Acute promyelocytic leukemia (APL) is characterized by a specific t (15;17) translocation which produce a PML/RAR-alpha fusion messenger RNA and by effectiveness of all-trans retinoic acid (ATRA) differentiation therapy. Breakpoints within PML intron 3 (bcr 3) produce a short PML/RAR-alpha isoform (S-isoform), whereas breakpoints within PML intron 6 (bcr 1) result in a longer form (L-isoform). Additionally, breakpoints within PML exon 6 (bcr 2) make a variable length transcript (V-isoform) in a small number of patients. The influence of breakpoint site on patient outcome remains controversial. Previous reports showed that patients with S-isoform have an increased incidence of clinical relapse and shorter survival compared to those with L-isoform. Others reported no difference in DFS between these patients groups. In this issue, Lee et al. reported that there were 58 L-isoform (62.1%), 32 S-isoform (34.0%), 4 V-isoform (4.3%) and, no significant prognostic factor for EFS from induction therapy using anthracycline plus ATRA among 94 patients with APL. They concluded pretreatment clinical characteristics and treatment outcomes were not significantly different according to PML/RAR-alpha isoform types in this induction group. Recently, it was reported that FLT3/ITD mutation was frequently associated with S-isoform and with the M3v form of leukemia and CNS relapse in APL was mostly related to S-isoform. With previous studies including this article, outcomes of different types of PML/RAR-alpha isoforms are not conclusive. Future researches need to be focused not only on clinical outcomes of different types of PML/RAR-alpha isoforms, but also clinical relevance of PML/RARA-alpha mRNA isoforms with other prognostic factors and particular clinical characteristics.
Exons ; Humans ; Incidence ; Introns ; Leukemia ; Leukemia, Promyelocytic, Acute ; Protein Isoforms ; Recurrence ; RNA Isoforms ; RNA, Messenger ; Tretinoin

Alternative splicing (AS) is an important mechanism of producing transcriptome diversity and microarray techniques are being used increasingly to monitor the splice variants. There exist three types of microarrays interrogating AS events-junction, exon, and tiling arrays. Junction probes have the advantage of monitoring the splice site directly. Johnson et al., performed a genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays (Science 302:2141-2144, 2003), which monitored splicing at every known exon-exon junctions for more than 10,000 multi-exon human genes in 52 tissues and cell lines. Here, we describe an algorithm to deduce the relative concentration of isoforms from the junction array data. Non-negative Matrix Factorization (NMF) is applied to obtain the transcript structure inferred from the expression data. Then we choose the transcript models consistent with the ECgene model of alternative splicing which is based on mRNA and EST alignment. The probe-transcript matrix is constructed using the NMF-consistent ECgene transcripts, and the isoform abundance is deduced from the non-negative least squares (NNLS) fitting of experimental data. Our method can be easily extended to other types of microarrays with exon or junction probes.
Alternative Splicing ; Cell Line ; Exons* ; Humans ; Least-Squares Analysis ; Protein Isoforms ; RNA Precursors ; RNA, Messenger ; Transcriptome

BACKGROUNDS: The inferior pharyngeal constrictor muscle (IPC), which consists of the thyropharyngeus (TP) and cricopharyngeus (CP) muscles, plays an important role during deglutition, but their function is different when analysed by radiographic, manometric and electromyographic studies. OBJECTIVES AND MATERIALS: The purpose of this study is to quantify the expression levels of MHC mRNA isoforms (2B, 2X, 2A, 2L, beta-cardiac, neonatal and embryonic) in thyropharyngeus and cricopharyngeus muscles of rats using competitive PCR. RESULTS: The thyropharyngeus muscle was mainly consisted of three fast-twitching MHC isoforms, mostly 2X isoform (85.2%). On the other hand, the cricopharyngeus muscle contained two-third of fast-twitching isoforms(65.1%) and one-third of neonatal MHC(34.9%). CONCLUSIONS: The thyropharyngeus muscle could be characterized as a fast-twitching muscle and the cricopharyngeus muscle is probably considered as a sarcomeric regenerating muscle that is caused by frequently mechanical damage on deglutition.
Animals ; Deglutition ; Gene Expression ; Hand ; Muscles ; Myosin Heavy Chains* ; Myosins* ; Pharyngeal Muscles* ; Polymerase Chain Reaction ; Protein Isoforms ; Rats* ; RNA Isoforms ; RNA, Messenger*

