Objective To construct a tyrosine kinase B(TrkB) targeted RNA interference (RNAi) lentiviral vector.Methods Four oligonucleotides targeting rat TrkB gene were synthesized and cloned into lentiviral vector pXZRNAi 1.0 to construct recombinant lentiviral vectors pXZRNAi-shTrkB-1,2,3,4.Neural stem cells prepared from rat hippocampus were infected with these high-titer viruses.Real-time PCR was employed to detect the TrkB mRNA expression and western blot was used to assess the gene silencing efficacy of these recombinants.Results Enzyme digestion and DNA sequencing results demonstrated that these shRNAs were correctly inserted into lentiviral vectors and the four recombinants were constructed successfully with the titer of 8.6 × 105cfu/ml.The infection efficiency of the letivirus on neural stem cells reached 80％.Compared with the uninfection group,the expression levels of TrkB mRNA in neural stem cells decreased significantly after transfected with pXZRNAi-shTrkB-3 and 4((66.7 ± 5.5) ％ and(76.8 ± 4.9) ％ respectively,P ＜ 0.05) ; and the protein expression levels were also significantly decreased ((68.5 ± 4.3)％ and (78.2 ± 5.1)％ respectively,P ＜ 0.05).Conclusion The lentiviral vectors for TrkB have been successfully constructed with high yield of lentivirus,which provides versatile method for assessing gene function in neural stem cells.

OBJECTIVETo observe the vasodilating effect of adrenomedullin 2 (ADM2) by antagonizing angiotensin 1 (Ang II), and to explore its mechanism.

METHODSEighteen male, 180-200 g SD rats were randomly divided into 3 groups (n = 6): control group, Ang II (150 ng/(kg x min)) group and Ang II (150 ng/(kg x min)) + ADM2(500 ng/(kg x h)) group. Mini-osmotic pumps filled with peptide were implanted in the back of rats subcutaneously. After two weeks, the blood pressure was measured by the way of carotid intubation. The plasma was collected for the detection of nitric oxide (NO) content and the activity of endothelial nitric oxide synthase (eNOS). The in situ oxidation of fluorescent dye dihydroethidium (DHE) was used for detecting superoxide in rat arteries. The rat isolated arterial rings were made for studying the vasodilating effect of ADM2. Human umbilical vein endothelial cell line EA. hy 926 cells were cultured and their intracellular reactive oxygen species (ROS) were evaluated by probe DCFH-DA.

RESULTSADM2 dramatically decreased the blood pressure in angiotensin II-induced hypertension rat model, enhanced plasma NO content and the activity of eNOS and reduced superoxide formation in vessel walls. ADM2 also induced relaxation of the vascular rings preconstricted by Ang II in a concentration-dependent and endothelium-dependent manner. In cultured vascular endothelium, ADM2 ameliorated the ROS generation induced by Ang II.

CONCLUSIONAdrenomedullin 2 relaxed blood vessels by antagonizing angiotensin II-induced oxidative stress and improving the vascular endothelial function.

Adrenomedullin ; pharmacology ; Angiotensin II ; pharmacology ; Animals ; Antihypertensive Agents ; pharmacology ; Blood Pressure ; drug effects ; Drug Antagonism ; Endothelium, Vascular ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Male ; Nitric Oxide ; blood ; Nitric Oxide Synthase Type III ; blood ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Vasodilation ; drug effects

