Alternative Splicing ; Fibronectins/genetics* ; Forensic Medicine ; Humans ; Protein Conformation ; Protein Isoforms/genetics* ; Wound Healing/physiology*

Wnt5a is a secreted Wnt ligand that plays a critical role in cellular pathways and inflammatory diseases. The WNT5A gene encodes two protein isoforms, Wnt5a-long and Wnt5a-short, which differ based on different promoter methylation and have distinct functions. However, the mechanisms of the promoter methylation are unclear. Depending on the extent of promoter methylation, Wnt5a exerts both anti-inflammatory and pro-inflammatory effects in inflammatory diseases, which may be involved in different Wnt5a isoforms. Therefore, the Wnt5a isoforms may be potential diagnostic markers for inflammatory diseases and the mechanisms of the WNT5A gene promoter methylation need to be further investigated.
DNA Methylation ; Wnt-5a Protein ; Promoter Regions, Genetic ; Protein Isoforms/genetics*

Titin, the largest known protein in the body expressed in three isoforms (N2A, N2BA and N2B), is essential for muscle structure, force generation, conduction and regulation. Since the 1950s, muscle contraction mechanisms have been explained by the sliding filament theory involving thin and thick muscle filaments, while the contribution of cytoskeleton in force generation and conduction was ignored. With the discovery of insoluble protein residues and large molecular weight proteins in muscle fibers, the third myofilament, titin, has been identified and attracted a lot of interests. The development of single molecule mechanics and gene sequencing technology further contributed to the extensive studies on the arrangement, structure, elastic properties and components of titin in sarcomere. Therefore, this paper reviews the structure, isforms classification, elastic function and regulatory factors of titin, to provide better understanding of titin.
Connectin/genetics* ; Muscle Proteins/metabolism* ; Protein Isoforms/genetics* ; Sarcomeres/metabolism* ; Muscle Fibers, Skeletal/metabolism*

Lipoxygenases (linoleate: oxygen oxidoreductase, EC 1.13.11.12; LOXs) are encoded by a multi-gene family in plants. The LOXs are monomeric non-heme, non-sulfur iron dioxygenases, which catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids containing a cis, cis-1,4-pentadiene moiety. The LOX isoforms are distinguished by differences in optimum pH of the reaction, pI, substrate and product specificity, spatial and temporal expression, and subcellular localization. The function of various LOXs in plants has been suggested. Some of the physiological processes in which lipoxygenases have been implicated include wounding, pathogen attack, seed germination, fruit ripening, plant senescence, and synthesis of Abscisic acid (ABA) and Jasmonic acid (JA). During normal vegetative and reproductive growth, lipoxygenases have also been suggested to act as vegetative storage proteins, participate in transference of lipoid, and response to nutrient stress and source/sink relationships. Significant progress in understanding LOX families will be beneficial to the application of the LOX in crop breeding, research on new-type phytoalexin and food industry.
Gene Expression Regulation, Plant ; Lipoxygenase ; genetics ; metabolism ; Multigene Family ; Plants ; enzymology ; Protein Isoforms ; genetics ; metabolism

OBJECTIVETo explore the mechanism of cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2).

METHODSThe expression and translocation of different isoforms of protein kinase C (PKC) in cells were observed with immunocytochemistry and Western blot techniques. The content of diacylglycerol (DAG) was analyzed with high performance thin layer chromatography (HPTLC) technique.

RESULTSTransfection of PEMT2 can inhibit the expression of cPKC alpha, but obviously promotes the expression and translocation from cytosol to plasma membrane of cPKC beta2. At the same time, the content of DAG was decreased in the transfected cells. Expression and translocation of other PKC isoforms were not changed by PEMT2 transfection.

CONCLUSIONEffects of overexpression of PEMT2 on the expression and translocation of different PKC isoforms might be related to the mechanism of cell proliferation inhibition and apoptosis induced by transfecting PEMT2.

Animals ; Liver Neoplasms, Experimental ; enzymology ; pathology ; Phosphatidylethanolamine N-Methyltransferase ; biosynthesis ; genetics ; Protein Isoforms ; Protein Kinase C ; biosynthesis ; genetics ; Rats ; Transfection

OBJECTIVETo investigate whether WT1 gene overexpressed in leukemic stem cells (LSCs) and its significance.

