Expression and Cellular Localization of Psx Gene in Rat Placenta / 체질인류학회지

Homeobox genes seem to play critical roles in regulating morphogenesis, patterning, organogenesis, and differentiation. They have the conserved sequence that codes the DNA-binding domain called homeodomain. The expression and cellular localization of rPsx mRNA in rat placenta during placental development were examined by in situ hybridization histochemistry at different embryonic stages (Embryonic days 7.5~16.5). rPsx mRNA was first detected in chorionic ectoderm of placenta at E 10.5. This transcript was localized in labyrinth trophoblast and trophoblast giant cells at E 11.5. Hybridization signals were observed in labyrinth trophoblast, spongiotrophoblast, and trophoblast giant cells at E 12.5, E 13.5, and E 14.5. At E 15.5, hybridization signal was detected in labyrinth trophoblast and spongiotrophoblast but not in trophoblast giant cells. Hybridization signal was only detected in labyrinth trophoblast at E 16.5. rPsx mRNA was not detected in decidua and any tissues of the embryo from E 7.5 to E 9.5 of gestations. From these results, a new rPsx homeobox gene is first expressed at E 10.5 and detected in chorionic ectoderm, labyrinth trophblast, spongiotrophoblast and trophoblast giant cells of the placenta. This gene may play a critical role in differentiation and development of trophoblast cells.

Enhanced Expression of Carbonic anhydrase II in Hypokalemic Rat Kidney / 체질인류학회지

A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3- reabsorption in the renal tubules. The present study was to examine the alterations of expression and distribution of Carbonic anhydrase II in the kidneys of normal and potassium-depleted rats using Western blot analysis and immuno-histochemistry. Western blot analysis demonstrated that CA II protein, ~30 kDa at molecular mass, was abundantly expressed in normal group. All potassium-depleted groups showed slightly increased CA II protein compared to normal group. In control group, immunoreactivity of CA II protein was detected in the entire collecting duct. Signal intensity was prominent in the intercalated cells and weak in the principal cells of the cortical collecting ducts. In potassium-depleted groups, the pattern of cellular labeling of CA II protein was identical to that of normal group, but the signal intensity was decreased in cortical collecting duct, markedly increased in the inner stripe of outer medullary and inner medullary collecting ducts, and unchanged in the outer stripe of outer medullary collecting duct. These results suggest that chronic hypokalemia impact the expression pattern of CA II protein depending the portion of the collecting duct.

Alterations of Aquaporin mRNA and Protein Expression in Hypokalemic Rat Kidney / 대한해부학회지

There has been a general agreement that potassium depletion causes metabolic alkalosis and substantial morphological changes in kidney structure, and is associated with renal functional abnormalities, including a decrease in urinary concentrating ability. The present study was to examine the alterations of expression and distribution of AQP-1, 2, 3 and 4 mRNAs and proteins in the kidneys of normal and K-depleted rats using RT-PCR, Western blot analysis, and immunohistochemistry. Predicted size of AQP-1, 2, 3, and 4 mRNAs was 119, 822, 539, and 642 bp, respectively. AQP-1 mRNA expression was gradually decreased in K-depleted rats, particularly LK 2W. AQP-2, 3 mRNAs were markedly decreased in K-depleted rats. AQP-4 mRNA expression was markedly increased in K-depleted rats, particularly LK 2W. Western blot analysis demonstrated that AQP-1 protein expression was only decreased in LK 3D and others were comparable with normal rat. AQP-2, 3 proteins expression was markedly decreased in K-depleted rats, compared with normal rat. But, AQP-4 protein expression was markedly increased in K-depleted rats, particularly LK 3W. In immunohistochemistry, AQP-1 was detected in the apical membranes of proximal tubules and thin limb of Henle loop. In potassium-depleted kidney, the pattern of cellular labeling and signal intensity of AQP-1 protein is identical to that of normal rat. AQP-2 was detected in apical region and cytoplasm of the principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-2 protein is identical to that of normal rat, but signal intensity is markedly decreased. AQP-3 was detected in the bosolateral plasma membrane of principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-3 protein is identical to that of normal rat, but signal intensity is markedly decreased. AQP-4 was detected in the bosolateral plasma membrane of principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-4 protein is identical to that of normal rat, but signal intensity is markedly increased in outer and inner medullary collecting ducts. In summary, these results demonstrate that chronic hypokalemia shows the different expression pattern of AQP-1, 2, 3, and 4 mRNAs and proteins. These results suggest that a decrease in urinary concentrating ability is a major factor in the decreased AQP-2, 3 expression, and that is partly compensated by increased expression of AQP-4.

