• Journal of Veterinary Science
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• v.24 no.4
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• pp.53.1-53.12
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• 2023

Background: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. Objectives: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. Methods: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. Results: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. Conclusions: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

• Applied Microscopy
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• v.35 no.2
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• pp.57-64
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• 2005

The ultrastructure and histochemical characteristics of the submandibular gland was examined in the big white-toothed shrew, Crocidura lasiura. A submandibular gland of Crocidura lasiura was a mixed gland composed of serous and mucous acinar cells. Secretory granules from the acini were discharged through the intercalated duct, the granular duct and the striated duct into the oral cavity. Serous and mucous acinar cells and granular duct cells had large amount of rough endoplasmic reticulum, free ribosome and prominent Golgi apparatus at the basal cytoplasm of the cell, and many granules at the apical cytoplasm. Oval type serous granules had a homogeneously pale round shape of bead at the center. Mucous granules were distinct from those of the other mammalian species having variety patterns with several dense bands into homogeneous pale matrix. A serous-like secretory granules and myelin-like body were observed in the cytoplasm and the lumen of granular duct cells. The myelin-like body is a characteristic structure only reported in the salivary glands of two shrews, Suncus murinus and C. dsinezumi. Striated duct cell had numerous well-developed mitochondria but secretory granule was not shown at all.

• International Journal of Oral Biology
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• v.31 no.4
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• pp.141-148
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• 2006

The effects of adenosine triphosphate(ATP) on salivary glands have been recognized since 1982. The presence of purinergic recepetors(P2Rs) that mediate the effects of ATP in various tissues, including parotid and submandibular salivary gland, has been supported by the cloning of receptor cDNAs and the expression of the receptor proteins. P2Rs have many subtypes, and the activation of these receptor subtypes increase intracellular $Ca^{2+}$, a key ion in the regulation of the secretion in the salivary gland. The apical pores of taste buds in circumvallate and foliate papillae are surrounded by the saliva from von Ebner salivary gland(vEG). Thus, it is important how the secretion of vEG is controlled. This study was designed to elucidate the roles of P2Rs on salivary secretion of vEG. Male Sprague-Dawley rats (about 200 g) were used for this experiment. vEG-rich tissues were obtained from dissecting $500-1,000\;{\mu}m$ thick posterior tongue slices under stereomicroscope view. P2Rs mRNA in vEG acinar cells were identified with RT-PCR. To observe the change in intracellular $Ca^{2+}$ activity, we employed $Ca^{2+}-ion$ specific fluorescence analysis with fura-2. Single acinar cells and cell clusters were isolated by a sequential trypsin/collagenase treatment and were loaded with $10\;{\mu}M$ fura -2 AM for 60 minutes at room temperature. Several agonists and antagonists were used to test a receptor specificity. RT-PCR revealed that the mRNAs of $P2X_4$, $P2Y_1$, $P2Y_2$ and $P2Y_3$ are expressed in vEG acinar cells. The intracellular calcium activity was increased in response to $10\;{\mu}M$ ATP, a P2Rs agonist, and 2-MeSATP, a $P2Y_1$ and $P2Y_2R$ agonist. However, $300\;{\mu}M\;{\alpha}{\beta}-MeATP$, a $P2X_1$ and $P2X_3R$ agonist, did not elicit the response. The responses elicited by $10\;{\mu}M$ ATP and UTP, a $P2Y_2R$ agonists, were maintained when extracellular calcium was removed. $10\;{\mu}M$ suramin, a P2XR antagonist, and reactive blue 2, a P2YR antagonist, partially blocked ATP-induced response. However, when extracellular calciums were removed, suramin did not abolish the responses elicited by ATP. These results suggest that P2Rs play an important role in salivary secretion of vEG acinar cells and the effects of ATP on vEG salivary secretion may be mediated by $P2X_4$, $P2Y_1$, $P2Y_2$, and/or $P2Y_3$.

