Alteration of Nrf2 and p-Nrf2 Protein Expression in Hypokalemic Rat Kidney / 체질인류학회지

Potassium (K) balance is regulated not only by ion channels and ion transporters, but also by various genes including NF-E2-related factor 2 (Nrf2). Although mRNA distribution and role of Nrf2 has been studied in hypokalemic kidney, the distribution of Nrf2 and phosphorylated-Nrf2 (p-Nrf2) proteins are not known. The present study was planned to examine the alteration of expression and distribution of Nrf2 and p-Nrf2 protein in the kidney of normal and K-depleted rats using immunohistochemistry. In normal rat kidneys, Nrf2 was highly expressed in the proximal convoluted tubule and proximal straight tubule, moderately in cortical thick ascending limb, and weakly in cortical collecting duct, outer medullary thick ascending limb, and outer medullary collecting duct. In K-depleted groups, the pattern of cellular labeling of Nrf2 protein was identical to that of normal group, but the signal intensity was prominently increased in proximal convoluted tubule and proximal straight tubule especially in rats at K-free diet 3 weeks. In normal rat kidneys, p-Nrf2 was highly expressed in nucleus of cortical thick ascending limb, cortical collecting duct, and glomerular endothelial cell, moderately in distal convoluted tubule and outer medullary collecting duct, and weakly in proximal convoluted tubules and outer medullary thick ascending limb. In K-depleted groups, the pattern of cellular labeling of p-Nrf2 protein was similar to that of normal group, but signal intensity was significantly increased in the nucleus of outer medullary collecting duct from of K-free diet 2 and 3 weeks groups. These results suggest that Nrf2 and p-Nrf2 expression was gradually increased in K-depleted groups of kidney, but Nrf2 and p-Nrf2 expression patterns were not exactly matched. In addition, it is suggested that enhanced expression of Nrf2 and p-Nrf2 in hypokalemic condition may affect the regulation of ion channels and ion transporters and subsequent intracellular signal transduction.

Efficacy of the Mineral Oil and Hyaluronic Acid Mixture Eye Drops in Murine Dry Eye

PURPOSE: To investigate the therapeutic effects of mineral oil (MO) and hyaluronic acid (HA) mixture eye drops on the tear film and ocular surface in a mouse model of experimental dry eye (EDE). METHODS: Eye drops consisting of 0.1% HA alone or mixed with 0.1%, 0.5%, or 5.0% MO were applied to desiccating stress-induced murine dry eyes. Tear volume, corneal irregularity score, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured at 5 and 10 days after treatment. Ten days after treatment, goblet cells in the conjunctiva were counted after Periodic acid-Schiff staining. RESULTS: There was no significant difference in the tear volume between desiccating stress-induced groups. The corneal irregularity score was lower in the 0.5% MO group compared with the EDE and HA groups. The 0.5% and 5.0% MO groups showed a significant improvement in TBUT compared with the EDE group. Mice treated with 0.1% and 0.5% MO mixture eye drops showed a significant improvement in fluorescein staining scores compared with the EDE group and the HA group. The conjunctival goblet cell count was higher in the 0.5% MO group compared with the EDE group and HA group. CONCLUSIONS: The MO and HA mixture eye drops had a beneficial effect on the tear films and ocular surface of murine dry eye. The application of 0.5% MO and 0.1% HA mixture eye drops could improve corneal irregularity, the corneal fluorescein staining score, and conjunctival goblet cell count compared with 0.1% HA eye drops in the treatment of EDE.

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.

