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.

Expression and localization of Kuzbanian-DN mRNA in trans-gene NOD mouse pancreas cells / 中国糖尿病杂志

Objective Kuzbanian (KUZ) is the functional homologue of the vertebrate ADAM10. ADAMs are a protein family of cell surface-anchored membrane, whose members have extensive biological activities. This paper was to explore KUZ-DN (the dominant negative construct of KUZ) mRNA expression and localization in the trans-gene NOD mouse pancreas tissue and to provide molecular basis for the mechanism of the gene intervened stem cell differentiation to treat diabetes. Methods Pancreas was frozen and paraffin sections of pancreas in eight trans-gene NOD mice were made. Non-radioactivity in situ hybridization experiment was conducted on the tissue sections by the cRNA probes of KUZ-DN (360 bp) sense and antisense with DIG labeled. Results There was the strong positive KUZ-DN mRNA signal report of in situ hybridization in the trans-gene NOD mouse pancreas cells. Sense chain probes and blank comparison sections were negative. Conclusion The positive expression and localization of KUZ-DN mRNA in the trans-gene NOD mouse pancreas cells provides a vital data for the gene therapy research project of the KUZ-DN intervened Notch/Delta signaling pathway by the stem cell differentiation to treat diabetes

Changes in Osteopontin Expression in the Rat Lumbar Spinal Cord Following the Avulsion of Lumbar Nerve Roots / 대한해부학회지

This study investigated the expression of osteopontin (OPN) in rat lumbar spinal cords after lumbar nerve root avulsion, using in situ hybridization histochemistry, immunocytochemistry and western blot analysis. Cells expressing OPN were motoneurons and interneurons in the ventral horn, but no signals were observed in neurons in the dorsal horn of the normal lumbar spinal cord. From day 1 after avulsion injury, OPN mRNA-labeled neurons increased in the ventral horn and the intermediate zone. By day 3, relatively strong OPN mRNA signals were found throughout the gray matter of the injured side of the spinal cord with OPN mRNA-labeled cells scattered in the superficial dorsal horn. By day 7, the labeling patterns for OPN mRNA were similar to those on day 3, but the numbers of OPN mRNA-labeled cells in the ventral horn and the intermediate zone peaked. At this point, these labeled cells were also more densely packed and the intensity of signals was stronger. Interestingly, these labeled cells were neurons, but not glial cells such as astrocytes or microglia. This OPN mRNA-labeled cell profile in the dorsal horn had nearly disappeared by day 14 after avulsion injury, and the labeling pattern became similar to that on day 1. By day 28, after avulsion injury, the numbers of OPN mRNA-labeled cells decreased further below control values. These results suggest that increased expression of OPN in the rat lumbar spinal cord after avulsion injury might play an important role in the pathogenesis of damaged neurons.

Localization of neural specific transcription factor LMO3 mRNA in adult mouse central nervous system / 第三军医大学学报

Objective To explore the localization of neural specific transcription factor LMO3 mRNA in the central nervous system of the mice.Methods Five male adult mice,weighing 60-80 g,were sacrificed,and the brain and spinal cord were fixed and sliced.In situ hybridization histochemistry staining method with digoxigen-lablled cRNA probe was used in detection of LMO3 mRNA.Results The expression of LMO3 mRNA could be observed in cerebrum,cerebellum,thalamus,brain stem and spine.They were localized in somatic and dendritic profiles at most regions of adult mouse central nervous system.The intense hybridization signal could be seen in Ⅱ-Ⅵ layers of cerebrum,piriform cortex,entorhinal cortex,hippocampus CA1 and dentate gyrus,substantia nigra,motor nucleus of trigeminus,nucleus of hypoglossal,red nucleus,paraventricular nucleus,gigantocellular reticular nucleus,lateral reticular nucleus.The moderate staining was detected in olfactory bulb,hippocampal CA2,amygdala nucleus,cingulated anterior cortex,piriform nucleus etc..The faint signal showed in globus pallidus,spinal nucleus of trigeminal,interposed cerebellar nucleus,ambiguous nucleus,lateral cerebellar nucleus,Purkinje cell of cerebellum,thalamus,caudate putamen,amygdaloid body,nuclei raphe.Conclusion The wide presence of LMO3 mRNA in adult mice central nervous system suggests a role of regulation for this gene in learning,memory and olfaction besides in dopamine neurotransmitter regulation.

