• Title/Summary/Keyword: in vitro cultivation

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Monitoring of Nervous Necrosis Virus (NNV) in the Broodstock of Seven Band Grouper Hyporthodus septemfasciatus (신경괴사증바이러스(Nervous Necrosis Virus, NNV) 모니터링을 통한 무감염 능성어(Hyporthodus septemfasciatus)친어의 선발)

  • Kim, Si-Woo;Kim, Wi-Sik;Seo, Han-Gill;Kim, Kyong Min;Oh, Myung-Joo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.50 no.5
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    • pp.527-533
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    • 2017
  • We investigated the infection of nervous necrosis virus (NNV) in seven band grouper Hyporthodus septemfasciatus broodstocks, which have been reared in aquaculture farms in South Korea during 2012-2014. To investigate the prevalence of NNV within the broodstock, egg, sperm, and blood were sampled in the spawning season. The egg and sperm samples were subjected to a nested reverse transcription (RT) polymerase chain reaction (PCR) assay to detect NNV and were inoculated on SSN-1 cells to culture the virus. Blood samples were used to detect antibodies against NNV using enzyme linked immunosorbent assay (ELSIA). Positive values from ELISA were found in 39 of 162 samples (24%) in 2012, and 13 of 28 samples (46%) in 2014. Additionally, 4 of 34 broodstocks (11%) investigated in 2013-2014 were determined to be carriers from the nested RT-PCR and in vitro cultivation. The broodstocks in which antibodies against NNV were detected by ELISA, or in which NNV was detected by the nested RT-PCR assay, posed a risk of vertical transmission of NNV. Therefore, it is necessary to select virus-free broodstocks in seed production to reduce the possibility of the vertical transmission of NNV.

Viability and Functions of Alginate-microencapsulated Islets Isolated from Neonatal Pigs

  • Lin, Yi-Juain;Wang, Jui-Ping;Chung, Yu-Tung;Sun, Yu-Ling;Chou, Yu-Chi
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.5
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    • pp.795-801
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    • 2007
  • Patients with Type I diabetes mellitus have been treated with porcine insulin for several decades and pigs have recently been deemed an ideal source of microencapsulated islet cells for clinical xenotransplantation. In this study, neonatal pigs were anesthetized and sacrificed prior to a pancreatectomy. Islet cells were isolated from pancreas via collagenase digestion. Islet cells were separated and collected by hand under microscopic guidance. These cells were suspended in 1.4% sodium alginate solution and encapsulated by dropping them into 1.1% calcium chloride solution and in which the round gel in size was 250-400 ${\mu}m$ in diameter. Viability of the microencapsulated islet cells cultured in medium at $37^{\circ}C$ was assessed by MTT assay. Furthermore, insulin released in response to glucose challenge was investigated using an enzyme-linked immunosorbent assay. Secretion of insulin was low in response to the basal glucose solution (4.4 mM) in medium and was significantly higher in response to the high glucose solution (16.7 mM). The viability of microencapsulated islet cells did not differ significantly over a period of 7 days; that is, the increasing pattern of insulin concentration in the culture medium after glucose stimulation interval day was similar throughout the 7 days cultivation. In summary, experimental evidences indicated that the effects of alginate-microencapsulation prolonged survival of the neonatal porcine islets in vitro cultures and the insulin response to glucose of the islets was maintained.

Changes in Gut Microbial Community of Pig Feces in Response to Different Dietary Animal Protein Media

