Actins ; genetics ; Adolescent ; Cardiomyopathy, Dilated ; genetics ; Child ; Child, Preschool ; Desmin ; genetics ; Female ; Humans ; Infant ; Male ; Mutation

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 optimal transfection method for human umbilical vein endothelium, so as for us to establish a basic method for the further transfection with other objective genes.

METHODSThe pEGFP-Actin plasmid was employed as an ectogenetic gene and was transfected into ECV-304 endothelium cells by lipofection, DEAE-dextran or electroporation transfection methods, respectively. The cell death rate and transfection rate after the transfection were compared among the three methods.

RESULTS(1) Twelve hours after the transfection, the cell death rate was 4% by all three methods, however, the cell death rate increased up to 50% at 60 hours after the transfection by DEAE-dextran. (2) The transfection rate of ECV-304 could reach 95% by all the three methods. But the fluorescent intensity was stronger in lipofection group compared with electroporation method.

CONCLUSIONGene transfection into ECV-304 with lipofection and electroporation methods exhibited better stability and repeatability.

Actins ; genetics ; Cells, Cultured ; Electroporation ; Endothelium, Vascular ; physiology ; Genetic Vectors ; genetics ; Humans ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; Transfection ; methods

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

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

OBJECTIVETo investigate the expression of vitamin D3 upregulated protein-1 (VDUP1) in peripheral eosinophils at different stages of asthma and its correlation with clinical manifestations of asthma.

METHODSFourteen normal volunteers and 51 mild to moderate asthma patients, including 16 untreated patients with asthma attack and 35 with asthma remission by continuously corticosterone inhalation. The symptom severity and pulmonary function indices were evaluated and induced sputum eosinophil counts and blood eosinophil count measured. VDUP1 and beta-actin gene fragments were amplified simultaneously by RT-PCR from the total RNA of peripheral eosinophils, and 10 microl of the RT-PCR product underwent agarose gel electrophoresis and the VDUP1/beta-actin ratio was obtained by Gel-Pro software.

RESULTSVDUP1/beta-actin ratio significantly decreased in untreated patients with asthma attacks in comparison with normal volunteers (0.314+/-0.242 vs 0.532 +/-0.279) but not in patients with asthma remission (0.612+/-0.381). In the former patients, a positive correlation of VDUP1/beta-actin ratio was found with FEV1.0% (r=0.587, P=0.046) and %PEF (r=0.563, P=0.033), whereas an inverse one observed with sputum eosinophil count (r=-0.436, P=0.049).

CONCLUSIONVDUP1 expression in the eosinophils correlates to eosinophil activation and may influence the disease severity of asthma patients.

Actins ; biosynthesis ; genetics ; Asthma ; blood ; metabolism ; Carrier Proteins ; biosynthesis ; genetics ; Eosinophils ; metabolism ; Humans ; Maximal Expiratory Flow-Volume Curves ; Peak Expiratory Flow Rate ; Respiratory Function Tests ; Thioredoxins ; biosynthesis ; genetics

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

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 explore the feasibility of real-time RT-PCR of housekeeping mRNA degradation in dead rats in order to seek new suitable techniques for post-mortem interval (PMI). METHODS: The levels of GAPDH mRNA and beta-actin mRNA in the brain and spleen of the rats were measured at different times after death by the SYBR Green I real-time RT-PCR. The relative mRNA level was indicated with the Ct value, then the relationship between the Ct value and PMI were analyzed and the corresponding regression equation was obtained. RESULTS: The Ct values of GAPDH mRNA and beta-actin mRNA by SYBR Green I real time RT-PCR correlated well with the PMI. CONCLUSION The SYBR Green I real-time RT-PCR is a reliable technique for studying mRNA degradation. Adoption of the housekeeping genes eliminates systematical errors of other genes which have individual difference. As an objective and dynamic indication, Ct value has a good correlation with PMI, and could be applied for estimating PMI, especially in late period.
Actins/genetics* ; Animals ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Postmortem Changes ; RNA Stability/genetics* ; RNA, Messenger/metabolism* ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction/methods* ; Time Factors

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.
Real-Time Polymerase Chain Reaction/methods* ; Paeonia/genetics* ; Actins/genetics* ; Reproducibility of Results ; Transcriptome ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Reference Standards ; Gene Expression Profiling/methods*