Prasert Sobhon - Academia.edu (original) (raw)

Papers by Prasert Sobhon

Research paper thumbnail of Characterization and expression of cathepsin B2 in Fasciola gigantica

Experimental Parasitology, 2012

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in inv... more Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites' antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.

Research paper thumbnail of Funding Policy and Strategies for Basic Research by Academic Division of The Thailand Research Fund

** ABSTRACT: The Academic Division of The Thailand Research Fund (TRF), is responsible for provid... more ** ABSTRACT: The Academic Division of The Thailand Research Fund (TRF), is responsible for providing funds to support basic research in science and technology, and social science and humanities. Three categories of research are currently being supported: (1) Investigator-initiated research is the research proposed by an investigator, which may be on any topics, but must be novel in idea and approach. Emphasis is on the creation of knowledge, which results in publications in reputable national and international journals, or peer- reviewed books. The support also aims to create career paths for professional researchers and is hence divided into three levels of grants: for young researchers, mid-career researchers, and senior researchers. The recipient of the last level is also named the TRF Senior Research Scholar, which is considered to be highly prestigious. (2) Strategic basic research, which is initiated and defined by TRF, covers areas that are deemed necessary for national development needs and for solving immediate problems. The research is target-based, and includes thematic programs on Pharmaceuticals, Nutraceuticals and Functional Foods, Sustainable Agriculture, Production of Aquatic Animals, Production of Land Animals, Stem Cell Biology and Regenerative Medicine, Nanoscience and Nanotechnology. The outputs are both publications, and patents or utilization for public benefits. (3) Translational research is the research initiated and defined by industry as the end user, in partnership with the researcher. It aims to utilize knowledge and research findings that are generated from the first two categories of basic research for industrial and commercial purposes. Output could be in the forms of patent, development of products or processes, especially those that will help in empowering and creating competitiveness for the small and medium enterprises.

Research paper thumbnail of Classification of Germ Cells, Reproductive Cycle and Maturation of Gonads in Haliotis asinina Linnaeus

Germ cells in the gonads of Haliotis asinina, a species of abalone found along the coast of Thail... more Germ cells in the gonads of Haliotis asinina, a species of abalone found along the coast of Thailand, were classified basing on light and electron microscopies. Germ cells in oogenetic units could be classified into six stages according to their histological and ultrastructural characteristics: oogonium and five stages of oocytes, ie, Oc1 with light to intense basophilia and abundant polyribosomes,

Research paper thumbnail of Molecular cloning and characterization of two genes encoding 2Cys peroxiredoxins from Fasciola gigantica

Experimental Parasitology, 2010

In Fasciola species, peroxiredoxin (Prx) serves as the major antioxidant enzyme to remove hydroge... more In Fasciola species, peroxiredoxin (Prx) serves as the major antioxidant enzyme to remove hydrogen peroxide that is generated from various metabolic reactions, because the parasites lack catalase, and only express glutathione peroxidases at minimal levels. We have cloned and characterized two genes, FgPrx-1 and FgPrx-2, belonging to the 2-Cys Prx family, by immunoscreening of an expressed adult stage Fasciola gigantica

Research paper thumbnail of Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice

Acta tropica, Jan 24, 2015

In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed... more In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro-F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rproFgCatL1 and rmatFgCatL1 combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls...

Research paper thumbnail of Research Articles Classification of Germ Cells, Reproductive Cycle and Maturation of Gonads in Haliotis asinina Linnaeus ..... 3-21

Research paper thumbnail of Involvement of c-Met/Hepatocyte Growth Factor Pathway in Cholangiocarcinoma Cell Invasion and Its Therapeutic Inhibition With Small Interfering RNA Specific for c-Met

Journal of Surgical Research, 2006

Materials and methods. The expression of c-Met and their signaling cascades were determined in RM... more Materials and methods. The expression of c-Met and their signaling cascades were determined in RMCCA1 and HuCCA1 cholangiocarcinoma cell lines by Western blotting. Small interfering RNA (siRNA) specific for c-Met was used to suppress the expression of c-Met. The proliferation, migration and invasion assay were tested in these cholangiocarcinoma cells treated with hepatocyte growth factor (HGF).

