OBJECTIVETo observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.

METHODSThe recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.

RESULTSInfection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.

CONCLUSIONThere is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.

Adenoviridae ; genetics ; Centrosome ; metabolism ; Genes, p53 ; genetics ; Genetic Vectors ; Humans ; K562 Cells ; Transfection ; Tubulin ; metabolism ; Tumor Suppressor Protein p53 ; 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 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 order to further study the potential interaction between PrP protein and the tubulin and identify the binding region in PrP with tubulin, native tubulin was extracted from rabbit brian tissues, while various recombinant PrP proteins were expressed and purified. The possible molecular interaction between various PrP fusion proteins and tubulin was tested by pull-down and immunoprecipitation assays. Remarkable molecular interaction between the full length PrP and tubulin was observed by pull-down and immunoprecipitation assays. Subsequently, the binding regions within PrP with tubulin were firstly mapped in the aa 23 -- 91 region within N-terminus of PrP peptide. The studies of the association of PrP with tubulin may further provide insight into the unresolved mechanism of active transport of PrP protein in neurons and possible cellular pathways by which prion causes disease.
Animals ; Binding Sites ; genetics ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Humans ; Immunoprecipitation ; Prions ; genetics ; isolation & purification ; metabolism ; Protein Binding ; Rabbits ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tubulin ; metabolism

OBJECTIVE: To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy. METHODS: The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology. RESULTS: The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, P<0.001). TS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all P<0.001). Multivariate analysis revealed that ECOG (Eastern Cooperative Oncology Group)≥2 (HR=2.42, P=0.009) and TUBB3 (HR=2.34, P=0.036) mRNA expression significantly impacted on OS. CONCLUSION High TUBB3 mRNA expression is correlated with resistance to DCF regimen chemotherapy. TUBB3 might be a predictive and prognostic factor in patients with advanced gastric cancer treated with TXT-based chemotherapy. The combined evaluation of TUBB3, TS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.
Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Biomarkers, Tumor ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Drug Resistance, Neoplasm ; Endonucleases ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Stomach Neoplasms ; drug therapy ; genetics ; Thymidylate Synthase ; genetics ; metabolism ; Treatment Outcome ; Tubulin ; genetics ; metabolism

AIMTo perform screening, related to A-kinase anchoring proteins 4 (AKAP4) and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure.

METHODSAn immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carried out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope (TEM) and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes.

RESULTSImmunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group II, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group III, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM.

CONCLUSIONWhile the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-labelling seems to be associated with absent or weak sperm motility.

A Kinase Anchor Proteins ; Humans ; Infertility, Male ; genetics ; physiopathology ; Male ; Microscopy, Electron ; Polymerase Chain Reaction ; Protein Precursors ; genetics ; metabolism ; Semen ; physiology ; Sperm Motility ; Spermatozoa ; physiology ; ultrastructure ; Tubulin ; genetics ; metabolism

Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.
Animals ; Cell Differentiation ; Dependovirus ; genetics ; Epithelial Cells ; cytology ; metabolism ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Lung ; cytology ; Membrane Potentials ; Mucins ; metabolism ; Swine ; Transduction, Genetic ; Tubulin ; metabolism

OBJECTIVETo investigate the protective effects of c-jun antisense gene recombinant transfection on rat cardiomyocytes inflicted by hypoxia and burn serum.

METHODSCardiomyocytes from Wistar rat were isolated and cultured before being divided into normal control (C), transfection (T) and non-transfection (NT) groups. C-jun antisense gene recombinant was constructed and transfected into cardiomyocytes, which were then treated by hypoxia and burn serum in T group, while those in NT group were simply treated by hypoxia and burn serum. The changes in the c-jun mRNA expression were determined by RT-PCR at 1, 3 and 7 hours after the cardiomyocytes being stimulated, and the changes in the expressions of c-jun protein, troponin-T (TnT) and beta-tubulin were assayed by Western blot. The morphological changes in the cardiomyocytes were observed by LM and EM.

RESULTS1) The expressions of c-jun mRNA and protein in the NT group were increased evidently when compared with those in C and T groups. 2) The expressions of TnT and beta-tubulin in NT group were decreased evidently in contrast to those in C and T groups. In addition, there exhibited evident structural derangement, dissolution and fragmentation to granules of cardiomyocytes in NT group, while the myocardial cytoskeletal structure was well preserved with scarce fragmentation and dissolution in T group.

