OBJECTIVE: To observe the expression of aquaporin 4 (AQP4) in diffuse brain injury (DBI) of rats and to explore the corresponding effect of AQP4 for brain edema. METHODS: The rat model of DBI was established using Marmarou's impact-compression trauma model. Brain water content was measured by dry-wet weight method. Blood-brain barrier permeability was evaluated by Evans blue (EB) staining. Immunohistochemical method was used to observe the expression of AQP4. RESULTS: Brain water content increased after 3 h and peaked at 24 h after DBI. Brain EB content significantly increased and peaked at 12 h after DBI. The expression of AQP4 significantly increased after 3 h and peaked at 24 h after DBI, and the number of AQP4 positive astrocytes increased. CONCLUSION The increment of the permeability of blood-brain barrier and the expression of AQP4 may contribute to the development of brain edema in rat DBI. The change of AQP4 expression in astrocytes may also contribute to determine DBI.
Animals ; Aquaporin 4/metabolism* ; Astrocytes ; Blood-Brain Barrier/metabolism* ; Brain ; Brain Edema/metabolism* ; Brain Injuries/metabolism* ; Cell Membrane Permeability/genetics* ; Disease Models, Animal ; Permeability ; Rats ; Water

OBJECTIVETo observe the effect of 25-hydroxyvitamin D3 on the permeability and ZO-1 expression in normal human airway epithelial cells.

METHODSMTT assay was used to assess the viability of human airway epithelial cell line 16HBE following a 24-hour exposure to different concentrations of 25-hydroxy vitamin D3, and the transepithelial electrical resistance (TER) of the cell monolayer was measured using a Millicell-ERS voltohmmeter. Real-time quantitative RT-PCR was employed to determine the changes of ZO-1 mRNA expression in the cells following the exposures.

RESULTSExposure to 25-hydroxyvitamin D3 resulted in significantly increased permeability of 16HBE cells, but the exspression of ZO-1 showed no obvious changes. 25-hydroxyvitamin D3 at 4×10(-9) mol/L showed the strongest effect in increasing the permeability of cell monolayer.

CONCLUSION25-hydroxyvitamin D3 increases the permeability of normal bronchial airway epithelial cell monolayer in vitro, but this effect is not mediated by upregulation of ZO-1 expression.

Bronchi ; cytology ; metabolism ; Calcifediol ; pharmacokinetics ; pharmacology ; Cell Line ; Cell Membrane Permeability ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Zonula Occludens-1 Protein ; genetics ; metabolism

To construct, express, purify and identify the Myc-R9-EGFP fusion protein and validate its transduction activity in the cultured porcine embryo fibroblasts. cDNA of pig c-Myc gene was amplified by RT-PCR with specific primers of 9 arginine (R9) from the primordial genital ridges and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the recombinant plasmid was then transformed into BL21 (Escherichia coli) strain. After IPTG induction, the target fusion protein was efficiently induced to express, successfully purified by Novagen His-Bind kit, identified by SDS-PAGE and Western blotting. Finally, its high transduction activity in the porcine embryo fibroblasts was validated. The purified Myc-R9-EGFP fusion protein and the validation of its transduction activity in fibroblasts have provided an experimental foundation for further studies on the biological characterization of Myc protein, and soundly facilitated the further study of establishing pig induced pluripotent stem cells by recombinant protein.
Animals ; Arginine ; genetics ; metabolism ; Cell Line ; Cell Membrane Permeability ; drug effects ; Escherichia coli ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacokinetics ; Swine

OBJECTIVETo deliver the naked genes into cells through the bioeffects of cell membrane porous produced by low-frequency ultrasound (US) and to investigate the safety by determining the threshold of cell damage and membrane permeability.

METHODSThe suspension of red cells from chickens, rabbits, rats, and S180 cells was exposed to calibrated US field with different parameters in still and flowing state. Laser scanning confocal microscopy, fluorescent microscopy, scanning electron microscopy, flow cytometry and spectrophotometry were used to examine cell morphology, membrane permeability, enzymes, free radicals, naked gene expression efficiency, threshold of cell damage and cell viability.

RESULTSThe plasmid of green fluorescent protein (GFP) as a reporter gene was delivered into S180 cells under optimal conditions without cell damage and cytotoxicity. The transfection rate was (35.83 +/- 2.53)% (n = 6) in viable cells, and the cell viability was (90.17 +/- 1.47)% (n = 6). Also, malondialdehyde, hydroxyl free radical, alkaline phosphatase, and acid phosphatase showed a S-shaped growth model (r = 0.98 +/- 0.01) in response to the permeability change and alteration of cell morphology. The constant E of energy accumulation in US delivery at 90% cell viability was an optimal control factor, and at 80% cell viability was the damage threshold.

