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 investigate the changes of the mRNA expressions of myocardial cytoskeletal proteins in endotoxemic rats.

METHODSThirty-seven Wistar rats were randomized into two groups with injection of 10 mg/kg lipopolysaccharide (LPS) or normal saline through the femoral vein. The cardiac function of the rats was monitored continuously for 24 h, and the morphological changes of the cardiac myocytes were observed with HE staining and electron microscope. The mRNA levels of myocardial cytoskeletal proteins including actin, tubulin and desmin were determined by RT-PCR.

RESULTSNo significant difference was found in the number of CD3(+)T lymphocytes in the TILs between different groups. After the immunotherapy, the peLPS injection resulted in significant impairment of the cardiac function and myocardial microstructure of the rats with reduced heart rate and left ventricular systolic pressure (LVSP). The mRNA expression of actin in the cardiac myocytes measured by fluorescence optical density was reduced significantly 8 h after LPS injection, and that of tubulin was decreased significantly 24 h after LPS treatment; desmin mRNA expression showed no significant variation after LPS injection.

CONCLUSIONLPS can significantly impair the cardiac function of the rats possibly by inducing damages of the myocardial cytoarchitecture and causing changes in the mRNA expressions of such cytoskeletal proteins as actin and tubulin.

Actins ; genetics ; metabolism ; Animals ; Cytoskeletal Proteins ; genetics ; metabolism ; Endotoxemia ; chemically induced ; metabolism ; Female ; Lipopolysaccharides ; Myocardium ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Tubulin ; genetics ; metabolism

Nuclear actin which plays a key role in many nucleic processes has become a research hotspot. Baculovirus is the only reported pathogen using nuclear actin to replicate and proliferate. However, little is known about the mechanism of monomeric G-actin accumulation within nuclei of baculovirus-infected cells. It has been reported that AcMNPV ie-1, pe38, ac4, he65, ac102, and ac152 could be required for mediating nuclear localization of G-actin from transiently transfected results in TN-368 cells. In this paper, we found that IE1, AC152, PE38, AC102 localized in the whole cell and PE38, AC102 localized in the nuclear mainly, while both AC4 and HE65 localized in cytoplasm and could be mediated into the nucleus by AC102 and IE1 respectively for the first time. And ie-1 or pe38, ac4, he65 could mediate nuclear G-actin to accumulate partly, while these four genes were sufficient for recruiting G-actin accumulation within the nucleus when driven by promoter OpIE2. Determining the functions of each of these AcMNPV NLA gene products will advance our understanding of baculovirus biology and function of nuclear actin.
Actins ; genetics ; metabolism ; Animals ; Cell Nucleus ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Nucleopolyhedrovirus ; genetics ; metabolism ; Promoter Regions, Genetic ; Protein Transport ; Spodoptera ; metabolism ; virology ; Viral Proteins ; genetics ; metabolism

In mankind, the circulation system is a closed pressure-loaded system; the pressure in circulation flow field would change with the variation of natural or pathological geometry of the local bloodvessel, and the pressure shift induced by the variation of vascular geometry would lead to a series of physiological and pathological changes in the endothelial cells (ECs). This experiment is designed to elucidate the effects of different pressure shift on F-actin alignment and expression in cultured endothelial cells in vitro, and to investigate the relationship between the altered pressure shift and the expression intensity of Vascular adhesion molecule (VCAM) and Integrin alphaVbeta3. Non-activated cultured ECs and single shear stress loaded ECs as control group were set, the double-immuno-fluoro-cytochemistry, laser confocal scanning microscopy and image analysis system were used to observe the expression of VCAM, Integrin alphaVbeta3 and F-actin in endothelial cells which were exposed to levels of pressure shift in an improved parallel plate flow chamber. When exposed to different decreased pressure shift, the expression intensity of VCAM, Integrin alphaVbeta3 and F-actin showed regular changes. The decreased pressure shift resulted in changes in cell alignment and cytoskeleton F-actin, and also affected ECs adhesion function and transmembrane mechanotransduction function which were represented by VCAM and Integrin alphaVbeta3 respectively.
Actins ; genetics ; metabolism ; Cell Adhesion ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Hemodynamics ; Humans ; Integrin alphaVbeta3 ; genetics ; metabolism ; Pressure ; Umbilical Veins ; cytology ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

OBJECTIVE: To investigate the role of IL-17A in promoting the activation of lung fibroblasts and the secretion of chemokine CXCL12, and to explore the possible mechanism. METHODS: Lung tissues of BALB/c mice were collected after intraperitoneal injection of recombinant mouse IL-17A (rmIL-17A). Real-time RT-PCR and Western blotting were used to detect the expression levels of α-smooth muscle actin (α-SMA) and collagen I in lung tissues, and immunohistochemical staining and real-time RT-PCR were used to determine the expression of CXCL12. Normal mouse primary lung fibroblasts were isolated and cultured, and identified by immunofluorescence staining with optical microscopy. Cells and supernatant of culture medium were collected after stimulation with rmIL-17A at different concentrations. mRNA levels of α-SMA, collagen I, and CXCL12 in the cells were determined by real-time RT-PCR, and the levels of collagen I and CXCL12 in the supernatant of culture medium were determined by ELISA. RESULTS: The mRNA and protein levels of α-SMA and collagen I in the lung tissue of mice injected with rmIL-17A were significantly increased compared with the control group (all CONCLUSIONS s: IL-17A can promote the activation of lung fibroblasts and translation into myofibroblast. The secretion of collagen is increased, which promote the deposition of extracullular matrix, and leads to the occurrence and development of lung fibrosis. CXCL12, a chemokine secreted by activated fibroblasts, may be involved in this process.
Actins/genetics* ; Animals ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Fibroblasts/metabolism* ; Interleukin-17/pharmacology* ; Lung/metabolism* ; Mice ; Mice, Inbred BALB C