• Childhood Kidney Diseases
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• v.10 no.1
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• pp.8-17
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• 2006

Purpose : We describe the changes of rat glomerular epithelial cells when exposed to high levels of glucose and advanced glycosylation endproducts(AGE) in the in vitro diabetic condition. We expect morphological alteration of glomerular epithelial cells and permeability changes experimentally and we may correlate the results with a mechanism of proteinuria in DM. Methods : We made 0.2 M glucose-6-phsphate solution mixed with PBS(pH 7.4) containing 50 mg/mL BSA and pretense inhibitor for preparation of AGE. As control, we used BSA. We manufactured and symbolized five culture dishes as follows; B5 - normal glucose(5 mM) + BSA, B30 - high glucose(30 mM) + BSA, A5 - normal glucose(5 mM) + AGE, A30 - high glucose(30 mM) + AGE, A/B 25 - normal glucose(5 mM) + 25 mM of mannitol(osmotic control). After the incubation period of both two days and seven days, we measured the amount of heparan sulfate proteoglycan(HSPG) in each dish by ELISA and compared them with the B5 dish at 2nd and 7th incubation days. We observed the morphological changes of epithelial cells in each culture dish using scanning electron microscopy(SEM). We tried the permeability assay of glomerular epithelial cells using cellulose semi-permeable membrane measuring the amount of filtered BSA through the apical chamber for 2 hours by sandwich ELISA. Results : On the 2nd incubation day, there was no significant difference in the amount of HSPG between the 5 culture dishes. But on the 7th incubation day, the amount of HSPG increased by 10% compared with the B5 dish on the 2nd day except the A30 dish(P<0.05). Compared with the B5 dish on the 7th day the amount of HSPG in A30 and B30 dish decreased to 77.8% and 95.3% of baseline, respectively(P>0.05). In the osmotic control group (A/B 25) no significant correlation was observed. On the SEM, we could see the separated intercellular junction and fused microvilli of glomerular epithelial cells in the culture dishes where AGE was added. The permeability of BSA increased by 19% only in the A30 dish on the 7th day compared with B5 dish on the 7th day in the permeability assay(P<0.05). Conclusion: We observed not only the role of a high level of glucose and AGE in decreasing the production of HSPG of glomerular epithelial cells in vitro, but also their additive effect. However, the role of AGE is greater than that of glucose. These results seems to correlate with the defects in charge selective barrier. Morphological changes of the disruption of intercellular junction and fused microvilli of glomerular epithelial cells seem to correlate with the defects in size-selective barrier. Therefore, we can explain the increased permeability of glomerular epithelial units in the in vitro diabetic condition.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.521-527
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• 1998

An important property of the intestine is the ability to secrete fluid. The intestinal secretion is regulated by a number of substances including vasoactive intestinal peptide (VIP), ATP and different inflammatory mediators. One of the most important secretagogues is adenosine during inflammation. However, the controversy concerning the underlying mechanism of adenosine-stimulated $Cl^-$ secretion in intestinal epithelial cells still continues. To investigate the effect of adenosine on $Cl^-$ secretion and its underlying mechanism in the rabbit colon mucosa, we measured short circuit current ($I_{SC}$) under automatic voltage clamp with DVC-1000 in a modified Ussing chamber. Adenosine, when added to the basolateral side of the muocsa, increased $I_{SC}$ in a dose-dependent manner. The adenosine-stimulated $I_{SC}$ response was abolished when $Cl^-$ in the bath solution was replaced completely with gluconate. In addition, the $I_{SC}$ response was inhibited by a basolateral Na-K-Cl cotransporter blocker, bumetanide, and by apical $Cl^-$ channel blockers, dephenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), glibenclamide. Amiloride, an epithelial $Na^+$ channel blocker, and 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS), a $Ca^{2+}-activated$ $Cl^-$ channel blocker, had no effect. In the mucosa pre-stimulated with forskolin, adenosine did not show any additive effect, whereas carbachol resulted in a synergistic potentiation of the $I_{SC}$ response. The adenosine response was inhibited by 10 ${\mu}M$ H-89, an inhibitor of protein kinase A. These results suggest that the adenosine-stimulated $I_{SC}$ response is mediated by basolateral to apical $Cl^-$ secretion through a cAMP-dependent $Cl^-$ channel. The rank order of potencies of adenosine receptor agonists was $5'-(N-ethylcarboxamino)adenosine(NECA)＞N^6-(R-phenylisopropyl)adenosine(R-$ PIA)＞2-[p-(2-carbonylethyl)-phenyl-ethylamino]-5'-N-ethylcarboxaminoadenosine(CGS21680). From the above results, it can be concluded that adenosine interacts with the $A_{2b}$ adenosine receptor in the rabbit colon mucosa and a cAMP-dependent signalling mechanism underlies the stimulation of $Cl^-$ secretion.

