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.
Animals ; Digoxigenin ; DNA, Complementary ; Extremities ; Gene Expression ; Hypokalemia* ; In Situ Hybridization ; Kidney ; Nephrons ; Potassium ; Protein Isoforms* ; Rats ; RNA, Complementary ; RNA, Messenger

PURPOSE: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-E1 osteoblastic cell line, especially on the osteonectin and bone sialoprotein. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.5, 1, 4 and 8 Gy at a dose rate of 5.38 Gy/min using Cs-137 irradiator. After specimens were harvested, total RNA was extracted on the 3rd, 7th, 14th, 21st day after irradiation. The total RNA was reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR with a pair of primers. RESULTS: The irradiated cells showed a dose-dependent increase in osteonectin mRNA expression when compared with the unirradiated control group. The irradiated cells showed no difference in bone sialoprotein mRNA expression when compared with the unirradiated control group. In accordance with the duration of culture period after irradiation, the level of osteonectin mRNA expression showed no difference, but it increased a little at the 21st day in the 4 and 8 Gy exposure groups. In the case of bone sialoprotein, however, the level of mRNA expression increased significantly at the 3rd and 7th day after irradiation, but it showed no difference at the 14th and 21st day when compared with the control group. CONCLUSION: These results showed that each single dose of 0.5, 1, 4 and 8 Gy influenced the mRNA expression of osteonectin and bone sialoprotein at the calcification stage of osteoblastic cells, suggesting that single dose of irradiation affected the osteoblastic bone formation at the cell level.
Cell Line* ; DNA, Complementary ; Integrin-Binding Sialoprotein* ; Osteoblasts* ; Osteogenesis ; Osteonectin* ; Polymerase Chain Reaction ; RNA ; RNA, Messenger*

PURPOSE: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-E1 osteoblastic cell line, particularly on the expression of osteocalcin and osteopontin. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.5, 1, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. After the specimens were harvested, RNA was extracted on the 3rd, 7th, 14th, and 21st day after irradiation. The RNA strands were reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR. RESULTS: The irradiated cells demonstrated a dose-dependent increase in osteocalcin and a dose-dependent decrease in osteopontin mRNA expression compared with the non-irradiated control group. The amount of osteocalcin mRNA expression decreased significantly at the 3rd day after irradiation of 0.5, 1, 4, and 8 Gy, and also decreased significantly at the 3rd, 14th, and 21st day after irradiation in the 8 Gy exposed group compared with the control group. The degree of osteopontin mRNA expression increased significantly at the 7th day after irradiation of 0.5, 1, 4, and 8 Gy. CONCLUSION: These results showed that each single dose of 0.5, 1, 4, and 8 Gy influenced the mRNA expression of osteocalcin and osteopontin associated with the calcification stage of osteoblastic cells, suggesting that each single dose affected bone formation at the cell level.
Cell Line* ; Cesium ; DNA, Complementary ; Osteoblasts* ; Osteocalcin* ; Osteogenesis ; Osteopontin* ; Polymerase Chain Reaction ; RNA ; RNA, Messenger*

RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site.]]>
Beekeeping ; Bees ; Diagnosis ; DNA, Complementary ; Methods ; Polymerase Chain Reaction ; RNA ; RNA, Viral ; Sensitivity and Specificity

PURPOSE: To verify the expression of the egg activator oscillin in mouse testis and adult organs. MATERIALS AND METHODS: Genomic PCR using primers for oscillin was conducted to confirm that the PCR product was derived from cDNA. Total RNA isolated from developing, immature, and mature testis was subjected to RT-PCR and restriction analysis. In situ hybridization of adult testis was performed to localize the oscillin transcript using cRNA probe. RESULTS: Genomic PCR using the primer for RT-PCR revealed no amplification product, suggesting that the oscillin gene consists of at least two exons. The RT-PCR product of oscillin mRAN was detected in testis beginning 2 weeks after birth. Oscillin mRAN was detected in both germ and somatic cells such as Sertoli and Leydig cells by in situ hybridization. The testis showed al high level of oscillin mRAN compared with other adult organs. CONCLUSION: Oscillin is not a testis-specific transcript and therefore may have another function intracellularly as well ad serving as a trigger for egg activation.
Adult ; Animals ; DNA, Complementary ; Exons ; Humans ; In Situ Hybridization ; Leydig Cells ; Male ; Mice* ; Ovum ; Parturition ; Polymerase Chain Reaction ; RNA ; RNA, Complementary ; Testis*

