Faculty of Science - Mahidol University

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270 strong reactivity to the 58 kDa putative polyhedrin protein in Western blots. Altogether, the results suggest that the 58 kDa protein is Thai MBV polyhedrin and that a previously reported MBV polyhedrin gene sequence may represent another protein or polyhedrin from a different variety of MBV. (Dis Aquat Org. 2005 65:79-84.) SURVIVAL AND INTEGRATION OF SOX-1-GFP MOUSE EMBRYONIC STEM CELLS IN AUDITORY BRAIN STEM SLICE CULTURES (NO. 692) A. Glavaski-Joksimovic, C. Thonabulsombat, A. Jonsoon, M. Eriksoon, B. Palmgren , M Hagglund an dOlivius. Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand. For a clinical therapeutic approach the use of stem cells in a cell replacement therapy is promissing for the regeneration of sensory function. Previously we have used embryonic neuronal tissue and stem cells to initiate a neuronal repair paradigm into the inner ear. Here we have studied the differentiation and incorporation of Sox1-GFP mouse embryonic stem-(ES) cell into the rat postnatal brainstem slice culture. Sox-1 is the earliest known specific marker of neuroectoderm in the mouse embryo. GFP is not detectable in undifferentiated ES cells but becomes evident after neural differentiation that allows direct analysis of the conversion of pluripotent ES cells into neuroectoderm in vitro. Previously it was shown that in adherent monoculture Sox1-GFP mouse ES cells can differentiate into neurons and glial cells. We have implanted Sox1- GFP mouse ES cells into auditory brainstem slice from SD postnatal rats (P 11-15). Using tissue chopper 300-ตm-thick slices encompassing vestibulocochlear nerve and cochlear nucleus were prepared and propagated using membrane interface methods described by Stoppini. After placing slices on membranes the cochlear nucleus was labeled by DiI. In vitro transplantation was conducted at day 5±2 in culture. A total volume of 0.5ตl of Sox1 cell suspension in concentration of 1x10 4 /ตl was deposited next to the vestibulocochlear nerve and cochlear nucleus. Two weeks after Sox-1 cell transplantation co-cultures were fixed and immunostained with antibodies raised against neuronal and glial markers. We have demonstrated that following deposition next to the slices Sox1 cells actively migrate toward the cochlear nucleus pre-labeled with DiI. The morphological and immunohistochemical data indicated that grafted Sox1 cells were able to differentiate into neurons and glial cells by two weeks after implantation. These results demonstrate that organotypic slice co-cultures of the auditory brainstem with Sox1 cells present a useful model to study the regeneration of the respective neurons and that co-cultured Sox1 cells generate neurons and glial cells capable of integrating into the brain circuitry. (Poster Presentation at Inner Ear Biology Meeting, Germany. September 2005) EVIDENCE FOR THE PRESENCE OF NONODON- SLOW GROWTH AGENT IN THE PACIFIC WHITE SHRIMP (NO. 693) G. Anatasomboon, A. Akrajamorn, W. Panphut, W. Saeng-Oum, K. Sritunyaluksana and B. Withachumnarnkul Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand. In Thailand, the relatively sudden, nation-wide occurrence of unusually slow-growth in the black tiger shrimp Penaeus monodon (called monodon slow growth syndrome or MSGS) was a major factor in causing the majority of Thai shrimp farmers to convert to farming the Paci.c white shrimp Penaeus vannamei. One possible cause of MSGS was an infectious agent that could be referred to as monodon slow growth agent (MSGA). Thus, morphological and ultrastructural studies were carried out to search for pathogens with MSGS shrimp . Although a number of known viral, bacterial and protozoan pathogens were found, a substantial fraction of the MSGS shrimp were free of known pathogens or unusual histopathology. In addition to known viruses, some unknown spherical viral-like particles (~25 nm) were frequently detected by transmission electron microscopy (TEM) in the lymphoid organ and gills of MSGS shrimp. Lymphoid organ extracts (LOE) passed through 0.45 μm membrane .lters induced MSGS in healthy P. monodon suggesting that the unknown viral particles might be associated with MSGS. In another test, Paci.c white shrimp P. vannamei were co-cultured with MSGS P. monodon and they grew normally. However, membrane .ltered LOE extracts from these