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

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: 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*

SUMMARY: The present study was performed to analyze the expression of LH genes in the rat ovary. Expression of LH subunit genes in the rat ovary was demonstrated by amplification of ovarian RNA by RT-PCR. The ovarian LHbeta transcripts contained at least two parts of the published cDNA structure, the pituitary exons 1, 2 and 3 and the part of testicular exon 1 in the major trancripts form in rat testis. Using RIA, significant amount of LH-like molecules were detected in crude ovarian extracts, and the competition curves with increasing amount of tissue extracts were parallel with those of standard peptide, indicating that the ovarian immunoreactive LH-like material is similar to authentic pituitary LH molecule. The administration of PMSG to immature rats resulted in a sharp decrease of the ovarian LH contents after 24 h post-injection. In conclusion, these findings demonstrate that genes for LH subunits are expressed in the rat ovary, and suggest that LH can play a central role in regulation of female reproduction with both endocrine (by pituitary LH) and auto- and/or para-crime (by ovarian LH) manner.
Animals ; DNA, Complementary ; Exons ; Female ; Humans ; Lutein* ; Luteinizing Hormone* ; Ovary* ; Rats* ; Reproduction ; RNA ; Testis ; Tissue Extracts

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.
Animals ; DNA, Complementary ; chemistry ; isolation & purification ; Gene Library ; RNA ; isolation & purification ; Sea Anemones ; genetics

BACKGROUND: Although relative quantification by real-time PCR may be easier to perform than absolute quantification, there is a risk of errors associated with standard curve construction. The aim of this study was to evaluate the effects of standard curves on relative quantification using real-time PCR in Candida albicans. METHODS: The reproducibility of real-time PCR-based standard curves for target genes and a reference gene generated from PCR amplicons (10-fold serial dilution, 10(-4) to 10(-9)) was evaluated. In addition, the effects on standard curves were evaluated by running the same cDNA samples. RESULTS: The within-run variation (CV) by crossing point (Cp) was 0.12-1.05% for ERG11 and ACT1, whereas the between-run CV was 2.07-6.84% for ERG11, CDR1, MDR1 (target gene) and ACT1 (ref-erencegene). The differences in PCR efficiency between targets and reference may be attributable to variations in relative quantification. CONCLUSIONS: To achieve reliable relative quantification of mRNA in real-time PCR, a feasible guide-line and standardization are of major importance.
Candida albicans ; DNA, Complementary ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction* ; RNA, Messenger ; Running

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