  • Jeong, Yujeong;Park, Jongbin;Kim, Eun Bae
    • Journal of Microbiology and Biotechnology
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    • v.30 no.9
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    • pp.1321-1334
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    • 2020
  • Beef, pork, chicken and milk are considered representative protein sources in the human diet. Since the digestion of protein is important, the role of intestinal microflora is also important. Despite this, the pure effects of meat and milk intake on the microbiome are yet to be fully elucidated. To evaluate the effect of beef, pork, chicken and milk on intestinal microflora, we observed changes in the microbiome in response to different types of dietary animal proteins in vitro. Feces were collected from five 6-week-old pigs. The suspensions were pooled and inoculated into four different media containing beef, pork, chicken, or skim milk powder in distilled water. Changes in microbial communities were analyzed using 16S rRNA sequencing. The feces alone had the highest microbial alpha diversity. Among the treatment groups, beef showed the highest microbial diversity, followed by pork, chicken, and milk. The three dominant phyla were Proteobacteria, Firmicutes, and Bacteroidetes in all the groups. The most abundant genera in beef, pork, and chicken were Rummeliibacillus, Clostridium, and Phascolarctobacterium, whereas milk was enriched with Streptococcus, Lactobacillus, and Enterococcus. Aerobic bacteria decreased while anaerobic and facultative anaerobic bacteria increased in protein-rich nutrients. Functional gene groups were found to be over-represented in protein-rich nutrients. Our results provide baseline information for understanding the roles of dietary animal proteins in reshaping the gut microbiome. Furthermore, growth-promotion by specific species/genus may be used as a cultivation tool for uncultured gut microorganisms.

Infection and Pathogenesis Mechanisms of Marek's Disease Virus (마렉병 바이러스 감염과 병원성 발현 기전)

  • Jang, H.K.;Park, Y.M.;Cha, S.Y.;Park, J.B.
    • Korean Journal of Poultry Science
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    • v.35 no.1
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    • pp.39-55
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    • 2008
  • Like the other herpesviruses, the virion of MDV consists of an envelope, which surrounds an amorphous tegument. Within the tegument, and icosahedral capsid encloses a linear double-stranded DNA core. Although the genome structure of MDV indicates that it is an ${\alpha}-herpesvirus$ like herpes simplex and varicella-zoster viruses, biological properties indicate MDV is more akin to the ${\gamma}-herpesvirus$ group, which includes Epstein-Barr and Kaposi's sarcoma herpesviruses. These herpesviruses replicate lytically in lymphocytes, epithelial and fibroblastic cells, and persist in lymphoblastoid cells. MDV has a complex life cycle and uses two means of replication, productive and non-productive, to exist and propagate. The method of reproduction changes according to a defined pattern depending on changes in virus-cell interactions at different stages of the disease, and in different tissues. Productive (lytic) interactions involve active invasion and take-over of the host cell, resulting in the production of infectious progeny virions. However, some herpesviruses, including MDV, can also establish a non-productive (abortive) infection in certain cell types, resulting in production of cell-associated progeny virus. Non-productive interactions represent persistent infection, in which the viral genome is present but gene expression is limited, there is no structural or regulatory gene translation, no replication, no release of progeny virions and no cell death. Reactivation of the virus is rare, and usually the infectious virus can be re-isolated only after cultivation in vitro. MDV establishes latency in lymphoid cells, some of which are subsequently transformed. In this review article, recent knowledges of the pathogenesis mechanisms followed by MDV infection to sensitive cells and chickens are discussed precisely.

Alteration of cellular events in tooth development by chemical chaperon, Tauroursodeoxycholic acid treatment

  • Lee, Eui-Seon;Aryal, Yam Prasad;Kim, Tae-Young;Pokharel, Elina;Kim, Harim;Sung, Shijin;Sohn, Wern-Joo;Lee, Youngkyun;An, Chang-Hyeon;Kim, Jae-Young
    • International Journal of Oral Biology
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    • v.45 no.4
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    • pp.190-196
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    • 2020
  • Several factors, including genetic and environmental insults, impede protein folding and secretion in the endoplasmic reticulum (ER). Accumulation of unfolded or mis-folded protein in the ER manifests as ER stress. To cope with this morbid condition of the ER, recent data has suggested that the intracellular event of an unfolded protein response plays a critical role in managing the secretory load and maintaining proteostasis in the ER. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone and hydrophilic bile acid that is known to inhibit apoptosis by attenuating ER stress. Numerous studies have revealed that TUDCA affects hepatic diseases, obesity, and inflammatory illnesses. Recently, molecular regulation of ER stress in tooth development, especially during the secretory stage, has been studied. Therefore, in this study, we examined the developmental role of ER stress regulation in tooth morphogenesis using in vitro organ cultivation methods with a chemical chaperone treatment, TUDCA. Altered cellular events including proliferation, apoptosis, and dentinogenesis were examined using immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, altered localization patterns of the formation of hard tissue matrices related to molecules, including amelogenin and nestin, were examined to assess their morphological changes. Based on our findings, modulating the role of the chemical chaperone TUDCA in tooth morphogenesis, especially through the modulation of cellular proliferation and apoptosis, could be applied as a supporting data for tooth regeneration for future studies.

