Long-Term Outcome of Treatment with Topical Corticosteroids for Severe Dry Eye Associated with Sjogren's Syndrome / 전남의대학술지

This retrospective study was performed to analyze the long-term outcome of topical corticosteroid treatment for severe dry eye associated with Sjogren's syndrome (SS). Patients who had severe dry eye associated with SS were topically treated with loteprednol etabonate 0.5% (group A, n=66) or fluorometholone 0.1% (group B, n=67) twice daily and were followed up for 2 years. Visual acuity (VA), intraocular pressure (IOP), Schirmer test, tear film breakup time (BUT), keratoepitheliopathy, and symptom scores were measured at baseline and 6, 12, 18, and 24 months after treatment. VA and IOP were not changed significantly during follow-up in either group. Schirmer test results, keratoepitheliopathy, and symptom scores at 6, 12, 18, and 24 months (p<0.05) and tear film BUT at 12, 18, and 24 months (p<0.05) significantly improved after treatment compared with baseline in both groups. No significant differences between the groups were found in any parameter during follow-up. At 24 months, the number of patients with IOP elevation of more than 2 mmHg compared with baseline was 4 in group A (6.1%) and 9 in group B (13.4%). The mean IOP in these patients was lower in group A than in group B (15.00+/-0.82 mmHg versus 16.50+/-1.12 mmHg; p=0.04). Long-term application of low-dose topical corticosteroids is effective for controlling signs and symptoms of chronic, severe dry eye associated with SS. Loteprednol etabonate 0.5% may have a lower risk for IOP elevation than fluorometholone 0.1%.

Long-Term Outcome of Treatment with Topical Corticosteroids for Severe Dry Eye Associated with Sjogren's Syndrome / 전남의대학술지

This retrospective study was performed to analyze the long-term outcome of topical corticosteroid treatment for severe dry eye associated with Sjogren's syndrome (SS). Patients who had severe dry eye associated with SS were topically treated with loteprednol etabonate 0.5% (group A, n=66) or fluorometholone 0.1% (group B, n=67) twice daily and were followed up for 2 years. Visual acuity (VA), intraocular pressure (IOP), Schirmer test, tear film breakup time (BUT), keratoepitheliopathy, and symptom scores were measured at baseline and 6, 12, 18, and 24 months after treatment. VA and IOP were not changed significantly during follow-up in either group. Schirmer test results, keratoepitheliopathy, and symptom scores at 6, 12, 18, and 24 months (p<0.05) and tear film BUT at 12, 18, and 24 months (p<0.05) significantly improved after treatment compared with baseline in both groups. No significant differences between the groups were found in any parameter during follow-up. At 24 months, the number of patients with IOP elevation of more than 2 mmHg compared with baseline was 4 in group A (6.1%) and 9 in group B (13.4%). The mean IOP in these patients was lower in group A than in group B (15.00+/-0.82 mmHg versus 16.50+/-1.12 mmHg; p=0.04). Long-term application of low-dose topical corticosteroids is effective for controlling signs and symptoms of chronic, severe dry eye associated with SS. Loteprednol etabonate 0.5% may have a lower risk for IOP elevation than fluorometholone 0.1%.

Long-Term Outcome of Treatment with Topical Corticosteroids for Severe Dry Eye Associated with Sjogren's Syndrome / 전남의대학술지

This retrospective study was performed to analyze the long-term outcome of topical corticosteroid treatment for severe dry eye associated with Sjogren's syndrome (SS). Patients who had severe dry eye associated with SS were topically treated with loteprednol etabonate 0.5% (group A, n=66) or fluorometholone 0.1% (group B, n=67) twice daily and were followed up for 2 years. Visual acuity (VA), intraocular pressure (IOP), Schirmer test, tear film breakup time (BUT), keratoepitheliopathy, and symptom scores were measured at baseline and 6, 12, 18, and 24 months after treatment. VA and IOP were not changed significantly during follow-up in either group. Schirmer test results, keratoepitheliopathy, and symptom scores at 6, 12, 18, and 24 months (p<0.05) and tear film BUT at 12, 18, and 24 months (p<0.05) significantly improved after treatment compared with baseline in both groups. No significant differences between the groups were found in any parameter during follow-up. At 24 months, the number of patients with IOP elevation of more than 2 mmHg compared with baseline was 4 in group A (6.1%) and 9 in group B (13.4%). The mean IOP in these patients was lower in group A than in group B (15.00+/-0.82 mmHg versus 16.50+/-1.12 mmHg; p=0.04). Long-term application of low-dose topical corticosteroids is effective for controlling signs and symptoms of chronic, severe dry eye associated with SS. Loteprednol etabonate 0.5% may have a lower risk for IOP elevation than fluorometholone 0.1%.