Immunohistochemical Localizaion of Carbonic Anhydrase Isozymes IV and IX in Rat Salivary Gland / 대한해부학회지

This study presents distribution of carbonic anhydrase (CA) isozymes IV and IX, membrane associated forms, and CA I and II, cytoplasmic forms, in rat parotid and submandibular glands using Western blot analysis and immunohistochemical staining. Western blot analysis demonstrated that CAs I, II and IX were found to be abundantly expressed, but CA IV was weakly expressed in parotid gland. Submandibular gland expressed abundant CAs I and II, weak CA IX, and undetectable level of CA IV. In hematoxylin-eosin staining, parotid gland was entirely composed of serous acini and their ducts while submandibular gland was mixed population of serous and mucous lobules. Most of lobules (submandibular gland proper type) contained mostly serous acini and their ducts with granular convoluted duct. Some lobules (sublingual gland type) contained mostly mucous acini with serous demilune and their ducts without granular convoluted duct. In parotid gland, CAs IV and IX were immunolocalized in duct cells and not in serous acinar cells. Immunoreactivity for CAs I and II was also detectable in duct cells. Serous acinar cells were positive for CA II, and negative for CA I. In submandibular gland, CAs IV and IX were immunolocalized in duct cells but not in acinar cells of both types of lobules. Immunoreactivity for CAs I and II was also detectable in duct cells of both types of lobules. Cells of serous acini and serous demilune were positive for CA II, and negative for CA I. Mucous cells were negative for both CAs I and II. These results demonstrate the distribution of CA isoenzymes in parotid and submandibular glands of the rat, and suggest CAs IV and IX as well as CAs I and II are related to electrolytes metabolism of saliva in duct cells.

Expression and Localization of General Transcription Factor IIA Gene in Hypokalemic Rat / 대한해부학회지

Potassium balance in chronic hypokalemia is regulated by ion channels, ion transporters, and various related genes. We isolated general transcription factor IIA (GTF IIA) gene using a DNA chip microassay, a useful method in cloning genes. Northern analysis and in situ hybridization (ISH) were carried out to analyze the expression and localization of GTF IIA mRNA in rat in relation to the amount of potassium in the diet. Isoform-specific 32P-labeled cDNA (Northern analysis) or digoxigenin-labeled cRNA (ISH) probes were used. Northern analysis demonstrated that GTF IIA mRNA was expressed abundantly in testis; modestly in heart, kidney, lung, adrenal gland, liver, and spleen; and weakly in brain, distal colon, duodenum, salivary gland, and stomach. In potassium-restricted animals, GTF IIA expression was decreased in the kidney, adrenal gland, and spleen, but expression was restored to normal levels in L3w. The expression level in the lung was decreased in L3d and L2w, and increased in L1w and L3w. ISH showed that mRNA for the GTF IIA gene was detected in the distal convoluted tubule, S3 segment of the proximal tubule, and cortical collecting duct in the normal group. In potassium-restricted groups, the hybridization signal was detected in the distal convoluted tubule, S3 segment of the proximal tubule, and entire collecting tubule. The signal intensity of the outer and inner medullary collecting ducts was higher in the potassium-restricted group than in the normal group but was decreased in the distal convoluted tubule and S3 segment of the proximal tubule. In the normal group, mRNA of the GTF IIA gene was detected in the zona glomerulosa cells of the adrenal gland, lymphocytes of the marginal zone, germinal center of the spleen, and bronchial epithelium and lymphocytes of the lung. mRNA for the GTF IIA gene was also detected in the cells of the basal portion of the intestinal glands of the distal colon and stomach, and in spermatogonia and spermatocytes of the seminiferous tubule. These results suggest that expression of GTF IIA differs between various tissues and that increased expression of the GTF IIA gene in the outer and inner medullary collecting ducts of the hypokalemic kidney might regulate the ion transporter genes in these segments.

