Anti-osteoclastogenic, Estrogenic, and Antioxidant Activities of Cell Suspension Cultures and Tuber Root Extracts from Pueraria mirifica

Tanatorn Saisavoey,Tanapat Palaga,Suchinda Malaivijitnond,Sukanya Jaroenporn,Nuttha Thongchul,Polkit Sangvanich,Aphichart Karnchanatat 한국식품과학회 2014 Food Science and Biotechnology Vol.23 No.4

The flavonoid contents of intact tubers and cellculture media were determined and physiological activitiesof Pueraria mirifica extracts were investigated. The totalflavonoid contents from cell culture media (PMC) werehigher than from tubers (PMT). Results from in vivoestrogenic activity assays indicated that PMT had a strongestrogenic activity in ovariectomized rats. The same amountof PMC exhibited a weak activity. In vitro osteoclastsuppression investigations indicated that both PMT andPMC extracts exhibited anti-osteoclastogenic activities withlow toxicities in a standard test cell line. Determination ofthe antioxidant potential using the DPPH assay revealedthat the IC50 value for PMT was lower than for PMC. P. mirifica cell cultures produce more flavonoids and exhibita mild estrogenic and more antioxidant activities than tubers.

Production and Characterization of a Monoclonal Antibody Against Enrofloxacin

( Chusri Manaspong ),( Pitikarn Wongphanit ),( Tanapat Palaga ),( Songchan Puthong ),( Sarintip Sooksai ),( Kittinan Komolpis ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.1

Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration (IC50) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

Propolis from the Stingless Bee Trigona incisa from East Kalimantan, Indonesia, Induces In Vitro Cytotoxicity and Apoptosis in Cancer Cell lines

Kustiawan, Paula M,Phuwapraisirisan, Preecha,Puthong, Songchan,Palaga, Tanapat,Arung, Enos T,Chanchao, Chanpen Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.15

Background: Previously, stingless bee (Trigona spp.) products from East Kalimantan, Indonesia, were successfully screened for in vitro antiproliferative activity against human cancer derived cell lines. It was established that propolis from T. incisa presented the highest in vitro cytotoxicity against the SW620 colon cancer cell line (6% cell survival in $20{\mu}g/mL$). Materials and Methods: Propolis from T. incisa was extracted with methanol and further partitioned with n-hexane, ethyl acetate and methanol. The in vitro cytotoxicity of the extracts was assessed by the MTT assay against human colon (SW620), liver (Hep-G2), gastric (KATO-III), lung (Chago) and breast (BT474) cancer derived cell lines. The active fractions were further enriched by silica gel quick column, absorption and size exclusion chromatography. The purity of each fraction was checked by thin layer chromatography. Cytotoxicity in BT-474 cells induced by cardanol compared to doxorubicin were evaluated by MTT assay, induction of cell cycle arrest and cell death by flow cytometric analysis of propidium iodide and annexin-V stained cells. Results: A cardol isomer was found to be the major compound in one active fraction (F45) of T. incisa propolis, with a cytotoxicity against the SW620 ($IC_{50}$ of $4.51{\pm}0.76{\mu}g/mL$), KATO-III (IC50 of $6.06{\pm}0.39{\mu}g/mL$), Hep-G2 ($IC_{50}$ of $0.71{\pm}0.22{\mu}g/mL$), Chago I ($IC_{50}$ of $0.81{\pm}0.18{\mu}g/mL$) and BT474 (IC50 of $4.28{\pm}0.14{\mu}g/mL$) cell lines. Early apoptosis (programmed cell death) of SW620 cells was induced by the cardol containing F45 fraction at the $IC_{50}$ and $IC_{80}$ concentrations, respectively, within 2-6 h of incubation. In addition, the F45 fraction induced cell cycle arrest at the G1 subphase. Conclusions: Indonesian stingless bee (T. incisa) propolis had moderately potent in vitro anticancer activity on human cancer derived cell lines. Cardol or 5-pentadecyl resorcinol was identified as a major active compound and induced apoptosis in SW620 cells in an early period (${\leq}6h$) and cell cycle arrest at the G1 subphase. Thus, cardol is a potential candidate for cancer chemotherapy.

Development of Gold Nanorods Stabilized with Drug-conjugated Polymer for Synergistic Cancer Therapy

Phim-on Khunsuk,Supattra Chawalitpong,Pritsana Sawutdeechaikul,Tanapat Palaga,Voravee P. Hoven 한국고분자학회 2016 한국고분자학회 학술대회 연구논문 초록집 Vol.41 No.2

Comparative production of a monoclonal antibody specific for enrofloxacin in a stirred-tank bioreactor

Kittinan Komolpis,Chatchawan Udomchokmongkol,Songchan Phutong,Tanapat Palaga 한국공업화학회 2010 Journal of Industrial and Engineering Chemistry Vol.16 No.4

A hybridoma producing monoclonal antibody (MAb) specific for enrofloxacin was cultivated in a 2-L stirred-tank bioreactor in various modes and the performances of each mode were compared. In batch mode, a maximum viable cell and MAb concentration of 9.21 105 cells mL1 and 67.3 mg L1,respectively, were obtained. When the hybridoma was cultivated in a fed-batch culture with the addition of specific nutrients, no improvement in either the viable cell number or MAb concentration was observed. On the other hand, an increase in the production of toxic metabolites, i.e. ammonia and lactate, was observed with growth inhibition of the hybridoma cells occurring at ammonia and lactate concentrations of 2.0 mM and 2.0 g L1, respectively. However, the best performance of hybridoma cultivation was achieved in a perfusion culture mode using a spin filter, which was installed in the stirred-tank reactor as a cell retention device with a perfusion rate of 0.80 vvd. Under these conditions a steady viable cell concentration of 1.57 106 cells mL1 was obtained within five days with an overall productivity and yield of 73.7 mg L1 d1 and 61.4 mg d1, respectively, which was a significant increase over that attained with the batch process.