BACKGROUND AND OBJECTIVES: Human Troponin T & I (TnT, TnI) has several isoforms which have different functional property. This study was designed to describe the isoform expression of TnT & TnI in failing and hypertrophic human heart and during normal development. MATERIALS AND METHOD: Myocardium was attained from hypertrophic hearts (n=10) of TOF patients who underwent myomectomy, from failing hearts (n=10) of transplant recipients, from normal hearts (n=5) of patients in brain death and from aborted fetal hearts (n=5). After the extraction of RNA, RT-PCR was performed for TnT & TnI isoforms and GAPDH to evaluate the isoform expression qualitatively and quantitatively. RESULTS: In terms of TnI, slow skeletal TnI was expressed more than cardiac TnI in fetal hearts[ratio of Troponin over GAPDH (R)=1.3:0.5] but cardiac TnI was dominant in adult hearts (r=0.3:1.1) (p 0.05). Failing hearts showed similar pattern with adult hearts (r=0.3:1.2) and hypertrophic hearts showed the intermediate pattern (r=0.9:1.3). In terms of TnT, T1 and T3 were expressed in fetal hearts (r=0.04, 0.8) but only T3 was expressed in adult hearts (r=1.1). Failing hearts and hypertrophic hearts showed similar pattern with adult hearts and no differences in the amount of expression (r=1.4, 1.3). CONCLUSION: There is isoform switch from fetal to adult form during development and it might be responsible for the differences of myocardial functional property between fetal and adult heart. Failing and hypertrophic hearts showed no differences with normal hearts, which means the isoform switch of TnT & I might have no significant role in functional disturbances in these conditions.
Adult ; Brain Death ; Fetal Heart ; Heart* ; Humans* ; Myocardium ; Protein Isoforms ; RNA ; Transplantation ; Trinitrotoluene ; Troponin T* ; Troponin*

Hypertonicity imposes a great deal of stress to cells since it causes rise in cellular ionic strength, which can be reduced by the accumulation of compatible osmolytes. TonEBP plays a central role in the cellular accumulation of compatible osmolytes via transcriptional stimulation of membrane transporters and aldose reductase. Alternatively spliced forms of TonEBP mRNA have previously been reported and two of them showed different transcriptional activity. In the present study, isoform-specific antibodies were produced to confirm the translation of the spliced mRNA to protein. TonEBP was immunoprecipitated by using anti-TonEBP antibody and then immunoblotted using anti-TonEBP or isoform specific antibodies to find out the expression profile of TonEBP isoforms in basal or stimulated condition. From these results, we conclude that all TonEBP isoforms are expressed in mammalian cells and their expression patterns are not same in every cells.
Aldehyde Reductase ; Alternative Splicing ; Antibodies ; Membrane Transport Proteins ; Osmolar Concentration ; Protein Isoforms* ; RNA, Messenger

Previous studies have demonstrated the role of hydroquinone (HQ), a hydroxylated benzene metabolite, in modulating various immune responses; however, its role in macrophage-mediated inflammatory responses is not fully understood. In this study, the role of HQ in inflammatory responses and the underlying molecular mechanism were explored in macrophages. HQ down-regulated the expression of interferon (IFN)-β mRNA in LPS-stimulated RAW264.7 cells without any cytotoxicity and suppressed interferon regulatory factor (IRF)-3-mediated luciferase activity induced by TIR-domain-containing adapter-inducing interferon-β (TRIF) and TANK-binding kinase 1 (TBK1). A mechanism study revealed that HQ inhibited IRF-3 phosphorylation induced by lipopolysaccharide (LPS), TRIF, and AKT by suppressing phosphorylation of AKT, an upstream kinase of the IRF-3 signaling pathway. IRF-3 phosphorylation is highly induced by wild-type AKT and poorly induced by an AKT mutant, AKT C310A, which is mutated at an inhibitory target site of HQ. We also showed that HQ inhibited IRF-3 phosphorylation by targeting all three AKT isoforms (AKT1, AKT2, and AKT3) in RAW264.7 cells and suppressed IRF-3-mediated luciferase activities induced by AKT in HEK293 cells. Taken together, these results strongly suggest that HQ inhibits the production of a type I IFN, IFN-β, by targeting AKTs in the IRF-3 signaling pathway during macrophage-mediated inflammation.
Benzene ; HEK293 Cells ; Inflammation ; Interferons ; Luciferases ; Macrophages ; Phosphorylation ; Phosphotransferases ; Protein Isoforms ; RNA, Messenger