BACKGROUND: Since heightened microglial activation was shown to play a role in the pathogenesis of many brain disorders, understanding the molecular mechanisms of microglial activation may lead to new treatment strategies. The microarray system permitted screening of large numbers of genes in biological or pathological processes. Therefore, we evaluated the gene expression pattern during microglial activation using microarray analysis. METHODS: Primary microglial cultures were prepared from postnatal Swiss Webster mice. The cells were activated by lipopolysaccharide (LPS, 10 microgram/ml) for 5 hours prior to cell harvesting. From the cultured cells, we isolated mRNA, synthesized cDNA, converted to biotinylated cRNA and then reacted with GeneChips (Affymetrix MU74A-v2). The data were normalized and analyzed. RESULTS: After microglial activation with LPS, we found >4 fold increases in the expression of 139 genes and >4 fold decreases of 16 genes expression compared with control. Most of the induced or suppressed genes were known to regulate inflammation, immune reactions, injury responses, cell death or survival related mechanisms. CONCLUSIONS: These results suggest that microarray analysis of gene expression may be useful for screening novel molecular mediators of microglial activation and making profound understanding of the cellular mechanisms as a whole. Such screening techniques should provide insights into the molecular basis of brain disorders and help to identify potential targets for therapy.
Animals ; Brain Diseases ; Cell Death ; Cells, Cultured ; DNA, Complementary ; Gene Expression* ; Inflammation ; Mass Screening ; Mice ; Microarray Analysis ; Microglia* ; Pathologic Processes ; RNA, Complementary ; RNA, Messenger

PURPOSE: We aimed to elucidate the expression and intracellular localization of sperm-specific cation channel CatSper in human spermatozoa. Moreover, the relationship between the expression of CatSper mRNA and the motility of ejaculated human spermatozoa were investigated. MATERIALS AND METHODS: Using cDNAs extracted from the ejaculated sperm of patients (n=39), the expression of CatSper mRNA was observed by RT-PCR. Semi-quantitative analysis of the CatSper mRNA expression was performed by comparing with the expression of GAPDH mRNA. To elucidate the expression and intracellular localization of CatSper protein, double fluorescent immunocytochemistry for CatSper and beta-tubulin was performed. RESULTS: The CatSper mRNA was expressed in all of the sperm samples. Using semi-quantitative analysis for the amount of CatSper mRNA expression, no significant difference was found between the normozoospermia and asthenozoospermia groups (1.5+/-0.6 vs. 1.4+/-0.6, p=0.623). Polyclonal antiserum, generated against a recombinant protein of the N-terminal 160 amino acids of human CatSper, was used. In double fluorescent immunocytochemistry, CatSper protein was found to be expressed in the flagellum of the ejaculated human spermatozoa, and localized in the connecting piece, mid-piece and principal piece, with the exception of the end piece of the flagellum. Moreover, the proportion of CatSper-positive sperm was similar in both the normozoospermia and asthenozoospermia groups. CONCLUSIONS: To the best of our knowledge, this is the first time ejaculated human spermatozoa have been shown to express the mRNA and protein of CatSper. The results of our RT-PCR and immunocytochemistry suggest that CatSper may play a role in the motility of ejaculated human spermatozoa.
Amino Acids ; Asthenozoospermia ; DNA, Complementary ; Flagella ; Humans* ; Immunohistochemistry ; RNA, Messenger ; Sperm Motility ; Spermatozoa* ; Tubulin