Research paper thumbnail of Biochemical and ultrastructural characterizations of nucleoprotamine in human sperm heads treated with micrococcal nuclease and salt

Research paper thumbnail of Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica

Acta tropica, 2014

Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1)... more Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1. All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30kDa and with the native CatL1 at MW 27kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory-secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including...

Research paper thumbnail of Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica

Veterinary parasitology, Jan 18, 2006

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments ... more Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostaining with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evid...

Research paper thumbnail of Alterations in the levels and distribution of octopamine in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei, and its possible role in ovarian development

General and Comparative Endocrinology, 2015

Octopamine (OA) is a major neurotransmitter that has not been studied in the Pacific white shrimp... more Octopamine (OA) is a major neurotransmitter that has not been studied in the Pacific white shrimp, Litopenaeus vannamei. Therefore, we investigated changes in OA levels, its distribution in regions of the central nervous system (CNS) and ovary during the ovarian maturation cycle, as well as its possible role in regulating ovarian maturation. OA exhibited the highest concentration in the brain and thoracic ganglia at ovarian stage II, and then declined to the lowest concentration at ovarian stages III and IV. In the cerebral ganglia, OA-immunoreactivity (OA-ir) was present in neurons of clusters 6, 17, the anterior and posterior medial protocerebral, olfactory, antenna II, and tegumentary neuropils. In the circumesophageal, subesophageal, thoracic ganglia and abdominal ganglia, OA-ir was detected in several neuropils, neurons and fibers. The high level of intensity in OA immunostaining was observed in early developmental stage of oocyte by comparison with low level of OA-ir in late stages of oocyte development. Functionally, OA-injected female shrimps at doses of 2.5×10(-7) and 2.5×10(-6)mol/shrimp, showed significantly decreased gonado-somatic indices, oocyte diameters, and hemolymph vitellogenin levels, compared with control groups. This study showed changes of OA in the CNS and ovary reaching the highest level in early ovarian stages and declining in late stages, and it decreased hemolymph vitellogenin levels, suggesting significant involvement of OA in female reproduction in this species.

Research paper thumbnail of Identification of Genes Associated with Reproduction in the Mud Crab (Scylla olivacea) and Their Differential Expression following Serotonin Stimulation

PLoS ONE, 2014

The central nervous system (CNS) is often intimately involved in reproduction control and is ther... more The central nervous system (CNS) is often intimately involved in reproduction control and is therefore a target organ for transcriptomic investigations to identify reproduction-associated genes. In this study, 454 transcriptome sequencing was performed on pooled brain and ventral nerve cord of the female mud crab (Scylla olivacea) following serotonin injection (5 mg/g BW). A total of 197,468 sequence reads was obtained with an average length of 828 bp. Approximately 38.7% of 2,183 isotigs matched with significant similarity (E value , 1e 24 ) to sequences within the Genbank non-redundant (nr) database, with most significant matches being to crustacean and insect sequences. Approximately 32 putative neuropeptide genes were identified from nonmatching blast sequences. In addition, we identified full-length transcripts for crustacean reproductive-related genes, namely farnesoic acid o-methyltransferase (FAMeT), estrogen sulfotransferase (ESULT) and prostaglandin F synthase (PGFS). Following serotonin injection, which would normally initiate reproductive processes, we found up-regulation of FAMeT, ESULT and PGFS expression in the female CNS and ovary. Our data here provides an invaluable new resource for understanding the molecular role of the CNS on reproduction in S. olivacea.