Potassium Depletion Upregulates Expression of Nrf2 Transcription Factor in Rat Kidney / 대한신장학회지

PURPOSE: The purpose of this study was to analyze the expression and localization of Nrf2 mRNA in rats according to the changes of K-diet. METHODS: Nrf2 gene was isolated using DNA chip microassay. Northern and Western blot analysis and in situ hybridization (ISH) were performed. RESULTS: Northern analysis of normal rat demonstrated that Nrf2 mRNA was abundantly expressed in stomach, moderately in testis, kidney, distal colon, duodenum, and adrenal gland, and weakly in brain, heart, spleen, salivary gland, liver, and lung. In the kidney of K-restricted groups, Nrf2 mRNA and protein expressions were gradually increased in K-restricted kidney. By ISH, hybridization signal for Nrf2 gene of normal group was prominent in the S3 segment of proximal tubule, distal tubule, and cortical collecting duct, and weak in outer and inner medullary collecting duct. In K-restricted groups, the localization of hybridization signal was the same as in normal group. Signal intensity of K-restricted groups was markedly increased in outer and inner medullary collecting ducts compared with normal group. But, that of the distal tubule and cortical collecting duct was decreased. mRNA for Nrf2 gene of normal group was detected in the cells of the basal portion of intestinal gland of distal colon and stomach, spermatogonia and spermatocytes of seminiferous tubule of testis, small lymphocytes of germinal center of spleen, and adrenal medulla cells of adrenal gland. CONCLUSION: These results suggest that expression of Nrf2 is different in various tissues and increased expression of Nrf2 gene in outer and inner medullary collecting ducts of hypokalemic kidney could regulate the ion transporter genes by these segments.

Gene expression profiling of mouse aborted uterus induced by lipopolysac charide / 대한해부학회지

To identify genes that participate in the abortion process, normal pregnant uteri were compared to lipopolysaccharide (LPS)-induced abortion uteri. At day 6 of pregnancy, mice were treated with LPS at various time points to induce an abortion. Total RNAs were applied to a cDNA microarray to analyze genes with altered expression. At the early stage (2 hours) of LPS-induced abortion, upregulated genes were mainly composed of immune responsive genes, including Ccl4, Ccl2, Cxcl13, Gbp3, Gbp2, Mx2, H2-Eb1, Irf1 and Ifi203. Genes related to toll-like receptor signaling were also overexpressed. At late stages of abortion (12-24 hours), many genes were suppressed rather than activated, and these were mainly related to the extracellular matrix, cytoskeleton, and anti-apoptosis. Altered expression of several selected genes was confirmed by real time reverse transcription-polymerase chain reaction. The results demonstrated that many known genes were altered in the LPS-treated pregnant uterus, implying that the molecular mechanisms of the genes involved in LPS-induced abortion are complicated. Further analysis of this expression profile will help our understanding of the pathophysiological basis for abortion.

Bilateral asymmetric supernumerary heads of biceps brachii / 대한해부학회지

Anatomical variations of the biceps brachii have been described by various authors, but the occurrence of bilateral asymmetric supernumerary heads is rare and has not been reported. We found three accessory heads of the biceps brachii muscle on right arm and an anomalous third head of biceps brachii on left arm. The third, fourth, and fifth heads of right arm originated from the body of humerus at the insertion site of coracobrachialis and inserted into the distal part of biceps brachii short head in order. The third head of left arm originated from humerus at the insertion site of coracobrachialis and combined with the distal part of biceps brachii and continued to the proximal part of common biceps tendon. Understanding the existence of bilateral asymmetric supernumerary heads of biceps brachii may influence preoperative diagnosis and surgery on the upper limbs.

Distribution of Interstitial Cells of Cajal in Menopausal Rat Urinary Bladder Showing Detrusor Overactivity / 대한배뇨장애요실금학회지

