Role of Cardiac Transcription Factor Nkx2.5 on Cardiomyoplasty Model in vitro / 대한해부학회지

Despite therapeutic advance, the prevalence of ischemic heart disease continues to increase. Recently, cell transplantation of stem cell has been proposed as a strategy for cardiac repair following myocardial damage. However, low differentiation efficiency into cardiomyocyte and poor cell viability associated with transplantation have limited the reparative capacity of these cell. In this study, we engineered P19 embryonal carcinoma cells using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction of P19 in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. An P19 embryonal carcinoma cell line expressing GFP, MEF2c, Nkx2.5 was generated by gene transfection and clonal selection. Nkx2.5 overexpression induced connexin43 expression level decrease. Electron microscopy revealed myofibril organization and immunostaining with cTnT showed positive staining in P19-Nkx2.5, consistent with early stage cardiomyocyte. Connexin43 and N-cadherin was expressed between P19-MEF2c and cardiomyocyte, P19- Nkx2.5 and cardiomyocyte in co-culture. And beating rate of cardiomyocyte co-cultured with P19-Nkx2.5 increased much more than other group, even if P19-Nkx2.5 did not have synchronous contraction with cardiomyocyte. Additionally, P19-Nkx2.5 had a resistance against hypoxia. These result suggest that overexpression of Nkx2.5 induced differentiation of P19 into cardiomyocyte and would be electro-mechanical coupling with cardiomyocyte after transplantation. Futhermore, Nkx2.5 overexpression had protection potential to hypoxic injury. Therefore, P19 cell overexpressed Nkx2.5 would be promising cell source for further study of new therapy of myocardial disease and building up in vitro model.

Formation of Intercellular Junction between Cardiomyocyte and H9c2 Cell Line in Co-Culture / 체질인류학회지

Recently, new treatments for human heart disease such as ischemia, infarction, cardiomyopathy, coronary heart disease have been developed. transplantation various kinds of cells from skeletal muscle, endothelium, mesenchyme, hemopoietic tissue to injured area after infarction were challenged. It's so called 'Cell Transplantation'. This therapeutic strategy already adopted and got a good result in clinical trial. But several limitations are still remained, including ethics, donor cell numbers, side effects, therapeutic efficiency. In this research, we investigated the formation of intercellular junction and synchronous contraction between cardiomyocyte and H9c2 cell line in co-culture to establish experimental model in vitro for cell transplantation. For this purpose, two kinds of cells, primary cultured cardiomyocyte and H9c2 (cardiomyoblast cell line) were used. Cultured cardiomyocytes had repetitive contraction-relaxation pattern along longitudinal axis both in single and coculture. But their contractions were slower, less regular, less strong in co-culture than in cardiomyocyte culture only. H9c2 cells did not contracted actively themselves, but moved toward cardiomyocyte passively coincided with contraction. In contact region between two kinds of cells, there was no signal after immunocytochemical staining labeled with connexin43 (gap junction), desmoplakin (desmosome), N-cadherin (adherent junction) even though they had membrane contact. Moreover, F-actin and striation were less developed. These results suggested that co-culture system interfere with remodelling of contractile apparatus, intercellular junction formation as well as contraction-relaxation. Furthermore cardiomyocyte could not induce H9c2 cells differentiation into cardiomyocyte. Therefore, much more research would be essential for clinical application of cell transplantation and this study would be the basic source for further study of new therapy of myocardial disease and building up in vitro model.

