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.

Apoptosis-Induced Gene Profiles of a Myeloma Cell P3-X63-Ag8.653

BACKGROUND: Apoptosis is a physiologic phenomenon involved in development, elimination of damaged cells, and maintenance of cell homeostasis. Deregulation of apoptosis may cause diseases, such as cancers, immune diseases, and neurodegenerative disorders. The mouse myeloma cell P3-X63-Ag8.653 (v653) is an HGPRT deficient (HGPRT) mutant strain. High dependency on de novo transcription and translation of aminopterin induced apoptosis of this cell seems to be an ideal experimental system for searching apoptosis-induced genes. METHODS & RESULTS: For searching apoptosis-related genes we carried out GE-array (dot blot), Affymetrix GeneChip analysis, Northern analysis and differential display-PCR techniques. The chip data were analyzed with three different programs. 66 genes were selected through Affymetrix GeneChip analyses. All genes selected were classified into 8 groups according to their known functions. They were Genes of 1) Cell growth/maintenance/death/ enzyme, 2) Cell cycle, 3) Chaperone, 4) Cancer/disease-related genes, 5) Mitochondria, 6) Membrane protein/signal transduction, 7) Nuclear protein/nucleic acid binding/ transcription binding and 8) Translation factor. Among these groups number of genes were the largest in the genes of cell growth/maintenance/death/enzyme. Expression signals of most of all groups were peaked at 3 hour of apoptosis except genes of Nuclear protein/nucleic acid binding/ transcription factor which showed maximum signal at 1 hour. CONCLUSION: This study showed induction of wide range of proapoptotic factors which accelerate cell death at various stage of cell death. In addition apoptosis studied in this research can be classified as a type 2 which involves cytochrome c and caspase 9 especially in early stages of death. But It also has progressed to type 1 in late stage of the death process.

Effect of Chronic Hypokalemia on Expression of Colonic H/K-ATPase a Subunit mRNA in Rat Uterus / 대한해부학회지

Potassium (K+) balance is achieved by the control of urinary K+ excretion and by the control of K+ absorption from the digestive tract. While it is well established that colonic H/K-ATPase a subunit mRNA is expressed in the kidney, distal colon, and uterus, little is known about the cellular localization and expression levels of this gene in chronic hypokalemia. Accordingly, Northern analysis and in situ hybridization (ISH) were carried out to analyze the expression of mRNA encoding the colonic H/K-ATPase a subunit in normal and potassium-restricted (2 weeks) rats. Northern analysis demonstrated that colonic H/K-ATPase a subunit mRNA was abundantly expressed in normal and potassium-restricted rat uterus. Abundance of colonic H/K-ATPase a subunit mRNA in potassium-restricted rat uterus was increased but, not statistically significant compared to that of controls. By ISH, mRNA for colonic H/K-ATPase a subunit was detected in the endometrial epithelial cells and the uterine glands. Both groups exhibited comparable cellular patterns of labeling, but signal intensity of potassium- restricted rats was higher than normal rats. The endometrial epithelial cells exhibited a mixture of hybridization signal intensity. Most cells had intense hybridization signal for colonic H/K-ATPase a subunit mRNA, some cells had moderate, and a few cells had weak. In summary, colonic H/K-ATPase a subunit mRNA is expressed in the endometrial epithelial cells and the uterine glands. These results suggest that two (or more) H/K-ATPase a subunit isoforms are present in rat uterus and this gene contributes to potassium reclamation during chronic hypokalemia to regulate the pH and/or electrolyte concentration of uterine fluid.

Effects of Chronic Hypokalemia on Rat Distal Colon / 대한해부학회지

Recent molecular and physiological studies suggested that at least two distinct H/K-ATPase activities are present in the mammalian colon. Potassium (K+) balance is achieved by the control of urinary K+ excretion and by the control of K+ absorption from the digestive tract. The colon also participates substantively in the regulation of systemic K+ homeostasis. Northern analysis and in situ hybridization (ISH) for analyzing the expression of mRNA encoding the colonic H/K-ATPase a subunit and EM study for morphologic adaptations were carried out in normal and potassium-deprived (2 weeks) rats. Northern analysis demonstrated that colonic H/K-ATPase a subunit mRNA is abundantly expressed in normal rat distal colon. Abundance of colonic H/K-ATPase a subunit mRNA in potassium-deprived rat distal colon was not significantly increased compared to controls. By ISH, mRNA for colonic H/K-ATPase a subunit was detected in the surface epithelial cells, Goblet cells, and upper third of the intestinal gland. Both groups exhibited comparable cellular patterns of labeling and signal intensity. The surface epithelial cells exhibited a mixture of hybridization signal intensity. Most cells had intense hybridization signal for colonic H/K-ATPase a subunit mRNA and some cells had moderate, and a few cells had weak. Occasionally, strong hybridization signal was detected in the lower portion of the intestinal gland. EM study demonstrated that two types of surface columnar epithelial cells were present in normal distal colon and included type 1 cells with more abundant vesicles in supranuclear cytoplasm and type 2 cells with moderate amount of vesicles. In potassium-deprived distal colon, type 2 cells were only present in surface columnar epithelial cells. Others were not significant differences between two groups. These results suggest that two (or more) H/K-ATPase a subunit isoforms are present in rat distal colon, and colonic H/K-ATPase asubunit gene does not significantly contribute to potassium conservation during chronic hypokalemia in spite of abundant expression of this gene.

