• Applied Microscopy
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• v.38 no.3
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• pp.175-183
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• 2008

Liver regeneration is a result of highly coordinated proliferation of hepatocytes and non-parenchymal liver cells. At this process, induction of metallothionein (MT), which is low molecular and cysteine rich, has been reported. The present study was carried to find the ultrastructure of hepatocytes and determine the expression of MT in regenerating rat liver after partial hepatectomy. As a result, the remnant liver after PH grew fast from 1 day until 7 days. Various changes were morphologically observed. Disintegration of cell plates and liver lobule appeared shortly after PH. And hepatocytes showed the rapid proliferation, characterized by high nuclear cytoplasmic ratio, weak intercellular junctional complexes, chromatin condensation, increase of ribosomes and mitochondria, and temporary increase of lipid droplets. Finally, remodeling of the liver lobule was completed through the rearrangement of blood vessels and cell plates by 7 days after PH. On histochemistry, immunoreactivity indicating the presence of MT appeared moderately throughout the cytoplasm of control rat hepatocyte. After PH, positive reactions for MT increased at the cytoplasm and the nucleus. These results suggest that the remnant liver cells immediately entered cell proliferation and increase of MT expression after PH. It is thought that MT protein might be associated with transfer of some factors needed to cell division from the cytoplasm to the nucleus for regeneration of the liver after PH.

• Journal of Korean Ophthalmic Optics Society
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• v.9 no.2
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• pp.333-343
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• 2004

Impression cytology refers to application of cellulose acetate filter material to the ocular surface to remove the superficial layers of the conjunctival epithelium. The technique is non-invasive, is easy to perform, causes minimal discomfort to the patient, and can be used to follow changes in the conjunctival ocular surface over time. With this method, the morphology of the conjunctival ocular surface can be studied and the degree of squmaous metaplasia assessed. This study was performed to evaluate the conjunctival surface by impression cytology in dry eye patients. A total of 70 students with no contact lens wearing history were recruited. Subjects were required to fill in a McMonnies dry eye symptom questionnaire. The non-invasive tear thinning time(TIT) test of each subject was measured, followed by Schirmer tear test(STI), tear film break-up time(TBUT) tests and Rose-bengal staining were performed as a baseline. Conjunctival epithelial cells from the inferior bulbar conjunctiva were harvested by the impression cytology technique. The specimens collected were labelled and stained with PAS(Periodic Acid Schift)-haematoxylin. The goblet cells and conjunctival epithelial cells were observed under a light microscope of 400x magnification. The specimens were classified according to the Nelson Grading scale which was based on the degree of squamous metaplasia such as changes of goblet cells density, size/form, N:C(nucleus : cytoplasm) ratio. Dry eye patients were observed morphological changes of the epithelial cells, different nuclear alterations, decrease of the goblet cells density. The degree of cytological changes was related to severity of dry eye conditions. When the epithelial cell morphology was graded according to the system described by Nelson, specimens from the control group revealed 91.43% of the eyes to be grade 0 and 8.57% to be grade 1, whereas of the dry eye patients, 20% were grade 0, 42.86% grade 1, 34.29% grade 2 and 2,86% grade 3. Impression cytology represents a non- or minimally invasive biopsy of the ocular surface epithelium with no side effects or contraindications. It has demonstrated to be a useful diagnostic aid for a wide variety of processes involving the ocular surface. This technique is a safe, simple method and may help increase understanding of various ocular surface alterations in dry eye patients.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.169-175
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• 2006

The objective of this study was carried out to examine the polar body extrusion of in vitro matured porcine follicular oocytes as a non-invasive marker of oocyte quality to know the developmental competence in advance. The porcine oocytes matured for 48 hours were examined the polar body extrusion and some parts were stained. The examined oocytes were matured for additional $16{\sim}18$ hours and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 hours for diploid formation. The treated oocytes were washed and cultured for 7 days. The polar body extrusion and degeneration rates were varied with $9.9{\sim}52.4%$ and $21.4{\sim}61.8%$ by repetition. The polar body extruded oocytes were shown the polar body chromosome and metaphase II plate by staining. However the non-extruded oocytes were shown expanded nucleus with 39.1%, premature chromosome condensation with 19.6%, metaphase I plate with 10.9 %, metaphase II with 13%, condensed chromatin with 6.5%, and absent nuclear material with 8.7%. The oocytes that were not examined for the polar body extrusion were cleaved 45.0%, and developed to blastocyst stage with 11.3%. In examined oocytes for polar body extrusion,. the polar body extruded oocytes were cleaved 94.2% and developed with 42.5%. This result suggests that discarding of the degenerating oocytes and oocytes that not extruded polar body will be effective for experiments of culturing effect in porcine embryos and embryo biotechnology.