DISTRIBUTION AND MORPHOLOGY OF NESTIN-POSITIVE CELLS IN HUMAN FETAL LIVER / 解剖学报

Objective To observe the morphological and distribution feature of nestin-positive cells in liver during various stages of human fetal development.Methods 10-20 weeks old fetal liver tissues were obtained and fixed for paraffin sections as usual.Digoxigenin-labeled oligo-DNA probe was used for detect nestin by means of in situ hybridization histochemistry(ISHH).Results Nestin-positive cells are small,spherical cells with a large nucleus and sometimes a dual nuclei,and distribute mainly in limiting plate and surround the developing bile duct.Conclusion There are some nestin-positive cells in the liver.They can divide and proliferate and may be a group of cells hided in various tissues during development,which have the same function.They may play an important role in hepatocyte injure repair and tumor occur during the later days.

Localization of phospholipase D genes on the human chromosome and various tissues of rat / 대한해부학회지

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. A variety of signal molecules such as hormones, neurotransmitters, extracellular matrix molecules, and growth factors are known to induce the activation of PLD in a wide range of cell types. Hence PLD is implicated in a broad spectrum of physio-logical processes and diseases, including mitogenesis, cell differentiation, metabolic regulation, secretion, neural and cardiac stimulation, inflammation, oncogenesis, and diabetes. The signal-dependent activation of PLD has been observed in a variety of brain and neural-derived cells. In this paper, human chromosomal locations and developmental neural expression patterns in rat of PLD1 and PLD2 were investigated with fluorescent in situ hybridization (FISH) and in situ hybridization histochemistry, respectively. The PLD1 was assigned to human chromosome 3q26 and expressed most strikingly in selected ventricular neural cells lining spinal cord and brain during neuronal differentiation and migration period. The PLD2 was assigned to human chromosome 17p13.1 and expressed in differentiating ventricular neural cells and multiple regions of the postnatal rat brain.

Cloning and Localization of Developmentally Regulated Gene, Glutamate/Aspartate Transporter (GLAST) Gene, in the Developing Rat Cerebellum / 대한해부학회지

In this study, S59158, a gene of GLAST (L-Glutamate/L-Aspartate transporter), was cloned by ordered differential display PCR with developing rat brains. The mRNA expression of this gene in the developing rat brain and the effect of kainic acid (KA), glutamate analogue, on this gene were investigated with in situ hybridization histochemistry. The expression of S59158 was restricted to nervous system and observed from E12 (embryonic day 12), peaked at E20, and gradually decreased to adult level. In embryos, S59158 was prominently expressed in the subventricular zones throughout the brain. After birth, strong expression was observed in the purkinje cell layer of cerebellum and moderate level of expression was observed in the subventricular zone, olfactory bulb, hippocampal formation, and cerebral cortex. In the KA treated rat brains, the expression of S59158 was significantly increased in dentate gyrus, hippocampus, and cerebral cortex. From these results, it may be suspected that S59158 is related to the development of the brain and is induced by increased extracellular glutamate level.