• Journal of Life Science
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• v.26 no.2
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• pp.164-173
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• 2016

Isotocin (IT), a nonapeptide homolog of oxytocin in mammals, has been suggested to be involved in physiological processes including social behaviors, stress responses, and osmoregulation in teleost fish. To study its structure and function, the gene encoding the IT precursor was cloned from the genomic DNA and brain cDNA of the cinnamon clownfish, Amphiprion melanopus. The IT precursor gene consists of three exons separated by two introns, and encodes an open reading frame of 156 amino acid (aa) residues, comprising a putative signal peptide of 19 aa, a mature IT protein of 9 aa, a proteolytic processing site of 3 aa, and 125 aa of neurophysin. Tissue-specific analysis of the IT precursor transcript indicated its expression in the brain and gonads of A. melanopus. To examine its osmoregulatory effects, the salinity of the seawater (34 ppt) used for rearing A. melanopus was lowered to 15 ppt. Histological analysis of the gills indicated the apparent disappearance of an apical crypt on the surface of the gill lamella of A. melanopus, as pavement cells covered the surface upon acclimation to the lower salinity. The level of Na⁺/K^+-ATPase activity in the gills was increased during the initial stage of acclimation, followed by a decrease to its normal level, suggesting its involvement in osmoregulation and homeostasis. The only slight increase in the level of IT precursor transcript in the A. melanopus brain upon low-salinity acclimation suggested that IT played a minor role, if any, in the process of osmoregulation.

• The Korean Journal of Malacology
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• v.22 no.1 s.35
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• pp.87-95
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• 2006

Immunohistochemical and ultrastuructural experiments were conducted to find out heavy metal accumulation in the intestine of an Antarctic gastropod Nacella concinna. According to the immune-histochemical experiment the apical cytoplasm of the intestinal epithelium showed positive reactions to anti-MT (rnotallothionein), indicating the presence of MT, a metal-binding protein involved in metal detoxifying process. In the transmission electron microscopic observations, the epithelial cells of the intestine exposed to Cd for over three hours showed irregular nuclear membranes, secretory granules, and probable metal granules. According to the SEM-EDS experiments on the intestine, concentration of Pb in the apical epithelium was in inverse proportion to that in the intestinal lumen. After exposing to Cd for over three days, S was rapidly reduced. Ca and Zn were rapidly increased after exposure to Cd. These elements are supposed to be concerned with the MT-reaction in the intestine. laken together, these data suggest that N. concinna could be used as a potential biomarker species.

• Archives of Pharmacal Research
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• v.24 no.6
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• pp.584-589
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• 2001

Isopropryl 2-(1-3-dithiethane-2-ylidene)-2 [N-(4-methyl-thiazole-2-yl) carbamoyl] acetate (YH439) is currently under phase ll clinical trials by the Yuhan Research Center for use as a hepatoprotective agent. Unfortunately, the oral bioavailbility of YH439, which is sparingly soluble in water (i.e., $0.3{\;}\mu\textrm{g}/ml{\;}or{\;}0.91{$\mu}M$ at room temperature), reportedly, is negligibleregardless of the dose administered to rats in the 10-300 mg/kg range. The bioavailability of the compound increased up to 24%, when administered in the form of a micellar solution ($700{\;}\mu\textrm{g}/ml$or 2.1 mM for YH439) at a dose of 10 mg/kg, suggesting that its limited solubility is associated with its negligible bioavailability. In order to obtain additional informmation concerning the bioavailability of YH439, the mechanism(s) involved in gastrointestinal (Gl) absorption were investigated in the present study. For this purpose, the transport of YH430 across a Caco-2 cell monolayer was measured in a $Transwell^{\circledR}$. A permeability of $4.07{\times}10^{-5}{\;}cm/s$ was obtained for the absorptive (i.e., apical to basolateral direction) transport of $0.42{\mu}M$ YH439, implicating that the in vivo Cl absorption is nearly complete. The absorptive transport exhibited a slight concentration-dependency with an intrinsic clearance ($CL_{i}$) of $0.38{\mu}L/{\textrm{cm}^2}/sec$, which accounted for 28.1% of the total intrinsic clearance (i.e., $CL_i$ plus the intrinsic clearance for the linear component) of the transport. Thus, saturation of the absorption process appears to be a minor factor in limiting the bioavailability of the compound. The apparent permeability of YH439 from the basolateral to the apical direction (i.e., efflux, $6.67{\times}10^{-5}{\;}cm/s$) was comparable to that for absorptive transport, but, interestingly, a more distinct concentration-dependency was observed for this transport. However, the efflux does not appear to influence the bioavailability of the compound, as evidenced by the sufficiently high permeability in the absorption direction. Rather, a reportedly extensive first-pass hepatic metabolism appears to be a principal factor in limiting the bioavailability. In this respect, reducing the first-pass metabolism by some means would lead to a higher bioavailability of the compound. Thus, elevation of the absorption rate of YH439 becomes a necessity. From a practical point of view, increasing the concentration of YH439 in the Cl fluid appears to be a feasible way to increase the absorption rate, because the compound is primarily absorbed via a linear mechanism. In summary, the solubilization of YH439, as previously demonstrated for a micellar solution of the compound, appears to be a practical way to increase the oral bioavailability of YH439.