The Effect of Green Tea Catechin on Epithelial Cancer Cell Lines / 대한해부학회지

Catechin is main component of polyphenol extracts from green tea, it is associated with prevention of hypertension and atherosclerosis, anti-diabetic effect, antioxidant, antitumor. The purpose of this research is to investigate the effect and its mechanism of green tea catechin on epithelial cancer cell lines in various concentrations and durations. For this study, epithelial cancer cell lines, A549 (lung cancer), EATC (Ehrlich-Lettre ascites tumor cell) were used. Inverted, light, confocal and electron microscopes were applied to find morphological changes. MTT assay, flowcytometric analysis, gel electrophoresis were used to compare severity of cellular damages to control after exposure to 1, 10, 100 and 500 microgram/ml catechin for 48 hours. In the A549 cells, after 1 microgram/ml and 10 microgram/ml catechin treatments, there was no notable changes. However, exposure to 100 microgram/ml catechin induced increase of cytoplasmic granules, destruction of lamellar body, inhibition of cell cycle, especially G0/G1. In the early phase of 500 microgram/ml catechin administration, decrease of cell population, severe destruction of lamellar bodies and mitochondria, derangement of cell cycle were shown. In the EATC, such as those effects occurred after exposure to lower concentration of catechin than in that of A549 cells. After exposure of 10 microgram/ml catechin, rounded-up cells and necrotic cells were found. Whereas, most of cells were under apoptotic changes-cytoplasmic condensation, nuclear fragmentation, cellular shrinkage, ladder pattern in the electrophoresis, when administrated 100 microgram/ml catechin. These results suggested that exposure of catechin induced severe cellular damage and growth inhibition in dose- and time-dependent manner. And we confirmed that these effects of catechin were involved with apoptosis, necrosis and cell cycle arrest and were quite different according to cancer type. Therefore, much more research would be demanded before clinical application of catechin to human cancer therapy and this study would be the basic source for further study of green tea.

The Measurement of Double Staining for Senescence Associated-beta-Galactosidase Activity and Keratin 10 or Involucrin in Monolayer and Organotypic Cultured Keratinocytes / 대한해부학회지

This experiment developed the methodology of double staining for senescence associated-beta-galactosidase (SA-beta-gal) activity and keratin 10 (K10) or involucrin. To prove the usefulness of the double staining, the author investigated the relationship between senescence and differentiation in monolayer and organotypic cultured keratinocytes. The results were as follows: K10 and involcrin together with SA-beta-gal were doubly stained in most of monolayer cultured keratinocyte. This fact indicated that the senescence and differentiation had simultaneously occurred in the same keratinocyte. In spite of the advantages to preserving structures, the paraffin specimen was not suitable for double staining because of the limitation of SA-beta-gal reactivity. Although the cryosectioned specimen did not have the morphology as good as the paraffin specimen, it was suitable for double staining due to the goodness of SA-beta-gal reactivity. Double staining well reflected the disturbances of senescence and differentiation which could be caused by deranged organizations of the organotypic cultured skin. The organotypic cultured skin which showed deranged organizations such as stratified basal layer, no typical cell features in each epdermal layer, and wide intercellular spaces had SA-beta-gal activity in epidermis and K10 or involucrin reaction in basal cell. But the skin which showed well arranged organizations resembling in vivo skin had no SA-beta-gal activity and no K10 or involucrin reaction in basal cells. In conclusion, it might be suggested that the double staining for SA-beta-gal activity and K10 or involucrin could be used for detecting the extent of senescence and differentiation in the same cell.

Replicative Senescence and Differentiation Properties of Reconstituted Skin and Monolayer Human Keratinocytes in Vitro / 대한해부학회지