Immunohistochemical Distribution of Carbonic Anhydrase in Rat Exorbital Lacrimal Gland / 대한해부학회지

There are several carbonic anhydrase (CA) isozymes, which differ in their kinetic properties, tissue distribution, and subcellular localization. In this study, the distribution of CA isozymes I, II, IV, and IX was investigated in the rat exorbital lacrimal gland using Western blotting analysis and immunohistochemical staining. In the Western blotting analysis of the rat lacrimal gland, CA II and CA IX were expressed abundantly and CA IV was expressed weakly. Hematoxylin-eosin staining of the exorbital lacrimal gland showed a multilobular tubuloacinar gland composed of acinar and ductal cells. Immunohistochemical reaction revealed no CAI staining in acinar cells and positive staining in intercalated and small duct cells. CA II reactivity was detected in the supranuclear cytoplasm of acinar cells and appeared to vary between acini. The intercalated and collecting duct cells showed weak or no immunoreactivity for CA II. CA IV was detected in the intercalated and collecting duct cells but not at the acinar cells. CA IX was detected in the intercalated and collecting duct cells, and in only a few acinar cells. These results demonstrate the differential distribution of CA isoenzymes in the exorbital lacrimal gland of the rat and suggest that CA II is related mainly to the electrolyte metabolism of tears in the acinar cells and that CAs I, IV, and IX are related to the electrolyte metabolism of tears in the duct cells.

Application of Tyramide Signal Amplification (TSA) Both to Biochip Platform and to the Immunoelectron Microscopy to Label Proteins within the Organelle / 대한해부학회지

The tyramide signal amplification (TSA) technique, based on the ability of HRP to catalyze the deposition of tyramide onto the surrounding proteins, has been proved to detect scarce tissue antigens. In this study we applied this technique to a biochip platform and an immunocytochemistry at the electron microscopic level. First, in the optical fluorescence sensing, the signal was amplified by Dako Envision(TM) (goat anti-mouse immunoglobulins IgG conjugated to peroxidase labelled-dextran polymer) and tyramide-Cy3, which was then compared to the non-amplified control using goat antimouse IgG-Cy3 conjugate instead. The result showed that the tyramide method produced a more sensitive signal than the control method. Secondly, in the pre-embedding immunocytochemistry, we investigated to see whether it is possible to label proteins within a organelle in the cell using the TSA method. The signal was amplified by a primary antibody, a biotinylated secondary antibody, streptavidin-HRP, biotinyl-tyramide, and streptavidinnanogold followed by silver enhancement and gold toning. Then, this protocol was compared to the non-amplified or simple protocol that does not include the steps of streptavidin-HRP and biotinyl-tyramide. With the TSA protocol, the labeling for a membrane bound antigen (gp100) that is known to be exclusively localized to melanosomes in melanocyte, was tested in a melanoma cell line (G361) and found to be highly sensitive and more enhanced than with the simple protocol. Moreover, the gold particles were well localized to the subcellular structures or melanosomes both in the TSA and simple protocols, which indicates that resolution of the signals remains high. Control experiment with omission of the primary antibody demonstrated that background levels or nonspecific bindings are negligible. This result showed that the TSA method can be successfully applied to label the intra-organelle protein that is known to be labeled only in the specific fixation condition with the optimal permeability.

Regulations of Bicarbonate Ions in Pregnant Rat Kidney / 대한해부학회지

The marked hemodynamic and hormonal changes of normal pregnancy are associated with striking alterations in renal physiology involving structure, dynamics, tubular function, and volume homeostasis. A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3-reabsorption in the renal tubules. The present study was to examine the alterations of expression and distribution of Na+/HCO3-cotransporter (NBC), Na+/H+ exchanger-3 (NHE-3), and carbonic anhydrase I and II (CA I, II) proteins in the kidneys of non-pregnant (NP) and pregnant rats using Western blot analysis and immunohistochemistry. Sprague-Dawley female rats were studied on days 10 (P 10), 12 (P 12), 14 (P 14), 17 (P 17), and 19 (P 19) of pregnancy. Western blot analysis demonstrated that the expression of NBC, ~110 kDa at molecular mass, was increased in pregnant rats, particularly P 12, compared with NP rat. The expression of NHE-3, ~83 kDa at molecular mass, was increased in pregnant rats, particularly P 12 and P 14. The expression of CA I, ~30 kDa at molecular mass, was decreased in pregnant rats, particularly P 14, but, CA II protein, ~30 kDa at molecular mass, was similar NP rat. In immunohistochemistry, strong immunoreactivity of NBC of NP rat was exclusively detected in the basolateral membranes of S1 and S2 segment of proximal tubules whereas not in S3 segment. In pregnant rats, the pattern of cellular labeling of NBC was identical to that of NP rat, but signal intensity was increased, particularly P 12. In NHE-3, strong immunoreactivity was detected in apical membranes and brush borders of S3 segments and moderate in S1 and S2 segments. In pregnant rats, the pattern of cellular labeling was identical to that of NP rat, but the signal intensity was increased, particularly P 12 and P 14. Expression of CA I and II proteins was detected in entire collecting duct. Signal intensity was prominent in type A intercalated cells and moderate in type B intercalated cells. In pregnant rats, the pattern of cellular labeling of CA I and II proteins was identical to that of non-pregnant rat, but the signal intensity of CA I was decreased in cortical collecting duct, particularly P 14 and CA II was identical to that of NP rat. These results suggest that the regulation of NBC and NHE-3 expressions in the proximal tubules and CA I expression in cortical collecting duct may maintain HCO3-concentration during the pregnancy.

