Carrier Proteins ; metabolism ; Humans ; Microfilament Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology

High expression of Fightless I (FLII) is associated to multiple tumors. Based on our previous study that FLII might be involved in the nuclear export, we assessed the possible interaction of FLII with the nuclear envelop associating proteins Importin β and Nup88. We first constructed GST-FLII, GST-LRR recombinant plasmids and transformed them into the Rosetta strain to produce GST-FLII, GST-LRR fusion protein. After purification of these proteins, GST-pull down, as well as co-immunoprecipitation, were used to test the interaction of FLII with Importin β and Nup88. FLII interacted with Importin β and Nup88, and FLII LRR domain is responsible for these interactions. Thus, FLII may play a role in nuclear export through interaction with Importin β and Nup88.
Humans ; Microfilament Proteins ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Recombinant Fusion Proteins ; metabolism ; beta Karyopherins ; metabolism

OBJECTIVETo explore the relationships between the expression of transgelin in dendritic cells (DCs) pulsed with hepatocellular carcinoma lysates and the functions of the DCs.

METHODSDCs derived from healthy human white blood cells were divided into 3 groups: one was pulsed with high metastatic potential hepatocellular carcinoma cell line (MHCC97H) lysates, one with lysates of a low metastatic potential cell line (MHCC97L), and one un-pulsed DCs served as the control. The morphology of the DCs was observed by confocal microscopy and scanning electron microscopy. The phenotypes of the DCs were detected by flowcytometric analysis. The mixed leucocyte reaction (MLR) test and IL-12 secretion of DCs in the supernatants of MLR were employed to determine the functions of the DCs; the expression of transgelin was detected by Western blot.

RESULTSThere were no morphological changes in the different DCs, but the levels of HLA-DR, CD80, CD83, CD86, MLR and IL-12 and transgelin were significantly higher in the two pulsed groups than those in the control group (P less than 0.01). In MHCC97H pulsed DCs, their CD80, CD83, CD86, and the expression of transgelin were also higher than those in the control group (P less than 0.05). The expression of transgelin was significantly higher in the MHCC97H pulsed group than in the MHCC97L loaded group, but CD80, CD83, CD86 and the level of IL-12 were all lower in the MHCC97H loaded DC group in comparison with those in the MHCC97 pulsed group (P less than 0.05).

CONCLUSIONThe expression of transgelin in DCs pulsed with HCC lysates is related to the functions of the DCs.

Carcinoma, Hepatocellular ; metabolism ; Cell Line, Tumor ; Dendritic Cells ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; Microfilament Proteins ; biosynthesis ; Muscle Proteins ; biosynthesis

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. At present, no definite ALS biomarkers are available. In this study, exosomes from the plasma of patients with ALS and healthy controls were extracted, and differentially expressed exosomal proteins were compared. Among them, the expression of exosomal coronin-1a (CORO1A) was 5.3-fold higher than that in the controls. CORO1A increased with disease progression at a certain proportion in the plasma of patients with ALS and in the spinal cord of ALS mice. CORO1A was also overexpressed in NSC-34 motor neuron-like cells, and apoptosis, oxidative stress, and autophagic protein expression were evaluated. CORO1A overexpression resulted in increased apoptosis and oxidative stress, overactivated autophagy, and hindered the formation of autolysosomes. Moreover, CORO1A activated Ca²⁺-dependent phosphatase calcineurin, thereby blocking the fusion of autophagosomes and lysosomes. The inhibition of calcineurin activation by cyclosporin A reversed the damaged autolysosomes. In conclusion, the role of CORO1A in ALS pathogenesis was discovered, potentially affecting the disease onset and progression by blocking autophagic flux. Therefore, CORO1A might be a potential biomarker and therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/pathology* ; Calcineurin/metabolism* ; Motor Neurons/pathology* ; Microfilament Proteins/metabolism* ; Cytoskeletal Proteins/metabolism*