CONCLUSIONUS under optimal conditions is a versatile gene therapy tool. The intensity of GFP expression in US group has a higher fluorescent peak than that in AVV-GFP group and control group (P < 0.001). The optimal gene uptakes, expression of gene and safety depend on E, which can be applied to control gene delivery efficiency in combination with other parameters. The results are helpful for development of a novel clinical naked gene therapeutic system and non-hyperthermia cancer therapeutic system.

Animals ; Cell Membrane ; chemistry ; metabolism ; ultrastructure ; Cell Survival ; genetics ; Cells, Cultured ; Chickens ; DNA Damage ; genetics ; Free Radicals ; metabolism ; Genes, Reporter ; genetics ; Green Fluorescent Proteins ; genetics ; Malondialdehyde ; metabolism ; Permeability ; Plasmids ; genetics ; Porosity ; Rabbits ; Rats ; Risk Assessment ; Transfection ; methods ; Ultrasonics

OBJECTIVETo determine the relationship of thick greasy tongue fur formation and permeability of vascular endothelial cells (ECs) with the protein expression of zonula occludens-1 (ZO-1).

METHODSSprague Dawley rats were randomly divided into a model group of severe acute pancreatitis (SAP) and a sham-operated (SO) group. The SAP rats were further divided into two subgroups on the basis of tongue-coating status: a thick greasy tongue fur group (SAP-TGF) and a normal tongue fur group (SAP-NF). Six rats were chosen randomly from every group mentioned above for an Evans blue assay 5 days after model establishment. For the histomorphology analysis, the expressions of ZO-1 protein and mRNA were studied by hematoxylin-eosin (H&E) staining, transmission electron microscope, Western blot, and Q-PCR using blood and tongue tissues, which were collected from 8 rats randomly chosen from each group.

RESULTSThe papillae density of the rat tongue surface and the caryocinesis frequency of the basal layer were significantly increased in the SAP-TGF group compared with the SO group (P<0.05). Evans blue levels in the tongue tissue of the SAP-TGF group were significantly higher than that of the SO and SAP-NF groups (P<0.05). Vascular ECs were wider and obviously fissured in the SAP-TGF group under transmission electron microscope observation. The protein and mRNA expression of ZO-1 in the SAP-TGF group were lower than those in the SAP-NF (P<0.05).

CONCLUSIONSReproductive activity enhancement of glossal epithelial cells was one of the main characteristics of thick greasy tongue fur formation. An increase in vasopermeability was closely associated with thick greasy tongue fur formation. Tight junction structural variation of vascular ECs might play an important role in the pathological and physiological process of thick greasy tongue fur formation.

Animals ; Blotting, Western ; Cell Membrane Permeability ; Endothelial Cells ; cytology ; Evans Blue ; metabolism ; Gene Expression Regulation ; Male ; Membrane Proteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tongue ; pathology ; ultrastructure ; Zonula Occludens-1 Protein

OBJECTIVETo study the effect of hypoxia-inducible factor-1alpha (HIF-1alpha) inhibition caused by RNA interference on permeability of hypoxic vascular endothelial (VE) cells.

METHODSPlasmid pcDNA6.2-GW/EmGFP-miR was applied to construct the RNA interference expression vector targeted to human HIF-1alpha gene. VE cells were divided into normal control group (NC), hypoxia group (H, cells were treated for hypoxia in mixed gas with 1% O(2) for 6 hours), transfection group (T), and transfection hypoxia group (TH, transfected with vector and treated with hypoxia). Expression of HIF-1alpha mRNA in NC and T groups were determined with RT-PCR. Expression of HIF-1alpha protein in each group was determined with Western blot. The permeability of VE cell monolayer was detected by fluorospectrophotometer. Another sample of VE cells were divided into dimethyloxallyl glycine (DMOG) group, transfected with DMOG group (TD), normal control group (NC), and transfection group (T), with 1 mmol/L DMOG (HIF-1alpha specific derivant) replacing hypoxia treatment. The expression of HIF-1alpha protein in each group was determined with Western blot. All data were recorded as density value ratio except for permeability data, which was recorded as fluorescence intensity value. Data were processed with t test (pairwise comparison among groups).

RESULTSThe relative content of HIF-1alpha mRNA of cells in NC group (0.765 +/- 0.069) was significantly higher than that of cells in T group (0.093 +/- 0.007, t = 16.696, P < 0.05). Content of HIF-1alpha protein of cells in TH group (0.591 +/- 0.029) was significantly lower than that of cells in H group (2.612 +/- 0.259, t = 13.415, P < 0.05). Content of HIF-1alpha protein of cells in TD group (0.566 +/- 0.008) was significantly lower than that of cells in DMOG group (3.243 +/- 0.551, t = 6.975, P < 0.05). The permeability of cell monolayer in H group (41.6 +/- 11.1) was significantly higher than that of cell monolayer in NC group (9.4 +/- 1.5, t = 6.238, P < 0.05). The permeability of cell monolayer in TH group (13.3 +/- 4.5) was markedly lower than that of cell monolayer in H group (t = 5.430, P < 0.05).