PURPOSE: Recent studies have showed that interstitial cells of Cajal (ICCs) are widely distributed in the genitourinary tract and have suggested their involvement in spontaneous electrical activity and muscle contraction. The purposes of this study were to investigate the effect of estrogen on ICCs in rat urinary bladder from the detrusor overactivity induced by ovariectomy. MATERIALS AND METHODS: Female Sprague-Dawley rats (230-240 g, N=60) were divided into three groups: control (N=20), bilateral ovariectomy (Ovx, N=20), and bilateral ovariectomy followed by subcutaneous injections of 17 beta-estradiol (50 mg/kg/day, Ovx + Est, N=20). After 4 weeks, urodynamic studies measuring contraction interval and contraction pressure were done. The cellular localization of ICCs was determined by immunohistochemistry in the rat urinary bladder. RESULTS: Filling cystometry studies demonstrated a reduced interval between voiding contractions and an increased voiding pressure in Ovx group. The approximate the contraction interval (min) was (3.9+/-0.25) significantly decreased in the Ovx group compared to the control group (6.7+/-0.15), which was increased after estrogen treatment (9.7+/-0.22) (p<0.05). Conversely, the average contraction pressures (mmHg) were increased in the Ovx group (28.9+/-2.1) compared to the control group (21.2+/-1.45), and decreased after estrogen treatment (24.8+/-2.21) (p<0.05). The population of c-Kit immunoreactive ICCs was decreased in both the urothelial and muscle layers in Ovx bladders, which increased to the control value after estrogen treatment. CONCLUSIONS: These results demonstrated an decreased immunoreactivity of ICCs in the menopausal rat model and suggest that thedecreased population of ICCs expression may contribute to the modulation of bladder overactivity induced by menopause.

Influences of Chronic Indirect Cigarette Smoking on the Expression of TGF-beta1 and eNOS in Rat Vagina / 대한남성과학회지

PURPOSE: The objectives of this study were to evaluate the effects of chronic indirect cigarette smoking on vaginal blood flow and on histological change in a rat model. MATERIALS AND METHODS: Female Sprague-Dawley rats (12 weeks old, n=40) were devided into smoking and control group. For the exposure to passive smoking, the rat, in plastic enclosure, had a constant influx of cigarette smoke using a smoking generator for 8 weeks in smoking group. The experimental group was exposured to cigarette smoke for 1 hour, twice a day, daily for 8 weeks. Vaginal blood flow was measured by laser Doppler flowmeter. Serum estrogen concentration was measured using competitive radioimmunoassay. Immunohistochemistry and western blot analysis was done to observe the expression of TGF-beta1 and e-NOS. RESULTS: Mean vaginal blood flow (ml/min/100g tissue) significantly decreased in smoking group (13.4+/-1.6) compared to control (19.6+/-5.9)(p<0.05). The estimated concentration of serum estradiol (pg/ul) was similar between smoking (1.1+/-0.8) group and control (1.1+/-0.3) group. Vaginal histology of the cigarette smoking group was similar to the control. In the cigarette smoking group, the immunoreactivity of TGF-beta1 increased in the smooth muscle and fibroblasts. The protein expression of TGF-beta1 was increased in the smoking group (p<0.05). There was no significant differences in expression of e-NOS between two groups. CONCLUSIONS: A chronic indirect exposure to cigarette smoke significantly reduces vaginal blood flow and appears to cause vaginal tissue fibrosis in the female rat model. This suggest that cigarette smoking has adverse effects on female sexual functions and may cause sexual arousal disorder in women.

