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

To investigate the effects of physiological shear stress on the vasodilator-stimulated phosphoprotein (VASP) location and expression changes associated with actin remodeling, we isolated and cultured human umbilical endothelial cells(HUVECs) with trypsin digestion. A parallel-plated flow chamber device was used to create laminar shear stress in vitro. The distributions of VASP and microfilaments in cells were observed by double staining with Alexa488 and rhodamine-phalloidin. Changes of VASP expression and phosphorylation were analyzed quantitatively with Western blot before and after exposure to shear flow for different times. We found that, under a shear stress of 10 dyn/cm2, HUVECs were elongated and oriented gradually to the flow direction. Microfilaments were recruited and oriented also to the flow direction with thicker VASP, specially targeted to their extremities. Western blotting data showed a rapid phosphorylation of VASP, and an increase of total VASP expression which peaked at 2 h (2 folds), then recovered until 8 h, followed by a slow increase again. These results suggest that VASP is a potential component which participates in the regulation of cell actin remodelling induced by shear flow.
Cell Adhesion Molecules ; biosynthesis ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Microfilament Proteins ; Phosphoproteins ; biosynthesis ; Stress, Mechanical ; Umbilical Veins ; cytology ; metabolism

AIMThe recombinant human smooth muscle 22 alpha (SM22alpha) was expressed by using Pichia pastoris.

METHODSUsing pGEM3z-SM22alpha as the template, SM22alpha coding region was amplified by PCR, and was inserted the expression vector pPIC9. Then the recombinant plasmid pPIC9-SM22alpha was transfected into Pichia pastoris. The products induced by methanol were precipitated by ammonium sulfate, then CM-cellulose chromatography was performed for SM22alpha. Polyclonal antibody against SM22alpha was produced by immunizing a rabbit with purified recombinant SM22alpha.

RESULTSThe positive clone with SM22alpha got high output at 84 hours after induction by methanol. The SM22alpha prepared by ammonium sulfate fractionation and chromatographic separation showed a single band whose apparent molecular weight was 22 kD on SDS-PAGE. Polyclonal antibody against SM22alpha could detect the SM22alpha expression in human or rat vascular walls.

CONCLUSIONHigh-level expression of SM22alpha is successfully achieved in Pichia pastoris. Antibody against SM22alpha can be used to explore the function of SM22alpha.

Amino Acid Sequence ; Animals ; Genetic Vectors ; Humans ; Microfilament Proteins ; biosynthesis ; genetics ; isolation & purification ; Muscle Proteins ; biosynthesis ; genetics ; isolation & purification ; Pichia ; metabolism ; Plasmids ; Rabbits ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

UNLABELLEDTo investigate the expressions of fibrillin-1, elastin and matrix metalloproteinase-1 and -9 (MMP-1, 9) in chronic actinic dermatitis in elderly patients and explore the pathogenesis of the disease.

METHODSTwenty-three patients with chronic actinic dermatitis were examined for the expressions of fibrillin-1, elastin, MMP-1, and MMP-9 with immunohistochemistry in the skin lesions. Image analysis was carried out to measure MMP-1 and MMP-9 expressions semi-quantitatively.

RESULTSIn the skin lesions of patients with chronic actinic dermatitis, elastin expression was obviously reduced or absent in the papillary dermis. The elastic fibers were disorderly arranged in the reticular dermis with local aggregation in some regions. Obvious fibrillin-1 deposition was found in the reticular dermis. Increased expressions of MMP-1, but not that of MMP-9, was found in the skin lesions of the patients.

CONCLUSIONElastin and fibrillin-1 deposition can be found in the skin lesions in patients with chronic actinic dermatitis, suggesting the association of increased MMP-1 expression with the elastic tissue degeneration in the lesions. MMP-9 does not exhibit an obvious association with the pathogenesis of chronic actinic dermatitis in elderly patients.

Aged ; Elastin ; biosynthesis ; Female ; Fibrillin-1 ; Fibrillins ; Humans ; Immunohistochemistry ; Male ; Matrix Metalloproteinase 1 ; biosynthesis ; Matrix Metalloproteinase 9 ; biosynthesis ; Microfilament Proteins ; biosynthesis ; Middle Aged ; Photosensitivity Disorders ; etiology ; metabolism ; Sunlight ; adverse effects

OBJECTIVETo search for hepatocellular carcinoma (HCC) invasion related biomarkers using the cell membrane proteomics approaches, and to validate the markers using experimental and clinical specimens.

