To investigate recovery, growth, and activity of hepatocyte in primary culture after cell separation, the authors followed up the marker enzyme activities of golgi complex, mitochondria and biologic membrane. Thiamine pyrophosphatase, the marker enzyme of golgi complex, activity approached the level of long term culture at 4th day. Succinate dehydrogenase, the marker enzyme of mitochondria, activity decreased with time, then it maintained constant level after 4th day. Alkaline phosphatase, the marker enzyme of biological membrane, activity increased from 3rd day, and after 5th day it showed strong reaction. These data suggested that hepatocytes were stabilized and recovered normal activity 4 day after cell separation. But the main secretory function was speculated to be reduced in culture.
Alkaline Phosphatase ; Animals ; Cell Separation ; Golgi Apparatus ; Hepatocytes* ; Membranes ; Mitochondria ; Organelles* ; Rats* ; Succinate Dehydrogenase ; Thiamine Pyrophosphatase

In transmembrane and intracellular sites of neurom, calcium ion(Ca(++)) has been known to have an important role of signalling process. It is naw well accepted that calcium binding proteins, calbindin D-28K (calbindin) and parvalbumin, modulate and mediate above aclcium ionss action as a second messenger. Although it has been reported that calbindinergic and parvalbuminergic neurons comprise different subpopulations in the cat and rat spinal cords, the studies of their morphology, topographical distribution and ultrastructural features have not been done extensively in the mammalian spinal cords until now. This study was conducted to localize calbidinergic and parvalbuminergic neurons and to define their morphology, topographical distribution and ultrastructural features in the rabbit cervical cord by the preembedding immunocytochemical method using anti-calbindin and anti-parvalbumin antisera. In the rabbit cervical cord, calbindin immunoreactive neurons were mainly distributed in the dorsal horn, especially in lamina II, and a smaI1 number of labelled neurons were observed in the intermediate gray matter (IGS), but calbindin immunoreactivities were not observed in the intermediate gray substance(IGS), but calbindin immunoreactiveties were not observed in thr ventral horn. The somata of calbindin immunoreactive neurons received synaptic inputs from non-immunoreactive axon terminals in the dorsal horn and in the IGS. Parvalbumin immunoreactive neurons were mainly observed in the IGS and in the ventral horn, but only a few of parvalbumin immunoreactive neurons were distributed in the dorsal horn. In the ventral horn, two types of parvalbumin immunoreactive neurons were identified according to the sizes of the somata and labelled motor cells received synaptic inputs from labelled and unlabelled axon terminals. These results demonstrate that calbindinergic neurons are a number of neurons located in lamina II of dorsal horn and a few of neurons located in the intermediate gray and parvalbuminergic ne.urons are laocated in the intermediate gray substance and in the ventral horn, and these neurons comprise different subpopulations of neurons. It was suggest that calbindinergic neurons might play an important role in the process of pain modulation and parvalbumiergic neurons in the control of motor activity with their specific synaptic circuitry in the spinal cord.
Animals ; Calbindins ; Calcium ; Calcium-Binding Proteins ; Cats ; Horns ; Immune Sera ; Motor Activity ; Neurons* ; Presynaptic Terminals ; Rats ; Second Messenger Systems ; Spinal Cord

We experienced a complication of brachial plexus palsy secondary to operative position during thoracoscopic thoracic sympathectomies. His general health was excellent and no previous histories vulnerable to peripheral nerve systems were observed. The thoracic sympathectomies were done under general anesthesia. The patient was placed left lateral position with his right arm abduced 150o on padded arm board. An operation was lasted 2 hours and 30 minutes at this position because of severe right apical lung adhesion. The controlateral side was performed same procedure and lasted 20 minutes. After the patient recovered from the anesthesia, the patient had a complete paralysis of right arm. There was also slightly diminished sensation to pinprick on the arm and hand. Neurologic examination and EMG study revealed brachial plexus palsy. Nerve blocks and physiotherapy were performed to treat brachial plexus injuries. His motor functions were improved day by day and he was discharged with a complete range of motion against gravity on 14th. postoperation day. However, there were loss of muscle powers against some resistances and tingling sensations of fingertips. Two months later, he was recovered completely and there was no residual disabilities.
Anesthesia ; Anesthesia, General ; Arm ; Brachial Plexus* ; Gravitation ; Hand ; Humans ; Lung ; Nerve Block ; Neurologic Examination ; Paralysis* ; Peripheral Nerves ; Range of Motion, Articular ; Sensation ; Sympathectomy*