Gene Therapy for Head and Neck Squamous Cell Carcinoma Using KITENIN (KAI1 COOH-Terminal Interacting Tetraspanin)-Antisense Therapy

PURPOSE: KAI1 COOH-terminal interacting tetraspanin (KITENIN) has been found to act as a promoter of metastasis in murine models of colon cancer and squamous cell carcinoma (SCC). The suppression of tumor progression and metastasis of established colon cancer in mice was observed after intravenous delivery of small interfering RNA (siRNA) targeting KITENIN. The purpose of this study was to investigate the efficacy of gene therapy targeting KITENIN in human head and neck SCC. MATERIALS AND METHODS: SNU-1041, a well-established human hypopharyngeal SCC cell line, was used. KITENIN expression in SNU-1041 was measured by Western blot analysis. The cells were prepared, maintained in culture dishes with media, and divided into two groups: the si-KITENIN group and the scrambled group (control). The siRNA targeting KITENIN (si-KITENIN) and scrambled DNA were transfected into the SNU-1041 cells in each group. The effect of gene therapy was compared by in vitro experiments to evaluate invasion, migration, and proliferation. RESULTS: KITENIN was strongly expressed in the SNU-1041 cells, and the number of invaded cells was reduced more in the si-KITENIN group than in the scrambled group (p<0.001). The speed for the narrowing gap, made through adherent cells, was lower in the si-KITENIN group (p<0.001), and the number of viable proliferating cells was reduced in the si-KITENIN group compared to the scrambled group (p<0.001, the third day). KITENIN protein expression was no longer identified in the si-KITENIN group. CONCLUSION: Gene therapy using an anti-KITENIN strategy might be effective for head and neck squamous carcinoma.

Intravenous KITENIN shRNA Injection Suppresses Hepatic Metastasis and Recurrence of Colon Cancer in an Orthotopic Mouse Model

KITENIN (KAI1 C-terminal interacting tetraspanin) promotes invasion and metastasis in mouse colon cancer models. In the present study, we evaluated the effects of KITENIN knockdown by intravenous administration of short hairpin RNAs (shRNAs) in an orthotopic mouse colon cancer model, simulating a primary or adjuvant treatment setting. We established orthotopic models for colon cancer using BALB/c mice and firefly luciferase-expressing CT-26 (CT26/Fluc) cells. Tumor progression and response to therapy were monitored by bioluminescence imaging (BLI). In the primary therapy model, treatment with KITENIN shRNA substantially delayed tumor growth (P = 0.028) and reduced the incidence of hepatic metastasis (P = 0.046). In the adjuvant therapy model, KITENIN shRNA significantly reduced the extent of tumor recurrence (P = 0.044). Mice treated with KITENIN shRNA showed a better survival tendency than the control mice (P = 0.074). Our results suggest that shRNA targeting KITENIN has the potential to be an effective tool for the treatment of colon cancer in both adjuvant and metastatic setting.

Myasthenia Gravis Presenting as Isolated Respiratory Failure: A Case Report

Myasthenia gravis (MG) is often complicated by respiratory failure, known as a myasthenic crisis. However, most of the patients who develop respiratory symptoms do so during the late course of disease and have other neurological signs and symptoms. However, in some patients respiratory failure is the initial presenting symptom. We report the case of a 68-year-old woman with MG who presented with isolated respiratory failure as her first presenting symptom. As illustrated by this case, it is important to consider neuromuscular disorders in cases of unexplained respiratory failure.