• Applied Microscopy
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• v.35 no.1
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• pp.15-22
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• 2005

Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, and non-enzymatic protein. The present study was carried out to investigate the expression of MT gene as well as the localization of MT in regenerating rat liver. In partial hepatectomized rats, MT mRNA was detected as early as 1 hr and reached a maximal level by 8 hr after the operation. Thereafter, this level decreased gradually until 24 hr, and it became similar to that of sham control. Meanwhile, time course of MT immunoreactivity using immunogold-labelling revealed that the number of gold particles in hepatocytes increased significantly by 12 hr, but decreased at 48 hr after partial hepatectomy. Ultrastructurally, immunogold particles indicating the presence of MT were distributed in both the cytoplasm and the nucleus of the rat hepatocytes, particularly the particles were distributed at rough endoplasmic reticulum and nucleolus and did not seem to adhere to mitochondria or lysosomes in proliferating hepatocytes. Briefly, high level of MT mRNA expression and the intense immunoreactivity and/or the specific localization of MT was observed during liver regeneration. These results suggest that MT possibly involves hepatocyte proliferation via the storage or the supply of various metal ions in the regenerating rat liver.

• Journal of Korean Ophthalmic Optics Society
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• v.8 no.1
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• pp.65-71
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• 2003

The corneal epithelium is constantly being shed. The mechanism of corneal desquamation is not fully understood. Apoptosis, programmed cell death, may play a role. Apoptosis can be induced by a number of factors and different mechanisms. The study was performed to examine the apoptotic index induced in human corneal epithelial cells maintained in tissue culture by various apoptotic inducers. Various inducers, recombinant human cytokines($INF{\gamma}$, $TNF{\alpha}$, FASAb), actinomycin D. camptothecin, cycloheximide, dexamethasone and etoposide, were purchased from commercial suppliers. Inducers at manufacturer-recommended concentration were added to the corneal epithelial cells for 48 hours. Cell viability was measured using MTT assay. The cells were then assessed for the level of apoptosis. Morphologic changes and quantification of apoptotic cells were determined and counted under fluorescence microscope after inducers-treated human corneal epithelial (HCE) cells for 48 hours with Hoechst 33342 staining. Annexin V-FITC/PI staining and DePsipher assay. The expression of Fas protein was studied by immunocytochemistry. All inducers induced apoptosis in HCE cells in a dose dependent manner. Actinomycin D. camptothecin and etoposide induced apoptosis at lower than manufacturer-recommended concentration, while cytokines, cycloheximide and dexamethasone induced apoptosis at higher concentrations at the end of 48 hours. All inducers elicited typical apoptotic morphologic changes (chromatin condensation, nucleus fragmentations non-orange-red colored mitochondria) and expresses Fas protein highly. Apoptotic index of HCE cells by these inducers was different from the other cell lines. RNA synthesis inhibitor and topoisomerase inhibitors induced apoptosis at lower concentration than manufacturer-recommended concentration. Cytokines, cycloheximide and dexamethasone were able to produce apoptosis at 10 times higher concentrations. RNA synthesis inhibitor and topoisomerase inhibitors are more sensitive than intracellular receptor-activators in apoptotic induction of HCE cells.

• Korean Journal of Plant Tissue Culture
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• v.28 no.5
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• pp.263-271
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• 2001

Inoculated anthers of Capsicum annuum L. were subjected to 4 and 32$^{\circ}C$ pretreatment and their influence on the microspore viability, early cytological changes and the induction frequency of microspore embryo was investigated. Viability of freshly isolated microspores was between 62 and 64%. During temperature pretreatment, microspore viability showed a rapid decrease and this tendency enhanced with the 32$^{\circ}C$ pretreatment. Irrespective of temperature pretreatment, microspore viability declined to nearly zero after nine days. Before temperature pretreatment, most of the microspores in anthers were at late uninucleate stage. Several types of multinuclear microspores appeared from the 2 day after culture onwards, together with many degenerated and non-induced microspores. The 32$^{\circ}C$ pretreatment gave higher proportions of embryogenic microspore than other treatment. However, the temperature pretreatment had no clear effect on the frequencies of symmetrical binucleate rnicrospore. The multinucleate grains might originate either by symmetrical or asymmetrical division. After 2 days of pretreatment at 25 and 32$^{\circ}C$ , degenerated microspore increased above 50%. In contrast, during 4$^{\circ}C$ treatment, nucleus of most microspores remained intact for 14 days. The 32$^{\circ}C$ pretreatment produced more embryos than 4$^{\circ}C$ treatment. The most effective period of 32$^{\circ}C$ pretreatment was 4 days. In contrast, effective period of 4$^{\circ}C$ pretreatment was 2 days and longer time had deleterious effect on induction of microspore embryo.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.59-67
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• 2000