To investigate differentiation and growth process of keratinocytes in organotypic cultured skin, we carried out immunohistochemical studies for cytokeratin (CK) 10, 14, 16, 17 and involucrin in the cultured skin. In normal skin CK14 and CK10 were detected in the basal and all suprabasal layer, respectively, whereas in artificial skin CK14 was detected up to the middle of spinous layer but CK10 expressed from the middle of spinous layer. The detection of involucrin in normal skin was from the upper spinous layer but found from lower spinous layer in the artificial skin. Both CK16 and CK17 did not expressed in in vivo skin but expressed weakly in the spinous layer of artificial skin. It is therefore concluded that the characteristics of basal cell were maintained in the several, lower layers of the sartificial skin. The growth and differentiation steps of the skin were similar to those of in vivo although differences were seen in the expression level.
Cells, Cultured* ; Epidermis* ; Immunohistochemistry ; Keratinocytes ; Keratins* ; Skin ; Skin, Artificial*

PURPOSE: To report two cases that showed clinical findings of recurrent keratoconus following penetrating keratoplasty. METHODS: A 39 year-old male and a 34 year-old female developed clinical signs of recurrent keratoconus in his left and her right eye 3 and 8 years after keratoplasty, respectively, and he underwent successful corneal regrafting. RESULTS: We confirmed recurrence of keratoconus histopathologically through the examination of the cornea of the recipient who underwent corneal regrafting and chromosomal study showed mosaicism, 47,XXY/46,XY. CONCLUSION: We report the histopathologic feature of one case of recurrent keratoconus for the first time in Korea.
Adult ; Cornea ; Corneal Transplantation ; Female ; Humans ; Keratoconus* ; Keratoplasty, Penetrating ; Korea ; Male ; Mosaicism ; Recurrence

Melatonin could be used as an anticancer agent to suppress the proliferation of tumor cells and induce the differentiation of cancer cells. HeLa, HepG2, A549, L929, and NIH/3T3 cell lines were cultivated in alpha-MEM with 0.2 mM/2 mM melatonin. The influences of melatonin on quantitative changes of glycoprotein, fibronectin, laminin and actin related to the metastasis of tumor cells investigated with PAS or PAP at light microscopic level. To elucidate the possibility of antitumor actions of melatonin, the changes of cell organelles were observed under transmission electron microscope. Cell proliferation was suppressed after treatment with 2 mM melatonin for 2 or 3 days. Compared with control groups, the amounts of glycoprotein, fibronectin, laminin and actin in all cell lines at 1st, 2nd and 3rd day after treatment with 0.2 mM and 2 mM melatonin were generally increased. Heterochromatin in the nucleus formed clumps in all cell lines at 2nd and 3rd day after treatment with 0.2 mM and 2 mM melatonin. The numerical increase of rough endoplasmic reticulum and golgi complex observed in HeLa and L929 cells treated with 0.2 mM and 2 mM melatonin at 1st, 2nd and 3rd day. The number of lysosomes increased in HeLa, A549, and L929 cells treated with 0.2 mM and 2 mM melatonin at 3rd day. The number of vesicles increased in all cell lines treated with 0.2 mM and 2 mM melatonin at 1st, 2nd and 3rd day. Taken together, antimitotic effect of melatonin can be expected at least 2day after treatment with 2 mM melatonin. The production of fibronectin and laminin in all cell lines treated with 0.2 mM or 2 mM melatonin increased. Therefore, the increase of amounts of extracellular matrix proteins in the extracellular space can be expected. And the increase of amounts of actin connected to the extracellular matrix proteins through the integrin of plasma membrane seemed to strengthen cell attachment. In order to metastasize of cancer cells, it is important for them to secrete various enzymes to pass through the extracellular matrix proteins. Hence, it will be more difficult for the cells to metastasize into other regions due to the increase of the extracellular matrix proteins. It was postulated that the clumps of heterochromatin and the numerical increase of vesicles induced by treatment with 0.2 mM and 2 mM melatonin could be represented for the actions of melatonin as morpholgical criteria.
Actins ; Cell Line* ; Cell Membrane ; Cell Proliferation ; Endoplasmic Reticulum, Rough ; Extracellular Matrix Proteins* ; Extracellular Matrix* ; Extracellular Space ; Fibronectins ; Glycoproteins ; Golgi Apparatus ; Heterochromatin ; Laminin ; Lysosomes ; Melatonin* ; Neoplasm Metastasis ; Organelles