Expression of Heat Shock Protein mRNA Induced by Ischemia-Reperfusion in Skeletal Muscles

The heat shock response is an important homeostatic mechanism that enables cells of animals, plants, and bacteria to survive a variety of environmental stresses. In all organisms a small set of proteins(heat shock proteins, hsps) is preferentially synthesized in response to stress. By seeking the expression of mRNA of hsps to tissue ischemia-reperfusion injury, a guideline can be provided in its application. A total of 100 Sprague-Dawley rats weighing about 200 to 300 gm were used. The rats were divided into three groups; group1: sham operation group, group 2: ischemia induced only, and group 3: ischemia-reperfusion group. The ischemia group(group 2) was subdivided according to the duration of ischemia. 1, 2, 4, 8, and 12 hours each. Ischemia-reperfusion group(group 3) was subdivided according to the duration of ischemic insult lasting 1, 2, and 4 hours each(group 3-1, 2, 3). Samples were taken from both subgroups 15 min, 30 min, 1, 2, 4, 8 hours after reperfusion. Creatine phosphokinase levels were measured and mRNA expression was observed using in situ hydridization histochemistry. The elevation of creatine phosphokinase was correlated to the duration of ischemic insult and to the duration of reperfusion time. This postulated the fact that the amount of muscle injury had direct connection with time of ischemia and the injury continued even after reperfusion. The overall mRNA of hsp in the ischemia-reperfusion group showed faster and stronger expression compared to that of the ischemia-only group. Among the ischemia-reperfusion subgroups, as the ischemic time was increased, the mRNA expression demonstrated faster but with decreased amount. This findings suggest that there can be a limit in expression of hsp based on the extent of ischemic insult. This can be helpful in clinical applications. When the gastrocnemius and soleus muscle are compared, white muscle group(gastrocnemius) revealed faster and stronger expression of mRNA of hsps. This is most likely due to the fact that red muscle group has abundant blood supply and mitochondria, and implies decreased injury after ischemia-reperfusion. Although the precisive mechanism of hsp is not yet known, heat shock induced protection increases flap survival dramatically. But by demonstrating the pattern of expression based on ischemia time and reperfusion, this paper suggests a possible limit of hsp against ischemia- reperfusion injury.

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.

The effect of convulsive treatments on the expression of heat shock protein 72 mRNA in the rat brain / 대한해부학회지

Recently, there are numerous efforts to explain the psycho-, neurological events through molecular biological standards. Because of the property as a strong stimulant to neural cells, convulsions induced by electroconvulsive shock (ECS) or kainic acid are used for neurobiological research. In this study, the effect of systemic administration of kainic acid and ECS on the expression of hsp 72 mRNA in the rat brain was investigated with in situ hybridization histochemistry. The induction of hsp 72 mRNA was observed in the dentate gyrus from 2 hr after KA treatment. After that, the expression was gradually increased in the various areas including dentate gyrus, hippocampus, olfactory bulb, cerebral cortex, caudate-putamen, thalamus, and peaked at 9 hr after KA treatment. At the 72 hr after KA treatment, weak expression was found only in the CA3 area of hippocampus. However, the expression of hsp 72 mRNA was not detected in any ESC treated rat brains, we examined.The inducton of c-fos was observed from 15 min, peaked at 6 hr, and returned to basal level at 48 hr after KA treatment. The expression of c-fos was observed in the same areas that showed induction of hsp 72 mRNA. In the ECS treated rat brains, the induction of c-fos was found in the dentate gyrus, olfactory bulb and cerebral cortex at 15 min and 30 min after ESC. From these results, it may be suggested that the effects of KA treatment and ECS on the neuronal cells are different, and it is due to difference in induction mechanism of convulsion between KA and ECS. And, the similarity between the expression pattern of hsp 72 mRNA by KA and KA receptor suggests that the induction of hsp 72 mRNA is based on the direct effect of KA through KA receptor.

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.

Cloning and localization of novel stage-specific gene (PKrCb1) of the postnatal rat cerebellum / 대한해부학회지