OBJECTIVE: To investigate the expression of Calponin-1 and Transgenlin in the uterine smooth muscles during normal labor on-sets, and to evaluate their effect on initiating the normal labor. METHODS: A total of 14 uterine bodies and lower segments of human pregnancy were divided to a non-labor group (NIL) and a labor group(IL). Immunohistochemical technology and Western blot were used to determine the expression of Calponin-1 and Transgelin in the 2 groups. RESULTS: Immunohistochemical detection and Western blot showed that Calponin-1 protein in the uterine smooth muscle tissue of the body and the lower uterine segment of smooth muscle tissues had significant difference (P<0.05). The expression of Transgelin in the uterine body smooth muscle tissue in the IL was higher than that in the NIL(P<0.05). In the lower uterine segments of the smooth muscle, the expression of Transgelin was not significantly different in the 2 groups (P>0.05). CONCLUSION Calponin-1 of the uterine smooth muscle and Transgelin of the uterine body smooth muscle may involve in the regulation of uterine smooth muscle contractility, which is closely related to child birth launch.
Adult ; Calcium-Binding Proteins ; genetics ; metabolism ; Female ; Humans ; Labor Onset ; metabolism ; Microfilament Proteins ; genetics ; metabolism ; Muscle Proteins ; genetics ; metabolism ; Myometrium ; metabolism ; Pregnancy ; Uterine Contraction ; metabolism

To understand the function of Mayven and investigate the pathogenesis of multiple sclerosis, the gene sequences of different truncated Mayven were amplified from the gene library of human brain. These truncated fragments, including fragment P1 (1-902 bp), fragment P2 (1-523 bp), fragment P3 (507-182 bp) and fragment P4 (887-1782 bp), were cloned into pGEX-4T-2 vector to construct recombinant plasmids. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The expressed proteins were detected by SDS-PAGE and Western blot, and were purified by GST purifying system. The results showed that recombinant express vectors of different truncated GST-Mayven were successfully constructed and were expressed in soluble form protein induced by IPTG. The fusion proteins have good reactivity to GST antibody. The construction of recombinant express vectors of different truncated GST-Mayven lays a basis for further function study on Mayven.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Multiple Sclerosis ; genetics ; Nerve Tissue Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo detect the expression of protein 4.1 family members in mouse melanoma cell lines and evaluate their effect on cell proliferation.

METHODSPCR and Western blot were used to detected to the expression of protein 4.1 family members (4.1R, 4.1B, 4.1G, and 4.1N) at the mRNA and protein levels in B16 and B16-F10 cell lines. The expression plasmid vector pEGFP-N1-EPB41L3 carrying 4.1B gene sequence amplified from genomic RNA of mouse embryo fibroblasts was constructed and transiently transfected into mouse melanoma cells. The change in cell proliferation was assessed using MTT assay.

RESULTSThe mRNA and protein expressions of all the protein 4.1 family members, with the exception of 4.1B, were detected in both B16 and B16-F10 cells. Transfection of cells with the eukaryotic expression vector pEGFP-N1-EPB41L3 markedly inhibited cell proliferation as compared with the non-transfected cells.

CONCLUSIONThe eukaryotic expression vector carrying EPB41L3 sequence is capable of inhibiting the proliferation of mouse melanoma B16 and B16-F10 cells.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Genetic Vectors ; Melanoma, Experimental ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; Neuropeptides ; metabolism ; Plasmids ; Transfection

AIMTo investigate the molecular mechanisms of smooth muscle 22 alpha (SM22alpha) whereby cytoskeleton remodeling of vascular smooth muscle cells (VSMCs) is regulated.

METHODSSynthetic (dedifferentiated) VSMCs were converted to contractile (differentiated) VSMCs by serum deprivation. Cells were transfected with pEGFP-SM22alpha, and localization of SM22alpha and its relationship with F-actin were observed through fluorescence microscopy. Fractional extraction of proteins and Western blotting were used to detect polymerization of SM alpha-actin in antisense-pcD2-SM22alpha-transfected VSMCs.Furthermore, effect of SM22alpha on F-actin cross-linking was observed by F-actin polymerization experiment.

RESULTSFluorescence microscopy showed that SM22alpha co-localized with F-actin in contractile VSMCs. Western blotting of protein extracts from F-/G-actin fractions revealed that polymerization of SM alpha-actin was lower in antisense-pcD2-SM22alpha-transfected VSMCs, in which SM alpha-actin mostly existed as soluble G-actin. Moreover, F-actin polymerization in vitro also showed that GST-SM22alpha could promote cross-linking of F-actin to form thick and bundled stress fibres,while extracts from VSMCs transfected with antisense-pcD2-SM22alpha could not effectively induce the polymerization of F-actin.