This study was investigated the developmental potential of bovine embryos following nuclear transfer with bovine fetal fibroblasts (BFF). BFF were isolated from a male 45-day-old-fetus. Non-starved BFF labeled with MitoTracker were transferred into perivitelline space of enucleated oocytes. BFF-oocyte units were fused by electric pulse, and then fused oocytes were activated with calcium ionophore A23187 and subsequently 6-dimethylaminopurine (6-DMAP). The resulting zygotes were placed into CRlaa bovine embryo culture medium. Transfer of the nucleus into enucleated oocyte led to premature chromosome condensation, swelling and pronucleus formation. Remodeled oocytes were developed to the mitotic and 2-cell stage at 18 to 26 h after nuclear transfer. The incidence of in vitro development to the blastocyst stages was 21% of fused oocytes. Mitochondria of BFF eliminated rapidly and were not detected at 8 h after fusion. These results suggest that BFF can be successfully reprogrammed in enucleated bovine oocytes, and that reconstructed embryos can develop to the blastocyst stage.

• Journal of fish pathology
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• v.11 no.1
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• pp.13-22
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• 1998

To know the features of Amyloodinium sp. parasitized in Black seabream, Acanthopagrus schlegeli, morphology and reproduction type of the parasite were investigated. Infection mode and histopathology of the parasites were also studied. In the developmental and morphological observation, the parasite passed through parasitic and non-parasitic phases with three developmental stages termed trophont, tomont, and dinospore. The trophont, 30-$80{\mu}m$ ovoidal or pyriform, for the vegetative stage had a spherical nucleus with $1.5{\mu}m$ in diameter, many food vacuoles and starch grains in cytoplasm. The tomont, 80-$90{\mu}m$ spherical, for the reproductive stage resorbed a stalk, secreted a cyst wall and reproduced within it. A dinospore, small 10-$15{\mu}m$ biflagellated, for the infestation stage had a stigma. The parasite was propagated for 15 days by serial passage in Korean rockfish (Sebastes schlegeli) at $25{\pm}1^{\circ}C$. Trophonts began to detach from the fish about 3 days after infection and was completed within 5 days at $25{\pm}1^{\circ}C$. It took from 3 to 5 days to reproduce at the same temperature. Reinfected fish showed that parasites penetrated under the epithelia of gill filament and gill lamellae causing hyperplasia and degeneration at infected area.

• Animal cells and systems
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• v.15 no.3
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• pp.203-210
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• 2011

In the present study, we examined the effects of caffeine on food intake and body weight, and pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) expression in the hypothalamus. Rats were administered intraperitoneally with 100 mg/kg caffeine (a high, non-toxic dose) or saline during the light phase. Intraperitoneal administration of caffeine induced a significant reduction in food intake and body weight 12 hr after treatment. In addition, POMC expression was significantly increased and AgRP expression was decreased in the arcuate nucleus (Arc) after caffeine treatment. These results demonstrate that administration of caffeine up-regulates POMC expression and down-regulates AgRP expression in the Arc, suggesting that the activation of the hypothalamic POMC neurons and inhibition of the AgRP neurons might play a role in the regulation of food intake and body weight by caffeine.

• Journal of Life Science
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• v.23 no.10
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• pp.1199-1208
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• 2013

Fibroblastic reticular cells (FRCs) form the structural backbone of the T zone provide a guidance path for immigrating T cells in the lymph node (LN). FRCs may contribute directly to developing T-cell biology in the LN and allow analyses of fundamental aspects of FRC biology related to T cells. FRCs inhibited T-cell apoptosis, and FRC culture supernatants strongly induced the expression of Bcl-xL in T cells against doxorubicin. Coculture of FRC and T cells resulted in rearrangements of the actin cytoskeleton, as well as global changes in the morphology of the FRCs. In addition, when cocultured, the T cells adhered to the FRC monolayer, and the membrane intercellular adhesion molecule (ICAM)-1 was slightly increased by day-dependent manner. In contrast, the expression of soluble ICAM-1 was dramatically increased in a day-dependent manner. Several chemokines, such as CCL5, CXCL1, CXCL5, CXCL16, CCL8, CXCL13, and ICAM-1, and MMPs were expressed in FRCs sensed by tumor necrosis factor (TNF) families. Nuclear factor kappa B ($NF{\kappa}B$)-RelA of the $NF{\kappa}B$ canonical pathway was translocated into FRC nuclear by $TNF{\alpha}$. In contrast, p52 proteolyzed from p100, a counterpart of RelB of the noncanonical $NF{\kappa}B$ pathway, accumulated in the peripheral FRC nucleus by agonistic anti-$LT{\beta}R$ antibody. In summary, we propose a model in which FRCs engage in bidirectional crosstalk to increase the efficiency of T-cell biology. This cooperative feedback loop may help to maintain tissue integrity and function during immune responses.