In rat that is helpless at birth, the cerebellum is in a corresponding state of immaturity, and its histogenesis and morphogenesis mainly occur after birth. The times and sites of origin of the four types of cerebellar local-circuit neurons, as well as their migration routes to specific positions in the cortex, their distinctive patterns of differentiation and growth, and their synaptogenesis, have been well studied. The stage-specific genes in the postnatal rat cerebellum may be related with these kind of neural development in the cerebellum. To clone the genes related with neural development in the postnatal cerebellum, developmentally differentially expressed genes were screened from postnatal rat cerebellum with ordered differential display (ODD) and the developmental expression pattern in the postnatal rat cerebella was investigated with in situ hybridization histochemistry. One novel postnatal stage-specific gene (PKrCb1) was cloned by ODD with 7 cDNA pools (P0, P3, P7, P12, P18, P25, adult rat cerebella). To investigate the developmental expression pattern of this novel gene on the cell level, in situ hybridization histochemistry was performed in the developing and adult rat brain sections. The developmental expression pattern of PKrCb1 in the cerebellum was well matched with spatiotemporal migration pattern of granule cells and it may be suspected that PKrCb1 is related with migration of granule cells from external granular layer to internal granular layer. From the results, it is suggested that the methods used in this experiment will be the powerful methods for the cloning and primary function study of the genes related with cerebellar development.

Cloning and characterization of developmentally differentially expressed novel genes from developing rat cerebral cortex / 대한해부학회지

The structural complexity and heterogeneity of cerebral cortex have been obvious since the earliest days of light microscopy. In fact, if there is one word that captures many of the key attributes of cortical structures, it is diversity. Neurodevelopmental approach is the one of the effective ways to understand complicated structures of cerebral cortex. In this experiment, as a first step to clone the genes related with development of cerebral cortex, the developmentally differentially expressed genes were cloned from developing rat brain with ordered differential display PCR(ODD-PCR). Novel genes were screened from these cloned genes by sequencing and sequence analysis with blast search program. The developmental expression patterns of novel genes in the cerebral cortex were investigated with in situ hybridization histochemistry on the developing and adult rat brain sections. Among the forty one developmentally differentially expressed cDNA bands, amplified with InEGA primer and TEAC primer by ODD PCR, twenty novel genes were screened by sequencing and sequence analysis with blast search program. Through the investigation of developmental expression pattern with in situ hybridization histochemistry, specific expression of five novel genes in the developing rat cerebral cortex was identified. 20-E14-2 was highly expressed in the cerebral cortex during the period of neurogenesis. The expression of 20-E20-1, 20-E20-6b, and 20-P0-5 was relatively well matched with neuronal cell migration in the cerebral cortex. And the strong expression of 20-P0-8b was observed in the neuronal cells of cerebral cortex during the period of syanptogenesis. From these results, it may be suspected that the five novel genes play roles in the development of cerebral cortex.

The expression of Protein Kinase C Isozymes in the Rat Brain / 대한해부학회지

The mRNA expression of protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon and zeta) in the rat nervous system was investigated with in situ hybridization histochemistry. In the central nervous system of rat, each PKC isozyme mRNAs was expressed in isozyme-specific pattern. PKC alpha mRNA was highly expressed in the olfactory bulb, piriform cortex, hippocampus, substantia nigra compacta, and inferior olive. The expression of PKC beta was highest in the olfactory tubercle, piriform cortex, caudate putamen, accumbens nucleus, neocortex, hippocampus, basolateral amygdaloid nucleus, pontine nucleus, and cerebellum. PKC gamma mRNA was distributed in the caudate putamen, hippocampus and cerebellum and PKC delta was expressed in the thalamus. PKC epsilon had widespread distribution, with relatively high levels in the anterior olfactory nucleus, olfactory tubercle, tinea tecta, piriform cortex, dorsal lateral septal nucleus, neocortex, hippocampus and cerebellum. PKC zeta had widespread and low expression. The spacially differential expression of PKC isozymes (alpha, beta, gamma, delta, epsilon and zeta) suggests that each PKC isozyme may be related with specific cellular function in the nervous system.

