OBJECTIVETo establish a novel in-vitro culture device simulating distraction osteogenesis, and to understand the the changes of cytoskeleton of osteoblasts under different magnitudes of mechanical distraction.

METHODSA novel in-vitro culture device simulating distraction osteogenesis was developed with titanium and silicon membrane. The expressions of F-actin cytoskeleton subjected to different magnitudes of distraction were observed in situ under a confocal laser scanning microscope, and protein levels of actin and tubulin alpha were analyzed by electrophoresis of Western blot.

RESULTSThe mode of culture device simulating distraction osteogenesis was similar to that of distraction osteogenesis used in clinic. After 24 h mechanical distraction with physiological magnitude, F-actin of osteoblasts was organized and paralleled to the direction of distraction forces. After 24 h mechanical distraction with hyperphysiological magnitude, however, F-actin of osteoblasts became reorganized and disrupted, and protein levels of actin and tubulin alpha decreased.

CONCLUSIONSThe novel in vitro culture device simulating distraction osteogenesis is suitable for the study of distraction osteogenesis in vitro. Physiological mechanical distraction causes organization and rearrangement of actin cytoskeleton. However, hyperphysiological mechanical distraction results in reorganization and disruption of actin cytoskeleton.

Actins ; biosynthesis ; Animals ; Cells, Cultured ; Cytoskeleton ; physiology ; Models, Biological ; Osteoblasts ; cytology ; Osteogenesis, Distraction ; Rats ; Tubulin ; biosynthesis

OBJECTIVETo study the mechanism of integrin in hypertrophic scar.

METHODSFibroblasts from 10 samples of human hypertrophic scars was cultured, FQ-PCR assay was applied to detect mRNA expression of alpha-SMA in hypertrophic scar fibroblasts after integrin and FAK antibody blocking.

RESULTSmRNA of alpha-SMA in fibroblasts expressed obviously lower after integrin and FAK antibody blocking than that of control groups ( P < 0.05).

CONCLUSIONThrough accelerating the synthesis of alpha-SMA, integrin and FAK play an important role in contracture of hypertrophic scar.

Actins ; biosynthesis ; Adult ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Contracture ; metabolism ; pathology ; Fibroblasts ; metabolism ; Focal Adhesion Kinase 1 ; biosynthesis ; Humans ; Integrins ; biosynthesis ; RNA, Messenger ; metabolism ; Young Adult

The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRedl-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 micros electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.
Actins ; genetics ; metabolism ; Electroporation ; Luminescent Proteins ; biosynthesis ; genetics ; Physarum polycephalum ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Transcriptional Elongation Factors ; genetics

OBJECTIVETo screen the stage-specific expression proteins during rats spermatogenesis, and to investigate the beta-actin expression and localization in the tissues of rat testicular.

METHODSHighly enriched type A spermatogonia, pachytene spermatocytes and round spermatids were isolated by STAPUT method (sedimentation velocity at unit gravity, with 2% - 4% BSA gradient in DMEM/F12 medium) respectively to get the total proteins. The difference of protein expression between the three kinds of cells was analyzed by two-dimensional electrophoresis. Then the distribution of beta-actin in rat testicular tissues was investigated using specific anti-beta-actin antibodies by immunohistochemical method.

RESULTSbeta-actin was identified as a stage-specific expression protein by two-dimensional electrophoresis. beta-actin protein was more strongly expressed in type A spermatogonia and pachytene spermatocytes, but not in round spermatids. The immunohistochemical results showed that beta-actin was mainly located in the cytoplasm of type A spermatogonia and pachytene spermatocytes and in the nuclei of nearly mature spermatids.

CONCLUSIONbeta-actin protein is a stage-specific expressed protein and may play an important role in spermatogenesis.