OBJECTIVE: To explore the influence of visual attention in visual evoked potential (VEP) examination. METHODS: The pattern visual evoked potential (PVEP) and flash visual evoked potential (FVEP) were used to examine 110 normal subjects whose visual acuity or corrected visual acuity is in the range of 4.8-5.2, and age between 20 to 30 years old. In PVEP inspection, subjects were asked to watch with three ways, to stare at the central screen (at the same time count silently the number of alternating of black and white squares to improve the subjects' attention), to stare at the central screen at the same time thinking other things, to look sideways (the left eye stared at the left bottom corner of the screen or the right eye stared at the right one). In FVEP inspection, subjects were checked with eyes open and closed respectively. The latency and amplitude of P100 wave were recorded. The data were statistically analyzed by SPSS 13.0 software. RESULTS In PVEP inspection, the latency and amplitude of P100 wave were statistically different among three watching ways. In FVEP inspection, latency and amplitude of P100 wave were statistically different between eyes open and closed group. CONCLUSION Although the visual evoked potential test is an objective electrophysiological visual function test, it is susceptible to the influence of various subjective factors such as visual attention. It should arise the attention of forensic experts.
Adult ; Attention ; Cooperative Behavior ; Electroencephalography ; Electrophysiology ; Evoked Potentials, Visual/physiology* ; Female ; Forensic Medicine/methods* ; Humans ; Male ; Pattern Recognition, Visual ; Photic Stimulation ; Reaction Time ; Visual Acuity/physiology* ; Visual Fields/physiology* ; Young Adult

To investigate the anti-cardiac hypertrophic mechanism of statins, thirty-eight male Wistar rats were randomly allocated to four groups. Rats in model group received nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NNA) 15 mg/(kg.d) by peritoneal injection. Rats in simvastatin treatment groups were given simultaneously L-NNA as those in model group and simvastatin 5 or 30 mg/(kg.d) intragastrically respectively. Rats in control group received the same volume of normal sodium. Left ventricular function, left ventricular mass index (LVMI), the content of brain natriuretic peptide (BNP) in plasma and myocardium, myocardial hydroxyproline and heme oxygenase activity were determined after 6 weeks. The results showed that rats in model group developed significant cardiac hypertrophy associated with reduced left ventricular function compared with the control group. However, compared with the model group, L-NNA-induced cardiac hypertrophy of rats was significantly relieved in simvastatin treatment groups, associated with improved left ventricular function, decreased LVMI, lower BNP levels in plasma and myocardium, lower content of myocardial hydroxyproline, and increased myocardial heme oxygenase (HO) activity. In cultured rat neonatal cardiomyocytes, simvastatin (30 or 100 mumol/L) significantly increased heme oxygenase-1 (HO-1) mRNA expression, HO activity as well as the production of CO in cardiomyocytes. Cultured with zinc protoporphyrin, a HO inhibitor, or simvastatin alone did not change [(3)H]leucine uptake of cardiomyocytes. However, cocultured with simvastatin significantly inhibited the cardiomyocyte [(3)H]leucine uptake induced by angiotensin II in a concentration-dependent manner. Cotreatment with zinc protoporphyrin significantly abolished the suppressive effect of simvastatin on cardiomyocyte [(3)H]leucine uptake. These data suggest that the activation of HO-1/CO pathway may be one of the important mechanisms by which statins inhibit cardiac hypertrophy caused by hypertension.
Angiotensins ; antagonists & inhibitors ; pharmacology ; Animals ; Carbon Monoxide ; metabolism ; Cardiomegaly ; etiology ; prevention & control ; Cell Enlargement ; drug effects ; Heme Oxygenase-1 ; metabolism ; Hypertension ; complications ; drug therapy ; Male ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Simvastatin ; pharmacology ; therapeutic use

BACKGROUNDEndogenous hydrogen sulfide (H(2)S) plays an important role in hypertension. The aim of this study was to investigate the role of erythrocyte and serum H(2)S in patients with untreated essential hypertension.

METHODSWe recruited 62 patients (age 22 - 74 years) with untreated prehypertension or hypertension, and 64 normotensive subjects (age 18 - 64 years). We assessed the 3-mercaptopyruvate sulphurtransferase (MPST) protein expression in erythrocytes and measured the H(2)S production from erythrocytes and serum H(2)S levels, then analyzed the association of erythrocytic or serum H(2)S content and blood pressure or cardiovascular risk factors (e.g., age, body mass index (BMI) and dyslipidemia). A stepwise regression analysis was used to evaluate the possible relationship of erythrocytic H(2)S in hypertension.

