Objective To investigate the mechanism underlying the WNK4 kinasemediated inhibitory effect on BK channel. Methods Cos-7 cells were cotransfected with BK in combination with either CD4 (control group) or wild type WNK4 (WNK4-WT).Immunostaining and confocal microscopy,chemiluminescence,Western blotting analysis were then employed to determine the BK localization in cells,BK surface expression and total protein level,respectively.To further investigate whether the reduction of BK protein expression is due to an increase in degradation through a lysosomal pathway,BK protein level was determined after treated with bafilomycin A1(Baf A1),a proton pump inhibitor affecting lysosomal degradation. Results Immunostaining and confocal microscopic study showed that BK was localized both in plasma membrane and cytosol in the control group.After cells transfected with WNK4-WT,BK expression was markedly reduced.Chemiluminescent assay found that BK surface expression level was 299.9±18.6 in the control group,whereas it was significantly reduced (148.4±13.7,P＜0.01) in the WNK4-WT group.Western blotting analysis showed that total BK protein level was markedly reduced in the presence of WNK4-WT compared to the control group.WNK4-WT was shown to significantly reduce the BK total protein level (42.3％±15.2％) compared to the control group (100％) (P＜0.01).When the cells was treated with Bafilomycin A1 (Baf A1,0.5 μmol/L),WNK4-mediated reduction in BK protein was reversed (82.2％±12.1％,P＜0.05). Conclusions WNK4 inhibits total and surface protein expression of BK in Cos-7 cells whick is likely due to an increase in BK degradation through a lysosomal pathway.

Objective To study adrenomedullin (AM) mRNA and protein expression level in myocardium of autoimmune myocarditis animal models induced by immunization of mice with lactobacillus casei cell wall element(LCWE). Methods Forty-five Balb/c male mice were randomly divided into experimental group (n = 30) and control group (n = 15), which were intraperitoneally injected with LCWE and phosphate buffered solution(PBS) at day 0,3,5 and 10,respectively. Sera and myocardium samples were gained 14,21 and 28 days after the first immunization. AM expression levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction(RT- PCR) and immunchistochemistry,and mycardial histopathological lesions were observed. The anti- myosin antibodies in different stages were examined by an ELISA. Results There were myocardial necrosis or inflammatory infiltration in the experimental group, but myocardial lesions were not found in the control group. Anti - myosin antibodies were detected in sera of experimental mice,but not in control group. Immunchistochemistry findings demonstrated that AM expression level was higher in the experimental group than in the control group( P

OBJECTIVETo investigate the therapeutic effect of recombinant adeno-associated virus carrying anti-amyloid β peptide single-chain antibody gene on Alzheimer's disease (AD) in animal models.

METHODSThe recombinant adeno-associated viruses were injected to the leg muscle of mutant amyloid precursor protein transgenic AD mice. The latency of the mice in Morris water maze was tested before and 3, 7, 10 months after drug administration. The animal brains were harvested 10 months after drug administration and sectioned for amyloid plaques staining.

RESULTSThe learning and memory abilities of AD model mice were improved significantly 3 months after gene drug administration. Ten months after gene therapy, the numbers of amyloid plaque in hippocampus of model mice decreased.

CONCLUSIONThe adeno-associated virus carrying anti-amyloid β peptide single-chain antibody gene has therapeutic effect on AD in model mice.

Alzheimer Disease ; therapy ; Amyloid beta-Protein Precursor ; immunology ; Animals ; Dependovirus ; genetics ; Disease Models, Animal ; Female ; Genetic Therapy ; Male ; Mice ; Mice, Transgenic ; Single-Chain Antibodies ; genetics

A growing body of evidence suggests that p300 histone acetyltransferase plays important roles in cancer cell differentiation and proliferation. Here, we employed structure-based hierarchical virtual screening method to identify novel lead compounds of p300 histone acetyltransferase. From a screening library containing approximate 100 000 diverse druglike compounds, 33 compounds were chosen for experimental testing and one compound, 4-acetyl-2-methyl-N-morpholino-3,4-dihydro-2H-benzo[b][1, 4]thiazine-7-sulfonamide (17), showed as micromolar inhibitor. Based on its predicted binding pose, we investigated its binding characteristics by designing two series of structural modifications. The obtained structure-activity relationship results are consistent with the predicted binding model. We expect that the identified novel p300 histone acetyltransferase inhibitors will serve as starting points for further development of more potent and specific histone acetyltransferase inhibitors.
Drug Design ; Enzyme Inhibitors ; chemical synthesis ; chemistry ; Molecular Structure ; Morpholines ; chemical synthesis ; chemistry ; Structure-Activity Relationship ; Sulfonamides ; chemical synthesis ; chemistry ; p300-CBP Transcription Factors ; antagonists & inhibitors ; chemistry