Sleep Duration and Body Mass Index in Korean Children

OBJECTIVES: We conducted this study to investigate the relationship between sleep duration and body mass index (BMI), in Korean children. METHODS: We performed a cross-sectional analysis of data collected on 3,639 boys and girls (aged 7-12) in Daegu, Korea. The data included each child's age, sex, weight, height, extracurricular activities, bedtime, wake-up time, sleep latency, total sleep duration, parents' occupations, and parents' educational levels. The relationship between sleep duration and each variable was examined via analysis of variance (ANOVA). RESULTS: The analysis showed an association between short sleep duration and high BMI. Boys showed a graded inverse relationship between sleep duration and BMI. However, there was no significant corresponding result for girls. In the total sample, hours of computer use, time when the computer was turned off, time when the television was turned off, mother's bedtime, and hours of extracurricular activity were associated with longer sleep duration. No association was found between sleep duration and hours of watching television, child's wake-up time, or educational level of the parents. CONCLUSION: The results of this study show an inverse relationship between a child's sleep duration and BMI;thus, children with shorter sleep duration tend to have higher BMIs.

Inhibition of Corneal Neovascularization by Subconjunctival Injection of alphaVbeta5 Integrin Antibody in Rabbit

PURPOSE: To investigate the efficacy of a subconjunctival injection of alphaVbeta5 integrin antibody on corneal angiogenesis induced by chemical epithelial denudation in a rabbit eye model. METHODS: One week after debridement by heptanol, rabbits were treated with a subconjunctival injection of alphaVbeta5 integrin antibody or control immunoglobulin G weekly for 2 weeks. Rabbits that did not receive injection after debridement served as the untreated group. The percentage of neovascularized corneal area was calculated by biomicroscopy, and the sectioned area and number of new vessels were calculated by histological examinations. RESULTS: At 7 days after the first injection, alphaVbeta5 integrin antibody-treated eyes had 9.5% (P=0.02) and 6.8% (P=0.03) less neovascularized corneal area than vector-treated eyes and untreated eyes, respectively. At 7 days after the second injection, alphaVbeta5 integrin antibody-treated eyes had 21.1% (P=0.02) and 18.3% (P=0.02) less neovascularized corneal area than vector-treated eyes and untreated eyes, respectively. Light microscopic examination showed a smaller neovascularized corneal area and a reduced number of new vessels in the alphaVbeta5 integrin antibody-treated eyes compared to the control eyes. CONCLUSIONS: Subconjunctival injection of alphaVbeta5 integrin antibody effectively reduces experimental corneal neovascularization induced by chemical injury, and could be used as a corneal angiogenesis inhibitor in the future.

Effects of Intracavernosal IGF-1 Gene Delivery on Erectile Function in the Aging Rat / 대한비뇨기과학회지

PURPOSE: Insulin like Growth Factor-1 (IGF-1) promotes the proliferation and migration of penile cavernous smooth muscle cells in rats. The goal of this study was to investigate the effects of an intracavernosal injection of the IGF-1 gene on the erectile function in the aging rat. MATERIALS AND METHODS: Corpus cavernosum smooth muscle cells (CCSMCs) were primarily cultured from Sprague-Dawley rats (16 wks). IGF-1 cDNA was subcloned into pcDNA3.1, and this expression plasmid transfected into CCSMCs. Two, three and four days after transfection, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analyses were carried out to quantify the transfection efficiency. In an in vivo study, male F344 rats (n=28) were divided into 2 groups; the young (5 months; n=7) and the old (20-21 months; n=21) groups. The old group was further divided into control (n=7) and treatment groups (n=14). The treatment group was given a single intracavernosal injection of DNA-liposome complex (pcDNA alone and pcDNA/IGF-1; 2mug or 4mug in 7 animals each). After 4 weeks of treatment, the erectile function and corpus cavernosal histology were evaluated by hemodynamic study and histomorphometric analysis, respectively. RESULTS: When the CCSMCs were transfected with IGF-1, the IGF-1 mRNA and protein were overexpressed compared to the control. After 4 weeks of treatment, the ratio of the peak intracavernosal pressure to systemic arterial pressure increased in the treatment group (66.3+/- 4.3% with 2mug, 65.4+/- 10.2% with 4mug), but without statistical significance (p>0.05) compared to the control group (55.5+/- 16.7%). The percentage of cavernosal smooth muscle also slightly increased in the treatment group (14.1+/-1.7% with 2mug), but without statistical significance (p>0.05) compared to the control group (13.2+/-1.2%). CONCLUSIONS: IGF-1 mRNA and IGF-1 protein were overexpressed in the rat corpus cavernosum smooth muscle cells due to IGF-1 gene delivery. However, lipid-mediated intracavernosal gene delivery of IGF-1 did not significantly improve the erectile function in the aging rat.