Immunohistochemical Study on the Distribution of Carbonic Anhydrase Isozymes in Pancreatic Islet of Rat / 대한해부학회지

The distribution of carbonic anhydrase (CA) isoenzymes I, II, IV, and IX was investigated in pancreatic islet of the rat using Western blotting analysis and immunohistochemistry. Western blotting analysis demonstrated strong CAI and II expression, but weak CAIV and no CAIX expression. Immunohistochemical reaction of pancreatic islet revealed no staining for CAI and II. CAIV was detected in the peripheral cells of the islet. CAIX was detected in the peripheral cells and occasional in the centrally located cells. Signals for CAIV were observed at the plasma membrane and/or in the cytoplasm of islet cells. Location of CAIV in the A cells was confirmed by subjecting serial sections of pancreas to staining for CAIV and glucagon, which showed colocalization in the A cells. Immunohistochemical staining of pancreatic acinus revealed abundant staining for CAI in interacinar blood vessels and CAII in ductal and acinar cells. These results demonstrate the differential distribution of CA isoenzymes in pancreatic islet, and suggest that A cells of pancreatic islet might contain both CAIV and IX.

Adaptive Responses to Dexamethasone Treatment in Rat Kidney Proximal Tubules / 대한해부학회지

Excess accumulation of glucocorticoid increases acid secretion and HCO3- reabsorption in the kidney. Reabsorption of HCO3-, which almost occurs at the proximal tubule, is mediated Na+ / H+ exchanger-3 (NHE-3) and H+ -ATPase on the apical membrane and the Na + /HCO3- cotransporter-1 (NBC-1)on the basolateral membrane. Impact of glucocorticoid was investigated by immunohistochemistry and electron microscopy to correlate the changes with the effect of in vivo dexamethasone treatment for the rat kidney proximal tubule. In a control group, immunoreactivity of NHE-3 was detected in the apical membrane and the brush borders of S1, S2 and 3 segments of the proximal tubule. Immunoreactivity of NBC-1 was detected in the basolateral membrane of S1 and S2 segments of the proximal tubule. Immunoreactivity of NHE-3 and NBC-1 protein was more pronounced in dexamethasone treated groups than the control group. Dexamethasone 1 mg/kg caused most intense immunoreactivity for NHE-3 and NBC-1 protein, however, 0.01 mg/kg and 0.1 mg/kg produced less intense immunoreactivity with no appreciable differences between these lower doses of dexamethasone groups. By electron microscopy, the tubular cells of S1 segment of the control group revealed numerous mitochondria, endocytic apparatuses, lysosomes and many basal cytoplasmic processes. In dexamethasone treated groups, the cells of S1 and S2 segments of the proximal tubule had more mitochodria and more basolateral invaginations and had an increased number of more elongated microvilli, compared with the control group. The cells of the S3 segment of the control group showed scant lateral interdigitations and had a few smaller mitochondria. The cells of the S3 segment of dexamethasone treated groups had many mitochodria and an increased number of microvilli in the brush border, but revealed no difference of basolateral invaginations among the different groups of dexamethasone. These results indicate that prolonged administration of excess glucocorticoid increases NHE-3 and NBC-1 protein, and the up-regulation of these proteins could result in increased HCO3 - reabsorption in the rat renal proximal tubules. It also suggests that these adaptive responses closely correlate to morphological alterations of proximal tubular epithelial cells.