The expression of new rat Ulip mRNA in the developing rat brains / 대한해부학회지

One cDNA was cloned out of developmentally differentially expressed genes in developing rat brains with ordered differential display method (ODD). The expression of cloned cDNA was observed from embryonal day 12 (E12), peaked at postnatal day 7 (P7), decreased to undetectable level at adult. By sequencing and sequence search with GenBank data, it was revealed that cloned cDNA was very similar to mouse Unc-33-like phosphoprotein (Ulip), which is known to be involved in the axonal outgrowth, thus, named as new rat Ulip (nrUlip). In situ hybridization histochemistry of developing rat brains showed that nrUlip mRNA was highly expressed in the area for differentiating neurons and the expression was observed just after neurogenesis in the various brain areas including thalamus, septal area, cerebral cortex, caudate-putamen, hippocampus, cerebellum, and dentate gyrus in the order of neurogenesis. These developmental expression pattern was well matched with the result of ODD. This may justify ODD as one of the best way to clone the genes differen-tially expressed among samples. These results and high sequence homology of nrUlip to mouse Ulip related with axonal outgrowth suggest that nrUlip may be also involved in the outgrowth of axon.

The effect of kainic acid induced convulsions on the expression of inositol 1,4,5-trisphosphate receptor mRNA in the rat brain / 대한해부학회지

In this study, the effect of systemic administration of kainic acid (KA) on the expression of inositol 1,4,5-trisphosphate receptor mRNA in the rat brain was investigated with in situ hybridization histochemistry. After the injection of KA in a convulsive dose (10 mg/kg i.p.), inositol 1,4,5-trisphosphate receptor mRNA was reduced significantly in dentate gyrus, cerebral cortex, and caudate-putamen and moderately in CA1-CA3 areas of hippocampus and cerebellum. In dentate gyrus, the expression of inositol 1,4,5-trisphosphate receptor mRNA was significantly decreased at 6 h, lowest level at 9 h, after that the expression was gradually recovered and returned to basal level at 72 h after KA injection. However, in the CA1-CA3 areas of the hippocampus, cerebral cortex, and caudate-putamen, the expression of inositol 1,4,5-trisphosphate receptor mRNA was abruptly decreased at 9 h and almost return to basal level at 24 h after KA injection. The significant repression of inositol 1,4,5-trisphosphate receptor mRNA in cerebellum was only found at 9 h after KA injection. But significant change of inositol 1,4,5-trisphosphate receptor mRNA was not found in the brains of rats treated with NMDA receptor blocker, MK-801, followed by KA injection. These observations suggest that the inositol 1,4,5-trisphosphate receptor is one of the genes whose expression can be altered by KA treatment and the NMDA receptor is related with this alternation.

The developmental expression of voltage dependent calcium channel alpha1 subunits (alpha1D, alpha1B, alpha1A, alpha1E) mRNA in the rat brain / 대한해부학회지

Voltage dependent calcium channels (VDCCs) mediate Ca++ influx into cells and are responsible for regulation of a variety of physiological effects. The key functional property of VDCCs are attributed to the calcium-pore forming alpha1 subunit. In this study, distribution pattern of alpha1 subunit (alpha1D, alpha1B, alpha1A, alpha1E) mRNA of VDCCs in developing and adult rat brain was investigated by in situ hybridization histochemistry. In the adult rat brain, each alpha1 subunit mRNA displayed a specific and distinct distribution pattern. alpha1D was highly expressed in the olfactory bulb, dentate gyrus, pituitary gland, pineal gland, hypothalamus, superior colliculus and cerebellum. Relatively low level of alpha1B was expressed throughout the whole brain and strong expression of alpha1A was observed in CA3 area of Ammon's horn, medial geniculate body, inferior colliculus and cerebellum. High level of alpha1E was found in the olfactory bulb, hippocampus, dentate gyrus, medial habenular nucleus and cerebellum. Moreover, alpha1B, alpha1A and alpha1E were expressed only in the nervous system but alpha1D was expressed not only in the nervous system but also in other tissues including liver, heart, lung and skeletal muscle. Generally the expression of alpha1D, alpha1A, and alpha1E subunit was observed from E14 and thereafter the intensity of labeling was gradually increased to P14 and then decreased to the adult level. But the expression of alpha1B subunit was observed from E14 and gradually increased to E20 and P0 and then decresaed. From the differential expressions of VDCC alpha1 subunits in developing and adult rat brain, it is suggested that each type of VDCCs may play a distinct roles in neural and nonneural tissues, and the VDCCs may be related with development of nervous system.