Actins ; biosynthesis ; Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Mass Spectrometry ; Rats ; Rats, Sprague-Dawley ; Spermatogenesis ; physiology ; Testis ; cytology ; metabolism

To investigate the role and mechanism of mechanical stress on arterial remodeling, a new model of common carotid artery exposed to stress in vivo was established in rat, in which the change of pressure is the only influencing factor. The effect of high pressure on the morphology and expression of alpha-actin and proliferating cell nuclear antigen (PCNA) of vascular smooth muscle cells (VSMCs) in the artery was assessed. The arteries were perfused by both high pressure (160 mmHg) and normal pressure (80 mmHg) for 6 hours. The changes of morphology, expression of alpha-actin and PCNA in VSMCs were studied by histology and immunohistochemistry. The results showed that the new model could be controlled well in pressure and frequency. The euchromatin was increased and PCNA-positive particles were observed in the nuclei of VSMCs, but the expression of alpha-actin was decreased when the arteries were exposed to the high pressure. The new model has been successfully established, which provides a new tool for studying the effect of mechanical stress on arterial remodeling. In this experiment, VSMCs underwent a transformation from contractile phenotype into synthetic phenotype and tended to proliferate in response to the high pressure.
Actins ; biosynthesis ; Animals ; Carotid Artery, Common ; cytology ; physiology ; Chemotherapy, Cancer, Regional Perfusion ; Male ; Muscle, Smooth, Vascular ; cytology ; Pressure ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of 8-methoxypsoralen on human melanocytes [Ca(2+)]i and cytoskeleton actin organization in vitro.

METHODSHuman melanocytes were obtained from normal foreskins. Laser confocal microscope was employed to measure [Ca(2+)]i and rhodamine-conjugated phalloidin was used to visualize the cytoskeleton actin.

RESULTS8-methoxypsoralen increased [Ca(2+)]i and induced organization of actin stress fiber cytoskeleton.

CONCLUSION8-methoxypsoralen might influence the migration of melanocytes by increasing the intracellular free Ca(2+) concentration and cytoskeleton actin reorganization.

Actins ; biosynthesis ; genetics ; Calcium ; metabolism ; Cell Movement ; drug effects ; Cells, Cultured ; Cytoskeletal Proteins ; biosynthesis ; genetics ; Humans ; Melanocytes ; cytology ; drug effects ; metabolism ; Methoxsalen ; pharmacology ; Skin ; cytology

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.
Actins/biosynthesis* ; Cell Differentiation/physiology* ; Cell Movement/physiology* ; Electric Stimulation Therapy ; Electricity ; Fibroblasts/physiology* ; Humans ; Myofibroblasts/physiology* ; Proliferating Cell Nuclear Antigen/biosynthesis* ; Skin/cytology*