This experiment tried to elucidate the characteristics of senescence and differentiation in the reconstituted skin and the monolayer cultured human keratinocytes in vitro, respectively. While the keratinocytes were cultivated from undifferentiated state to completely senescent and differentiated, the monolayer cultured cells of every passage were doubly stained with SA-beta-gal initially, then keratins or involucrin. We also performed the SA-beta-gal enzyme staining and the immuno-reaction such as keratins or involucrin in the reconstituted skin. The results were as follows: Lack of reactivity against SA-beta-gal in the reconstituted skin indicated that there was no senescence occurred. The reconstituted skin showed decreased expression of K10 and preceded expression of involucrin compare to in vivo skin. Nevertheless, the reconstituted skin which did not express the K10 or involucrin in the basal cell maintained the differentiation system similar to that of in vivo skin. On the other hand, the monolayer cultured keratinocytes showed a thoroughly different pattern in the senescent and differentiating process. SA-beta-gal was colocalized with K10 or involucrin in the cells of high percentage ratio by the double staining method, and this indicated that the senescence and differentiation in the kratinocytes were simultaneously progressed. Reaching the nearer stage leading to the cell death, the cells choosed the one of senescence or differentiation pathway. It was supported by the fact that the percentage index of double staining together with SA-beta-gal and involucrin was lower at passage 5 than passage 1~4. The SA-beta-gal's reactivity was maximally reached at passage 4 and the involucrin maximally reached at passage 5. These trends suggested that the senescence was preceded by the differentiation. In conclusion, the reconstituted skin maintained only the differentiation system without the cell senescent process similar to the in vivo while the senescent and differentiating events were simultaneously processed in the monolayer cultured keratinocytes.

Skin Pigmentation and its Regulation: Role of Keratinocyte and Ovarian Hormones on Human Melanocytes in Vitro / 대한해부학회지

Estrogen and progesterone are thought to be responsible for the pigmentary changes in pregnancy and also melasma. To investigate the action mechanism of estrogen and progesterone on the facultative skin pigmentation, Human melanocytes and keratinocytes were cultivated in the forms of pure melanocyte culture, co-culture (melanocytes and keratinocytes were cultivated together in a vessel but they were separated with membrane) or mixed culture (melanocytes and keratinocytes were cultivated together in a vessel in mixed form). After 2 days of cultivation in the presence of hormones (estrogen, progesterone and melanocyte stimulating hormone), the author studied the cell proliferation, the cellular features (the number of dendrites, perimeter and area), and the tyrosinase activity of melanocytes. Progesterone or melanocyte stimulating hormone increased in both the cell growth and the tyrosinase activity in pure melanocyte culture but estrogen did not. However, mixed culture treated with estrogen lead to increases in the tyrosinase activity. Pure melanocyte culture treated with estrogen or progesterone increased in the cell perimeter and the area but not in the number of dendrites. Co-cultured melanocytes without hormones revealed more increases in the perimeter (p.0.01) and the area (p.0.01) even in the number of dendrites (p.0.01) compared to the pure cultured melanocytes treated with the hormones. It was postulated with these results that estrogen, progesterone and keratinocyte possibly induced hyperpigmentation of the skin via the keratinocytes stimulated by estrogen, via the proliferation of melanocytes induced by progesterone, and via the cellular features altered by keratinocytes.

The Investigation of the Melanocytes in a Cultured Skin Equivalent Model / 대한해부학회지

Melanocytes grown in the pure monolayer culture lack the three-dimentional organization and the cellular interactions that exist in vivo. These can be partially overcome by growing melanocytes together with other epidermal cells in cultured skin equivalent models. In this study, skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in ratio 10 : 1 onto artificially constructed dermis. They were cultured in DMEM/F12 (1 : 1) for 4 days and then lifted to the air-liquid interface and maintained in DMEM/F12 (3 : 1) for 10 days. Histological and electronmicroscopic examinations of the cultured skin equivalants revealed a structure that closely resembled human interfollicular epidermis; 1. Melanocytes, were identified by positive staining with melanoma-specific antibodies (NKI/C3 and S-100 protein) and prominent cytoplasm with rich cell organelles, were located in the basal layer. 2. Melanocytes contained predominently early stage melanosomes and prominent Golgi apparatus. Mature melanins, were usually abundant in normal skin, were hardly seen both in melanocytes and in neighbouring keratinocytes. 3. Melanocytes were surrounded by keratinocytes but did not form intercellular junctions with them. 4. Keratinocytes were charaterized by microfilament bundles and intercellular junctions such as desmosomes and hemidesmosomes with neighbouring keratinocytes and artificial dermis. The melanocyte in the above skin equivalents had a strong resemblance to the one of normal human skin and therefore this model can be used as artificial skin for the transplantation and in the investigation of melanocytes' role to the UV stimuli.