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.

Chromosomal localization and neural distribution of voltage dependent calcium channel alpha1A and alpha1E subunit genes / 대한해부학회지

Fluorescent in situ hybridization using human genomic DNA probes was performed to localize genes encoding the alpha1A and alpha1E of voltage dependent calcium channels (VDCCs) in the human chromosome and the mRNA expression of these two alpha1 subunits of VDCC was demonstrated in the 18 day old embryo (E18) and adult rat brain by in situ hybridization histochemistry. The genes for the VDCC alpha1A and alpha1E were specifically localized on human chromosome 19p13.1 and 1q25, respectively. In 18 days old rat embryos, the mRNAs of the VDCC alpha1A and alpha1E were predominently expressed in the nervous system including brain and spinal cord. In adult rat brain, the expression pattern of each subunit was extremely different. The expression of alpha1A mRNA was strong in the purkinje cells of cerebellum and CA3 area of hippocampus, relatively high level of expression was found in the dentate gyrus, CA1 area of hippocampus, superficial layer of cerebral cortex and olfactory mitral cells. Whereas alpha1E was highly expressed in the dentate gyrus, CA1-3 area of hippocampus, medial habenula nucleus of thalamus and olfactory mitral and internal granule cells and relatively high level of expression was found in the Purkinje cells of cerebellum, cerebral cortex and caudate-putamen. Until now, no neurological disorder has been mapped to 1q25, location of VDCC alpha1E gene. Recently, it has been reported that mutation of VDCC alpha1A gene causes episodic ataxia type 2 (EA-2) and spinocerebellar ataxia 6 (SCA6). These reports comfirm the our experimental results of chromosomal mapping and prominent cerebellar expression of VDCC a1A gene.

Cloning and localization of two forms of voltage-dependent calcium channel beta3 subunit gene / 대한해부학회지

Voltage-dependent calcium channel (VDCC) is composed of at least four subunits: alpha1, alpha2, beta, delta. Four mammalian beta subunit isoforms (beta1, beta2, beta3 and beta4) have been identified from nervous system. beta subunit accelerates the kinetics of activation (channel openning) and inactivation (channel closure), and regulates the channel activity by phosphorylation through various signal transduction mechanisms. We have cloned three cDNAs (RB8, RB10, and RB11) encoding beta3 subunit of voltage-dependent calcium channel from rat cDNA library using the oligonucleotides of which sequences obtained from the highly conserved regions of rat b subunits. The RB8 and RB10 (rtB3a) encode a same protein of 484 amino acids with estimated Mr of 54,571 Da, which was identical to beta3 subunit gene previously reported. The RB11 (rtbBb) is diffferent from RB10 at N-terminal region but shares common amino acid sequences from the glycine, the 16th amino acid of RB10, to the end of the gene. Open reading frame of RB10 encodes a 483 amino-acid protein with a predicted Mr of 54,473 Da. The RB10 and RB11 are suspected to be alternatively spliced variants from a single b3 subunit gene. The existence of the variants was confirmed by RT-PCR using the oligonucleotide primers from the specific sequences of each variant. The expression patterns of VDCC beta3 (rtB3a) and its specific variant (rtB3b) were investigated in the rat brain by in situ hybridization histochemistry. The mRNAs for rtB3a and rtB3b were exclusively expressed in the nervous system. In the brain, strong expression of both mRNAs (rtB3a and rtB3b) was found in the medial habenular nucleus of thalamus, hippocampus, dentate gyrus, olfactory bulb and cerebellum. But significant discrepancy of expression was found in the lateral posterior thalamic nucleus and olfactory bulb. From these results, it is suspected that newly cloned VDCC variant (rtB3b) should be the alternatively spliced variant of VDCC beta3 gene.