METHODSExpression of WT1(+17AA) and WT1(+KTS) gene isoforms in CD34(+)CD38(-)CD123(+) cells (LSCs) of 47 AML patients were determined by fluorescence quantitative RT-PCR. The ratio of the four splicing isoforms WT1(+/+), WT1(+/-), WT1(-/+) and WT1(-/-) in LSCs were calculated and compared with that in normal CD34(+)CD38(-)CD123(-) cells (HSCs). The relationship in AML patients between LSCs WT1 expression and remission rate, survival time and relapse rate was analyzed.

RESULTSThe expression of WT1 gene was highest (0.034 +/- 0.034) in LSCs, and higher in CD34(+)CD38(-)CD123(-) AML cells as compared with that in HSCs (P < 0.05). The proportion of +17AA isoform was predominant over -17AA in all the three cell subsets with no difference. The proportion of +KTS isoform was the highest in HSCs (0.57 +/- 0.04), while the lowest in CD34(+)CD38(-)CD123(-)AML cells (0.50 +/- 0.12) (P < 0.05). No significant difference in the four isoforms expression ratio was observed among the three groups. WT1 expression in LSCs was not correlated with sex, age, FAB subtype and blast cell ratio, while the ratio of CD34(+) cells in blast cell was significantly higher in the WT1 high expression group than in the low expression group (P < 0.01). The CR rate was significantly lower in WT1 high expression group (21.1%) than in the WT1 low expression group (59.1%) (P < 0.05). The follow-up data were available in 41 patients with a median follow-up duration of 118 (3 - 290) days. The median overall survival (OS) for WT1 high and low expression group were 77\[95% confident interval (CI) 45 - 108\], 158 (95%CI 100 - 215) days respectively (P = 0.041).

CONCLUSIONWT1 gene overexpressed in AML LSCs and the ratios of four WT1 isoforms have no difference in LSC compared with HSC. Patients with higher LSC WT1 expression have lower CR rate and shorter survival time.

Humans ; Leukemia, Myeloid, Acute ; genetics ; Neoplasm Recurrence, Local ; Protein Isoforms ; Stem Cells

OBJECTIVE: In order to supply an effective reference of early injury time estimation and explore the time limit of detection of EDA\EDB mRNA in human skin samples, the expression of alternative splicing segment of fibronectin--EDA\EDB in incised wound of human skin were studied. METHODS: Using in situ hybridization with DIG-labeled anti-sense RNA probe, the expression of FN EDA\EDB domain was detected in human skin incised wound at the early stage of injury (from 30 min to 3 h). RESULTS: The positive expression rates of FN-EDA\EDB immediately after injury and area far away from wound were same as the control group. The expression of FN-EDA\EDB in human skin incised wound showed a gradually increased tendency in early injury time (within 3 h). The positive expression cells were mainly distributed in basement cells of epidermis and the expression of EDA is much higher than EDB. It's difficult to detect EDA\EDB mRNA when the samples were deposited in air for 4 hour. CONCLUSION FN-EDA\EDB may be used as a sensitive mark for the estimation of early injury time. The in-situ hybridization technique is not applicable in the application.
Fibronectins/genetics* ; Forensic Medicine ; Humans ; In Situ Hybridization ; Protein Isoforms/genetics* ; RNA, Messenger/genetics* ; Skin/metabolism* ; Time Factors ; Wound Healing/physiology*

Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; Tumor Cells, Cultured

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

Periaxin, a protein of noncompact myelin, is specifically expressed in the peripheral nervous system (PNS). There are two protein isoform L-periaxin and S-Periaxin by alternative splicing of periaxin gene, playing an important role in the initiation of myelin formation. So far, 18 different mutation sites in L-periaxin gene have been found to induce the peripheral demyelinating neurological charcot-marie-tooth diseases subtype 4F (CMT4F). The technique of activation of transcription activator-like effector nucleases (TALENS) was used to knock out the L-periaxin gene in RSC 96 cell line of Rattus. According to the design principle, the knock-out site of L-periaxin was assured to NLS domain of L-periaxin, which is target sequence of left and right arms of TALEN. The knock-out vectors of TALEN-L and TALEN-R were established and transfected into RSC96 cell. After puromycin screening, L-periaxin was knocked out successfully in RSC96 cell, which is confirmed by DNA sequence. The mutation efficiency is 21.6%. S-periaxin, not L-periaxin can be detected by Western blotting in L-periaxin gene knock-out RSC96 cell. The cell growth rate was decreased and the number of cells in G1 increased and decreased in S phase in L-periaxin gene knock-out RSC96 cell by flow cytometry and MTT assay.
Animals ; Cell Line ; Charcot-Marie-Tooth Disease ; genetics ; Gene Knockout Techniques ; Membrane Proteins ; genetics ; Mutation ; Protein Isoforms ; Rats