Effect of Insulin Therapy on Vagina in Diabetic Rat / 대한해부학회지

The most commonly reported sexual problems in diabetic women are sexual arousal disorder and a lack of vaginal lubrication. The aims of this study were to investigate the vaginal structural changes and expressions of TGF-beta1, Ec-NOS and estrogen receptor alphaby histochemistry, immunohistochemistry and Western blot analysis in diabetic and insulin-treated diabetic rats. The mean blood glucose levels were significantly increased in the diabetic rats (453+/-88.4 mg/dL)compared to the control group (79+/-6 mg/dL)and insulin-treated diabetic rats (56.7+/-0.6 mg/dL).The vaginal wall in control rat showed 6~11 layered stratified squamous epithelial lining and submucosal smooth muscle, connective tissue and vasculatures. In diabetic rat, the vaginal epithelium was reduced to 2~6 layers and the submucosal vasculatures were decreased n size and number.Collagen fibers were increased and irregularly distorted arrangement. Insulin-treated diabetic rat showed similar morphologic features as control rat.In diabetic rat, TGF-beta1 expression was upregulated by 1.65 times and Ec-NOS expression was 40% downregulated compared to control and insulin-treated diabetic rats in Western blot analysis. In control and insulin-treated diabetic rats, TGF-beta1 immunoreactivity was detected in fibroblasts and the collagen fibers, Ec-NOS immunoreactivity in the endothelial cells of blood vessels, and estrogen receptor alphaimmunoreactivity in the basal and intermediate cell layers of stratified squamous epithelium, smooth muscle fibers, and nerve fibers. In diabetic rat, expression of TGF-beta1, Ec-NOS, and estrogen receptor alphawas exhibited comparable cellular patterns of labeling, but signal intensity was increased in TGF-beta1 and decreased in Ec-NOS and estrogen receptor alpha. These results suggest that vaginal tissue fibrosis in diabetes mellitus may be caused by altered expression of TGF-beta1, NOS and estrogen. It also mplies that sexual arousal disorder and lack of vaginal lubrication in the diabetic women could be protected or delayed by controlling blood glucose levels.

Alterations of Cyclooxygenase-1, 2 Expression in Hypokalemic Rat Kidney / 대한해부학회지

A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3- reabsorption in the renal tubules. There has been a general agreement that potassium depletion induces metabolic alkalosis and affects the expression of the several ion transporters in rats. The present study was to examine the alterations of expression and distribution of COX-1, 2 mRNAs and proteins in the kidneys of normal and K-depleted rats using RT-PCR, Western blot analysis, and immunohistochemistry. Predicted size of COX-1 mRNA was 306 bp. It's expression was increased in K-depleted rats, particularly LK 2W, but decreased in LK 3D. Predicted size of COX-2 mRNA was 356 bp and it's expression was increased in K-depleted rats, particularly LK 2W. Western blot analysis demonstrated that COX-1 protein, ~70 kDa at molecular mass, was increased in potassium-depleted rats, particularly LK 2W and decreased in LK 3D, compared with normal rat. COX-2 protein, ~72 kDa at molecular mass, was only increased in LK 3D and others were comparable with normal rat. In immunohistochemistry, COX-1 was detected in entire collecting duct, intraglomerular mesangial cells, arterial endothelial cells, medullary interstitial cells, papillary epithelial cells, and pelvic epithelium. Signal intensity of the collecting duct was more increased toward the papillary tip. In K-depleted rat, the pattern of cellular labeling of COX-1 protein was identical to that of normal rat. However, the signal intensity of LK 3D was only decreased in cortical and outer medullary collecting duct and that of LK 2W was increased particularly in the inner stripe of outer medullary collecting duct and proximal 1/3 inner medullary collecting duct. Immunoreactivity of COX-2 of normal rat was detected in the cortical thick ascending limb and macula densa. In K-depleted rat, the pattern of cellular labeling of COX-2 protein was identical to that of normal rat, but the signal intensity was only increased in LK 3D rat. These results suggest that chronic hypokalemia enhances the expression of COX-1, 2 mRNAs and proteins and the regulation of K reabsorption depends on COX-1 rather than COX-2 by the portions of expression.