Research paper thumbnail of Developments and applications of mass microscopy

Medical Molecular Morphology, 2010

We have developed a mass microscopy technique, i.e., a microscope combined with high-resolution m... more We have developed a mass microscopy technique, i.e., a microscope combined with high-resolution matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a powerful tool for investigating the spatial distribution of biomolecules without any time-consuming extraction, purification, and separation procedures for biological tissue sections. Mass microscopy provides clear images about the distribution of hundreds of biomolecules in a single measurement and also helps in understanding the cellular profile of the biological system. The sample preparation and the spatial resolution and speed of the technique are all important steps that affect the identification of biomolecules in mass microscopy. In this Award Lecture Review, we focus on some of the recent developments in clinical applications to show how mass microscopy can be employed to assess medical molecular morphology.

Research paper thumbnail of Changes of Phosphatidylcholine and Fatty Acids in Germ Cells during Testicular Maturation in Three Developmental Male Morphotypes of Macrobrachium rosenbergii Revealed by Imaging Mass Spectrometry

PLOS ONE, 2015

Testis maturation, germ cell development and function of sperm, are related to lipid composition.... more Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatographymass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in PLOS ONE |

Research paper thumbnail of Coating thickness testing

Research paper thumbnail of Ultrastructure of the differentiating male germ cells in Haliotis asinina Linnaeus

Invertebrate Reproduction & Development, 2001

Male germ cells in the testis of H. asinina can be divided into 14 stages based on the ultra-stru... more Male germ cells in the testis of H. asinina can be divided into 14 stages based on the ultra-structure and patterns of chromatin condensation. The spermatogonium is a spherical or oval-shaped cell with diameter about 8 μm. Its nucleus contains mostly euchromatin with only a thin rim of heterochromatin along the inner facet of the nuclear envelope. Primary spermatocytes (PrSc)

Research paper thumbnail of Electron microscopic and biochemical analyses of the organization of human sperm chromatin decondensed with Sarkosyl and dithiothreitol

Journal of Experimental Zoology, 1982

Human sperm chromatin was decondensed by treating the purified sperm heads with Sarkosyl for 60 m... more Human sperm chromatin was decondensed by treating the purified sperm heads with Sarkosyl for 60 minutes followed with dithiothreitol (DTT) for 20 minutes and overnight. Following Sarkosyl treatment all histones and nonhistones were removed; the remaining nucleoprotamines in the sperm heads exhibited two levels of higher-order structure in the forms of 900-1200 A thick and 380-520 A thin knobby cords, which were randomly coiled. Subsequent treatment with DTT resulted in the dissociation of the 380-520 A cords into subunits of 180-210 A fibers, which were further decondensed into beads-on-a-string structure with diameter of the beads about 120-150 A.

Research paper thumbnail of Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L

Vaccine, 2015

Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles... more Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its efficacy is currently being studied in the larger economic animals.

Research paper thumbnail of Saposin-like protein 2 has an immunodiagnostic potential for detecting Fasciolosis gigantica

Experimental Parasitology, 2015

Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigan... more Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigantica (Fg). It is one of the major proteins synthesized by the caecal epithelial cells and released into fluke's excretion-secretion. Therefore, FgSAP-2 is a plausible target for detecting fasciolosis. A polyclonal antibody (PoAb) against recombinant FgSAP-2 was produced by immunizing rabbits with the recombinant protein (rFgSAP-2), and used in sandwich ELISA assay to detect the circulating FgSAP-2 in sera of mice experimentally infected with F. gigantica metacercariae. The assay could detect rFgSAP-2 and the native FgSAP-2 in the excretory-secretory (ES) and whole body (WB) fractions of adult F. gigantica at the concentrations as low as 38 pg/ml, 24 ng/ml, and 102 ng/ml, respectively. As well, the sera from mice experimentally infected with F. gigantica were tested positive by this sandwich ELISA, which exhibited sensitivity, specificity, false positive rate, false negative rate and accuracy at 99.99, 98.67, 1.33, 0.01 and 99.32%, respectively. Therefore, this assay could be used for diagnosis of fasciolosis by F. gigantica.