• Korean Journal of Environmental Biology
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• v.40 no.4
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• pp.387-397
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• 2022

The germination characteristics of the resting cysts of Pheopolykrikos hartmannii collected from the southern coastal sediments of Korea were studied at different temperature conditions, and the morphology and phylogeny of the germlings were examined. The resting cysts of Ph. hartmannii were round and characterized by a red accumulation body and many arrow-like spines and could germinate at temperature of 10 to 30℃. High germination rates (>90%) were observed at 15 and 20℃, indicating that the resting cysts could act as seed populations for the bloom initiation of Ph. hartmannii in Korean coastal waters in early summer or early fall. The morphology of the germlings was generally consistent with the previous description, and an apical groove characterized by a fully enclosed loop was observed. Phylogenetic analysis based on large SubUnit (LSU) rRNA gene sequences revealed that the germlings shared an identical sequence with the Korean and American isolates of Ph. hartmannii and was a sister clade of Polykrikos species.

• The Korean Journal of Zoology
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• v.39 no.4
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• pp.400-409
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• 1996

A study on the ultrastructural changes In the epithellum of the vas deferens by season was conducted for the spdng and summer specimens of a slug Incilarfa fruhstoiferl. The vas deferens of the spdng spedmen was muscular tube about 0.4mm in diameter. Its lumen was divided into three flat grooves and the each groove was subdivided into two subbranches. The luminal epithellal celis of the Vas deferens which were irregular In shape showed strong methylenophilla in a double stain of methylene blue and basic fuchsln. The lumen of the vas deferens was filled with components strongly stained by methylene blue. The circular muscle layers surrounding the luminal epithellum of the vas deferens contained numerous granules arranged at regular intervals. The vas deferens of the summer specimen also was a thick muscular tube showing 0.4 mm in diameter. Its lumen was divided into four grooves but, the each of the grooves was not subdivided to form certain branclees unlikely to the spdng spedmen. The lining epfthelium of the lumen was consisted of simple ciliated columnar cells, irregular columnar cells and conical cells. The histological features were quiet different from those of the spring spedmen which showed irregular cell arrangement. According to electron microscopy the epithelium of the vas deferens in the spring specimen was composed of irregular columnar cells which had irregular shaped nuclei. The nuclei of the epitheilal cells were relatively large in comparison to their cytoplasm. The overall electric density of the cytoplasm was relatively high. The lumen of the vas deferens in the summer specimen was lined by a epfthelium with tail ciliated columnar cells and irregular cells. The unclei of the epithellal cells were long ellipsoid or irregular in shape. Both of the cytoplasm and the nuclei were showed low electric density. in consideration with the observable cell organelles were only ndoplasmic reticulum, lysosomes and microtutules, the cell organelles were poorly developed. The apical surfaces of the epithelial cells possessed brush borders with numerous microvilli and cilia with 9+2 arrangement of microtubules. The circular muscle layers surrounding the epithelium are usually thick and the degree of development of the circular muscle layers seems to be even in the both of the spring and summer specimens.