Cloning and Expression of Sodium-Bicarbonate Cotransporter Isoform in Rat / 대한해부학회지

The potassium depletion has remarkable and opposite effect on kidney and body growth and has affected the expression of the several ion transporters. Previously, Ahn et al. have reported that HK alpha 1 and 2 subunit gene were upregulated in the hypokalemic rat kidney. To clone the unreported genes expressed in potassium deficiency, differential display PCR-based cloning strategy was used in normal and potassium-depleted rat kidney and a novel gene was isolated. Sequence analysis with blast search program identified a cDNA clone encoding an isoform of kidney sodium bicarbonate cotransporter-1. The tissue and cellular expression pattern of this gene were investigated with Northern analyses and in situ hybridization histochemistry (ISH) in normal and hypokalemic rats. This novel transcript was highly expressed in kidney and brain and at lower levels in distal colon, urinary bladder, and heart but not in salivary gland, stomach, liver, and lung in normal rat. In potassium-depleted rat, this transcript was upregulated in kidney, brain, and distal colon. By ISH, cellular distribution of this gene was highly expressed in S3 segment of proximal tubule, distal convoluted tubule, and cortical collecting duct of kidney and lower third of intestinal glands of distal colon but at lower levels in cortical and medullary thick ascending limb and medullary collecting duct of kidney and middle third of intestinal glands of distal colon. From these results, this candidate gene may play an important role in HCO3-transport by these organs during potassium depletion.

The Expression of Carbonic Anhydrase Isozymes in Human Intestine / 대한해부학회지

The distribution of carbonic anhydrase (CA) isozymes I, II, IV, and IX was investigated in the human duodenum and colon using Western blotting analysis and immunohistochemical staining. A Western blotting analysis revealed an abundant expression of CAI and IX in the duodenum and colon. The expression of CAII and IV was detected in mucosa of duodenum and colon. The degree of expression, however, showed regional difference. The expression of CAII was strong in the duodenum and colon, and that of CAIV was weak in the duodenum and colon. Immunohistochemical staining of duodenum revealed no staining for CAI. CAII was detected at the columnar cells of surface epithelium and secretory cells and ductal cells of Brunner's gland. CAIV was detected at the ductal cells of Brunner's gland. CAIX was detected at the cells of intestinal gland and rare cells of the Brunner's gland. Immunohistochemical staining of colon showed a positive reaction of CAI and II at the columnar and goblet cells of surface epithelium, and CAIV and IX at the columnar cells of surface epithelium. These results demonstrate the differential distribution of CA isozymes in duodenum and colon, and suggest that Brunner's gland may contribute the control of acid-base balance in the duodenal lumen by secreting bicarbonate ion catalyzed by CAII and IV.

Regulation of Osteoclasts via Osteoblasts by Alendronate / 대한해부학회지

Bisphosphonates have been used for the prevention of pathological bone resorption and its related disease for their high affinity to hydroxyapatite and direct effects on osteoclasts. Several studies have shown that in vitro the potency of bisphosphonates for osteoclasts is not coincident with in vivo results, which proposes other mechanisms may be involved in their inhibition of bone resorption. Osteoblasts have paracrine regulation for osteoclast activities and formation. Thus, it is hypothesized that bisphosphonates may have indirect effects on osteoblasts to regulate osteoclastic bone resorption. This study was carried out to investigate molecular mechanism that alendronate, a nitrogen containing bisphosphponate, acts on osteoblasts to regulate osteoclastic bone resorption in vivo and in vitro system. In vitro MTS system using MC3T3-E1 osteoblastic cell line, absorbance values increased at 10(-10) to 10(-4) M concentration after a day treatment of alendronate. The values decreased at 3 and 5 days after treatment at 10(-5) M higher concentration. Gene expression levels of ALP, OPG and type I collagen were not changed at both 10(-5) M and 10(-6) M. However, MCP-1 and M-CSF notably decreased in their gene expression by the treatment. In vivo system using the rat pup tibia, M-CSF gene expression decreased a lot by alendronate treatment. The number of osteoclasts significantly decreased from trabeculae of femur and tibia in alendronate treated group (p < 0.01). Furthermore, trabeculae themselves underneath the epiphyseal plate were longer in the alendronate treated group (p< 0.01). These results suggested that bisphosphonates can indirectly inhibit osteoclastic bone resorption by affecting osteoblasts to regulate osteoclasts at molecular level.