co-cultured P. vannamei caused MSGS when injected into healthy P. monodon. These .ndings suggest that a pathogen called MSGA exists, that it is possibly a virus(es) located in the lymphoid organ of both P. vannamei and P. monodon, and that P. vannamei may act as an unaffected carrier. (Poster presentation at World Aqualculture, May 2005. Bali, Indonesia) RESEARCH PROGRESS ON MONODON-SLOW GROWTH SYNDROME (MSGS) IN THAILAND T.W. Flegel and B. Withyachumnarnkul Faculty of Science (NO. 694) Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailandã During 2002, slow growth of farmed P. monodon (monodon slow growth syndrome or MSGS) was reported throughout shrimp growing areas of Thailand and .gures indicated that annual production volume was down by approximately 36%. Although the etiology is still uncertain, a working case de.nition of MSGS has been developed for surveillance and epidemiological purposes . By this de.nition, a suspected population must have a coef .cient of variation (CV = Standard deviation/Mean) of more than 35% by weight and absence of hepatopancreatic parvovirus (HPV) or of other severe hepatopancreatic infections by known agents while also complying with any 3 out of the 5 following gross signs: 1) unusually dark color, 2) average daily weight gain of less than 0.1 g/day at 4 months, 3) unusually bright yellow markings, 4) “bamboo” abdominal segments, 5) brittle antennae. Preliminry trials using bacteria -free .ltrates from slow growing P. monodon caused slow growth in laboratory injection experiments with P. monodon but not with P. vannamei. This suggested that an infectious agent might be involved . An initial survey suggested that known pathogens were unlikely to be the cause. A new type of yellow head virus (YHV) was found in these shrimp, but preliminary tests revealed that was not correlated with the problem. Another candidate virus is lymphoid organ vacuolization virus (LOVV) .rst described in several captured P. monodon broodstock from Thailand by DV Lightner (personal communication) in late 2002. Ultracentrifuge bands of tissue homogenates from MSGS shrimp have shown 2 previously unreported viral-like particles, and extracts of these bands give products by random RT-PCR but not by PCR, suggesting possible involvement by an RNA virus (es). As of now, there is no tested management approach to tackle this problem. 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Mahidol University Annual Research Abstracts, Vol. 33 271 However, it is well known that exotic viruses can move easily amongst crustacean species. Implementing the following recommendations might help to reduce the impact of slow growth syndrome. Imported crustaceans and especially exotic species should be reared separately from native species particularly at the hatchery phase and importations should follow the full ICES protocol with the addition of co-habitation tests employing important, endemic crustacean species. This will reduce the risk of importing exotic viral pathogens that may damage local aquaculture or .sheries. (Poster presentation at World Aqualculture, May 2005. Bali, Indonesia) PROSTAGLANDINS IN THE POLYCHAETE Perinereis nuntia AND THEIR RECEPTORS IN THE OVARY OF THE BLACK TIGER SHRIMP Penaeus monodon. (NO. 695) P. Poltana, T. Lerkitkul, G. Anantasomboon, W. Wannapapho, K. Wongprasert, P.J.W. Olive and B. Withyachumnarnkul Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand. Nereid polychaetes e.g. Perinereis nuntia brevicirris are an important fresh feed for Penaeus monodon broodstock; the worms are believed to contain substance(s) that stimulate ovarian maturation of the shrimp. In addition to essential polyunsaturated fatty acids, the polychaetes may contain certain hormones, such as prostaglandins (PGs), that could stimulate ovarian maturation in the shrimp. This study was aimed at isolating PGs from adult (atokous) P. nuntia and finding their receptors in the ovarian tissue of P. monodon. Cultured P. nuntia were extracted for PGs, using ethanol as solvent; the extract was analyzed by fast-performance liquid chromatography (FPLC), and by comparing with standard PGs, the substance was found to be PGF2α, which was present at a concentration of 0.66 ng/g wet weight of the polychaete. Using mass spectrophotometry (MS), the molecular weight was found to be 368.5, which could be either PGF2α methylester or 15(S)-15-methyl