RESULTSIn hypertensive patients, erythrocyte H(2)S production ((111.04 ± 29.20) nmol/min per 10(8) erythrocytes) was higher than that in controls ((78.85 ± 19.38) nmol/min per 10(8) erythrocytes), and serum H(2)S was also higher. The erythrocytic H(2)S production was associated with increased systolic blood pressure (sBP), diastolic blood pressure (dBP), age, BMI, level of C-reactive protein (CRP), as well as triglycerides (TG) and high density lipoprotein cholesterol (HDL-C). Serum H(2)S was not associated with age or CRP. Stepwise regression analysis showed that erythrocytic H(2)S production was correlated with sBP, TG, HDL-C, low density lipoprotein cholesterol (LDL-C) and blood urea nitrogen (BUN) and serum H(2)S was correlated with dBP and TG. Results of receiver-operating characteristic curve analysis suggested that erythrocytic H(2)S production was a more sensitive predictor of hypertension development than serum H(2)S.

CONCLUSIONErythrocytic or serum H(2)S production is sensitive predictor of hypertension.

Adolescent ; Adult ; Aged ; Erythrocytes ; metabolism ; Female ; Humans ; Hydrogen Sulfide ; blood ; Hypertension ; blood ; metabolism ; Male ; Middle Aged ; Young Adult

Bone Wires ; Child ; Child, Preschool ; External Fixators ; Female ; Fracture Fixation ; methods ; Humans ; Humeral Fractures ; surgery ; Male

BACKGROUNDEndogenous hydrogen sulfide is a new neuromodulator which takes part in the regulation of central nervous system physiology and diseases. Whether endogenous hydrogen sulfide in the central nervous system regulates cardiovascular activity is not known. In the present study, we observed the hemodynamic changes of hydrogen sulfide or its precursor by intracerebroventricular injection, and investigate the possible roles of endogenous digitalis like factors and sympathetic activity in the regulation.

METHODSNinety-four Sprague-Dawley rats underwent a right cerebroventricular puncture, then the hydrogen sulfide saturation buffer or its precursor injected by intrcerebroventricular catheter. A heperin-filled catheter was inserted into the right femoral artery or into the left ventricle, and changes of blood pressure or cardiac function recorded by a Powerlab/4S instrument. Phentolamine or metoprolol were pre-injected to observe the possible role in autonomic nerve activity. After rats were sacrificed, plasma was collected and endogenous digitalis-like factors were measured with a commercial radioimmunoassay kit. The aortic, cardiac sarcolemmal vesicles were isolated and the activity of Na(+)-K(+)-ATPase was measured as ouabain-sensitive ATP hydrolysis under maximal velocity conditions by measuring the release of inorganic phosphate from ATP. Unpaired Student's t test for two groups or analysis of variances (ANOVA) for multiple groups were used to compare the differences of the changes.

RESULTSIntracerebroventricular injection of hydrogen sulfide induced a transient hypotension, then dramatic hypertenive effects in a dose-dependent manner. Bolus injection of L-cysteine or beta- mercaptopyruvate also increased mean arterial pressure (P < 0.01), whereas hydroxylamine-a cystathionine beta synthase inhibitor decreased the arterial pressure (P < 0.01). Hydrogen sulfide and L-cysteine increased mean arterial pressure, left ventricular develop pressure and left-ventricle maximal rate of systolic and diastolic pressure; these functions were decreased by hydroxylamine (P < 0.01). Glibenclamide (a K(ATP) channel blocker) blocked the transient hypotensive effect, phentolamine (an alpha-adrenergic receptor blocker) blocked the hypertensive effect, and metoprolol (a selective beta 1 receptor blocker) blocked the positive inoptropic effect of central nervous system hydrogen sulfide. The endogenous digitalis-like factors in plasma were elevated (P < 0.01) after treatment with L-cysteine, association with decreasing Na(+)-K(+)-ATPase activity in cardiac or aortic sarcolemmal vesicles (P < 0.01). Hydroxylamine injection reduced the endogenous digitalis-like factors level in plasma association with increasing Na(+)-K(+)-ATPase activity in cardiac and aortic sarcolemmal vesicles.