Immunogenicity of a DNA and Recombinant Protein Vaccine Combining LipL32 and Loa22 for Leptospirosis Using Chitosan as a Delivery System

( Supawadee Umthong ),( Arun Buaklin ),( Alain Jacquet ),( Noppadol Sangjun ),( Ruthairat Kerdkaew ),( Kanitha Patarakul ),( Tanapat Palaga ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.4

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4+ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.

Increased susceptibility against Cryptococcus neoformans of lupus mouse models (pristane-induction and FcGRIIb deficiency) is associated with activated macrophage, regardless of genetic background

Saowapha Surawut,Jiradej Makjaroen,Arthid Thim-uam,Jutamas Wongphoom,Tanapat Palaga,Prapaporn Pisitkun,Ariya Chindamporn,Asada Leelahavanichkul 한국미생물학회 2019 The journal of microbiology Vol.57 No.1

The severity of cryptococcosis in lupus from varying geneticbackgrounds might be different due to the heterogeneity of lupus-pathogenesis. This study explored cryptococcosis in lupus mouse models of pristane-induction (normal geneticbackground) and FcGRIIb deficiency (genetic defect). Because the severity of lupus nephritis, as determined by proteinuria and serum creatinine, between pristane and FcGRIIb-/- mice were similar at 6-month-old, Cryptococcus neoformans was intravenously administered in 6-month-old mice and were age-matched with wild-type. Indeed, the cryptococcosis disease severity, as evaluated by mortality rate, internal-organ fungal burdens and serum cytokines, between pristane and FcGRIIb-/- mice was not different. However, the severity of cryptococcosis in wild-type was less severe than the lupus mice. On the other hand, phagocytosis activity of peritoneal macrophages from lupus mice (pristane and FcGRIIb-/-) was more predominant than the wild-type without the difference in macrophage killing-activity among these groups. In addition, the number of active T helper cells (Th-cell) in the spleen, including Th-cells with intracellular IFN-γ, from lupus mice (pristane and FcGRIIb-/-) was higher than wildtype. Moreover, these active Th-cells were even higher after 2 weeks of cryptococcal infection. These data support enhanced macrophage activation through prominent Th-cells in both lupus models. In conclusion, an increased susceptibility of cryptococcosis in both lupus models was independent to genetic background. This might due to Th-cell enhanced macrophage phagocytosis with the interference of macrophage killing activity from Cryptococcal immune-evasion properties.

Oxidized Carbon Nanosphere-Based Subunit Vaccine Delivery System Elicited Robust Th1 and Cytotoxic T Cell Responses

( Pritsana Sawutdeechaikul ),( Felipe Cia ),( Gregory J. Bancroft ),( Supason Wanichwecharungruang ),( Chutamath Sittplangkoo ),( Tanapat Palaga ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.3

Subunit vaccines are safer and more stable than live vaccines although they have the disadvantage of eliciting poor immune response. To develop a subunit vaccine, an effective delivery system targeting the key elements of the protective immune response is a prerequisite. In this study, oxidized carbon nanospheres (OCNs) were used as a subunit vaccine delivery system and tuberculosis (TB) was chosen as a model disease. TB is among the deadliest infectious diseases worldwide and an effective vaccine is urgently needed. The ability of OCNs to deliver recombinant Mycobacterium tuberculosis (Mtb) proteins, Ag85B and HspX, into bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) was investigated. For immunization, OCNs were mixed with the two TB antigens as well as the adjuvant monophosphoryl lipid A (MPL). The protective efficacy was analyzed in vaccinated mice by aerosol Mtb challenge with a virulent strain of Mtb and the bacterial burdens were measured. The results showed that OCNs are highly effective in delivering Mtb proteins into the cytosol of BMDMs and BMDCs. Upon immunization, this vaccine formula induced robust Th1 immune response characterized by cytokine profiles from restimulated splenocytes and specific antibody titer. More importantly, enhanced cytotoxic CD8⁺ T cell activation was observed. However, it did not reduce the bacteria burden in the lung and spleen from the aerosol Mtb challenge. Taken together, OCNs are highly effective in delivering subunit protein vaccine and induce robust Th1 and CD8⁺ T cell response. This vaccine delivery system is suitable for application in settings where cell-mediated immune response is needed.

Detecting Allergens From Black Tiger Shrimp Penaeus monodon That Can Bind and Cross-link IgE by ELISA, Western Blot, and a Humanized Rat Basophilic Leukemia Reporter Cell Line RS-ATL8

Thanyapat Jarupalee,Pantipa Chatchatee,Kittinan Komolpis,Narissara Suratannon,Sittiruk Roytrakul,Yodying Yingchutrakul,Wanaporn Yimchuen,Patcharavadee Butta,Alain Jacquet,Tanapat Palaga 대한천식알레르기학회 2018 Allergy, Asthma & Immunology Research Vol.10 No.1

Purpose: Black tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8. Methods: Sera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens. Results: Allergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32-39 kDa and 91-230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens. Conclusions: The RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.