Cardiotrophin-1 (CT-1) activates a distinct form of cardiac muscle cell hypertrophy in which the sarcomeric units are assembled in series. The aim of the study was to determine the expression pattern of sarcomeric contractile protein α-actin, specialized cytoskeletal protein α-actinin and mitochondrial uncoupling protein-2 (UCP2) in myocardial remodeling induced by chronic exposure to CT-1. Kunming mice were intraperitoneally injected with carboxy-terminal polypeptide (CP) of CT-1 (CT-1-CP, 500 μg·kg(-1)· day(-1)) for 1, 2, 3 and 4 week (s), respectively (4 groups obtained according to the injection time, n=10 each, with 5 males and 5 females in each group). Those injected with physiological saline for 4 weeks served as controls (n=10, with 5 males and 5 females). The heart tissues of mice were harvested at 1, 2, 3 or 4 week (s). Immunohistochemistry (IHC) and Western blotting (WB) were used to detect the distribution and expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2 in myocardial tissues. IHC showed that α-actin was mainly distributed around the nuclei of cardiomyocytes, α-actinin concentrated around the striae and UCP2 scattered rather evenly in the plasma. The expression of α-actin was slightly greater than that of α-actinin and UCP2 in the control group (IHC: χ(2)=6.125; WB: F=0.249, P>0.05) and it gradually decreased after exposure to CT-1-CP. There was no significant difference in the expression of α-actin between the control group and the CT-1-CP-treated groups (χ (2)=7.386, P>0.05). But Western blotting revealed significant difference in the expression of α-actin between the control group and the 4-week CT-1-CP-treated group (F=2.912; q=4.203, P<0.05). Moreover, it was found that the expression of α-actinin increased stepwise with the exposure time in CT-1-CP-treated groups and differed significantly between CT-1-CP-treated groups and the control group (ICH: χ (2)=21.977; WB: F=50.388; P<0.01). The expression of UCP2 was initially increased (WB: control group vs. 1- or 2-week group, q values: 5.603 and 9.995, respectively, P<0.01) and then decreased (WB: control group vs. 3-week group, q=4.742, P<0.01; control group vs. 4-week group, q=0.558, P>0.05). It was suggested that long-term exposure to CT-1-CP could lead to the alteration in the expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2. The different expressions of sarcomeric structure proteins and mitochondrial UCP2 may be involved in myocardial remodeling.
Actinin ; biosynthesis ; Actins ; biosynthesis ; Animals ; Cardiomegaly ; chemically induced ; metabolism ; pathology ; Cytokines ; adverse effects ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Ion Channels ; biosynthesis ; Male ; Mice ; Mitochondrial Proteins ; biosynthesis ; Myocardium ; metabolism ; pathology ; Sarcomeres ; metabolism ; pathology ; Uncoupling Protein 2

Malignant fibrous histiocytoma(MFH) is a rare primary neoplasm that constitutes less than 1% of the malignant tumors of bone, and involvement of the skull is very rare. We present a case of malignant fibrous histiocytoma of the skull, presenting an intraosseous lesion in a 43-yr-old woman. She had a rapidly growing, tender mass in the right parietal region. A plain radiograph showed an osteolytic lesion of the right parietal bone. Magnetic resonance imaging revealed that the lesion showed heterogeneous low signal intensity on T1-weighted images and slightly high signal intensity on T2-weighted images. No evidence of an extraosseous extension to the adjacent dura and soft tissue was found, and a wide excision of the parietal bone was performed. Histologically, the tumor was a typical MFH displaying pleomorphic spindle cells in a storiform pattern. The results of immunohistochemical stainings revealed that the tumor cells were positive for vimentin, alpha-1-antitryp-sin, and p53, and negative for smooth muscle actin, S100 protein, desmin, and MyoD1. Three months later, a mainly cystic, recurrent mass was developed at the previously operated site. Before the resection, we first performed the percutaneous aspiration cytology, revealing diagnostic multinucleated pleomorphic cells. There-after, she had to receive repetitive resections of recurrent or residual lesions, and she died of postoperative meningoencephalitis two years after the first operation.
Actins/biosynthesis ; Adult ; Brain/pathology ; Desmin/biosynthesis ; Female ; Giant Cells/metabolism ; Histiocytoma, Fibrous/*diagnosis ; Human ; Immunohistochemistry ; Magnetic Resonance Imaging ; Mitosis ; Muscle, Smooth/metabolism ; MyoD Protein/biosynthesis ; Protein p53/biosynthesis ; S100 Proteins/biosynthesis ; Skull Neoplasms/*diagnosis ; Tomography, X-Ray Computed ; Vimentin/biosynthesis ; alpha 1-Antitrypsin/biosynthesis

Actins ; biosynthesis ; genetics ; Adult ; Aged ; Female ; Hepatitis B, Chronic ; complications ; Humans ; Liver Cirrhosis ; blood ; enzymology ; virology ; Male ; Matrix Metalloproteinase 1 ; biosynthesis ; genetics ; Middle Aged ; Plasminogen Activator Inhibitor 1 ; blood ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; Urokinase-Type Plasminogen Activator ; blood