Animals ; Dietary Carbohydrates ; administration & dosage ; Dietary Fats ; administration & dosage ; Dietary Proteins ; administration & dosage ; Humans ; Malnutrition ; complications ; Obesity ; etiology ; Trace Elements ; administration & dosage

AIM: To seek a small interfering RNA ( siRNA ) sequence targeting rat inhibitor of nuclear factor kappa Bα ( IκBα) that can specifically and effectively suppress IκBα mRNA expression of rat ciliary muscles in vivo.METHODS:Three IκBα specific double stranded siRNAs were designed and synthesized. They were transfected into rat A7r5 cells which express IκBα gene. Flow cytometry was used to assess transfected efficiency. The mRNA and protein levels of IκBα were examined by Real Time quantitative polymerase chain reaction ( Real Time-PCR ) and western blot to screen a candidate valid sequence with the highest inhibitory rate. The Cy3 labeled non-specific control siRNA or the candidate valid siRNA was then injected into rat anterior chamber. Distribution of Cy3- siRNA in rat ciliary muscles was viewed by fluorescence microscopy, and the inhibitory effect in vivo of the valid siRNA was identified via Real Time-PCR and immunofluorescence. RESULTS: The suppression effect of the siRNA targeting the CTACGATGACTGTGTGTTT of IκBα gene was most obvious by vitro screening. By anterior chamber injection, this valid siRNA could reach rat ciliary muscles and effectively suppress IκBα gene expression with the highest inhibitory rate of 59. 0% on mRNA level at 24h after RNAi, and 52. 3% on protein level at 72h after RNAi (P<0. 01).CONCLUSION: It proves that the siRNA targeting the CTACGATGACTGTGTGTTT of IκBα gene is the valid sequence to suppress rat IκBα expression of ciliary muscles by RNAi in vivo.

Teaching method for basal experiment, comprehensive experiment, design experiment and teach- ing practice in food microiological analysis were elaborated completely, and design experimental teaching was discussed stress. At the same time, Through introducing various experience of the design experiment teaching, resolvent and way of thinking against problem meeted in design experiment teaching were put forward.

In the present study, the correlated activities of adjacent ganglion cells of transient subtype in response to full-field white light stimulation were investigated in the chicken retina. Pharmacological studies and cross-correlation analysis demonstrated that application of the GABA(A) receptor antagonist bicuculline (BIC) significantly down-regulated the correlation strength while increasing the firing activities. Meanwhile, application of the GABA(A) receptor agonist muscimol (MUS) potentiated the correlated activities while decreasing the firing rates. However, application of the GABA(C) receptor antagonist (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) did not have a consistent influence on either the firing rates or the correlation strength. These results suggest that in the chicken retina, correlated activities among neighborhood transient ganglion cells can be increased while firing activities are reduced with the activation of GABA(A) receptors. The GABA(A)-receptor-mediated inhibitory pathway may be critical for improving the efficiency of visual information transmission.
Action Potentials ; Animals ; Bicuculline ; pharmacology ; GABA-A Receptor Antagonists ; pharmacology ; Mice ; Muscimol ; pharmacology ; Phosphinic Acids ; pharmacology ; Pyridines ; pharmacology ; Receptors, GABA-A ; metabolism ; Retina ; physiology ; Retinal Ganglion Cells ; physiology ; gamma-Aminobutyric Acid

In July 1997, a strain (GB30) of virus was isolated from 60 samples of brain tissues of Murina aurata (Chiroptera: Vespertilionidae) co llec ted in Gengma county, Yunnan province, China. Isolation of virus was negative fr om 4 samples of brain tissues of Rousettus leschenaulti (Chiroptera: Pteropo did ae) collected in Gengma. GB30 virus strain could regularly cause illness and dea th in suckling mice, produced evident CPE in BHK21 cells. It agglutinated red b lood cells of dove at pH5.75～7.4. This virus has been identified serologically by hemagglutination inhibition and immunofluorescent tests using Japanese enceph alitis (JE), dengue (DEN) type 1,2,3,4, and chikungunya (CHIK) viruses monoclona l antibodies, and JE and sindbis (SIN) viruses immune sera. It showed specific r eaction to JE virus only and no reaction with DEN 1～4, CHIK and SIN viruses. Th erefore it can be identified as JE virus. This is the first report on the isolat ion of JE virus from Murina aurata. The results showed that bats are conside red as the reservoir and amplifier host of JE virus transmission in nature.

The relationship between tyrosine phosphorylation (TP) and protein expression of insulin receptor (InsR) and insulin resistance (IR) in patients with gestational diabetes mellitus (GDM) was investigated. The InsR expression and TP in skeleton muscle tissue were determined by Western blotting and immunoprecipitation in women with GDM (GDM group, n=22), normal pregnant women (normal pregnancy group, n=22) and normal non-pregnant women (normal non-pregnant group, n=13). Fasting plasma glucose (FPG) and fasting insulin (FINS) were measured by oxidase assay and immunoradioassay. The results showed that the levels of FPG (5.61±0.78 mmol/L), FINS (15.42±5.13 mU/L) and Homeostasis model assessment-IR (HOMA-IR) (1.21±0.52) in GDM group were significantly higher than those in normal pregnancy group (4.43±0.46 mmol/L, 10.56±3.07 mU/L and 0.80±0.31 respectively) (P<0.01). The levels of FINS and HOMA-IR in normal pregnancy group were significantly higher than those in normal non-pregnant group (7.56±2.31 mU/L and 0.47±0.26 respectively) (P<0.01). There was no significant difference in the InsR expression level among the three groups (P>0.05). TP of InsR with insulin stimulation was significantly decreased in GDM group (0.20±0.05) as compared with normal pregnancy group (0.26±0.06) (P<0.01). TP of InsR with insulin stimulation in normal pregnancy group was lower than that in normal non-pregnant group (0.31±0.06) (P<0.01). TP of InsR with insulin stimulation was negatively related with HOMA-IR in GDM group (r=-0.525, P<0.01). There was no correlation between the protein expression of InsR and HOMA-IR in GDM group (r=-0.236, P>0.05). It was suggested that there is no significant correlation between the protein expression of InsR in skeletal muscle and IR in GDM, but changes in TP of InsR are associated with IR in GDM.