Enhancement of Shoot Regeneration by Ethylene Inhibitors from Cotyledon Explant of Brassica campestris L. ssp. pekinensis (에틸렌 저해제에 의한 배추 자엽조직의 기내 재분화율 향상)

  • 이혜승;조화진;김병동
    • Korean Journal of Plant Tissue Culture
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    • v.22 no.5
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    • pp.267-271
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    • 1995
  • To improve regeneration efficiency of Brassica campestris ssp. pekinensis (chinese cabbage) in vitro, the effect of ehtylene inhibitors [AgNO$_3$ and silver thiosulfate (STS)] and optimal age of explantse were investigated. On the effect of ethylene inhibitors either 100 $\mu$M of AgNO$_3$ or 5 $\mu$M of STS enhanced shoot regeneration from cotyledons when it was added in basal shoot induction media(MS salts, B5 vitamine, sucrose 2%, BA 2.0mg/L, NAA 1.0mg/L). But at higher concentrations, AgNO$_3$ induced abnormal shoots, and STS greatly reduced regeneration frequency. On the other hand, the maximum regeneration rate was obtained from the cotyledons taken from 3-day old seedlings. However there was no distinctive effect among the containers used for cultivation. The most optimal condition of root induction was a minimal Murashige and Skoog media containing 0.1 mg/L NAA. In order to induce bolting and flowering from in vitro regenerated chinese cabbage, the plant were healed at 4$^{\circ}C$ for weeks in a cold chamber. When they were planted in pots, the plane produced phenotipically normal flowers and seeds. The overall results suggest that ethylene inhibitors promote regeneration of shoot from cotyledons of chinese cabbage without alleviating fertility.

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Evaluation of Nematicidal Activity of Streptomyces yatensis KRA-28 against Meloidogyne incognita

  • Park, Eun-Jae;Jang, Hyun-Jae;Park, Chan Sun;Lee, Seung-Jae;Lee, Soyoung;Kim, Kang-Hoon;Yun, Bong-Sik;Lee, Seung Woong;Rho, Mun-Chual
    • Journal of Microbiology and Biotechnology
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    • v.30 no.5
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    • pp.700-707
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    • 2020
  • The root-knot nematode (Meloidogyne incognita) is an important pathogen in crop cultivation, however, few methods are available to control this parasitic roundworm. In this study, the nematicidal effects of approximately 30 Streptomyces strains isolated from soil samples of Mt. Naejang (Korea) were tested against Meloidogyne incognita, and the culture broth of the strains KRA-24 and KRA-28 exhibited approximately 75% and 85% insecticidal activity, respectively, in in vitro assays. In in vivo pot experiments, these strains reduced the number of nematodes in the soil and the number of egg masses in the roots of red peppers. The two strains also survived in the presence of insecticidal agents (0.1 to 3.0%) such as fosthiazate, ethoprophos and terbufos when they were used in parallel. The mixture of KRA-24 or KRA-28 culture broth and fosthiazate exhibited nematicidal effects that were similar to those observed when KRA-24 or KRA-28 were used alone. Our results clearly suggest that the Streptomyces strains KRA-24 and KRA-28 should be promoted as a biocontrol agent against Meloidogyne incognita.