Antioxidative Effect of Ginseng Saponin on Cardiac Endothelial Cells in Culture / 대한해부학회지

The cytotoxic effects of oxygen free radicals and the antioxidative effect of ginseng saponin (SPN) on cardiac endothelial cell cultures derived from 3-day old rats were studied. Reactive oxygen species were generated by hypoxanthine (HX) and xanthine oxidase (XO) mixture to the culture medium. Exposure of cardiac endothelial cells to this oxygen-radical-generating system resulted in significant time-dependent decrease of MTT activity and increase of lactate dehydrogenase (LDH) release. These results correlated well with the morphological examination of randomly selected cultured cardiac endothelial cells, which showed large cytoplasmic vacuoles, disordered organelles, pronounced increase of endoplasmic reticular swelling, and decreased maintenance of membrane integrity. The decrease in cell viability and increase of LDH release induced by the oxygen free radicals in cardiac endothelial cell cultures were blocked during the first two hours by antioxidants such as ginseng saponin (SPN), deferoxamine (DFX), and ginseng saponin/deferoxamine mixture (SPN/DFX). These antioxidative effects were significantly greater in the SPN-treated group than in the other antioxidant-treated groups. Especially, the cells of the SPN-treated group showed well developed cytoskeletons, which enabled them to firmly attach to the culture vessel. In conclusion, these results indicate that ginseng saponin has a significant antioxidative effect on cardiac endothelial cells in culture and plays an important role in stimulating the formation of cytoskeleton and maintaining the integrity of cell membrane.

Anti-tumor Effect of Green Tea Catechin on Cancer Cell Lines / 대한해부학회지

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.

Effects of Ischemic Preconditioning on Hypoxia-Reoxygenation Injury of Cardiac Myocyte in Culture / 대한해부학회지

Short period of ischemia and reperfusion protect heart against subsequent prolonged ischemia-reperfusion injury. This phenomenon was first described by Murry et al in 1986, who demonstrated that four 5-minute coronary artery occlusions followed by equal period of reflow at each time before a subsequent prolonged occlusion resulted in a reduction of infarct size in dog. Although the precise mechanism of preconditioning remains unknown, this phenome-non is present among different species of mammals, including dogs, rats, pigs, rabbits, and human. The objects of present study was to investigate effect of ischemic preconditioning on cell viability, structural changes and apoptosis during 60 min hypoxia and 60 min reoxygenation of the cell. In present study we investigated through cell culture system using myocyte of three days old neonatal rat cultured for three days. During hypoxia and reoxygenation, differences between preconditioned and nonpreconditioned of beating counts, morphological and structural changes are investigated through inverted phase contrast microscope and transmis-sion electron microscope. To detection of apoptotic cell, TUNEL (TdT-mediated dUTP-biotin nick end labeling) stain was accomplished, and through which we invesigate the effects of preconditioning on apoptosis. Viabiliy of each cell and it's mitochondria were measured quantitatively by MTT assay. After 60 min of hypoxia and 60 min of reoxygenation, beating rate decreased remarkably. But at the time of 60 min of reoxygenation, there was marked increase in beating count in pre-conditioned cell. Swollen mitochondria with amorphous granules in inner membrane, destroyed mitochondrial cristae, indented nuclear envelope, chromatin condensation, contracture of myofibril, fragmentation of myofilaments, cytoplasmic shrinkage were observed in both preconditioned cell and nonpreconditioned cell. But it is much less in pre-conditioned cell than in nonpreconditioned cell. MTT activity decreased in both experimental groups in compared with normal group, but in preconditioned group, MTT activity increased markedly in compared with nonpreconditioned group. And apoptosis is decreased by precontitioning in TUNEL staining. These results suggest that cardioprotective effects of ischemic preconditioning is mediated by attenuating structural destroy, increasing cell viability, decreasing apoptosis.