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.

Mitogenic effects of nicotine to human periodontal ligament(PDL) cells in vitro / 대한치과교정학회지

Nicotine is one of the major components of cigafette smoking which causes various systemic and local diseases to human body. Mitrogenic effects of nicotine to systemic disease are interesting factors in the results of cellular proliferation especially to vascular and pulminary tissus or cells. The study of local effects concerns with destruction of tissue and delayed healing rate after various surgicla treatment. Platelet-Derived Growth factor(PDGF) and Insulin-like growth factor(IGF) are known as major mitogens to human PDL cells. The purpose of this studywas to investgate the mitogenic effects of nicotine to human PDL cells. We studied the expression of PDGF-alpha receptor, PDGF-betareceptor, and IGF-1 receptor mRNA form the nicotine treated human PDL cells by northern analysis. The experimental groups were divided into different serum(1%, 10%) and nicotine(100ng/ml, 1000ng/ml) condentrations and each group was studied by time course. The results of this study showed upregulation of PDGF-alpha, beta receptor and IGF-1 receptor mRNA at 100ng/ml nicotine concentration and 10% serum group to the time course. These results suggest that physiologically attainable nicotine concentrations may stimulate mitogenic gene synthesis to human PDL cells in vitro.

Expression of Jun and p53 Genes from the Brain of Rats Irradiated with 60Co gamma-ray / 대한치료방사선과학회지

Damage produced by radiation elicits a complex response in mammalian cells. Including growth rate changes and the induction of a variety of genes associated with growth control and apoptosis.At doses of 10,000 cGy or greater, the exposed indivisual was killed in a matter of minutes to a couple of days. With symptoms consistent with pathology of the central nervous system(CNS) including degenerative changes. The nature of the damege in irradiated cells underlies the unique hazads of ionizing radiation. Radiation injury CNS is a rare event in clinical medicine, but it is catastrophic for the patient in whom it occurs. The incidience of cerebral necrosis has been reportes as high as 16% for doses greater than 6,000 cGy.In this study, the effect of radiation on brain tissues was studied in vivo. Jun and p53 genes in the rat brain were induced by whole body irradiation of rat with 60Co in doses between 1 Gy and analyzed for expression analyses were done using 1.8 Kb & 0.8 Kb-pGEM-2-JUN/Eco RI/Pst I fragments, 2.0 Kb-php53B/Bam HI fragment and 1.1 Kb-pBluescript SK-ACTIN/Eco RI fragment as the digoxigenin or [alpha32P]dCTPlabeled probes for Jun, p53 and beta-actin genes, respectively.Jun gene seemed to be expressed near the threshold levels in 1 hour after irradiation of 60Co in dose less than 1 Gy and was expressed in maximum at 1 hour after irradiation of 60Co in dose of 30 Gy. Jun was expressed increasingly with time until 5 or 6 hours after irradiation of 60Co in dose of 1 Gy and 10 Gy . After irradition of 60Co in dose between 20 Gy and 100Gy, the expression of Jun was however increased to peak in 2 hours and decreased thereafter.P53 gene in this study also seemed to be expressed near the threshold levels in 1 hours after irradiation of 60Co in less than 1 Gy and was expressed in maximum and 6 hours after irradiation of 60Co in dose of 1 Gy. P53 was expressed increasingly with time until 5 or 6 hours after irradition of 60Co in dose between 1 Gy and 40 Gy. After irradition of 60Co in dose of 50 Gy and 100Gy. The expression of p53 was however increased in peak in 2 hours and decreased thereafter. The expression of Jun and p53 genes was not correlative in the brain tissue from rats.It seemed to be very important for the establishment of the optimum conditions for the animal studies relevant to the response of genes inducible on DNA damage to ionizing radiation in mammalian cells. But there are many limitations to the animal studies such as the ununiform patterns of gene expression from the tissue because of its complex compositions. It is necessary to overcome the limitations for development of in situ Northern analysis.