Integration and Expression of Goat ${\beta}-Casein/hGH$ Hybrid Gene in a Transgenic Goat

  • Lee, Chul-Sang;Lee, Doo-Soo;Fang, Nan-Zhu;Oh, Keon-Bong;Shin, Sang-Tae;Lee, Kyung-Kwang
    • Reproductive and Developmental Biology
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    • v.30 no.4
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    • pp.293-299
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    • 2006
  • In order to generate transgenic goats expressing human growth hormone (hGH) in their mammary glands, goat ${\beta}-Casein/hGH$ hybrid gene was introduced into goat zygotes by pronuclear microinjection. DNA-injected embryos were transferred to the oviduct of recipients at 2-cell stage or to the uterus at morula/blastocyst stage after cultivation in glutathione-supplemented mSOF medium in vitro. Pregnancy and survival rate were not significantly different between 2-cell embryos and morula/blastocysts transferred to oviduct and uterus, respectively. One transgenic female goat was generated from 153 embryos survived from DNA injection. Southern blot analysis revealed that the transgenic goat harbored single-copy transgene with a partial deletion in its sequences. Despite of the partial sequence deletion, the transgene was successfully expressed hGH at the level of $72.1{\pm}15.1{\mu}g/ml$ in milk throughout lactation period, suggesting that the sequence deletion had occurred in non-essential part of the transgene for the transgene expression. Unfortunately, however, the transgene was not transmitted to her offspring during three successive breeding seasons. These results demonstrated that goat ${\beta}-casein/hGH$ gene was integrated into the transgenic goat genome in a mosaic fashion with a partial sequence deletion, which could result in a low level expression of hGH and a failure of transgene transmission.

Oxidation of Acridine by Laccase of Pycnoporus cinnabarinus SCH-3 (주걱송편버섯(Pycnoporus cinnabarinus SCH-3)의 Laccase에 의한 Acridine 산화)

  • Lee, Hyoun-Su;Han, Man-Deuk;Yoon, Kyung-Ha
    • Korean Journal of Microbiology
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    • v.44 no.2
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    • pp.110-115
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    • 2008
  • Acridine was not a substrate for fungal laccase but it was oxidized to acridone in the culture medium of P. cinnabarinus SCH-3. During the cultivation of P. cinnabarinus SCH-3, Laccase was the predominant extracellular phenoloxidase, and 3-hydroxyanthranilic acid (3-HAA) was produced in the early culture. Cinnabarinic acid (CA) was observed to accumulate in the culture medium. When P. cinnabarinus was grown in the culture medium containing acridine, acridine was oxidized to acridone. But when the laccase purified from the culture medium of P. cinnabarinus directly reacted with acridine in sodium tartrate buffer (pH 3.0), The oxidation of acridine did not happen. In contrast, when 3-HAA was added to the buffer that was mixed with laccase and acridine, the acridine was oxidized to acridone. While in vitro studies, the CA was formed from 3-HAA in the presence of purified laccase. The results suggest that the acridine should be oxidized to the acridone through the mediation of 3-HAA by the laccase in the culture medium of P. cinnabarinus SCH-3.

Maintenance of Filamentous Fungi in Korean Agricultural Culture Collection (KACC) (농업미생물은행(KACC)의 곰팡이 보존)

  • Shin, Myeong-Suk;Hong, Seung-Beom
    • The Korean Journal of Mycology
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    • v.42 no.2
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    • pp.97-103
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    • 2014
  • A total of 7039 strains of filamentous fungi are preserved in Korean Agricultural Culture Collection (KACC). The 4065 strains (58%) of them, which produce many spores in cultivation on proper media, are preserved with freeze-drying method. They are also preserved with liquid nitrogen and deep-freezer storage in order to minimize loss by death. Aspergillus, Penicillium, Lichtheimia, Mucor, Rhizopus, etc. which are common in surrounding environments, are included in this category. The others which do not produce spores, or produce few spores in vitro, are preserved with liquid nitrogen, deep-freezer and mineral oil storage. Phytophthora, Pythium, Cercospora, Septoria, Rhizoctonia, etc. are included in this category. The authors also introduced various fungal preservation methods and provided detailed preservation procedures that are used in KACC.