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

OBJECTIVE: To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR). METHODS: By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique. RESULTS: The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected. CONCLUSION A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.
Exosomes ; Gene Expression ; Humans ; Leukemia, Promyelocytic, Acute ; Oncogene Proteins, Fusion ; analysis ; Polymerase Chain Reaction ; Protein Isoforms

An overview of the background for proteomics and a description of the present state of art are given with a description of the main strategies in proteomics. The advantages and limitations of the two major strategies, 2D-gel based and LC-MS based, are discussed and a combination for the two, CeLC-MS is described. A number of challenging problems which have been solved using different proteomics strategies including the advantage of organell enrichment or modifications specific peptide isolation to get deeper into the proteome are described. Finally the present status and future needs discussed.
Chromatography, High Pressure Liquid ; Electrophoresis, Gel, Two-Dimensional ; Mass Spectrometry ; Peptides ; analysis ; chemistry ; Phosphorylation ; Protein Isoforms ; analysis ; Proteome ; analysis ; Proteomics

The Wilms' tumor gene (WT1) is a transcription factor involved in tumorigenesis, especially in leukemogenesis. However, the role of WT1 expression in nonmalignant hematopoietic cells remains unclear. Furthermore, due to alternative splicing at two sites: 17 amino acid residues of exon 5 (+17AA) and 3 amino acid residues (+KTS) between exons 9 and 10, WT1 gene has four main isoforms (17AA+/KTS+, 17AA+/KTS-, 17AA-/KTS+, 17AA-/KTS-, abbreviation: +/+, +/-, -/+, -/-). The isoforms probably existed in hematopoietic cells, which make the research more complex. The aim of study was to elucidate the expression and its isoforms of WT1 gene in different cell subsets of healthy bone marrow donors. The fluorescence RT-PCR detection system was established to measure the expressions of full-length WT1, WT1 (+17AA) and WT1 (+KTS) in CD34(+)CD38(-) (stem cell), CD34(+)CD38(+) (progenitor cell), CD15(+)CD11b(+) (granulocyte), CD33(+)CD14(+) (monocyte), CD20(+)CD5(-) (B-lymphocyte) and CD20(-)CD5(+) (T-lymphocyte) subsets from 18 normal human bone marrow samples. The results showed that WT1 expressed in CD34(+)CD38(-), CD34(+)CD38(+), CD15(+)CD11b(+) and CD33(+)CD14(+), but not in CD20(+)CD5(-) and CD20(-)CD5(+) subsets. The highest expression was in CD34(+)CD38(-), but decreased gradually in CD15(+)CD11b(+) and CD33(+)CD14(+) subsets. WT1 (+17AA), WT1 (+KTS) and WT1 (+/+) isoforms were predominant in CD34(+)CD38(-) and CD34(+)CD38(+) primitive subsets, while in CD15(+)CD11b(+) and CD33(+)CD14(+) the dominant isoforms were WT1 (-17AA), WT1 (-KTS) and WT1 (-/-). It is concluded that the expression of WT1 in normal bone marrow decreases gradually with cell differentiation. Hematopoietic cells may adjust the ratios of WT1 isoforms to inhibit or promote cell differentiation.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; metabolism ; Cell Differentiation ; Gene Expression Regulation ; Humans ; Protein Isoforms ; metabolism ; WT1 Proteins ; metabolism