CONCLUSIONIncreased expression of c-jun in rat cardiomyocytes resulting in myocardial injury could be induced by combined treatment of hypoxia and burn serum, while c-jun antisense gene recombinant transfection might protect rat cardiomyocytes from injury.

Animals ; Burns ; blood ; complications ; genetics ; Cells, Cultured ; Hypoxia ; blood ; complications ; genetics ; Myocytes, Cardiac ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Transfection ; Troponin T ; metabolism ; Tubulin ; metabolism

OBJECTIVETo explore the biomarkers and mechanism of kidney toxicity induced by trimethyltin chloride (TMT-Cl) through analyzing the differences of protein expression profiles between vero cells and vero cells exposed to TMT-Cl.

METHODSThe differences of protein expression levels of three paired samples of vero cells and vero cells exposed to TMT-Cl were compared by two-dimensional gel electrophoresis (2-DE) and liquid chromatography-electrospray ionization-linear trap quadrupole (LC-ESI-LTQ). The differences of expression levels of Annexin A1 and α-Tubulin proteins were validated with western blot assay, and the differences of mRNA expression levels of Annexin A1 and α-Tubulin genes were detected with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).

RESULTSFifteen spots of differential expression in protein profiles between vero cells and vero cells exposed to TMT-Cl were found, and 9 of these spots were identified by LC-ESI-LTQ. The expression levels of 3 proteins (Annexin A1,similar to RAN protein and a hypothetical protein) increased and the expression levels of 6 proteins(growth factor receptor-bound protein 10, tubulin alpha 6, heterogeneous nuclear ribonucleoprotein, similar to elongation factor SIII p15 subunit, S-adenosylhomocysteine hydrolase and a hypothetical protein) reduced. The expression levels of α-Tubulin protein and mRNA significantly decreased in vero cells exposed to TMT-Cl, as compared with vero cells (P < 0.01). The expression of Annexin A1 protein in all exposure groups was significantly up-regulated, the expression of Annexin A1 mRNA in the groups exposed to 25 and 50 µmol/L TMT-Cl was significantly down-regulated, and The expression of Annexin A1 mRNA in the group exposed to 100 µmol/L TMT-Cl was significantly up-regulated (P < 0.01).

CONCLUSIONSThe results of present study suggest that 9 proteins with differential expression detected by LC-ESI-LTQ may be related to the kidney toxicity induced by TMT-Cl, which can serve as the biomarkers of early diagnosis and therapeutic effect for the kidney toxicity induced by TMT-Cl.

Animals ; Cercopithecus aethiops ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Profiling ; RNA, Messenger ; genetics ; Transcriptome ; Trimethyltin Compounds ; toxicity ; Tubulin ; genetics ; metabolism ; Vero Cells

ERα36 is a novel subtype of estrogen receptor alpha (ERα) known to play an important role in breast cancer development and widely expressed in normal tissues and cells including nerve cells. However, the expression and function of ERα36 in nerve cells have not been well elucidated. To examine whether ERα36 is involved in differentiation of nerve cells, the differentiated and undifferentiated PC12 (PC12D and PC12unD) cells were used. Transfection of ERα36-shRNA plasmid into PC12 cells was performed to establish the ERα36 gene knock-down cells model. Immunocytofluorescence and Western blot were used to analyze the expression of Nestin, β-tubulinIII and Neu-N in the PC12 cells. The results showed that ERα36 was expressed in both cell types. Compared with PC12D cells, PC12unD cells showed higher expression of Nestin and lower expression of β-tubulinIII. ERα36-shRNA-mediated knock-down of ERα36 expression enhanced the expression of β-tubulinIII and Neu-N, but attenuated Nestin expressions in PC12unD cells; ERα36 knock-down in PC12D cells mediated Nestin, β-tubulinIII and Neu-N in a contrary manner. These results indicate that ERα36 knock-down appear to be associated with inhibiting differentiation in differentiated cells and promoting differentiation in undifferentiated cells, suggesting that ERα36 is a dual regulator in nerve differentiation.
Animals ; Antigens, Nuclear ; metabolism ; Cell Differentiation ; Estrogen Receptor alpha ; genetics ; metabolism ; Gene Knockdown Techniques ; Nerve Tissue Proteins ; metabolism ; Nestin ; metabolism ; Neurons ; cytology ; metabolism ; PC12 Cells ; Rats ; Transfection ; Tubulin ; metabolism