Research paper thumbnail of Transmission and scanning electron microscopic studies of the human sperm chromatin decondensed by micrococcal nuclease and salt

Journal of Experimental Zoology, 1982

Research paper thumbnail of Characterization and expression of cathepsin B2 in Fasciola gigantica

Experimental Parasitology, 2012

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in inv... more Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites' antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.

Research paper thumbnail of Funding Policy and Strategies for Basic Research by Academic Division of The Thailand Research Fund

** ABSTRACT: The Academic Division of The Thailand Research Fund (TRF), is responsible for provid... more ** ABSTRACT: The Academic Division of The Thailand Research Fund (TRF), is responsible for providing funds to support basic research in science and technology, and social science and humanities. Three categories of research are currently being supported: (1) Investigator-initiated research is the research proposed by an investigator, which may be on any topics, but must be novel in idea and approach. Emphasis is on the creation of knowledge, which results in publications in reputable national and international journals, or peer- reviewed books. The support also aims to create career paths for professional researchers and is hence divided into three levels of grants: for young researchers, mid-career researchers, and senior researchers. The recipient of the last level is also named the TRF Senior Research Scholar, which is considered to be highly prestigious. (2) Strategic basic research, which is initiated and defined by TRF, covers areas that are deemed necessary for national development needs and for solving immediate problems. The research is target-based, and includes thematic programs on Pharmaceuticals, Nutraceuticals and Functional Foods, Sustainable Agriculture, Production of Aquatic Animals, Production of Land Animals, Stem Cell Biology and Regenerative Medicine, Nanoscience and Nanotechnology. The outputs are both publications, and patents or utilization for public benefits. (3) Translational research is the research initiated and defined by industry as the end user, in partnership with the researcher. It aims to utilize knowledge and research findings that are generated from the first two categories of basic research for industrial and commercial purposes. Output could be in the forms of patent, development of products or processes, especially those that will help in empowering and creating competitiveness for the small and medium enterprises.

Research paper thumbnail of Classification of Germ Cells, Reproductive Cycle and Maturation of Gonads in Haliotis asinina Linnaeus

Germ cells in the gonads of Haliotis asinina, a species of abalone found along the coast of Thail... more Germ cells in the gonads of Haliotis asinina, a species of abalone found along the coast of Thailand, were classified basing on light and electron microscopies. Germ cells in oogenetic units could be classified into six stages according to their histological and ultrastructural characteristics: oogonium and five stages of oocytes, ie, Oc1 with light to intense basophilia and abundant polyribosomes,

Research paper thumbnail of Molecular cloning and characterization of two genes encoding 2Cys peroxiredoxins from Fasciola gigantica

Experimental Parasitology, 2010

In Fasciola species, peroxiredoxin (Prx) serves as the major antioxidant enzyme to remove hydroge... more In Fasciola species, peroxiredoxin (Prx) serves as the major antioxidant enzyme to remove hydrogen peroxide that is generated from various metabolic reactions, because the parasites lack catalase, and only express glutathione peroxidases at minimal levels. We have cloned and characterized two genes, FgPrx-1 and FgPrx-2, belonging to the 2-Cys Prx family, by immunoscreening of an expressed adult stage Fasciola gigantica

Research paper thumbnail of Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice

Acta tropica, Jan 24, 2015

In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed... more In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro-F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rproFgCatL1 and rmatFgCatL1 combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls...

Research paper thumbnail of Research Articles Classification of Germ Cells, Reproductive Cycle and Maturation of Gonads in Haliotis asinina Linnaeus ..... 3-21

Research paper thumbnail of Involvement of c-Met/Hepatocyte Growth Factor Pathway in Cholangiocarcinoma Cell Invasion and Its Therapeutic Inhibition With Small Interfering RNA Specific for c-Met

Journal of Surgical Research, 2006

Materials and methods. The expression of c-Met and their signaling cascades were determined in RM... more Materials and methods. The expression of c-Met and their signaling cascades were determined in RMCCA1 and HuCCA1 cholangiocarcinoma cell lines by Western blotting. Small interfering RNA (siRNA) specific for c-Met was used to suppress the expression of c-Met. The proliferation, migration and invasion assay were tested in these cholangiocarcinoma cells treated with hepatocyte growth factor (HGF).