For investigation of the regulatory mechanism of cholecystokinin-octapeptide (CCK-8) on pulmonary circulation in rabbits with endotoxic shock (ES) induced by lipopolysaccharides (LPS), mean arterial pressure (MAP) and pulmonary arterial pressure (PAP) were evaluated for 5 h in five groups of rabbits: group of LPS (8 mg/kg, i.v.)-induced ES, group of CCK-8 pretreatment (15 microg/kg, i.v.) 15 min before LPS administration (8 mg/kg, i.v.), group of proglumide pretreatment (1 mg/kg, i.v.) 15 min before LPS administration (8 mg/kg, i.v.), group of CCK (15 microg/kg, i.v.) only, and normal saline (control) group. The pulmonary arterial tension was measured with isolated vascular ring technique. The results showed that LPS-induced pulmonary arterial hypertension was abolished by CCK-8. In contrast, proglumide, a nonspecific antagonist of CCK-8 receptor, potentiated the deleterious effect of LPS. The contractile response of isolated pulmonary artery to alpha-adrenoceptor agonist phenylephrine (PE) was enhanced and the relaxation response to acetylcholine (ACh) was depressed significantly after LPS was injected, but the effect could be reversed by CCK-8. These results suggest that pulmonary circulation is improved by CCK-8 in ES, and the regulatory effects of CCK-8 may be brought about by modulating the pulmonary arterial tension.
Animals ; Hypertension, Pulmonary ; etiology ; physiopathology ; Male ; Pulmonary Artery ; drug effects ; physiology ; Rabbits ; Shock, Septic ; complications ; physiopathology ; Sincalide ; pharmacology ; Vasodilation ; drug effects

Objective To develop an electrochemical impedance immunosensor based on polypyrrole modified microelectrodes for periodontal bacteria rapid quantification.Methods Mcirofluidic chip with embedded IDAM was prepared by conventional photolithography process.Then polypyrrole structure was deposited on microelectrodes through electropolymerization method.Polypyrrole was biofuncationalized with mouse anti-Porphyromonas gingivalis gingipain K IgG antibody using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide-hydrochloride (EDC) chemistry.The linear relationship between the impedance and bacterial concentration,the impedance characteristic and the feasibility of the developed impedic immunosensor in quantifing Porphyromonas gingivalis was investigated respectively.Results The polypyrrole membrane structure was observed on work electrodes,and the lowest detection limit of the immunosensor was 107 cells/L in pure culture.In the concentration range of 107-1012 cells/L,a linear relationship was found between the normalized impedance change and the logarithmic value of the cell concentration.The total detection time from sampling to measurement was less than 1 h.Conclusions The immunosensor developed in the present study offered some insight into chair-side quantifing periodontal bacteria.

AIM To explore the effects of Yupingfeng Powder on the binding of programmed death receptor 1(PD-1)and programmed death ligand 1(PD-L1),and the growth of lung cancer cells in vitro and in vivo.METHODS Yupingfeng Powder extract had its effect on the growth of Lewis lung carcinoma(LLC)cells in mice detected by MTT assay;its effect on eliminating target cells in the co-culture of T cells and LCC cells detected by lactate dehydrogenase(LDH)release test;its effect on blocking the combination of PD-1 and PD-L1,and its influence on the levels of interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)release detected by enzyme-linked immunosorbent assay(ELISA);its inhibitory effect on the growth of LLC cells in vivo analyzed by homologous tumor model;its effect on the infiltration of CD4+T cells and CD8+T cells in tumor tissues investigated by immunohistochemical staining.RESULTS Yupingfeng Powder inhibited the growth of LLC cells and the expression of endogenous PD-L1 protein in a dose-dependent manner(P＜0.05,P＜0.01),competitively blocked the combination of PD-1 and PD-L1(P＜0.05,P＜0.01),and promoted the release of IFN-γ and TNF-α from T cells(P＜0.05,P＜0.01).Yupingfeng Powder inhibited the growth of LLC tumor(P＜0.01),and increased the infiltration of CD4+T and CD8+T cells(P＜0.01).CONCLUSION Yupingfeng Powder exhibits its in vitro capability in blocking the PD-L1 inhibition on T cells,and its in vivo anti-tumor effect in terms of the improvement of CD4+T cells and CD8+T cells in this homologous mouse tumor model.

OBJECTIVETo study the effect of Tiaomaiyin injection on the experimental arrhythmia for analyzing its underlying mechanism in the treatment of cardiovascular disease.

METHODExperimental animals anesthetized with 20% urethane (6 mL x kg(-1)) were evenly randomized into control group, positive control group, low-dose and high-dose Tiaomaiyin group. The rate of ventricular fibrillation (VF) chloroform-induced in mice, and the epoch of ventricular extrasystole (VE), ventricular tachycardia (VT),VF and cardiac arrest (CA), actonitine-induced in rats (1.0 microg x mL(-1) x min(-1)), and vabain-induced in guinea pigs (10 microg x mL(-1) x min(-1)), were detected respectively. The result loas converted into cumulative dosage of actonitine or vabain. In ischemia-reperfusion model in rats, the duration of arrhythmia and activity of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected.

RESULTAfter venous injection of Tiaomaiyin, VF in mice was lower significantly (P < 0.01), VE, VT, VF in rats and VF in guinea pigs were lowered considerably (P <0.05). The duration of arrhythmia in ischemia-reperfusion model was reduced considerably (P < 0.05), and the activity of myocardial SOD was raised significantly (P <0.01).