PGF2α. To study PG receptors, ovaries were isolated from sexually mature P. monodon and divided into two parts; one for paraf.n sections followed by immuno-peroxidase identi.cation of the receptors and the other for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for Western immunoblotting with speci.c PG antibodies. The immuno-peroxidase technique revealed PGF2α receptor, or FP receptor, activity in the ovarian tissues. For the previtellogenic stage, the reaction was predominantly localized throughout the cytoplasm and in the cell membrane and nuclear membrane of the primary oocytes. For the vitellogenic stage, the activity was similarly localized, but with less intensity, and it was not observed within the cortical rods. In the spent stage, the activity could be similarly observed in the remaining oocytes, as well as in the irregular-shaped primary oocytes. The SDS-PAGE and Western immunoblotting revealed an FP protein band at 47 kDa in stages I and II and at 51 kDa in stage III and IV ovaries. By using an enhanced chemiluminescence (ECL) glycoprotein detection kit, no glycoprotein was observed in the 51 kDa band. The .ndings suggest that the polychaete P. nuntia contains PGF2α and this hormone might be an exogenous source of PGs that stimulate ovarian maturation in P. monodon broodstock. (Poster presentation at World Aqualculture, May 2005. Bali, Indonesia.) VIBRIO BACTERIN AND CARBOXYMETHYL b-1,3-GLUCANS PROTECT Penaeus monodon FROM Vibrio harveyi INFECTION (NO. 696) K. Wongprasert, S. Somapa Klannukarn, K. Khanobdee, P. Meeratana, P. Pongtippatee-Taweepreda and B. Withyachumnarnkul Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand. The aim of this study was to determine effects of a vibrio bacterin, with or without carboxymethyl β-1,3-glucans (CMBG), on the protection of Penaeus monodon against Vibrio harveyi infection. It also aimed to determine the mechanism underlying any observed protection. The study was carried out in 2 phases. In the .rst phase, healthy juvenile shrimp were exposed to short-term treatment in concrete tanks and in the second phase, they were exposed to long - term treatment in commercial ponds. In both phases, they were subsequently challenged with V. harveyi. In the .rst phase, the shrimp were fed for 10 days with commercial pellets top-dressed with a formalin-killed V. harveyi bacterin alone, with CMBG alone or with bacterin plus CMBG. They were then challenged with virulent V. harveyi and the relative percent survival (RPS) was determined. The shrimp haemolymph was also examined to determine haemocyte counts, phagocytic index and bactericidal and phenoloxidase activities. In the second commercial pond phase, shrimp groups were given the same feeds for two months before challeng with virulent V. harveyi and calculation of RPS. Controls in both test phases comprised shrimp fed with commercial shrimp pellets plus coated with un-supplemented coating material.. In the .rst-phase tank trials, shrimp groups receiving bacterin alone, CMBG alone or the combination survived better than shrimp in the control group . After 10 days of treatment, total haemocyte counts and counts of haemocyte types (i.e., hyalinocytes, semi-granulocytes and granulocytes) were signi.cantly increased in the treated shrimp groups when compared to the control group. Levels of phagocytosis, bactericidal activity of mixed cell and haemocyte fractions and prophenoloxidase activity of haemolymph lysate supernatant .uids were also signi.cantly higher in the treated shrimp groups. Treated shrimp groups from the commercial ponds were also protected against V. harveyi when compared to the control group . For all the parameters tested, there were no signi.cant differences among the shrimp groups treated with bacterin alone, CMBG alone or combined bacterin plus CMBG. Thus, either bacterin or CMBG could protect shrimp against V. harveyi challenge and this correlated with stimulation of the shrimp cellular and humoral defense factors. (Poster presentation at World Aqualculture, May 2005. Bali, Indonesia) SEROTONIN IMMUNOREACTIVITY IN THE CENTRAL NERVOUS SYSTEM AND OVARIES OF THE BLACK TIGER SHRIMP, Penaeus monodon (NO. 697) K. Wongprasert, S. Asuvapongpatana and B.Withyachamnarnkul Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand. PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
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