Research paper thumbnail of Biochemical and ultrastructural characterizations of nucleoprotamine in human sperm heads treated with micrococcal nuclease and salt

Research paper thumbnail of Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica

Acta tropica, 2014

Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1)... more Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1. All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30kDa and with the native CatL1 at MW 27kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory-secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including...

Research paper thumbnail of Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica

Veterinary parasitology, Jan 18, 2006

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments ... more Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostaining with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evid...

Research paper thumbnail of Alterations in the levels and distribution of octopamine in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei, and its possible role in ovarian development

General and Comparative Endocrinology, 2015

Octopamine (OA) is a major neurotransmitter that has not been studied in the Pacific white shrimp... more Octopamine (OA) is a major neurotransmitter that has not been studied in the Pacific white shrimp, Litopenaeus vannamei. Therefore, we investigated changes in OA levels, its distribution in regions of the central nervous system (CNS) and ovary during the ovarian maturation cycle, as well as its possible role in regulating ovarian maturation. OA exhibited the highest concentration in the brain and thoracic ganglia at ovarian stage II, and then declined to the lowest concentration at ovarian stages III and IV. In the cerebral ganglia, OA-immunoreactivity (OA-ir) was present in neurons of clusters 6, 17, the anterior and posterior medial protocerebral, olfactory, antenna II, and tegumentary neuropils. In the circumesophageal, subesophageal, thoracic ganglia and abdominal ganglia, OA-ir was detected in several neuropils, neurons and fibers. The high level of intensity in OA immunostaining was observed in early developmental stage of oocyte by comparison with low level of OA-ir in late stages of oocyte development. Functionally, OA-injected female shrimps at doses of 2.5×10(-7) and 2.5×10(-6)mol/shrimp, showed significantly decreased gonado-somatic indices, oocyte diameters, and hemolymph vitellogenin levels, compared with control groups. This study showed changes of OA in the CNS and ovary reaching the highest level in early ovarian stages and declining in late stages, and it decreased hemolymph vitellogenin levels, suggesting significant involvement of OA in female reproduction in this species.

Research paper thumbnail of Identification of Genes Associated with Reproduction in the Mud Crab (Scylla olivacea) and Their Differential Expression following Serotonin Stimulation

PLoS ONE, 2014

The central nervous system (CNS) is often intimately involved in reproduction control and is ther... more The central nervous system (CNS) is often intimately involved in reproduction control and is therefore a target organ for transcriptomic investigations to identify reproduction-associated genes. In this study, 454 transcriptome sequencing was performed on pooled brain and ventral nerve cord of the female mud crab (Scylla olivacea) following serotonin injection (5 mg/g BW). A total of 197,468 sequence reads was obtained with an average length of 828 bp. Approximately 38.7% of 2,183 isotigs matched with significant similarity (E value , 1e 24 ) to sequences within the Genbank non-redundant (nr) database, with most significant matches being to crustacean and insect sequences. Approximately 32 putative neuropeptide genes were identified from nonmatching blast sequences. In addition, we identified full-length transcripts for crustacean reproductive-related genes, namely farnesoic acid o-methyltransferase (FAMeT), estrogen sulfotransferase (ESULT) and prostaglandin F synthase (PGFS). Following serotonin injection, which would normally initiate reproductive processes, we found up-regulation of FAMeT, ESULT and PGFS expression in the female CNS and ovary. Our data here provides an invaluable new resource for understanding the molecular role of the CNS on reproduction in S. olivacea.