Effect of beta-amyloid Peptide on Fine Structure of Cardiac Myocytes in Culture / 대한해부학회지

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.

Ultrastructural Injury and Its Mechanism of Cultured Cardiac Myocytes under Anoxia-Reoxygenation / 대한해부학회지

Abrupt reoxygenation (or reperfusion) after anoxia (or ischemia)-induced injury resulted in the loss of contractile property, destruction of cell organelles, and ultimately, cell death in cardiac myocytes. This phenomenon has been called 'oxygen paradox' or 'reperfusion injury'. The purpose of this study was to investigate the changes of fine structures and enzyme activities associated with oxygen paradox during 60 min. of anoxia, followed by a 30 min. of reoxygenation. Cardiac myocytes were dissociated from neonatal rat ventricles and cultured for three days. While they were exposed to anoxia and reoxygenation, the cardiac myocytes were investigated through beating counts, enzyme cytochemistry, immunofluorescence, electron microscopy for morphological study. Activity staining and Western blot for Cu, Zn-SOD, NADPH-diaphorase stain and nitrite concentration mesurement for nitric oxide synthase, and catalase activity measurement were performed. After 60 min. of anoxia, the beating rate increased remarkably. Swollen mitochondria with amorphous dense clumps, mild contracture of myofibrils and retraction of cytoplasmic processes were observed in cardiac myocytes. Under confocal microscope, weak reaction of Mn-SOD and myosin were observed, whereas reaction of Cu, Zn-SOD was enhanced in perinuclear region. Cu, Zn-SOD and catalase activity in cardiac myocytes increased markedly. Nitric oxide synthase activity increased gradually with time. After 30 min. of reoxygenation following 60 min anoxia, structural changes of myocardial cells was more pronounced than in the cells of anoxic group. Beating rate was variable but decreased gradually. Myocardial cells showed evidence of severe structural alterations, including marginal clumping of chromatids, varying-sized bleb formation, many vacuoles, mitochondrial matrix exposed to cytoplasm and fragmen-tation of cristae, myofibrillar hypercontracture. Decline of immunocytochemical reaction of Mn-SOD, myosin and Cu, Zn-SOD were observed under confocal microscope. The declines of activity and quantity of Cu, Zn-SOD were severe compared to control. In contrast, nitric oxide synthase activity significantly increased. Catalase activity was lower than in anoxic group, but still higher than in control activity. These results suggested that there were two possible mechanisms for the drastic morphological changes induced by anoxia-reoxygenation; 1) direct effect of oxygen free radicals, and 2) reaction of nitric oxide with superoxide radicals, which resulted in generation of toxic metabolites of nitric oxide, exacerbated myocardial cellular damages.

Expression of cytokeratins and involucrin in cultured human keratinocytes / 대한해부학회지

To evaluate the maturation and differentiation state of cultured keratinocytes, the author investigated expression of differentiation markers in cultured keratinocytes. The specimens were divided into three experimental groups, 3rd passage keratinocytes cultured in serum free media (3rd SFM group), 6th passage keratinocytes cultured in serum free media (6th SFM group) and 3rd passage keratinocytes cultured in DMEM (DMEM group). CK14, marker of basal layer, expressed in all groups. The expression was localized and condensed in the SFM groups but spreade in the DMEM group. Most of the cells in both SFM groups were positive but a few cells in DMEM group were also positive. CK10, marker of initiation of differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. Involucrin, marker of terminal differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. CK16 and 17, markers of fast turnover of keratinocytes, were not expressed in SFM groups. Weak positive reactions were observed in DMEM group. With these results the authors concluded that the keratinocytes from 3rd passage to 6th passage, cultured in serum free media with calcium less than 0.1 mM, had highly homogeneous basal cell characteristics.