Effects of Ginsenosides on Relaxation of Rabbit Vaginal Smooth Muscle / 대한남성과학회지

PURPOSE: Ginseng has been used throughout the Far East, including Korea and China, as a tonic and restorative agent to maintain physical vitality. The main pharmacoactive molecules of ginseng are ginsenosides. The present study was designed to investigate whether ginsenosides relax rabbit vaginal smooth muscle and whether this effect is modulated by nitric oxide(NO) and the cGMP pathway. MATERIALS AND METHODS: Strips of rabbit vagina were mounted in organ chambers to measure isometric tension. The strips were contracted with phenylephrine(5 X 10(-5) M), and the responses to acetylcholine, nitric oxide inhibitor, and ginsenosides were examined. The cGMP content of the strips was measured by radioimmunoassay after various doses of ginsenosides. RESULTS: Ginsenosides(100~500microgram/mL) relaxed vaginal smooth muscle in a dose-dependent manner(5~25%). Acetylcholine-induced relaxation was significantly increased in the presence of ginsenosides(100, 200microgram/mL)(p0.05). Ginsenosides(400microgram/mL for 7 min) increased the accumulation of cGMP. CONCLUSIONS: These data suggest that ginsenosides have a relaxing effect on rabbit vaginal smooth muscle. This effect is at least in part mediated by the NO-cGMP pathway.

Downregulation of TGF-beta1 Expression in Penile Corpus Cavernosum by Antisense TGF-beta1 Oligonucleotides in Diabetic Rat / 대한남성과학회지

PURPOSE: We investigated the effect of antisense TGF-beta1 oligonucleotides on the expression of TGF-beta1 in the penile corpus cavernosum of streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Forty Sprague-Dawley male rats (200-210 g) were divided into two groups: control (n=10) and experimental group (n=30). The experimental group was received intravenous injection of streptozotocin (50 mg/kg). After 2 weeks, development of diabetes was verified by measuring body weight and blood sugar levels. The experimental group was divided further into 3 groups: vehicle (liposome only), sense oligomer group, and antisense oligomer (5'-CCGAGGGCGGCATGGGGGA-3') group. Penile tissues were used to perform Western analysis and immunohistochemistry for the TGF-beta1 expression. RESULTS: The mean glucose concentrations were 86 9 mg/dl in the control group and 485 119 mg/dl in the experimental group. Downregulation of TGF-beta1 protein expression was noted in the antisense oligomer group compared to sense oligomer group. In the immunohistochemistry, control group showed weak immunoreactivity whereas vehicle or sense oligomer group showed strong immunoreactivity both after 1 and 5 days of treatment. Antisense oligomer treated diabetic group showed weak immunoreactivity similar to control group after 1 day of treatment, even though they showed similar immunoreactivity as vehicle or sense oligomer group after 5 days of treatment. CONCLUSIONS: Antisense TGF-beta1 oligonucleotides suppressed the expression of TGF-beta1 in the corpus cavernosum of diabetic rats. It implies that penile corpus cavernosal fibrosis may be prevented by the application of antisense TGF-beta1 oligonucleotides.