OBJECTIVETo construct DNA methyltransferase 1 (DNMT1) low expression 16HBE cell line and observe the variation of cell cycle and global genomic DNA methylation.

METHODSThe method of Lenti-virus induced RNA interference was applied to introduce four different shRNA fragment into 16HBE cells. Flow cytometry and 5-mC immunofluorescence methods were used to observe the cell cycle and global DNA methylation status of DNMT1 low expression 16HBE cells.

RESULTSThe DNMT1 protein relative expression level of 16HBE-shDNMT1-4 cell line was down regulated about 44% (P < 0.05) compared with the control. No obvious differences of cell cycle and global genome DNA methylation status were observed between the 16HBE and 16HBE-shDNMT1.

CONCLUSIONThe DNMT1 gene low expression cell is successfully constructed, and there are no obvious changes happened on the cell cycle and global genomic DNA methylation.

Cell Cycle ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Methylation ; Down-Regulation ; Epithelial Cells ; metabolism ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics

In the present study, the modulatory effect of AMPA receptors on gamma-aminobutyric acid (GABA) transporter current was investigated on enzymatically isolated horizontal cells of carp retina. The GABA transporter current elicited by 1 mmol/L GABA was decreased immediately after pre-application of AMPA (30 mumol/L or 3 mmol/L) for 50 s. Application of 10 mmol/L BAPTA in intracellular solution inhibited the suppression effect of AMPA on GABA transporter current. The suppression effect induced by co-application of 3 mmol/L AMPA and 3 mmol/L NMDA was similar to that of 3 mmol/L AMPA or 3 mmol/L NMDA alone. These results suggest that the activation of AMPA receptors inhibits GABA transporter-mediated current by affecting intracellular Ca(2+) processes in the retinal horizontal cells, which is identical with the modulatory effect of NMDA receptors on GABA transporters.
Animals ; Carps ; Egtazic Acid ; analogs & derivatives ; pharmacology ; GABA Plasma Membrane Transport Proteins ; metabolism ; Receptors, Ionotropic Glutamate ; metabolism ; Retinal Horizontal Cells ; metabolism ; gamma-Aminobutyric Acid ; pharmacology

OBJECTIVETo study the expression and switching of Th1/Th2 cytokines gene in human gliomas and its effects on occurring and developing of human gliomas.

METHODSInterleukin (IL)-2 and interferon-gamma represent Th1 type cytokines. IL-4, IL-6, IL-10, and IL-13 represent Th2 type cytokines. The gene expressions of Thl/Th2 cytokines in human glioma cells, glioma infiltrating lymphocytes, and glioma cell lines were detected by reverse transcription polymerase chain reaction (RT-PCR). The biological activity of cytokines in the supernatant of glioma cell lines was assayed by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe total positive rates of Th1 and Th2 type cytokines gene in human glioma cells were 14.77% and 75%. The total positive rates of Th1 and Th2 type cytokines gene in glioma infiltrating lymphocytes were 22.73% and 68.17%. There was obviously predominant expression of Th2 type cytokines in human glioma tissues, glioma infiltrating lymphocytes, and glioma cell lines. There was no unbalanced expression of Th1/Th2 cytokines in normal brain tissues.

CONCLUSIONThere is a predominant expression of Th2 type cytokines in human glioma cells. The switching of Thl/Th2 cytokines gene may play an important role in the occurring and developing of human gliomas.

Adolescent ; Adult ; Aged ; Astrocytoma ; metabolism ; pathology ; Brain Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Child ; Child, Preschool ; Cytokines ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Glioblastoma ; metabolism ; pathology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-6 ; metabolism ; Male ; Middle Aged ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism

It was previously found that the efficacy of synaptic transmission between retinal cone systems and luminosity-type horizontal cells (LHCs) was activity-dependent. Repetitive activation of red-cone pathway increased the LHCos hyperpolarizing response to red light, and the response enhancement was reversible. In this study, intracellular recording and pharmacological method were applied to investigate the mechanism(s) underlying red-flickering-induced response enhancement. Lowering intracellular Ca(2+) in the LHC by intracellular injection of Ca(2+) chelator EGTA prevented the development of red-flickering-induced response enhancement, which implicates the importance of postsynaptic calcium signal. The response enhancement could also be eliminated by a potent antagonist of Ca(2+)-permeable AMPA receptor (CP-AMPAR), which suggests the possibility that Ca(2+) influx via glutamate-gated calcium channels is related to the changes of [Ca(2+)](i). Furthermore, the administration of ryanodine or caffeine also attenuated the phenomenon, which gives evidence that the local calcium signal caused by intracellular calcium-induced calcium release (CICR) may be involved. Taken together, our data implicate that postsynaptic CICR and CP-AMPAR are related to the activity-dependent response enhancement.
Animals ; Caffeine ; pharmacology ; Calcium ; metabolism ; Carps ; Neuronal Plasticity ; physiology ; Receptors, AMPA ; physiology ; Retina ; cytology ; Retinal Cone Photoreceptor Cells ; physiology ; Ryanodine ; pharmacology ; Ryanodine Receptor Calcium Release Channel ; physiology ; Signal Transduction ; physiology ; Synapses ; physiology