CONCLUSIONCentral nervous system endogenous hydrogen sulfide upregulated mean arterial pressure and cardiac systolic function by activation of sympathetic nerves or release of endogenous digitalis-like factors.

Animals ; Blotting, Western ; Cardenolides ; metabolism ; Central Nervous System ; drug effects ; metabolism ; Cystathionine beta-Synthase ; metabolism ; Cysteine ; analogs & derivatives ; pharmacology ; Hemodynamics ; drug effects ; Hydrogen Sulfide ; metabolism ; pharmacology ; Male ; Radioimmunoassay ; Rats ; Rats, Sprague-Dawley ; Saponins ; metabolism ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Sulfurtransferases ; metabolism

The prokaryotic expression plasmid pQE30-HN of hemagglutinin-neuraminidase (HN) protein gene of bovine parainfluenza virus type 3 (BPIV3) strain HJ-1 was expressed by IPTG induction in E. coli XL1Blue. The recombinant HN protein(rHN) was purified by electroeluting method, and used as coated antigen. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody valence of BPIV3. The best working conditions of ELISA were as follows: the antigen concentration was 6 microg/mL; the serum dilution was 1:50; the blocking reagent was 5% skimmed milk; the blocking time was 60 min at 37 degrees C; the second antibody concentration was 1:10 000; The cut-off value was 0.30. The method revealed a good specificity, no cross-reaction to the positive sera of BCV, IBRV or BRSV was observed. We applied the method to detect 323 serum samples of dairy cow in Heilongjiang Province, the seropositivity rate of BPIV3 was about 58%. The indirect ELISA established provided a technological basis for the development of ELISA kit.
Animals ; Antibodies, Viral ; blood ; Blotting, Western ; Cattle ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; Female ; HN Protein ; genetics ; Parainfluenza Virus 3, Bovine ; genetics ; immunology

OBJECTIVE: To investigate the changes of collagen fibers and the expression of osteopontin in the left ventricle in cases of hypertrophic cardiomyopathy (HCM), along with the significance of their potential forensic application. METHODS: Fifteen cases of HCM, 15 cases of coronary heart disease with cardiac hypertrophy and 20 cases of traffic accidents were selected as HCM group, coronary heart disease group and control group, respectively. Collagen volume fraction and osteopontin expression were observed and compared by HE staining, Masson trichrome staining and immunohistochemistry methods. Imaging and statistical methods were used for quantitative analysis. RESULTS: Collagen volume fraction in left ventricle of HCM and coronary heart disease were significantly higher than that in the control group (P < 0.05), which was not significantly different between the HCM group and the coronary heart disease group. The integral light density value of osteopontin in left ventricular cardiomyocytes of the HCM group and the coronary heart disease group were significantly higher than that of the control group (P< 0.05), and the value of the HCM group was also significantly higher than that of coronary heart disease group (P < 0.05). CONCLUSION The increased contents of collagen fibers and the overexpression of osteopontin may play an important role in myocardial fibrosis, and they can be used as markers in aid of diagnosing sudden death due to HCM.
Cardiomyopathy, Hypertrophic/physiopathology* ; Case-Control Studies ; Collagen/metabolism* ; Coronary Disease/physiopathology* ; Death, Sudden, Cardiac/etiology* ; Female ; Fibrosis ; Forensic Pathology ; Heart Ventricles/pathology* ; Humans ; Immunohistochemistry ; Male ; Myocardium/pathology* ; Osteopontin/metabolism* ; Staining and Labeling

Ziziphi spinosae semen mainly contains contents of saponins,flavonoids,alkaloids and aliphatic acids.Meanwhile,it has a variety of activities such as sedative-hypnotic,anti-anxiety,anti-depression,nerve protection,cardiovascular and cerebrovascular protection,liver protection,and antioxidant,which is widely used in medicine,food,health food and other fields.The chemical constituents,pharmacological action and clinical application of Ziziphi spinosae semen were systematically summarized in this paper by reviewing the literature,in order to provide theoretical guidance for the sustainable development of the resources and the rational use of Ziziphi spinosae semen.