The Expression of AQP1 and eNOS in Menopausal Rat Urinary Bladder / 대한배뇨장애요실금학회지

PURPOSE: Aquaporins (AQPs) have been reported to be expressed in rat and human urothelium. Nitric oxide (NO) is thought to play an important role in the bladder overactivity related to menopause. The purpose of this study was to investigate the effect of hormonal alteration on the expression of AQP1 and eNOS in menopausal rat urinary bladder. MATERIALS AND METHODS: Female Sprague-Dawley rats (230-240 g, N=30) were divided into three groups: control (N=10), bilateral ovariectomy (Ovx, N=10), and bilateral ovariectomy followed by subcutaneous injections of 17beta-estradiol (50 mg/kg/day, Ovx+Est, N=10). After 4 weeks, urodynamic studies measuring the contraction interval and contraction pressure were done. The expression and cellular localization of AQP1 and eNOS were determined by performing Western blotting and immunohistochemistry on the rat urinary bladder. RESULTS: The approximate contraction interval (min) was significantly decreased in the Ovx group (3.9+/-0.25) compared to the control group (6.7+/-0.15), and was increased after estrogen treatment (9.7+/-0.22) (p<0.05). The AQP1 and eNOS immunoreactivities were localized in the same areas: capillaries, arterioles, and venules of the lamina propria. The protein expression of AQP1 was not changed significantly, whereas eNOS expression was significantly decreased in the Ovx group and restored to the control value in the Ovx+Est group. CONCLUSIONS: This study showed that ovariectomy causes a significant change in e-NOS expression without a change in AQP1 in menopausal rat urinary bladder. This may imply that e-NOS has a functional role in the bladder overactivity that occurs in association with menopause.

HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression / 대한해부학회지

During the prostate cancer (PCa) development and its progression into hormone independency, androgen receptor (AR) signals play a central role by triggering the regulation of target genes, including prostate-specific antigen. However, the regulation of these AR-mediated target genes is not fully understood. We have previously demonstrated a unique role of HOXB13 homeodomain protein as an AR repressor. Expression of HOXB13 was highly restricted to the prostate and its suppression dramatically increased hormone-activated AR transactivation, suggesting that prostate-specific HOXB13 was a highly potent transcriptional regulator. In this report, we demonstrated the action mechanism of HOXB13 as an AR repressor. HOXB13 suppressed androgen-stimulated AR activity by interacting with AR. HOXB13 did neither bind to AR responsive elements nor disturb nuclear translocation of AR in response to androgen. In PCa specimen, we also observed mutual expression pattern of HOXB13 and AR. These results suggest that HOXB13 not only serve as a DNA-bound transcription factor but play an important role as an AR-interacting repressor to modulate hormone-activated androgen receptor signals. Further extensive studies will uncover a novel mechanism for regulating AR-signaling pathway to lead to expose new role of HOXB13 as a non-DNA-binding transcriptional repressor.

Alterations of COX-1, 2 mRNA and Protein Expression in Pregnant Rat Kidney / 대한해부학회지

The pregnancy causes the marked changes in maternal renal hemodynamic and volume homeostasis. During pregnancy, renal sodium and water retention result in an expansion of extracellular fluid and plamsma volume. This study was to examine the alteration of expression and localization of COX-1, 2, mRNAs and proteins in the kidneys of non-pregnant (NP) and pregnant rats using RT-PCR, Western blot analysis and immunohistochemistry. Pregnant Sprague-Dawley rats were evaluated on various time sets : days 10.5 (P10.5), 12.5 (P12.5), 17.5 (P17.5), and 19.5 (P19.5). In RT-PCR, COX-1 expression was gradually increased from P10.5 to P19.5 compared with NP rat. COX-2 expression was gradually decreased from P10.5 to P17.5 compared with NP rat, but restored NP level at P19.5. In Western blot analysis, COX-1, 2 proteins were detected in ~70, ~72 kDa, respectively. COX-1 expression was gradually increased from P10.5 to P17.5 and peaked at P19.5 compared with NP rat. COX-2 expression of pregnant rats was slightly decreased compared with NP rat. In NP rat, immunoreactivity of COX-1 was detected in entire collecting duct, glomerular epithelium, and medullary interstitial cells. In pregnant rats, the pattern of cellular labeling and signal intensity of COX-1 protein was identical to NP rat, but signal intensity was markedly increased in the inner stripe of outer medulla and inner medulla at P19.5. COX-2 immunoreactivity of NP rat was detected in the cortical thick ascending limb and macula densa. In pregnant rats, the pattern of cellular labeling of COX-2 protein was identical to NP rat, but signal intensity was slightly decreased. These results suggest that the expansion of extracellular fluid volume and water retention may be partly regulated by COX-1 rather than COX-2 during the pregnancy, especially at late stage.