Localization of Cyclooxygenase Isozymes in Skin Wound Healing in Mouse / 대한해부학회지

Cyclooxygenase (COX)-1 and -2 expressions in the incisional wound healing of mouse skin were determined by immunohistochemistry and Western blot analysis. By Western blotting, compared to normal skin, COX-2 activity was increased at days 1, 4, 8, and 12 and was maximal at 4 day after incisional wound of mouse skin whereas COX-1 was barely detectable. In normal skin, COX-1 immunostaining was observed among the basal cells of epidermis whereas COX-2 immunostaining was detected in the more differentiated, suprabasal keratinocytes. At 1~4 days after wound, COX-2 staining was particularly prominent in the inflammatory cells, and at day 8, many macrophage-like cells were stained positively. COX-2 immunoreactive fibroblast, macrophage-like cells, and newly formed vascular endothelial cells were increased in number at 12 days after incision. These data suggest that COX-2 is constitutively expressed, just as is COX-1, in epidermis and is associated with keratinocyte differentiation. In addition, these findings support the well-established role for COX-2, the prostaglandins that they generate, as mediators of inflammatory response.

Degenerate oligonucleotide primed PCR for the application to comparative genomic hybridization / 대한해부학회지

Degenerate oligonucleotide primed PCR is an useful technique to amplify whole genome and its the applications for fluorescent in situ hybridization and comparative genomic hybridization (CGH) were reported. For the CGH, topoisomerase and sequenase were recommended to use for the better hybridization. But adding the enzymes to PCR reaction per every cycle is labor-intensive and can easily contaminate PCR reaction. This study was carried out to prove the possibility of application of DOP-PCR to CGH without use of sequenase. Several combinations of CGH e.g., DOP-PCR amplified normal DNA vs. DOP-PCR amplified normal DNA, DOP-PCR amplified normal DNA vs. non-DOP normal DNA, DOP-PCR amplified normal DNA vs. DOP-PCR amplified MCF-600 cell line DNA, and non-DOP normal DNA vs. non-DOP MCF-600 DNA were performed. In addition, randomly selected microsatellite loci were tested to know whether DOP-PCR covers whole genome amplification. Apparently the DOP-PCR provides enough amount and size of DNA for CGH application and covers whole genome amplification. These results suggest that DOP-PCR can be used for CGH and genotyping.

Immunohistochemical Localization of Na/K-APTase Subunit Isoforms in Rat Inner Ear / 대한해부학회지

The endolymph and perilymph of the inner ear have unique ionic composition and electrical potential. It is widely accepted that normal auditory function depends on them and Na/K-ATPase plays a central role in production and maintenance of them. The distribution of five Na/K-ATPase subunit isoform (alpha1, alpha2, alpha3, beta1, and beta2) in rat inner ear was determined by immunohistochemistry after decalcifying the temporal bone with Gooding and Stewart's solution. In the cochlear regions, Na/K-ATPase alpha1beta1 isozyme was abundantly expressed in the infrastrial fibrocytes, suprastrial fibrocytes, spiral prominence, outer sulcus cells and spiral ganglion, and also detected in cochlear nerve and interdental cells. alpha1beta2 isozyme was abundantly expressed in all layers of stria vascularis and alpha3beta1 isozyme was detected in cochlear nerve and spiral ganglion. alpha3beta2 isozyme was expressed in spiral ganglion. In vestibular regions, Na/K-ATPase alpha1b1 isozyme was expressed in macular sacculi hair cell, transitional cells of ampulla, and vestibular ganglion, and alpha1b2 isozyme was abundantly expressed in ampullary dark cells and transitional cells and vestibular ganglion. a3b1 isozyme was abundantly expressed in crista ampularis, macula utriculi, and macula sacculi hair cells, and also moderately detected in ampullary, utricular, and saccular nerves, and vestibular ganglion. alpha3beta2 isozyme also detected in ampullary, utricular, and saccular nerves, and vestibular ganglion. But, alpha2beta1 and alpha2beta2 isozymes were not detected in any regions of inner ear. These findings suggest the possibility of four unique Na/K-ATPase isozymes deferentially expressed among the various cell types of the inner ear. This structural diversity imparts considerable biological versatility to the Na/K-ATPase and would be provided the explanations for the differences in fluid and ion transport and its regulation among the inner ear regions.