Endothelial cells were isolated from the aortic intima of Sprague-Dawley species, rat. These cells and each cancer cell line (HeLa, Hep G2, A549, L929 and NIH/3T3 cells) were co-cultivated in alpha-MEM with 3 micrometer or 30 micrometer nocodazole. To investigate the influences induced by nocodazole, the morphological changes were observed under inverted microscope and transmission electron microscope, the amounts of fibronectin produced by vascular endothelial cells and cancer cell lines and the activities of nitric oxide synthetase synthesized mainly by endothelial cells were analyzed in the aspects associated with fine structural changes. The vascular endothelial cells of control group at the 1st, 2nd and 3rd days extended the cell processes, which contacted with cells from all cell lines investigated, but the endothelial cells of nocodazole-treated groups didn't possess the processes. All cell lines in nocodazole-treated groups had a large number of micronucleated cells, but endothelial cells didn't show micronuclei. Compared with control group, the endothelial cells of nocodazole-treated groups at the 1st, 2nd and 3rd days showed the decrease of amounts of fibronectin because of the increase of heterochromatin area. The amounts of fibronectin increased in all cell lines of nocodazole-treated groups at the 2nd and 3rd days whereas the nuclear folding or the dilatation/numerical increase of rough endoplasmic reticulum didn't appear. The activities of nitric oxide synthetase heightened in endothelial cells of nocodazole-treated groups, and therefore the considerable changes in fine structures such as vesicles, lysosomes, liposomes, pyknosis and cell lysis occurred even though the extent of changes differed among the cell lines. Taken together, the materials such as fibronectin or nitric oxide synthetase produced by endothelial cells directly or indirectly acted on cancer cells, and the amounts of fibronectin and the vesicles, lysosomes, liposomes and cell lysis seemed to be much more increased or enforced. Therefore, co-culture system seemed to work better for the investigation of actions of nocodazole and the role of endothelial cells in cancer cells research. Also, the co-culture system was closer to the in vivo state and more favorable in studies for proliferation or metastasis of cancer cells.
Animals ; Cell Line* ; Coculture Techniques ; Endoplasmic Reticulum, Rough ; Endothelial Cells* ; Fibronectins ; Heterochromatin ; Liposomes ; Lysosomes ; Neoplasm Metastasis ; Nitric Oxide Synthase ; Nocodazole* ; Rats ; Rats, Sprague-Dawley

Lectins are glycoproteins that bind specifically to carbohydrates. Considerable interests in the lectins were encouraged by several reports that certain members of the family bind to the extracellular matrix proteins (ECM), such as fibronectin and laminin. However, the relations between lectin and ECM protein remain unclear. To elucidate the relations of lectin-matrix-cell, we treated three cancer cell lines, HeLa, L929, and EATC with ConA and PHA-P at low dose (4 microgram/ml) and high dose (20 microgram/ml) for 1, 3, 5 days. 1. Whether or not lectins significantly regulate the cell proliferation was evaluated by MTT assays. 2. Whether the amount of fibronectin and laminin which of cancer cells can be influenced by lectins was confirmed by immunocytochemical staining. 3. Whether, in turn, the lectins which can change the morphology were observed under inverted and electron microscopes. ConA and PHA-P inhibited cell proliferation rate of all cell lines in a dose- and time- dependent manner. The amount of fibronectin and laminin considerably reduced in the three cell lines after the lectins treatment in a dose- and time-dependent manner. The cancer cell lines showed various morphological changes such as cell aggregation, irregular-shaped cellular processes, rounded cells, cytoplasmic vacuolation, swollen RERs, dilation of mitochondria, margination of chromatin and cell death. In conclusion, our results showed ConA and PHA-P caused damages of the three cancer cell lines, but the effect of PHA-P was much stronger than ConA. Taken together, the present data strongly indicate that ConA and PHA-P influence the cell proliferation rate, reduce the amount of fibronectin and laminin and induce cell injuries of HeLa, L929, and EATC cell lines. Our results also suggest that the cancer cell proliferation and the morphological changes might be modulated by the specific interaction between lectins and ECM proteins associated with the cell surface.
Carbohydrates ; Cell Aggregation ; Cell Death ; Cell Line* ; Cell Proliferation ; Chromatin ; Cytoplasm ; Extracellular Matrix Proteins ; Extracellular Matrix* ; Fibronectins ; Glycoproteins ; Humans ; Laminin ; Lectins ; Mitochondria