Research paper thumbnail of Developments and applications of mass microscopy

Medical Molecular Morphology, 2010

We have developed a mass microscopy technique, i.e., a microscope combined with high-resolution m... more We have developed a mass microscopy technique, i.e., a microscope combined with high-resolution matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a powerful tool for investigating the spatial distribution of biomolecules without any time-consuming extraction, purification, and separation procedures for biological tissue sections. Mass microscopy provides clear images about the distribution of hundreds of biomolecules in a single measurement and also helps in understanding the cellular profile of the biological system. The sample preparation and the spatial resolution and speed of the technique are all important steps that affect the identification of biomolecules in mass microscopy. In this Award Lecture Review, we focus on some of the recent developments in clinical applications to show how mass microscopy can be employed to assess medical molecular morphology.

Research paper thumbnail of Changes of Phosphatidylcholine and Fatty Acids in Germ Cells during Testicular Maturation in Three Developmental Male Morphotypes of Macrobrachium rosenbergii Revealed by Imaging Mass Spectrometry

PLOS ONE, 2015

Testis maturation, germ cell development and function of sperm, are related to lipid composition.... more Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatographymass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in PLOS ONE |

Research paper thumbnail of Coating thickness testing

Research paper thumbnail of Ultrastructure of the differentiating male germ cells in Haliotis asinina Linnaeus

Invertebrate Reproduction & Development, 2001

Male germ cells in the testis of H. asinina can be divided into 14 stages based on the ultra-stru... more Male germ cells in the testis of H. asinina can be divided into 14 stages based on the ultra-structure and patterns of chromatin condensation. The spermatogonium is a spherical or oval-shaped cell with diameter about 8 μm. Its nucleus contains mostly euchromatin with only a thin rim of heterochromatin along the inner facet of the nuclear envelope. Primary spermatocytes (PrSc)

Research paper thumbnail of Electron microscopic and biochemical analyses of the organization of human sperm chromatin decondensed with Sarkosyl and dithiothreitol

Journal of Experimental Zoology, 1982

Human sperm chromatin was decondensed by treating the purified sperm heads with Sarkosyl for 60 m... more Human sperm chromatin was decondensed by treating the purified sperm heads with Sarkosyl for 60 minutes followed with dithiothreitol (DTT) for 20 minutes and overnight. Following Sarkosyl treatment all histones and nonhistones were removed; the remaining nucleoprotamines in the sperm heads exhibited two levels of higher-order structure in the forms of 900-1200 A thick and 380-520 A thin knobby cords, which were randomly coiled. Subsequent treatment with DTT resulted in the dissociation of the 380-520 A cords into subunits of 180-210 A fibers, which were further decondensed into beads-on-a-string structure with diameter of the beads about 120-150 A.

Research paper thumbnail of Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L

Vaccine, 2015

Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles... more Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its efficacy is currently being studied in the larger economic animals.

Research paper thumbnail of Saposin-like protein 2 has an immunodiagnostic potential for detecting Fasciolosis gigantica

Experimental Parasitology, 2015

Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigan... more Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigantica (Fg). It is one of the major proteins synthesized by the caecal epithelial cells and released into fluke's excretion-secretion. Therefore, FgSAP-2 is a plausible target for detecting fasciolosis. A polyclonal antibody (PoAb) against recombinant FgSAP-2 was produced by immunizing rabbits with the recombinant protein (rFgSAP-2), and used in sandwich ELISA assay to detect the circulating FgSAP-2 in sera of mice experimentally infected with F. gigantica metacercariae. The assay could detect rFgSAP-2 and the native FgSAP-2 in the excretory-secretory (ES) and whole body (WB) fractions of adult F. gigantica at the concentrations as low as 38 pg/ml, 24 ng/ml, and 102 ng/ml, respectively. As well, the sera from mice experimentally infected with F. gigantica were tested positive by this sandwich ELISA, which exhibited sensitivity, specificity, false positive rate, false negative rate and accuracy at 99.99, 98.67, 1.33, 0.01 and 99.32%, respectively. Therefore, this assay could be used for diagnosis of fasciolosis by F. gigantica.

Research paper thumbnail of Transmission and scanning electron microscopic studies of the human sperm chromatin decondensed by micrococcal nuclease and salt

Journal of Experimental Zoology, 1982