Relationship between Radiation Induced Activation of DNA Repair Genes and Radiation Induced Apoptosis in Human Cell Line A431 / 대한핵의학회잡지

PURPOSE: The purpose of this study was to evaluate the relationship between radiation-induced activation of DNA repair genes and radiation induced apoptosis in A431 cell line. MATERALS AND METHODS: Five and 25 Gys of gamma radiation were given to A431 cells by a Cs-137 cell irradiator. Apoptosis was evaluated by flow cytometry using annexin V-fluorescein isothiocyanate and propidium iodide staining. The expression of DNA repair genes was evaluated by both Northern and Western blot analyses. RESULTS: The number of apoptotic cells increased with the increased radiation dose. It increased most significantly at 12 hours after irradiation. Expression of p53, p21, and hRAD50 reached the highest level at 12 hours after 5 Gy irradiation. In response to 25 Gy irradiation, hRAD50 and p21 were expressed maximally at 12 hours, but p53 and GADD45 genes showed the highest expression level after 12 hours. CONCLUSION: Induction of apoptosis and DNA repair by ionizing radiation were closely correlated. The peak time of inducing apoptosis and DNA repair was 12 hours in this study model. hRAD50, a recently discovered DNA repair gene, was also associated with radiation-induced apoptosis.

Changes in Expression of BCL-2, BAX and ICE mRNA After Middle Cerebral Artery Occlusion in Rat

BACKGROUND: In order to delineate the involvement of apoptotic death in post-ischemic cerebral infarction, the temporal expressions of some apoptosis-regulating genes (BCL-2, BAX and ICE) were studied in the parietofrontal cortex of a rat following focal cerebral ischemia using a Northern blot analysis and RT-PCR. METHODS: Animals were decapitated at 0.5, 1, 2, 4, 8 and 24 hours following a middle cerebral artery occlusion (MCAO). RESULTS: With the use of Northern blotting, the expression of proto-oncogene BCL-2, the apoptosis-repressor gene, increased up to 4 hours after a MCAO and then recovered to the control level. In a RT-PCR analysis, the expression of BCL-2 increased up to 8 hours after the MCAO and then recovered to the control level at 24 hours. The expression of BAX, the apoptosis-effec-tor gene, was not altered by a MCAO in a Northern blot analysis. The expression of ICE, an cysteine protease gene, markedly decreased at 1 and 2 hours after the MCAO and then recovered to the control level. CONCLUSIONS: These results show that ischemic insults by a MCAO alter the expression of the apoptosis-regulating genes and support the notion that the apoptosis may have occurred in the post-ischemic cerebral infarction.

Influence of Intracerebroventricular Kallikrein and Lys-bradykinin on the Rabbit Renal Function / 대한신장학회잡지

The renal function is under regulatory influence of central nervous system, in which various neurotransmitter and neuromodulator systems take part, and it has been known that kallikrein-kininogen- kinin system exists also in the brain, but its physiological role remains to be explored. This study was, therefore, undertaken to delineate the possible role of central kinin system in the regulation of renal function. Kallikrein given into a lateral ventricle(icv) of rabbit brain in doses ranging from 3 to 30 microgram/kg icv elicited increases in Na excretion and the fraction of filtered sodium excreted(FENa), as well as in urine flow rate. K excretion, however, did not parallel the Na excretion, but tended to decrease when the natriuresis reached its peak. Renal blood flow and glomerular filtration did not significantly change. Neither did free water reabsorption significantly change, but tended to decrease. The systemic blood pressure slightly increased. When 30 microgram/kg kallikrein was given intravenously, all the parameters of renal function and systemic blood pressure did not show any increase but decrease, primarily by decreased renal hemodynamics, resulting from transient hypotension. In experiments in which the plasma ANP was measured, the ANP level markedly increased, reaching more than 5 times the control value 25min after 30 microgram/kg icv, and lasting until the end of the experiment at 80min. The renal nerve activity increased with kallikrein, 30 microgram/kg icv, peaking at 1 min but it remained slightly increased until about 40 min, and then slightly declined. This indicates that the increased renal nerve activity may have antagonized or ameliorated the natriuretic effect of icv kallikrein. Lys-bradykinin(kallidin), a cleavage product from kallidinogen by kallikrein, when given icv in doses of 0.3 to 30 microgram/kg also produced increased Na excretion and diuresis. When CHA, a kallikrein inhibitor, was given icv in doses of 3-30 microgram/kg, elicited antidiuresis and antinatriuresis. However, pretreatment with CHA tended slightly to suppress the kallikrein effect. These results indicate that the central kallikrein- kinin system is involved in the central regulation of renal function, the activation of the system in the CNS resulting in increased natriuresis and diuresis, which are related to increased plasma ANP level, with the possible antagonistic effects of increased renal nerve activity.