METHODSThe HCCLM9 and MHCC97L cells with a similar genetic background and remarkably different metastasis behaviors were used for comparative membrane proteome profiling using sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry technologies. Candidate protein makers were further validated by western blot on cells, immunohistochemistry (IHC) on animal tumor tissues, and tissue micro-array on clinical specimens.

RESULTSThe membrane proteins of MHCC97L and HCCLM9 cells were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. 14 proteins were identified by ESI-MS/MS among the differential bands. Coronin-1C was overexpressed in HCCLM9 (7.31+/-0.73) versus MHCC97L (2.84+/-0.99) validated by western blot. Elevated coronin-1C expression was observed in liver cancer tissues of HCCLM9 nude mice. IHC study in 115 human HCC specimens demonstrated that patients with higher coronin-1C expression had more advanced stage.

CONCLUSIONThe study suggests that coronin-1C could be a potential molecule to predict HCC invasive behavior.

Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microfilament Proteins ; biosynthesis ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis

Gastric cancer is a very serious disease and is naturally resistant to many anticancer drugs. To reduce the mortality and improve the effectiveness of therapy, many studies have tried to find key biomarkers. Proteomic technologies are providing the tools needed to discover and identify disease-associating biomarkers. The proteomic study of gastric cancer establishes any specific events that lead to cancer, and it provides a direct way to define the true function of genes. Using two dimensional (2-D) electrophoresis of the stomach cancer tissue, we have gained about 1,500 spots in each gel, and 140 protein spots also were identified. Among the identified proteins, there were seven over-expressed proteins in stomach cancer tissue: NSP3, transgelin, prohibitin, heat shock protein (hsp) 27 and variant, protein disulfide isomerase A3, unnamed protein product and glucose regulated protein. There were also seven under-expressed proteins in stomach cancer: Apolipoprotein A-1, p20, nucleoside diphosphate isomerase A, alpha 1 antitrypsin, desmin, serum albumin and sero-transferrin.
Aged ; Carrier Proteins/biosynthesis ; Cell Line, Tumor ; Electrophoresis, Gel, Two-Dimensional ; Female ; Human ; Male ; Microfilament Proteins/biosynthesis ; Middle Aged ; Muscle Proteins/biosynthesis ; Neoplasm Proteins/biosynthesis ; Proteins/biosynthesis ; *Proteome ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stomach Neoplasms/*metabolism ; *Tumor Markers, Biological

The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Genetic Vectors ; genetics ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Membrane Proteins ; biosynthesis ; genetics ; physiology ; Mice ; Mice, Transgenic ; Microfilament Proteins ; genetics ; Muscle Proteins ; genetics ; Mutant Proteins ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the expression of FSCN1 in human epithelial tumors and their clinical significance.

METHODSFSCN1 expression was examined immunohistochemically in specimens of human epithelial tumors, including 26 cases of lung cancer, 33 cervical cancer, 22 ovarian cancer, 38 esophageal cancer, 24 pancreatic cancer, 23 gastric cancer, 29 laryngocarcinoma, 17 primary hepatocellular carcinoma, 34 colorectal cancer, 33 breast cancer, 24 nasopharyngeal carcinoma and their corresponding normal tissues.

RESULTSThe positivity rates of FSCN1 expression in epithelial tissues and epithelial tumors were 6.3% (5/80) and 58.7% (178/303), respectively. FSCN1 showed higher expressions in cervical cancer, ovarian cancer, esophageal cancer, pancreatic cancer, gastric cancer, laryngocarcinoma, colorectal cancer, breast cancer and nasopharyngeal carcinoma, but lower or no expression in the corresponding normal tissues (P<0.05). In gastric cancer and nasopharyngeal carcinoma, the edges of the tumors were more strongly stained for FSCN1 than the interior of the tumor.

CONCLUSIONFSCN1 expression is significantly upregulated in human epithelial tumors in close correlation with tumor occurrence and progression.

Carrier Proteins ; biosynthesis ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Laryngeal Neoplasms ; metabolism ; pathology ; Microfilament Proteins ; biosynthesis ; Neoplasms ; metabolism ; pathology ; Neoplasms, Glandular and Epithelial ; metabolism ; pathology ; Uterine Cervical Neoplasms ; metabolism ; pathology

OBJECTIVEFurther studies have been conducted to evaluate the roles of Ngn3 in adult islet maintenance and renewal.