Light and Electron Microscopic Study on the Mode of Cell Death Induced by Methotrexate Administration in the Crypt and Villus of Rat Small Intestine / 대한해부학회지

Methotrexate (MTX), folic acid antagonist, is effective against the cancer, but is often accompanied by damage to the small intestine due to its inhibition of cell division. The nature of cell damage induced by MTX on the surface lining and crypt cells of the small intestine was investigated in the rat receiving MTX administration. MTX was injected once in every other day intraperitoneally to rats [0.03, 0.1, and 0.3 mg per 100 g body weight in small (SD), large (LD), and over dose (OD) groups, respectively] and the animals were sacrificed with various intervals after single, three-, five-times administrations of SD, single, three-, four-times administrations of LD, and single administration of OD. In case of three times injection of LD, intravenous injection was also made. Three portions, duodenum, jejunum, and ileum of the small intestine were studied by light and electron microscopy. Histologically, there were no appreciable morphological changes in villi and crypts after single and three times administrations of SD. The cells contained dense nucleus with clear cytoplasm, however, were rarely presented among the crypt cells. The duodenal villi were relatively normal in shape, but the number of crypts were somewhat decreased in five times administrations of SD. On the other hand, the villi of jejunum and ileum were atrophied and the epithelial cells became flattened. Crypts were atrophied and decreased in number. In single administration of LD, the shape of villi were normal in all of three portions. The cellular damages were noted in crypts of all of them, and the severity was most predominant at 6 hours than at 2, 4, or 24 hours after administration. In three times administrations of LD, more serious damages were noted in intravenous injection groups than intraperitoneal injection groups. Villi and crypt of the jejunum and ileum became atrophy, and epithelial cells were vacuolated. In four times administrations of LD, loss of villi and atrophied glands contained cellular debris within their lumen were noted in all three portions. In single administration of OD, there was no remarkable changes in histological structures of duodenum, jejunun, and ileum. With TUNEL staining, positive cells were present mainly in the crypt. The positive cells appeared among the glandular cells and were consistent with dense nucleus contained cells in hematoxylin-eosin stain. In small dose group the number of apoptotic cells in duodenum and ileum was increased in proportion to the times of administration, but that in jejunum was decreased after three times of administrations because of the destruction of crypts. Most prominent apoptotic changes of crypt cells were noted at 6 hours compare with 2, 4, and 24 hours after three times administrations of SD. Positive cells appeared most numerous at 6 hours after single administration of LD and OD. Destructive changes of crypt were found in jejunum and ileum after three times intravenous injection of LD, and were found in all portions after four times administrations of LD. Electron microscopic observation generally confirmed the findings of light microscopy. The apoptotic cells appeared in the crypt and was characterized by chromatin condensation and fragmentation, cytoplasmic condensation. Notable changes were observed in epithelial and crypt cells after five- and four-times administrations of SD and LD, respectively. The epithelial cells became flat and had many fat droplets in the cytoplasm. The crypts were atrophied and the glandular cells were very flat. These changes were more severe in jejunum and ileum than in duodenum. These results indicate that the damage of small intestine by MTX is more severe in jejunum and ileum than in duodenum, and also suggest that the cell death of the crypt is mediated by apoptosis, and that of the epithelial cell is due to fatty degeneration.

Light and Electron Microscopic Study on Epithelial Cell Death During Implantation in Rat Uterus / 대한해부학회지

The loss of the uterine epithelium surrounding blastocyst is important in bringing the trophoblast into close association with the endometrial stroma during embryonal implantation in several laboratory rodents. It is usually assumed that the epithelial cells die during this process, but the cause of epithelial cell death remains speculative. This study was performed to investigate the mechanism of epithelial cell death during implantation in the endometrium of rat uterus throughout days 5-9 of pregnancy by light and electron microscopy. Cathepsin-D reactivity, detected by imunohistochemistry, was localized to all viable epithelia, some degenerating epithelial and decidual cells between days 5-9 of pregnancy. DNA fragmentation, detected by TUNEL staining, was observed in degenerating epithelial and decidual cells throughout days 6-9 of pregnancy. Cellular fragments within lumen and phagosome within trophoblasts were reacted positively to TUNEL staining. Electron microscopy revealed that the epithelium lining the antimesometrial chamber was sloughed off into the lumen on day 6 of pregnancy. Its cell organelles appeared healthy but its nucleus was condensed and irregular shaped. The detached epithelial cells lining the antimesometrial chamber on day 7 of pregnancy contained condensed and fragmented nuclei, but exhibited different morphological pattern according to the intactness or loss of cytoplamic organelles and membrane. And these cells were surrounded and phagocytosed by trophoblasts. On day 7 of pregnancy, syncytial knots were formed, and later these knots and most of detached epithelial cells were severely degenerated. Some decidual cells nearby the lumen were also degenerated at this period. These results indicate that the epithelial cell death of rat uterine endometrium during implantation is mainly due to the process of apoptosis and these dying cells are removed by trophoblasts.