PURPOSE: A cDNA microarray is a systematic method to identify key molecules for prognosis and for treatment response by profiling thousands of genes expressed in a single cancer. The clinical value of cDNA microarray is still being investigated in various fields. This technique could be used in detecting molecules important for cancer to develop, to monitor the effect of new cancer therapeutics, and to give a prognosis for cancer patients. We now report the results of our initial cDNA microarray data to analyze the genome pattern of colorectal cancer tissues and to evaluate the possibility of using cDNA microarrays in a clinical setting for cancer patients. METHODS: We used the general cDNA microarray technique with a 2.4 K cDNA chip provided by Macrogene company. RNA extracted from seven colorectal cancer tissues was amplified by using RT-PCR (reverse transcriptase-polymerase chain reaction), and applied to a cDNA chip to produce an antigen-antibody reaction. The results were analyzed individually and hierarchically. RESULTS: All seven tested cancer tissues were harvested from operative specimens at the Korea Cancer Center Hospital. The male-to-female ratio was 4 to 3. Five patients were TNM stage II, and two patients were stage III. Eighteen genes were upregulated in stage II patients, and 51 in stage III patients. The number of genes discriminating stage was 69, including 8 control genes, 4 ribosomal genes, 5 EST genes, 10 known non-functional genes, 23 genesof unknown function, and 19 possible cancer-related genes. A hierarchial graph showed similar patterns within a stage, which suggests that genetic patterns might affect clinical characteristics. CONCLUSIONS: Seven colorectal cancer tissues were analyzed with the cDNA microarray technique using 2.4 K cDNA chip. Authors could identify 69 genes that showed the significant change of expression. Although our reports presented the preliminary results, we think that the cDNA microarray will be able to offer an informative results to predict cancer development and progression in colorectal cancer.
Antigen-Antibody Reactions ; Colorectal Neoplasms* ; DNA, Complementary* ; Genome ; Humans ; Korea ; Oligonucleotide Array Sequence Analysis* ; Prognosis ; RNA

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease characterized by neurological, cutaneous, and ophthalmological manifestations. A 33-year-old woman with typical symptoms of NF1 visited Ajou University Hospital. Screening of the whole-messenger RNA region of NF1 at the complementary DNA level by polymerase chain reaction-direct sequencing confirmed the presence of an NF1 mutation at the genomic level. The mutation analysis revealed an in-frame skipping of exon 46 (c.6757_6858del) caused by a point mutation (c. 6792C>A) in exon 46. In this report, we have described the first Korean case of a proband with NF1 that carries an allele with an exon 46 deletion caused by an exonic splicing enhancer site mutation, leading to the skipping of the whole of exon 46 (c.6757_6858del).
Adult ; Alleles ; DNA, Complementary ; Exons* ; Female ; Humans ; Mass Screening ; Neurofibromatosis 1* ; Point Mutation ; RNA

PURPOSE: To evaluate the pathogenesis of keratoconus through differentially expressed genes in human keratocyte of keratoconus. METHODS: Total RNAs extracted from primary cultured keratocytes in normal cornea and keratoconus were used for the synthesis of cDNA. Differentially expressed genes were screened by ACP-based PCR method using GeneFishingTM DEG kits. The differentially expressed bands were sequenced and analyzed, and identified genes were further evaluated by RT-PCR. RESULTS: Over-expressions of BMP4 and CFL1 and under-expression of CFL1, GRCC10, and TIMP3 were verified, and comfirmed by RT-PCR. All confirmed genes were concerned with cytoskeleton, wound healing, and apoptosis. CONCLUSIONS: The identified differentially expressed genes seem to be important role in the mechanism of keratoconus, and apoptosis as well as cytoskeleton and wound healing may be attributed to the underlying stromal keratocyte.
Apoptosis ; Cornea ; Cytoskeleton ; DNA, Complementary ; Humans ; Keratoconus* ; Polymerase Chain Reaction ; RNA ; Wound Healing