OBJECTIVETo investigate whether the alterations of microtubule and microfilament expression are responsible for the neurotoxicity of carbon disulfide.

METHODSWistar rats were administered with carbon disulfide by gavage at a dosage of 300 or 500 mg/kg for continuous 12 weeks (five times per week). Spinal cords of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and ultracentrifuged to yield a pellet and a corresponding supernatant fraction. Then, the contents of alpha-tubulin, beta-tubulin, and beta-actin in both fractions were determined by immunoblotting. In the meantime, their mRNA levels in spinal cords were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSIn the supernatant fraction, the contents of beta-tubulin and beta-actin in both treated groups increased significantly (P < 0.01) the content of beta-tubulin increased by 141% and 158% respectively, and the content of beta-actin increased by 19% and 32% respectively. In the pellet fraction, the content of beta-tubulin in both groups increased by 107%(P < 0.01) and 118%(P < 0.01) respectively, and the others keep unaffected. In the meantime, the levels of of mRNA expression of beta-tubulin and beta-actin gene were elevated consistently in CS(2)-treated groups (P < 0.01) the levels of mRNA expression of beta-tubulin increased by 207% and 212% respectively, and the levels of mRNA expression of beta-actin increased by 94% and 91% respectively.

CONCLUSIONCarbon disulfide intoxication results in alternations of microtubule and microfilament expression, and the alternations might be related to its neurotoxicity.

Actins ; genetics ; metabolism ; Animals ; Carbon Disulfide ; poisoning ; Disease Models, Animal ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Spinal Cord ; drug effects ; metabolism ; Tubulin ; genetics ; metabolism

OBJECTIVEWhite matter damage (WMD) in preterm infants is a well-recognized serious complication of prematurity. The collapse of cell skeleton of growth cone after hypoxia-ischemia (HI) is considered as the basic neuropathologic change of the long-term residuals of premature white matter damage. F-actin is the major component of cell skeleton and maintains the normal form of cells, its function and potential mechanism of WMD have not been reported. In this study, changes of F-actin and its influencing factor RhoA were investigated.

METHODSTotally 184 Sprague-Dawley (SD) rats (age 2 days, body weight 6 to 8 grams) were randomly divided into 14 groups: 7 different time WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d, 14 d, 28 d) and 7 corresponding control groups. The 2 day-old SD rats were subjected to ligation of right carotid artery (ischemia), and then they were put into a box full with 6% oxygen and 94% nitrogen for 4 hours (hypoxia). The light microscopy was used to observe the brain pathological changes and the electron microscopy was used to detect the brain ultrastructural changes after hypoxia and ischemia. Eighty SD rats were used for flurescent-immunohistochemical method to detect the distribution of F-actin in cell membrane and cytoplasm of both WMD groups and the control groups at 12 h, 24 h, 48 h, 72 h, 7 d after HI respectively. The distribution of F-actin was reflected by the percentage of non-integrity cells. Another 80 SD rats were used for real time RT-PCR to detect the expression of RhoAmRNA in the white matter tissue of both WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d) and the control groups.

RESULTS(1) Necrosis of lateral ventricle tissue was observed by 72 h after HI. Dilatation of ventricle and formation of capsular space beneath white matter had been observed by 14 d after HI. (2) Disregulation, pyknosis, mitochondrion swelling and chromatin agglutination were observed in WMD groups. The maldevelopment of myelins in WMD groups was detected at 1 h after HI. (3) The fluorescent stains decreased on cellular membrane, but increased in cytoplasm with time. The percentage of non-integrity cells was significantly higher (P < 0.05) in HI groups (0.32 +/- 0.04, 0.43 +/- 0.04, 0.56 +/- 0.03, 0.65 +/- 0.04, 0.87 +/- 0.03) than the controls (0.02 +/- 0.01, 0.02 +/- 0.01, 0.01 +/- 0.01, 0.02 +/- 0.01, 0.02 +/- 0.01). (4) The expression of RhoA mRNA was significantly increased (P < 0.05) in HI groups (1.205, 2.415, 4.830, 1.500) in the white matter tissue compared with the controls (0.300, 0.375, 0.375, 0.530) at 12 h, 24 h, 48 h, 72 h after HI. The expression of RhoA mRNA reached the peak value at HI 48 h, and then gradually decreased. The expression of RhoA mRNA at HI 7 d in WMD group (0.500) was not significantly different from the control (P > 0.05).