BACKGROUND/AIMS: There are three types of PML-RAR alpha mRNA fusion transcripts associated with acute promyelocytic leukemia (APL): the short (S)-form, the long (L)-form and the variable (V)-form. No study on the Korean population has addressed the clinical significance of the specific types of PML-RAR alpha mRNA fusion transcripts for APL patients who receive the combination therapy of all-trans-retinoic-acid and idarubicin (AIDA regimen). METHODS: We performed a retrospective analysis on 94 patients with APL to evaluate differences in the therapeutic outcomes, such as the response rate, an event-free survival (EFS), and overall survival (OS), after remission following the induction of chemotherapy. We also analyzed whether differences in the pretreatment clinical characteristics depend on the PML-RAR alpha isoform. RESULTS: The median age of the patients was 41 years (range 15-85). Among the 94 patients, there were 58 L-form cases (62.1%), 32 S-form cases (34.0%), and 4 V-form cases (4.3%). The CR rate following remission induction treatment was 84.9%. The CR rate was higher in patients with an initial WBC <10.0x109/L, as compared to patients with an initial WBC higher than 10.0X109/L (93.5% vs. 65.4%, p=0.001). The AIDA induction regimen was associated with a better EFS than non-AIDA induction regimens (81.9% vs. 49.6%, p=0.006). The induction group was also a significant prognostic factor for EFS in the multivariate analysis (p=0.020). There were no differences in OS and EFS in patients with either isoform L or isoform S in the AIDA induction group. CONCLUSIONS: This retrospective study demonstrated that pretreatment clinical characteristics and treatment outcomes were not significantly different among patients with varying PML-RAR alpha isoform types in the AIDA induction group.
Disease-Free Survival ; Humans ; Idarubicin ; Leukemia, Promyelocytic, Acute ; Multivariate Analysis ; Protein Isoforms ; Remission Induction ; Retrospective Studies ; RNA, Messenger

OBJECTIVETo study the epidemic status of human immunodeficiency virus type 1 (HIV-1) subtypes and sequence variations in Henan province and to explore their epidemic characteristics and transmission sources and routes.

METHODSHIV-1 env gene was amplified by nested PRC from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 1157 HIV-1 carriers confirmed in Henan from 2005 to 2006. The C2-V3 region (about 350450 bp) of HIV-1 env was sequenced.

RESULTSOf 1157 samples, there were 4 HIV-1 strains including subtype B', C and recombinant subtype BC and AE, accounting for 96.456% (1116/1157), 0.346% (4/1157), 2.593% (30/1157) and 0.605% (7/1157), respectively. In comparison with the sequences of the international strains of RL42, C.95in21068, 07BC.CN.97.C54A and 01AE.TH.90.CM240, the genetic divergence was (8.971 +/- 3.182)%, (5.109 +/- 0.112)%, (3.568 +/- 0.254)% and (4.775 +/- 0.128)%, respectively. The phylogenetic tree analysis showed that 1116 Henan B' strain was closely related to those of Thailand B' and was far different from other international subtypes. The major transmit route in subtype B' strain was mainly found among former blood donators, while sexual transmission was the major route for BC spreading. For AE, the major transmission was sex and blood transfusion, and sex was defined as the major route for C.

CONCLUSIONThere are four HIV-1 strains including subtype B', C and recombinant subtype BC and AE identified in Henan province currently, and the dominant subtype B' is closely related to HIV-1 strains of Thailand B'. While, for non-B' subtype, there exists the aggregating phenomenon in some local areas. Therefore, it is necessary to attach importance and to strengthen the HIV test and surveillance on migrant populations.

Adolescent ; Adult ; Base Sequence ; China ; DNA, Viral ; Female ; HIV-1 ; classification ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Protein Isoforms ; Sequence Analysis, Protein ; Viral Proteins

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3beta) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3beta kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.
Alzheimer Disease ; Amyloid ; Catalytic Domain ; Down-Regulation ; Glycogen Synthase Kinase 3 ; Negotiating ; Neurofibrillary Tangles ; Phosphorylation* ; Phosphotransferases ; Protein Isoforms ; Protein Phosphatase 2* ; Spectrum Analysis ; tau Proteins*

OBJECTIVE: Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of Obox4 in oocyte maturation by evaluating downstream signal networking. METHODS: The Obox4 dsRNA was prepared by in vitro transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by in vitro maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix GeneChip(R) Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. RESULTS: Total 424 genes were up (n=80) and down (n=344) regulated after Obox4 RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by Obox4 RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition. CONCLUSION: From the results of this study, it is concluded that Obox4 is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.
Animals ; Cell Cycle ; Chimera ; Chromosome Segregation ; Cytoplasm ; Gene Expression ; Genes, Homeobox ; Genome ; Meiosis ; Metabolic Networks and Pathways ; Mice ; Microarray Analysis ; Oocytes ; Protein Isoforms ; Protein Kinase C ; Protein Kinases ; RNA ; RNA Interference ; Statistics as Topic ; Ubiquitin-Protein Ligase Complexes