Renal Adaptive Responses of HK alpha2 Gene(HK alpha 2a, HK alpha 2b) to the Changes of Potassium(K) Diet / 대한신장학회잡지

Recent molecular and physiological studies suggested that at least two H/K-ATPase isozymes are expressed in the rat kidney, and two distinct isoforms(HK alpha 2a, 2b) are resulted from alternative splicing of the 5-end of HK alpha 2 in distal colon by sequence analysis. Northern analysis and in situ hybridization(ISH) were carried out to analyze the expression of HK alpha 2a. and HK alpha 2b, mRNAs in rat kidney according to the changes of K-diet. Isoform specific 32P-labeled cDNA(for Northern) or digoxigenin labeled cRNA(for ISH) probes were used. Northern analysis demonstrated that HK a z. mRNA is abundantly expressed in normal(group 1: normal diet 2W) renal cortex, modestly in normal outer medulla, and weakly in normal inner medulla. The potassium-deprived rats(group 2: K-free diet 1W, and group 4: K-free diet 2W) expressed 40N lower levels in cortex and 2-4 fold higher levels of HKalpha 2a. mRNA in outer and inner medulla compared to normal rat. The potassium loading rats after potassium-deprivation(group 3: normal diet 1W after K-free diet 1W, and group 5: normal diet 1W after K-free diet 2W showed almost normal levels of HK alpha 2a. mRNA. HK alpha 2b, mRNA was not detected in any tissues of groups. By ISH, mRNA for HK alpha 2, was detected in the thick ascending limb, distal convoluted tubule, and the entire collecting duct. All groups exhibited comparable cellular patterns of labeling. Signal intensity of group 2 and 4 was less in cortical collecting duct(CCD), especially principal cells and much higher in the inner stripe of the outer medullary collecting duct(OMCDi) and the proximal inner medullary collecting duct(IMCD) compared to group 1. Group 3 and 5 exhibited signal intensity of group l. These results indicate that chronic hypokalemia enhances expression of HK alpha 2a, gene in OMCDi and proximal IMCD, decreases in CCD, and restores at normal levels in potassium-loading after potassium-deprivation and suggest that this isoform plays an important role in potassium balance by these segments accordiog to the changes of K-diet.

Expression of the nm23 Gene in Primary & Metastatic Gastric Cancer Tissues

BACKGROUND: The nm23 gene was originally identified by screening of cDNA libraries from murine melanoma cell lines of varying metastatic potential. Gene expression of nm23 has been investigated in a number of tumors. Its down-regulation has been shown to be associated with metastasis or disease progression in some of the tumors. METHODS: We evaluated the nm23 mRNA levels in 23 surgically resected primary gastric cancers, in the matched adjacent mucosa, and in lymph nodes or distant metastatic foci by using Northern blot analyses and immunohistochemical staining. RESULTS: The expression of nm23 mRNA was lower in the matched normal adjacent mucosa than in the primary tumor. The expressions of the nm23 gene were higher in normal lymph nodes and in lymph nodes with metastasis than in primary tumors. This result was due to the high expression in normal lymph nodes. The expression of nm23 in distant metastatic foci was lower than it was in primary tumor tissues (p<0.05). The expression of the nm23 protein in a primary tumor with distant metastasis was higher than it was in a primary tumor with lymphnode metastasis only (p<0.05). CONCLUSIONS: It is suggested that down-regulation of the nm23 gene might have a role in distant metastasis in gastric cancer, possibly leading to a poor prognosis.