CONCLUSIONSThe expression of HIF-1alpha gene in vascular endothelial cells is effectively inhibited by specific RNA interference, which significantly prevents the hypoxia-induced increase in vascular endothelial cell permeability.

Base Sequence ; Cell Hypoxia ; Cell Line ; Cell Membrane Permeability ; Endothelial Cells ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Molecular Sequence Data ; RNA Interference ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo study the synergistic effects of hypothermia and hypoxia on the damage of pulmonary microvascular endothelial cells (PMVEC) in rat.

METHODSPrimary PMVECs were obtained by complex phosphoesterasum digesting from isolated lung tissues of Wistar rats, the PMVECs were identified by phase contrast microscope and immunofluorescence studies for CD31 antigen and bandeiraea simplicifolia isolectin (BSI) binding test. Factorial design was adopted in trial according to hypothermia and hypoxia existing or not. Using corresponding kit measured the levels of lactate dehydrogenase (LDH) activity in cell medium. Level of nitric oxide (NO) concentration was measured by Griess Assay. RT-PCR was used to examine the expression of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) mRNA in PMVECs.

RESULTSThe monolayer of cultured PMVECs displayed the shape of pavingstone. CD31 antigen and binding BSI results by fluorescence microscope identified the cultured cells were PMVECs. Compared to the control group, LDH activity and VEGF, ET-1 expression levels were significantly increased in hypothermia group, hypoxia group and hypoxia combined with hypothermia group. And the levels of NO concentration were reduced in these three groups. The results of One-way ANOVA showed that there was a synergistic effect between hypothermia and hypoxia.

CONCLUSIONHypothermia and hypoxia both have an effect on PMVECs whether in altering the cell permeability or in releasing of vasoactive substances including NO and ET-1. In addition, there is a synergistic effect between hypothermia and hypoxia.

Animals ; Cell Hypoxia ; Cell Membrane Permeability ; Cells, Cultured ; Cold Temperature ; Endothelial Cells ; cytology ; metabolism ; Endothelin-1 ; metabolism ; Endothelium, Vascular ; cytology ; Lung ; blood supply ; Male ; Nitric Oxide ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; metabolism

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
Animals ; Cell Membrane Permeability ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Cells, Cultured ; Gene Knockdown Techniques ; Mice ; Mice, Knockout ; Podocytes ; drug effects ; physiology ; Puromycin Aminonucleoside ; pharmacology ; TRPC Cation Channels ; genetics ; metabolism

HIV-1 Tat is considered to be one of key players to facilitate monocyte entry into the CNS, which is characteristic feature of AIDS-related encephalitis and dementia. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the HIV-1 Tat-induced signaling pathways leading to NF-kappaB activation, expression of adhesion molecules, and monocyte adhesion in CRT-MG human astroglioma cells by using cell-permeable SOD. When cell-permeable SOD was added to the culture medium of CRT-MG cells, it rapidly entered the cells in dose- and time-dependent manners. Treatment of astrocytes with cell-permeable SOD led to decrease in Tat-induced ROS generation as well as NF-kappaB activation. Cell-permeable SOD inhibited the activation of MAP kinases including ERK, JNK and p38 by HIV-1 Tat. Treatment of CRT-MG cells with cell-permeable SOD significantly inhibited protein and mRNA levels of ICAM-1 and VCAM-1 up-regulated by HIV-1 Tat, as measured by Western blot analysis and RT-PCR. Furthermore, enhanced adhesiveness of monocyte to astrocyte by HIV-1 Tat was significantly abrogated by pretreatment with cell-permeable SOD fusion proteins. These data indicate that SOD has a regulatory function for HIV-1 Tat-induced NF-kappaB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.
Astrocytes/*enzymology ; Cell Adhesion/*physiology ; Cell Membrane Permeability ; Gene Products, tat/*pharmacology ; HIV Infections/metabolism ; HIV-1/*chemistry ; Humans ; Monocytes/cytology/*drug effects ; Signal Transduction ; Superoxide Dismutase/genetics/*physiology

Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X(L) overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met(mut)) and Bcl-X(L)-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-X(L) in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X(L) overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X(L) appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X(L) may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X(L) overexpression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.
Animals ; Apoptosis ; Cell Line, Tumor ; Cell Membrane Permeability ; Humans ; Membrane Potential, Mitochondrial ; physiology ; Mice ; Mitochondrial Membrane Transport Proteins ; physiology ; Mitochondrial Membranes ; metabolism ; Mutation ; Necrosis ; Proteins ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Rhodamine 123 ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; physiology ; bcl-X Protein ; metabolism