Several predetermined concentrations of beta-amyloid peptide, (betaA) were administered to the rat cardiac myocyte cultures for three days to determine the effects of betaA. Stainings with congo red and crystal violet were used to evaluate the deposition of betaA in the cardiac myocytes and MTT assay was used to elucidate the cytotoxic effects of betaA by anlaysis of cell viability. Beating rates and morphological changes were investigated with inverted microscope and TEM was used to study the fine structures. Administration of 0.5 microgram/ml of betaA to cardiac myocytes induced the reduction of beating rate, however, it did neither affect the viability nor fine structures. No significant differences in cell viability or fine structures were noted in the experimental groups which were exposed to 5 microgram/ml or higher concentration of betaA. Deposition of betaA was confirmed in the cytoplasm of betaA treated cardiac myocytes with congo red and crystal violet amyloid stains. The viability of cardiac myocytes exposed to betaA was found to be reduced significantly (19%) compared to control cultures with the MTT assay. Cardiac myocytes treated with betaA presented a reduced cytoplasmic area that appeared very condensed under inverted microscope. Mitochondrial abnormalities in betaA treated cardiac myocytes included their significant enlargement, vacuolization, disorganization or paucity of cristae, paracrystalline inclusion, and accumulation of amorphous material in mitochondrial space. Mitochondrial abnormalities were present sometimes in betaA treated cardiac myocytes without disorganization of myofibils or degeneration of other cell organelles. To understand the mechanism involved in amyloid deposit and its role in pathogenesis of the diseases such as Alzheimer and inclusion body myositis (IBM), a need for in vitro model is imperative. This model of betaA treated cultured cardiac myocytes represent a amyloidosis model, and it offers several advantages for future studies of betaA to help elucidate the pathogenesis of amyloid diseases. For example, cardiac myocytes can be easily accessible, and since cardiac myocytes can be cultured for quite a long time, it is possible to study morphological and physiological changes consequent to amyloid deposits.
Amyloid ; Amyloidosis ; Animals ; Cell Survival ; Coloring Agents ; Congo Red ; Cytoplasm ; Gentian Violet ; Myocytes, Cardiac* ; Myositis, Inclusion Body ; Organelles ; Plaque, Amyloid ; Rats

In this research, the author investigated antitumor effects of green tea catechin on cancer cell lines in various concentrations and durations. Additionally, antitumor mechanism of catechin associated with apoptosis and necrosis, especially their onset and duration were invesigated. Cancer cell lines, L1210 (lymphocytic leukemia), L929 (fibrosarcoma), HepG2 (hepatoblastoma) were used. In each group, intensity of morphological changes and cell damage was observed under inverted, light, confocal and electron microscopes, and MTT and flowcytometric analysis, gel electrophoresis were used to elucidate the effects of catechin after exposure to 1, 10, 100 and 500 microgram/ml catechin until 72 hours. In all cancer cell lines, catechin induced cellular injury and inhibition of proliferation in concentration- and duration-dependent manner. The effects of catechin were the strongestt in L1210 cells and L929 and HepG2 cells in order. Dual phenomena, of apoptosis and necrosis, were shown after catechin treatment. In necrotic cells, cellular swelling, cell organelles destruction, nuclear disintegration and random DNA fragmentation were observed. In apoptotic cells, apoptotic bodies, nuclear and cytoplasmic condensations, periodic DNA fragmentation were seen. In L1210 cells, necrotic and apoptotic cells were co-existed earlier, after exposure to catechin 100 microgram/ml and then apoptosis predom-inated later. In the same concentration, catechin induced apoptosis of L929 cells. but after exposure to 500 microgram/ml catechin, They were involved with apoptosis and necrosis simultaneously. On the other hand, HepG2 cells were damaged less than other cell lines but were involved with necrosis and inhibition of G2/M phase after treatment with 500 microgram/ml catechin. These results suggested that anti-tumor mechanism of catechin, associated with apoptosis, necrosis and cell cycle arrest, were quite different according to cancer type, concentration and duration of catechin treatment. Therefore, much more research would be essential for clinical application of catechin and this study would be the basic source for further study of green tea.
Apoptosis ; Catechin* ; Cell Cycle Checkpoints ; Cell Line* ; Cytoplasm ; DNA Fragmentation ; Electrophoresis ; Hand ; Hep G2 Cells ; Necrosis ; Organelles ; Tea*