Development of Psoriasis Model in Vitro / 대한해부학회지

Psoriasis is a disease caused by hyperproliferation of keratinocytes. Pathogenesis of psoriasis is still unclear, but many reports suggest that psoriatic keratinocytes themselves may have some factors of pathogenesis. The author developed artificial psoriatic skin by culturing keratinocytes of psoriasis skin over collagen lattice which was constructed with collagen and normal fibroblasts. After the keratinocytes had grown to full layers of stratification, expression patterns of differentiation marks and ultrastructural changes were investigated by immunohistochemistry and electron microscope. The results were very similar to those of psoriasis skin in vivo as follows. Cytokeratin (CK)10, marker of initiation of differentiation of keratinocytes, was expressed in the spinous layer. CK14, marker of basal cells of stratified squamous epithelium, was expressed in the basal and spinous layer. CK16 and CK17, markers of fast turnover of squamous epithelium, were expressed in the spinous layer. Involucrin, marker of terminal differentiation of squamous epithelium, was expressed weakely over the lower spinous layer. In immuno electron microscopical study, involucrin was expressed but confined to cornified cell envelops in the horney layer. Mitochondria, rER and ribosomes were abundant in the basal layer. They continued to appear in the upper spinous layer but intermediate filaments were scarce. Keratohyalin granules were visible in some parts of the granular layer zone, but the granules were smaller and fewer. In the horney layer, cells were thicker than normal and there were many lipid droplets within the cells. Intercellular spaces were enlarged at the basal layer but disappeared in the upper spinous layer. In these results, non systematic expression of differentiation markers and ultrastructural changes suggest that psoriasis is a disease caused by hyperproliferation of keratinocytes concurrent with unstable maturation and degeneration. Artificial psoriatic skin, in exclusion of systemic or dermal effects, showed very similar results with psoriasis skin in vitro. So it was concluded that psoriasis keratinocytes had some factors of pathogenesis and this kind of model on artificial psoriatic skin can be used for further studying of psoriasis.

Role of nitric oxide in penile erection

The present study was undertaken to investigate the role of nitric oxide (NO) in erectile physiology by correlating its action with the existence and activity of nitric oxide synthase (NOS), which produces NO. We applied Western blot analysis in both human and rat penile tissue. In the rat, reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining and spectrophotometric assay were also performed, in addition to in vivo electroerection study with pharmacological manipulation. Western blot analysis identified a protein of 155 KDa identical to the neural form of NOS in the human and rat penis. The NOS blot densities in the two species were similar, and both were lower than that in the rat cerebellum. Histochemical staining localized NOS to neurons innervating the corpora cavernosa, including the pelvic plexus, the cavernosal nerves and their terminal fibers within the corporeal erectile tissue, and dorsal penile nerves. NOS activity was also found in the cerebellum, urethra, penis, and urinary bladder, in decreasing order of intensity. Intracavernous injections of NOS inhibitor (L-NOARG or L-NAME in concentrations from 10(-6) M to 10(-3) M suppressed electrostimulation-induced erection in a concentration-dependent manner. Subsequent intracavernous injection of L-Arginine (10(-2) M) partially restored the erection. The neural form of constitutive NOS in the corpora cavernosa synthesizes NO, which mediates penile erection. Determination of cavernosal NOS expression or activity may permit characterization of certain pathological conditions that cause impotence.

Correlation of Neuronal Nitric Oxide Synthase with Penile Erection in Diabetic Rats / 대한비뇨기과학회지

