepatocarcinogenesis is still controversial. We have established an inducible (tet-off system) HBx-expressing cell line, Chang-HBx. Compared with the original of Chang liver cell line (ATCC CCL13), Chang-HBx grows faster in serum-containing medium but slower in serum-free medium. Chang-HBx colony formation in soft agar shows an anchorage-demanding character and its tumorigenicity potential in BALB/c nude mice were substantially inhibited. HBx also causes the induction of G1 phase arrest of cell growth in early infection of HBV and therefore plays a negative role in tumorigenicity. An excellent mice animal model for producing hepatoma was also provided in this study.
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http://dx.doi.org/10.1016/j.virusres.2004.01.023DOI Listing
June 2004

Detection of the hepatitis B virus X protein (HBx) antigen and anti-HBx antibodies in cases of human hepatocellular carcinoma.

J Clin Microbiol 2003 Dec;41(12):5598-603

Department of Biology, Life Science Research Center, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China.

Hepatitis B virus X protein (HBx) expressed in Escherichia coli DH5alpha by recombinant DNA technology was purified to homogeneity by use of glutathione-Sepharose beads. Immunological characterization of the recombinant HBx protein was performed. Specific binding between the anti-HBx monoclonal antibody and HBx protein showed the specificity of the recombinant HBx protein. The intact HBx protein of the factor Xa-digested glutathione S-transferase-HBx fusion protein was further purified and was used as an antigen for screening the titers of anti-HBx antibodies in sera. Titers of anti-HBx in sera from 20 patients with hepatocellular carcinoma (HCC), 20 patients with chronic hepatitis (CH), and 20 healthy individuals were evaluated by Western blotting and a quantitative enzyme-linked immunosorbent assay. The results indicated that 70% of sera from HCC patients and 5% of sera from CH patients contained antibodies with significant binding to the HBx protein. Western blotting of HBx protein in liver extracts from 20 HCC patients was also performed by using the anti-HBx monoclonal antibody. Results showed that 85% of HCC patients' liver tissues contained a specific HBx protein with the same molecular size as the purified intact HBx. Full correlation was found between anti-HBx antibody positivity in serum and HBx protein positivity in HCC tissues. The data demonstrated that the etiology of HCC is involved with hepatitis B virus (HBV) infection and that HBx in particular plays a role in the development of HBV-related HCC.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC309044PMC
http://dx.doi.org/10.1128/jcm.41.12.5598-5603.2003DOI Listing
December 2003

Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus.

Curr Microbiol 2003 Jul;47(1):40-5

Department of Biology, Tung-Hai University, 181 Taichung-Kan Road, Taichung, Taiwan.

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His(6)-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60 degrees C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His(6)-tagged enzyme was stimulated by Co(2+) ions, but was strongly inhibited by Cu(2+) and Hg(2+) and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co(2+) ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.
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http://dx.doi.org/10.1007/s00284-002-3950-zDOI Listing
July 2003