Renal Adaptive Responses of Na+-K+-APTase Subunit Isoforms to Chronic Hypokalemia / 대한해부학회지

Chronic hypokalemia alters Na+-K+-ATPase gene expression in several tissues. While it is established that Na+-K+-ATPase activity and alpha1 and beta1 subunit protein levels increase during K depletion in the outer medullary collecting duct (OMCD) and do not significantly change in the cortical collecting duct (CCD), little is known about the adaptive responses of the other isoforms in these other nephron segments. Accordingly, this study was performed to characterize the relative levels of expression and cellular distribution of mRNAs encoding the Na+-K+-ATPase subunit isoforms in normal and K-deprived (2 weeks) rats using the Northern analysis and in situ hybridization (ISH). Isoform specific 32P-labeled cDNA (for Northerns) or digoxigenin labeled cRNA (for ISH) probes were used. In normal rats, the order of expression amounts of all isoforms mRNAs from highest was outer medulla > cortex > inner medulla, and that of K-deprived rats was outer medulla > inner medulla > cortex. alpha1 mRNA levels were much greater than those of alpha2 or alpha3 in cortex, outer and inner medulla. mRNA levels for all isoforms were 2~3 folds greater in inner medulla of K-deprived rats compared to controls. In contrasts, the levels of all isoforms mRNAs in cortex and outer medulla were comparable between the two gruops. By ISH, mRNAs for all isoforms were observed in the S3 segment of proximal tubule, the cortical thick ascending limb (CTAL), medullary thick ascending limb (MTAL), distal convoluted tubule (DCT), connecting tuble (CNT), and the entire collecting duct. Both groups exhibited comparable cellular patterns of labeling, but the signal intensity of K-deprived rats was much greater in the proximal portion of the inner stripe of outer medullary collecting duct (OMCDi) and proximal portion of the inner medullary collecting duct (IMCD), and less in the MTAL compared to controls. The signal intensity of alpha1, alpha3, and beta1 isoforms was less in the CTAL, DCT, and CCD of K-deprived rats, but alpha2 isoform was slightly increased. These results suggest that chronic hypokalemia enhances expression of Na+-K+-ATPase subunit isoforms in the proximal portion of OMCDi and proximal IMCD, but not other nephron segments, and that these isoforms may participate in potassium conservation by these segments during potassium deprivation.

Expression of Several Cell Cycle Regulatory Genes in Cultured AT-1 Cardiomyocytes after Formation of Synchronously Beating Sheets

BACKGROUND AND OBJECTIVE: AT-1 cells have been derived from the left atrial tissue in which the ANF promoter targeted SV40 large T antigen expression. When cultured, clusters of spontaneously contracting cells were observed after 4-5 days and contiguous sheets of synchronously beating cardiomyocytes were formed after 10 days. In this study, expression of several cell cycle regulatory genes were monitored through Northern blot analyses in AT-1 cells during beating and after formation of beating sheets (BS). MATERIALS AND METHOD: AT-1 RNAs were obtained in 3 days after plating, during beating and after formation of BS, and used for Northern blot analyses. RESULTS: alpha-Cardiac myosin heavy chain expression was prominent in beating cells, as would be expected for this contractile protein isoform but ANF was decreased after beating. Gax was not expressed in cultured AT-1 cells but in AT-1 tumor and murine heart. p53 and p21 were decreased after beating which indicate transcription level of p53 and p21 correlated well in AT-1 cells. In contrast, pRB and p107 were increased after beating but p68 (2.4 kb) which arose by alternative splicing of p107 and lacks the pocket domain B was decreased in beating cells. pTCS2, murine tuberous sclerosis gene, represented similar levels during beating but a little was decreased after formation of BS. mRAD50, the murine homologue of yeast DNA recombinational repair gene RAD50, was increased in beating cells, a similar pattern to p107 and pRB. But the p50 arose by alternative splicing of mRAD50 and has 3' half of mRAD50 had unexpectedly appeared and maintained after beating. CONCLUSION: The expression of cell cycle regulatory genes after beating and formation of BS in AT-1 cells showed gene-specific pattern and the p50 which has homology to the mRAD50 may participate in differentiation of cardiomyocytes.