Diabetes mellitus (DM) is an important cause of organic impotence in man. The exact pathogenesis remains debatable although it has been focused on cavernosal neuropathy and/or endothelial dysfunction. This study was designed to investigate the effect of DM on penile erection, especially in association with nitric oxide synthase (NOS) in corpus cavernosum of diabetic rats. NOS studies of rat penis were performed in diabetic (DM was induced for 3, 6, 9, 12 weeks, respectively, by intraperitoneal administration of streptozotocin, 60mg/kg), in control and neurotomy group (3 weeks after bilateral cavernous nerve transection). The experiments consisted of nicotinamide dinucleotide phosphate (NADPH) diaphorase activity with spectrophotometric assay for NOS catalytic activity, NADPH diaphorase staining for the identification of NOS containing nerve fibers, and Western blotting analysis with anti-brain NOS antibody for the expression of neuronal NOS. Finally, these results were compared with erectile response to cavernous nerve stimulation in diabetic and in control rats. In assay of NADPH diaphorase activity, NOS activity decreased significantly in penis of diabetic rat as compared to that of controls. Between the diabetic groups, NOS activity was not seen significantly different, and in neurotomy groups it was similar to that of diabetic groups. On histochemical staining of penile tissues, the number of NADPH-positive nerve fibers in control group (a mean of 127+/-6 fibers recorded in 4 random fields on each corporal side) contrasted significantly with that of the bilateral cavernous nerve ablation group (a mean of 12+/-2). In diabetic group, the number of NOS-containing nerve fibers was gradually reduced along with duration of diabetes (from 92+/- 3 at 3 weeks to 28+/-3 at 12 weeks). In addition, analysis of blot density of neuronal NOS by Western blotting showed similar findings: 16% at 3 weeks and 8% at 12 weeks in diabetic group, 5% in neurotomy group and 27% in controls, based on the density of the rat cerebellum. Furthermore, erection response to cavernous nerve stimulation was also decreased in diabetic rats along with DM duration. The results, indicated that reduction of cavernous NOS, particularly, its neuronal form at the level of NO production plays an important role on the pathogenesis of erectile impotence in diabetic rats although the role of endothelial N0S in DM remains to be elucidated. Furthermore, cavernosal NADPH diaphorase staining and/or NOS activity may allow to characterize certain pathological condition, which comprise neurogenic impotence.

The effects of high glucose concentration on the synthesis of extracellular matrix and the fine structures of mesangial cells in culture / 대한해부학회지

No abstract available.

Distribution patterns of cytoskelectal proteins in cardiac endothelial cells : Investigation using monoclonal antibodies / 영남의대학술지

To investigate the changing patterns of microfilament and microtubule arrangement and influence of myocardial cells and colchicines to microfilament and microtubule formation in cardiac endothelial cells the authors carried out indirect immunofluorescence stain for actin and tubulin with supernatant monoclonal antibodies. Secondary antibodies were IgG FITC conjugate. The results were summarized as follows. Fiberform reactions were stronger in the cells with many processes and spread cytoplasm and they became weaker after the endothelial cells formed monolayer. In the endothelial cells cocultured with myocardial cells the fiberform of the microtubule became less visible compared to control group but fiberform of the microtubule maintained strong intensity as endothelial cells formed monolayer. In the group treated with colchicines, there were no visible differences in microfilaments compared to control group but fiberform of microtubule revealed weaker intensity after colchicines treatment. The intensity of microtubule fiberform returned to control level after 2 days.

Transition of Marker Enzymes of Rat Hepatocyte Organelles in Culture / 영남의대학술지

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.

Effects of Dimethyl Sulfoxide on the Differentiation of Myocardial and Endothelial Cells / 영남의대학술지

To elucidate the effects of dimethyl sulfoxide on of myocardial and endothelial cells in culture, the cells were exposed to 10% dimethyl sulfoxide in culture medium for 1 hour at 48 hours after cell isolation. The general morphology and the cytochemical reaction of marker enzymes for mitochondria and Golgi complexes were investigated. The results were summarized as follows 1. DMSO induced elongation and narrowing of the cells and increase of mitochondrial reaction in myocardial cells. 2. DMSO induced destruction and disruption of myofibrils in myocardial cells resulting in increase of contractile activities. 3. In the endothelial cells, DMSO suppressed proliferative activities but thiamine pyrophosphatase reactions were enhanced indicating increase of Golgi complex activity. 4. DMSO seemed to hamper with the adhesiveness and motility of the endothelial cells causing the decrease of the number of cells in vitro.