CONCLUSIONSM22alpha acts as a modulator to participate in VSMC cytoskeleton remodeling. It can not only induce polymerization of G-actin to F-actin, but also promote cross-linking of F-actin to bundled stress fibres, indicating its vital role in cytoskeleton remodeling.

Actins ; metabolism ; Animals ; Cytoskeleton ; metabolism ; Male ; Microfilament Proteins ; metabolism ; Muscle Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo assess the pathologic markers for evaluation of reversibility in pulmonary hypertension (PAH) related to congenital heart disease.

METHODSTwenty-eight patients with congenital heart disease complicated by PAH were subclassified into reversible pulmonary hypertension (RPAH) and irreversible pulmonary hypertension (IPAH), according to post-operative mean pulmonary artery pressure (MPAP). Pulmonary vascular lesion was analyzed according to Ruan's method. Mean medium thickness percent, mean medium area percent and pulmonary arteriolar density were measured by quantitative morphometry. Immunohistochemical study for transgelin and filamin A was carried out.

RESULTSAmongst the 28 cases studied, 24 were RPAH and 4 were IPAH. Of the 24 patients with RPAH, 13 (54.2%, 13/24) had pulmonary vascular lesion of grade 0, 9 (37.5%, 9/24) of grade 1 and 2 (8.3%, 2/24) of grade 2. Of the 4 patients with IPAH, 1 had lesion of grade 1, 1 of grade 2 and 2 of grade 3. Both preoperative and postoperative MPAP were higher in IPAH patients than that in RPAH patients[(53.3±23.4) mmHg versus (34.1±12.7) mmHg, P=0.020 and (35.0±8.8) mmHg versus (17.8±3.9) mmHg, P<0.01]. Compared to patients with pulmonary vascular lesion of grades 0 and 1, the preoperative MPAP in patients with grades 2 and 3 showed no significant difference, but the postoperative MPAP was higher (P<0.05 or 0.01). Compared to control group, mean medium thickness percent and mean medium area percent were significantly higher in RPAH and IPAH categories (12.0±3.5, 8.5±2.0 versus 5.7±1.0, P<0.01 and 55.8±11.1, 49.0±9.4 versus 34.0±5.5, P<0.01). Mean medium thickness percent was significantly higher in IPAP group than that in RPAH group (12.0±3.5 versus 8.5±2.0, P=0.001). Correlation analysis demonstrated that mean medium thickness percent and mean medium area percent had positive correlation with preoperative and postoperative MPAP. There was no correlation between grading of pulmonary vascular lesion and reversibility. Transgelin and filamin A had stronger staining in pulmonary vascular smooth muscle cells in IPAH than those in RPAH and controls(P<0.05).

CONCLUSIONSPathologic assessment of lung biopsy remains the gold standard for evaluation of the reversibility in PAH related to congenital heart disease. Mean medium thickness percent, mean medium area percent and immunoreactivity for transgelin and filamin A are useful parameters.

Biomarkers ; metabolism ; Biopsy ; Filamins ; metabolism ; Heart Diseases ; complications ; pathology ; Humans ; Hypertension, Pulmonary ; complications ; diagnosis ; pathology ; Lung ; pathology ; Microfilament Proteins ; metabolism ; Muscle Proteins ; metabolism

OBJECTIVETo investigate the role of fascin, an actin bundling protein, in the development and progression of human esophageal squamous cell carcinoma (ESCC), and explore its association with the clinicopathologic characteristics and 5-year survival of the patients.

METHODSUsing tissue array and immunohistochemistry, the expression of fascin was determined in 241 ESCC tissues and the corresponding normal esophageal mucosal tissues.

RESULTSESCC tissues showed a significantly higher overexpression rate of fascin than the corresponding normal esophageal mucosal tissues (68.9% vs 15.5%, P<0.05). The overexpression of fascin was correlated to lymph node metastasis and TMN stage, but not to the patients' age, gender, tumor differentiation and general classification. Survival analysis showed that abnormal expression of fascin was associated with the 5-year survival rate of patients with ESCC.

CONCLUSIONSThe abnormal expression of fascin may play an important role in the progression of ESCC, and detection of fascin expression may have important prognostic values.

Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Carrier Proteins ; metabolism ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Male ; Microfilament Proteins ; metabolism ; Middle Aged ; Prognosis