CONCLUSIONTiaomaiyin shows the reduction of experimental arrhythmia and protect effect to ischemia-reperfusion injury of the heart, which indicates that the effect mechanism may have the relationship with inhabition of lipid peroxidation and damnification of the free radical.

Animals ; Anti-Arrhythmia Agents ; administration & dosage ; isolation & purification ; pharmacology ; Arrhythmias, Cardiac ; etiology ; metabolism ; physiopathology ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Electrocardiography ; Guinea Pigs ; Injections ; Malondialdehyde ; metabolism ; Mice ; Myocardial Ischemia ; complications ; Myocardial Reperfusion Injury ; etiology ; metabolism ; physiopathology ; Myocardium ; metabolism ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the possible mechanism of L-arginine supplementation on the angiogenesis of burn wounds in diabetic rats.

METHODSOne hundred male Sprague-Dawley (SD) rats were used in the study and were randomly divided into A (scalding control, n = 25), B (scalding of the rats with diabetes, n = 25), C (L-glycine control, n = 25) and D (L-arginine supplementation, n = 25) groups. Diabetes was produced by intra-peritoneal injection of streptozotocin (STZ) in B, C and D groups. The rats in C and D groups were gavaged with L-glycine and L-arginine in dose of 200 mg.kg(-1).d(-1), respectively. The glucose content of the back skin tissue was determined for five rats in each group eight weeks after STZ administration. Deep partial thickness scalding of 20% TBSA was engendered on the back in the other 80 rats. The wound area, wound healing rate, and microvascular density with CD34 immunohistochemistry staining were determined on 3rd, 7th, 14th, and 21st post scalding days (PSDs), In addition, the amount of nitric oxide (NO) released from the wound tissue and the tissue contents of vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1) from wound were determined at the above time points.

RESULTSCompared to those in group B, the wound healing rate in group D increased significantly since the 7th PSD [(44.10 +/- 3.50)%, P < 0.05], and the wound MVD value was increased significantly at all postburn time points. Furthermore, the levels of VEGF, NO and TGF-beta1 in the wound tissue was also increased significantly, while the glucose content in the cutaneous tissue was decreased to (1.380 +/- 0.120) mg/g.

CONCLUSIONL-arginine supplementation could be beneficial to the angiogenesis in the burn wound of the rats with diabetes, as well as to wound healing by increasing the synthesis and the release of VEGF, NO and TGF-beta1 from burn wound and by decreasing the glucose content in the cutaneous tissue of diabetic rats.

Animals ; Arginine ; therapeutic use ; Blood Glucose ; metabolism ; Burns ; metabolism ; therapy ; Diabetes Mellitus, Experimental ; metabolism ; therapy ; Male ; Neovascularization, Physiologic ; drug effects ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Wound Healing ; physiology

BACKGROUNDThe morphological and electrophysiological characteristics of cardiac cells in Koch triangle are still disputed. We studied the appearance and electrical properties of these diverse myocytes to elucidate their complex electrophysiological phenomena.

METHODSExperiments were conducted using cooled charge coupling device (CCD) system and whole cell, patch clamp technique to determine the morphology, action potential and sodium current density of single viable myocytes enzymatically isolated from the Koch triangle of rabbit hearts.

RESULTSMorphologically, cardiac cells in shape of spider, tiny spindle, slender spindle, rod and strip were observed in percentage of 3.0 +/- 0.3, 35.0 +/- 5.0, 15.0 +/- 2.0, 40.0 +/- 5.0 and 6.0 +/- 0.7 respectively. The cellular dimensions and capacitance gradually increased in the above order (all P < 0.05). Electrophysiologically, action potential configurations recorded from them were similar respectively to nodal (N), atrial nodal (AN), nodal Hisian (NH), atrial (A) and Hisian like potentials obtained from the intact atrioventricular nodal preparations. Diastolic depolarization appeared in all myocytes except for rod cells. Sodium current density increased in the order of tiny spindle, strip, rod, slender spindle cell (all P < 0.05), but could not be detected in spider-shaped cells. Linear regression analysis revealed that membrane capacitance was correlated negatively to the rate of diastolic depolarization r = -0.70, P < 0.001, but positively to maximum depolarization potential, amplitude of action potential, upstroke velocity and maximum peak value of sodium current density r = 0.84, 0.80, 0.87 and 0.75, respectively; all P < 0.001.

CONCLUSIONSThe results demonstrated that spider-shaped, spindle, rod and strip cells in Koch triangle might correspond to pacemaking, transitional, atrial and Purkinje like cells, respectively. Furthermore, tiny spindle and slender spindle cells were referred to transitional cell alpha (TCalpha) and beta (TCbeta) accordingly considering their distinctive electrical properties. Different myocytes with diverse electrical properties constituted the infrastructure of sophisticated electrophysiological phenomena in Koch triangle. In view of the prominent percentage and electrical properties, tiny spindle and slender spindle cells were presumed to play important roles.

Action Potentials ; Animals ; Atrioventricular Node ; cytology ; physiology ; Female ; Male ; Myocytes, Cardiac ; cytology ; physiology ; Rabbits ; Sodium Channels ; physiology