CONCLUSION(1) The pathological and ultrastructural changes of white matter in WMD groups after HI suggest that the WMD model was successfully set up in premature 2 days SD rats. (2) F-actin is redistributed within cells after HI: expression in membrane is decreased and expression in plasma was increased. The redistribution possibly results in the collapse and retraction of cells. (3) The expression of RhoA mRNA is increased significantly after HI, which may lead to the redistribution of F-actin. (4) The increase of the expression of RhoA mRNA is not persistent, but the redistribution of F-actin is continued, which suggests that RhoA may not be the only factor affecting the redistribution of F-actin.

Actins ; genetics ; metabolism ; Animals ; Humans ; Hypoxia-Ischemia, Brain ; metabolism ; Infant ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; rhoA GTP-Binding Protein ; genetics ; metabolism

OBJECTIVETo investigate the effect of Smad7 on the expressions of collagen I and alpha-smooth muscle actin (alpha-SMA) in HSC-T6 cell line activated by transforming growth factor-beta1 (TGF-beta1).

METHODSHSC-T6 cells stably expressing M2-flag protein were selected after co-infection of the cells with pTRE-Smad7-M2-flag and pTet-on. The optimal dose of doxycycline for inducing Smad7 was determined, and the effects of Smad7 over-expression on the expressions of collagen I and alpha-SMA in the cells activated by TGF-beta1 and on Smad2/3 phosphorylation were evaluated using Western blotting.

RESULTSThe optimal dose of doxycycline for inducing Smad7 expression was 2 mg/L. Smad7 over-expression induced by doxycycline decreased the expressions of collagen I and alpha-SMA in HSC-T6 cells activated by TGF-beta1, and down-regulated the level of Smad2/3 phosphorylation.

CONCLUSIONSmad7 over-expression inhibits Smad2/3 phosphorylation, and decreases the expression of collagen I and alpha-SMA in HSC-T6 cells induced by TGF-beta1 to inhibit the progression of liver fibrosis.

Actins ; genetics ; metabolism ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Genetic Therapy ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; therapy ; Smad7 Protein ; pharmacology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the role of lung fibroblast activation in radiation-induced lung injury.

METHODSThirty-five male Wistar rats were exposed to a single-dose 30 Gy irradiation of the right hemithorax or sham right lung irradiation. At 1, 7, 14, 28, 56 or 84 days after the irradiation, the rats were sacrificed for examination of alpha-smooth muscle actin (alpha-SMA) expression in the bilateral lung tissues using immunohistochemistry.

RESULTSalpha-SMA expression in fibroblast increased significantly in the out-field and in-field lung tissues within 24 h after irradiation after the irradiation (P<0.001).

CONCLUSIONActivation of the lung fibroblasts occurred within 24 h after irradiation and found in ont-field and in-field lung tissues, suggesting that radiation-induced lung injury may not have an obvious latency.

Actins ; genetics ; metabolism ; Animals ; Fibroblasts ; metabolism ; pathology ; Lung ; cytology ; pathology ; Male ; Radiation Injuries ; pathology ; Rats ; Rats, Wistar

OBJECTIVETo explore the changes in the mRNA expression of adiponectin (Adp), adiponectin receptors(AdpR), and leptin in different adipose tissues of Wannanhua pigs at different stages of development, and their sexual dimorphism.

METHODSFive Wannanhua boars and five Wannanhua gilts were sampled at birth, 30, 45, 90, and 180 days of age respectively. The delta delta Ct relative quantification real-time PCR was used to detect the transcription levels of Adp, AdpR1, AdpR2, and leptin mRNAs in subcutaneous (SC) and perirenal (PR) adipose tissues, and beta-actin were used as internal standards.

RESULTSThe expression level of Adp, AdpR1, AdpR2, and leptin mRNA in SC and PR adipose tissue were changed with age significantly (P < 0.01). In general, Adp mRNA expression in SC adipose tissue was significantly lower than that in PR adipose tissue (P < 0.05), while AdpR1, AdpR2, and leptin mRNA expression in SC adipose tissue were significantly higher than those in PR adipose tissue (P < 0.05 or P < 0.01). Although the sexual dimorphism were found in apart genes or apart days of age, Adp, AdpR1, AdpR2, and leptin mRNA expression both in SC adipose tissue and PR adipose tissue had no significant differences between Wannanhua gilts and boars in general. Significant positive correlation was found between Adp and AdpR1, AdpR2 (P < 0.05 or P < 0.01), and significant negative correlation was found between Adp and leptin (P < 0.05) in SC adipose tissue and PR adipose tissue respectively (P < 0.05).

CONCLUSIONThe expression of Adp, AdpR1, AdpR2, and leptin mRNA in adipose tissue of Wannanhua pigs followed specific developmental patterns and tissue specificity. Adp correlated with its receptors.

Actins ; metabolism ; Adiponectin ; metabolism ; Adipose Tissue ; growth & development ; metabolism ; Animals ; Female ; Leptin ; metabolism ; Male ; RNA, Messenger ; genetics ; Receptors, Adiponectin ; metabolism ; Swine