METHODSIslets were isolated from 6 - 8 week old male C57BL/6 mice. After common bile duct cannulation, the pancreas was resected and digested in collagenase V (2.5 mg/ml). Islets were then handpicked and 10 - 12 islets were plated in 60 mm culture dish and cultivated with RPMI-1640, which contained 12.5 mmol/L HEPES, 5.2 mmol/L glucose and 2% fetal bovine serum (FBS). Islet cells were analyzed by immunocytochemistry methods for A6, insulin, glucagon, nestin, Ngn3 and 5-bromo-2'-deoxy-uridine (BrdU).

RESULTSThe results of these studies indicated that less than 15 percent of proliferated islet cells were Ngn3 expressing cells, in which about one third of the Ngn3 positive cells co-expressed A6. The existence of Ngn3 in cultured islet cells is consistent with the results from other's findings both in embryogenesis and adult islet studies. A significant finding of our study is that the existence of A6 and Ngn3 co-expressing cells in the cultured islet. A6 is a marker for identifying bile duct epithelial cell oriented hepatic progenitor cells. Islet-derived A6 cells are possibly born in the adult pancreatic duct and migrate into islets. A6 cells co-express Ngn3 when these cells commit to endocrine lineage within the islets. More interestingly, islet-derived A6 positive cells have the potential to transdifferentiate into hepatic cells.

CONCLUSIONThe presence of Ngn3(+) and A6(+) cells in the cultured islets suggests that the four established islet cell types arise from a common endocrine lineage residing within the adult islets. A6 and Ngn3 are useful markers for understanding intra-islet adult stem cell lineages in our future studies. This approach may allow for significant advances in understanding the IPC proliferation and differentiation, and open the possibility of using intra-islet adult stem cells for diabetes treatment.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; Cell Differentiation ; Cell Lineage ; Cells, Cultured ; Islets of Langerhans ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Microfilament Proteins ; Nerve Tissue Proteins ; biosynthesis ; Protein-Tyrosine Kinases ; biosynthesis ; Stem Cells ; cytology ; metabolism

OBJECTIVETo search and identify the non-steroid receptor binding cis-acting elements in the L-plastin promoter in prostate cancer, and the correlative regulation pathway and transcription factors.

METHODSOn the basis of construction of the L-plastin promoter luciferase vectors which were removed the steroid hormone receptor AR and ER binding elements, the promoter on the vector was nest-deleted by Exonuclease III and the relative luciferase plasmids were constructed. Transfected these twelve plasmids into prostate cancer cell line LNCaP under dihydrotestosterone-stimulated situation or not and test the intensity of luciferase, then we got the regulation message of every 200 bp part of the promoter in prostate cancer. After the analysis of relative programme, we got the possible regu- lation pathway of non-steroid hormone transcription factors. After removing the possible transcription factors binding site sequence by site-specific mutagenesis, the changes luciferase of activities proved our reasoning.

RESULTSWe succeed in segmental deletion of the L-plastin promoter, and constructing the relative plasmids containing part L-plastin promoter on luciferase vector pGL3-basic. After testing the luciferase activities of constructed plasmids, we found the sequence from 206 to 1 of L-plastin promoter had significant luciferase activity. The software TRANSFECT showed that there were binding elements for transcription factors AP-4 at seq-198 to 192 and SP-1 at seq-54 to 41 on the short part promoter (206 to 1). The recombinant plasmids deleted the AP-4 and SP-1 binding elements had lower luciferase activity than the wild-type.

CONCLUSIONThere are some other non-steroid hormone pathway to regulate the expression of L-plastin except the steroid hormone pathway in prostate cancer. The main binding sites of the non-steroid hormone regulator lies in the sequence from 206 to 1. Transcription factors AP4 and SP-1 may up-regulated the expression of L-plastin by binding these sites.

Animals ; DNA-Binding Proteins ; physiology ; Gene Expression Regulation, Neoplastic ; Luciferases ; metabolism ; Male ; Membrane Glycoproteins ; Mice ; Microfilament Proteins ; Phosphoproteins ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; Prostatic Neoplasms ; metabolism ; Response Elements ; Transcription Factors ; physiology ; Transfection ; Tumor Cells, Cultured ; Up-Regulation