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

This study was purposed to investigate the expression of ADAR1 isoforms of P110 and P150 during the development of murine leukemia. A Notch1 over-expressing murine T cell acute lymphoblastic leukemia model was used to study the expression of ADAR1. BMMNC were isolated at different stages of disease and CD45.2(+)GFP(+) leukemia cells were sorted by flow cytometry at late stage. The expression of ADAR1 was detected by real time quantitative PCR. The results showed that mouse bone marrow cells from both leukemia and control groups expressed P110 and P150. Difference of P110 and P150 mRNA expression were observed during the development of leukemia. The expression of P110 dramatically increased and was significantly higher than that in control group. However, the expression level of P150 in leukemia group decreased stably and reached one-fourth of that in control group at 14 day. Furthermore, similar expression patterns could be detected in sorted CD45.2(+)GFP(+) leukemia cells. It is concluded that the mRNA expressions of P110 and P150 show diverse patterns in the development of leukemia, suggesting that RNA editing mediated by ADAR1 isoforms may play different roles in leukemia.
Adenosine Deaminase ; genetics ; Animals ; Gene Expression ; Mice ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Protein Isoforms ; genetics ; RNA Editing ; RNA, Messenger ; genetics ; RNA-Binding Proteins

OBJECTIVE: The object of this study was to provide a better understanding of the roles played by sex hormones in ovarian carcinogenesis. METHODS: 1) The expressions of estrogen receptor-alpha and -beta, progesterone receptor A and B, androgen receptor in normal ovarian tissue, benign, borderline, and malignant ovarian tumor were analyzed using immunohistochemical stains. 2) Expression of mRNA of sex hormone receptors of cell lines from postmenopausal normal surface epithelial cells, ovarian cancer, and breast cancer were studied using real time quantitative PCR methods. 3) Expression of PR isoforms after treatment with estradiol, expression of AR after treatment with testosterone were analyzed using RT-PCR and immunoblotting methods. 4) Apoptosis after treatment with levonorgestrel in cell lines from ovarian cancer were analyzed using flowcytometry. RESULTS: There was no significant difference in results shown by immunohistochemical staining between sex hormonal receptors of normal ovarian tissue (n=8), benign tumor (n=10), borderline tumor (n=8) and malignant tumor (n=24) according to malignant change. But, PRA was significantly reduced in epithelial ovarian cancer. 1) Expressions of ER-alpha and ER-beta, PRA, PRB and AR mRNA were seen in normal ovarian epithelial cells. 2) Deletion of exons of ER-alpha, ER-alpha wild type and variant, ER-beta variant were seen in many cell lines. 3) Down regulation of PR mRNA isoforms (especially PRA) in cell lines of ovarian cancer. 4) Flowcytometry showed that apoptotic cells markedly increased during exposure of progestins in ovarian cancer cell lines. CONCLUSION: These results suggest that down-regulation of ER-alpha and PRA is associated with the development of ovarian epithelial carcinoma. Progestins can activate the apoptotic pathway in the ovarian epithelium for protection of normal tissue from neoplastic transformation suggests that progestins deserve further evaluation as potential ovarian cancer preventive agents.
Apoptosis ; Breast Neoplasms ; Carcinogenesis ; Cell Line ; Coloring Agents ; Down-Regulation ; Epithelial Cells ; Epithelium ; Estradiol ; Estrogens* ; Exons ; Gonadal Steroid Hormones ; Humans* ; Immunoblotting ; Levonorgestrel ; Ovarian Neoplasms ; Polymerase Chain Reaction ; Progesterone* ; Progestins ; Protein Isoforms ; Receptors, Androgen* ; Receptors, Progesterone* ; RNA Isoforms ; RNA, Messenger ; Testosterone

Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by immunohistochemistry. The results showed that survivin mRNA and protein were expressed in gastric cancer and para-cancerous tissues. The survivin-2B was dominantly expressed in para-cancerous tissues, whereas the survivin-DeltaEx3 was more frequently detected in cancer tissues. The positive rate of survivin-2a was 100% in both cancer and para-cancerous tissues, but its relative transcript expression level was not significantly increased in cancer tissues in comparison with para-cancerous tissues. The correlation analysis revealed that the expression of survivin-2a mRNA was significantly associated with that of total survivin (r (s)=0.4178, P=0.0018), whereas inversely to that of survivin-DeltaEX3 (r (s)=-0.4506, P=0.0007). It was suggested that survivin-2a may act as an antagonist of survivin-DeltaEX3. The balance between antiapoptotic survivin iso-forms and nonantiapoptotic ones may play an important role in tumorigenesis and tumor progression. Promising value is hinted to analyze survivin and its variants in tumor early diagnosis and distinguishing malignant tumors from benign ones.
Microtubule-Associated Proteins/genetics ; Microtubule-Associated Proteins/*metabolism ; Protein Isoforms/genetics ; Protein Isoforms/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Stomach Neoplasms/*metabolism ; Stomach Neoplasms/pathology ; Tumor Cells, Cultured ; Tumor Markers, Biological/genetics ; Tumor Markers, Biological/*metabolism