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.

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.

Alterations of Aquaporin-1, 2, 3 Protein Expression in Pregnant Rat Kidney / 대한해부학회지

The pregnancy causes the marked change in maternal renal hemodynamic and volume homeostasis. During pregnancy, renal sodium and water retention result in an expansion of extracellular fluid and plamsma volume. Although many studies suggested that water balance or water balance disorder was associated with regulation of Aquaporin (AQP) expression, the studies were only limited to AQP-2 expression during the pregnancy. The present study was to examine altered expression and distribution of AQP-1, 2, and 3 proteins in the kidneys of non-pregnant (NP) and pregnant rats using Westhern blot analysis and immunohistochemistry. Pregnant Sprague-Dawley rats were evaluated on various time sets: days 10.5 (P10.5), 12.5 (P12.5), 17.5 (P17.5), and 19.5 (P19.5). In Westhern blot analysis, expression of AQP-1, 2 was peaked at P17.5 and AQP-3 at 19.5. Immunoreactivity of AQP-1 of NP rat was detected in the apical membranes of proximal tubules and thin limb of Henle loop. In pregnant rats, the pattern of cellular labeling of AQP-1 protein was identical to NP rat, but signal intensity was continuously increased from P10.5 and peaked at P17.5. In NP rat, immunoreactivity of AQP-2 was the most prominent in apical region and moderate in cytoplasm of the principal cells of entire collecting duct. In pregnant rats, the pattern of cellular labeling of AQP-2 protein was identical to NP rat, but signal intensity was moderately expressed in P10.5 and P12.5 and most prominent signal was observed in P19.5. In NP rat, immunoreactivity of AQP-3 was most prominent in the bosolateral plasma membrane of principal cells of entire collecting duct. In pregnant rats, the pattern of cellular labeling of AQP-3 protein was identical to NP rat, but signal intensity was continuously increased from P10.5 to P17.5 and peaked at P19.5. These results suggest that the expansion of extracellular fluid volume and water retention are regulated by AQP-1, 2, and 3 during the pregnancy, especially at late stage.

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.

Effects of Intracavernosally Injected Embryonic Stem Cells in Aged Rat / 대한남성과학회지

PURPOSE: Stem cell-based cell therapy has recently been tried as a way to restore cavernosal function in an animal model. The aims of this study were to elucidate the effect of intracavernosally injected embryonic stem cells(ESCs) in aged rat. MATERIALS AND METHODS: Male Sprague Dawley rats were divided into 3 groups: young control(12 weeks old; n=10), old with vehicle injection(24 months old; n=7), and old with ESC injection(24 months old; n=8). ESCs were transfected with firefly luciferase attached to adenovirus and then injected intracavernously 2 times with a 1-week interval. Cell survival was assessed by optical molecular imaging 2 days after the last ESC injection. At 4 weeks after the last injection, intracavernosal pressure and systemic arterial pressure were recorded after pelvic nerve stimulation. Serum testosterone levels were measured by radioimmunoassay. RESULTS: We observed fluorescent signals around the external genitalia of animals injected with ESCs. The serum testosterone level of the old group(1.39+/-0.07 ng/ml) was significantly lower compared to the young control group(2.98+/-0.31 ng/ml)(p=0.03). The percentage of intracavernosal pressure/systolic blood pressure was significantly lower in the old group(58.5+/-8.6%) compared to the young control group(69.5+/-6.6%)(p=0.034). However, the old group with ESC injection(61.6+/-9.9%) did not show any significant differences from the old group(p>0.05). The old group with ESC injection showed histomorphometry similar to the old group. CONCLUSIONS: The presence of intracavernosal ESCs can be noninvasively monitored with optical molecular imaging. However, the intracavernosal injection of ESCs did not improve erectile function in the aging rat. Further studies are needed to elucidate the regulatory factors of stem cell differentiation in the corpus cavernosum.

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.