BACKGROUNDHemangiomas are the most common tumors in children. Some hemangiomas may require intervention because of their location, size, behavior, or potential for important complications. Pharmacological therapy with glucocorticoids is the mainstay treatment, but there is no consensus on therapeutic regimens or candidate selection, therapeutic efficacy varies, and the mechanism mediating the beneficial effects of glucocorticoids remains unclear. This study was performed to investigate the expression patterns of the glucocorticoid receptor (GR) and its alpha isoform (GRalpha) in cutaneous hemangiomas and vascular malformations.

METHODSSP immunohistochemical technique was used to examine the expression of GR(e-20) (GR) and GR(p-20) (GRalpha) on vascular endothelial cells in 80 specimens that included 33 proliferating hemangiomas, 32 involuting hemangiomas, 7 vascular malformations as well as 8 normal skin tissues, all obtained from infants and children. GR and GRalpha expression in prepared tissue slides were examined using automated computer-assisted microscopic analysis. Mean gray scale values were compared among the various tumor types.

RESULTSThe mean gray scale values of GR were 127.0 +/- 6.4 and 121.4 +/- 6.6 in hemangiomas and vascular malformations respectively, but this difference was not statistically significant (P = 0.104). However, these values were all markedly higher than that of normal skin, which was only 108.6 +/- 6.8 (P = 0.001 and P = 0.000 for comparison with hemangiomas and vascular malformations respectively). The gray scale of GR in proliferation and involuting hemangiomas were 127.9 +/- 4.8 and 126.0 +/- 5.8 respectively, but this difference was not significant (P = 0.146). However, GRalpha expression in hemangiomas, vascular malformations and normal skin declined gradually in stepwise fashion (127.3 +/- 5.4, 120.4 +/- 6.1 and 109.9 +/- 5.3 respectively; P < 0.001). GRalpha expression was higher in proliferating hemangiomas than in involuting hemangiomas (127.2 +/- 6.3 and 122.5 +/- 6.3; P = 0.004).

CONCLUSIONSGR and GRalpha are strongly expressed in hemangiomas and vascular malformations. The expression of GRalpha is closely related to the phase of the hemangioma. Determination of GR and GRalpha may be a positive significance to understand the information of hemangiomas and vascular malformations and may further help determining proper strategies of steroid therapy for hemangiomas and vascular malformations.

Blood Vessels ; abnormalities ; Child ; Child, Preschool ; Female ; Hemangioma ; chemistry ; pathology ; Humans ; Immunohistochemistry ; Infant ; Male ; Protein Isoforms ; Receptors, Glucocorticoid ; analysis ; Skin Neoplasms ; chemistry ; pathology

To explore the hematopoiesis inhibition mechanisms of interferon-gamma (IFN-gamma), the effects of IFN-gamma on the expression of the cyclin D in the umbilical cord blood hematopoietic stem/progenitor cells were observed. In the experiments the CD34(+) cells were isolated from the cord blood with MIDI-MACS system; semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; the expression levels of cyclin D isoforms were assayed by semi-quantitative RT-PCR, after the hematopoietic stem/progenitor cells were incubated with IFN-gamma. The results indicated that IFN-gamma could inhibit the formation of CFU-GM and down-regulate the expression of cyclin D2 and cyclin D3 at the mRNA level. It is concluded that the IFN-gamma could inhibit the proliferation of hematopoietic stem cells and down-regulate the expression of cyclin D, that may be one mechanism underlying the hematopoietic inhibition of IFN-gamma.
Cyclin D ; Cyclins ; genetics ; Fetal Blood ; cytology ; G1 Phase ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Protein Isoforms ; RNA, Messenger ; analysis