Objective To synthesize and identify artificial antigens of lung elastin degradation peptide and for the purpose of preparation of COPD test.Methods The artificial antigens were synthesized by Sulfo-SMCC and KLH.The complete antigens were identified by ultraviolet spectrum and SDS-PAGE.Immunize Balb/c mice was used to prepare antibody.The antiserum activity was evaluated by indirect competitive ELISA.Results The artificial antigens were identified by ultraviolet spectrum and SDS-PAGE. The protein concentration was 1.181 mg/mL.The titer of antiserum was 1∶64 000,and IC50 was 13.7 ng/mL.The antiserum had no cross-reaction with nonsense peptide.Conclusion The artificial antigens were acquired successfully,which had good immunoge-nicity.The results have laid basis for COPD test.

Stimulation of cardiac mAChRs by carbachol (CCh) produces a biphasic inotropic response. The mechanisms of the positive inotropic response by higher concentration of CCh appear to be paradoxical. This article was aimed to study the mechanism of the positive inotropic effect of CCh in guinea pig ventricular myocytes. The effects of CCh on L-type calcium current (I(Ca)) and Na/Ca exchange current (I(Na/Ca)) were observed in voltage-clamped guinea pig ventricular myocytes by using Axon 200A amplifier. The results showed that CCh (100 micromol/L) increased both forward mode and reverse mode I(Na/Ca) from (1.2+/-0.1) pA/pF to (2.0+/-0.3) pA/pF for forward mode (P<0.01) and from (1.3+/-0.5) pA/pF to (2.1+/-0.8) pA/pF for reverse mode (P<0.01), respectively. CCh had no effect on I(Ca). The stimulating effect of CCh on I(Na/Ca) could be blocked by application of atropine, a nonselective blocker of muscarinic receptors, which means that the stimulating effect of CCh is through the activation of muscarinic receptors. We made a further study by using methoctramine, a selective antagonist of M2 muscarinic receptors. It completely abolished I(Na/Ca) induced by 100 micromol/L CCh, indicating that the effect of CCh on I(Na/Ca) was mediated by M2 muscarinic receptors. It is generally accepted that contraction in cardiac myocytes results from elevation of intracellular Ca2+ concentration. Ca2+ enters the cells through two pathways: L-type Ca2+ channels and, less importantly, reverse mode Na/Ca exchange. The calcium influx via both pathways promotes the contraction of cardiac myocytes. Because CCh had no effect on L-type Ca2+ current, the increase in Na/Ca exchange current might be the main factor in the positive inotropism of CCh. These results suggest that the positive inotropic effect of CCh in guinea pig heart is through stimulation of Na/Ca exchange and is mediated by M2 muscarinic receptors.
Animals ; Calcium Channels, L-Type ; physiology ; Carbachol ; pharmacology ; Cardiotonic Agents ; pharmacology ; Diamines ; pharmacology ; Female ; Guinea Pigs ; Heart Ventricles ; Male ; Myocytes, Cardiac ; metabolism ; physiology ; Patch-Clamp Techniques ; Receptor, Muscarinic M2 ; physiology ; Sodium-Calcium Exchanger ; physiology

OBJECTIVETo compare the efficacy difference in the treatment of shoulder pain in post-stroke shoulder-hand syndrome among floating acupuncture, oral administration of western medicine and local fumigation of Chinese herbs.

METHODSNinety cases of post-stroke shoulder-hand syndrome (stage I) were randomized into a floating acupuncture group, a western medicine group and a local Chinese herbs fumigation group, 30 cases in each one. In the floating acupuncture group, two obvious tender points were detected on the shoulder and the site 80-100 mm inferior to each tender point was taken as the inserting point and stimulated with floating needling technique. In the western medicine group, mobic 7.5 mg was prescribed for oral administration. In the local Chinese herbs fumigation group, the formula for activating blood circulation and relaxing tendon was used for local fumigation. All the patients in three groups received rehabilitation training. The floating acupuncture, oral administration of western medicine, local Chinese herbs fumigation and rehabilitation training were given once a day respectively in corresponding group and the cases were observed for 1 month. The visual analogue scale (VAS) and Takagishi shoulder joint function assessment were adopted to evaluate the dynamic change of the patients with shoulder pain before and after treatment in three groups. The modified Barthel index was used to evaluate the dynamic change of daily life activity of the patients in three groups.

RESULTSWith floating acupuncture, shoulder pain was relieved and the daily life activity was improved in the patients with post-stroke shoulder-hand syndrome, which was superior to the oral administration of western medicine and local Chinese herbs fumigation (P < 0.01). With local Chinese herbs fumigation, the improvement of shoulder pain was superior to the oral administration of western medicine. The difference in the improvement of daily life activity was not significant statistically between the local Chinese herbs fumigation and oral administration of western medicine, the efficacy was similar between these two therapies (P > 0.05).

CONCLUSIONThe floating acupuncture relieves shoulder pain of the patients with post-stroke shoulder-hand syndrome promptly and effectively, and the effects on shoulder pain and the improvements of daily life activity are superior to that of the oral administration of western medicine and local Chinese herbs fumigation.

Acupuncture Therapy ; Aged ; Female ; Humans ; Male ; Middle Aged ; Reflex Sympathetic Dystrophy ; etiology ; rehabilitation ; therapy ; Shoulder Pain ; etiology ; rehabilitation ; therapy ; Stroke ; complications ; Treatment Outcome

The present study was aimed to investigate the positive inotropic mechanism of carbachol (CCh) on rat ventricular myocytes. The effects of CCh on L-type calcium current (I(Ca,L)) and Na(+)/Ca(2+) exchange current (I(Na/Ca)) were investigated in isolated rat ventricular myocytes. After loading myocytes with Fura-2/AM, electrically triggered Ca(2+) transient and cell shortening in single myocyte were measured simultaneously using ion imaging system with charge-coupled device (CCD) camera. CCh (100 mumol/L) increased I(Na/Ca) in forward mode from (1.18 +/- 0.57) pA/pF in the control group to (1.65 +/- 0.52) pA/pF (P<0.01) and that in reverse mode from (1.11 +/- 0.49) pA/pF in the control group to (1.53 +/- 0.52) pA/pF (P<0.01), respectively. CCh had no effect on I(Ca,L). The stimulatory effect of CCh on I(Na/Ca) was blocked by application of atropine, a non-selective M muscarinic receptor antagonist, and methoctramine, a selective M(2) muscarinic receptor antagonist. CCh (100 mumol/L) increased cell shortening from (3.00 +/- 0.67) mum in the control group to (3.55 +/- 1.21) mum. Ca(2+) transient was also increased from 203.8 +/- 50.0 in the control group to 234.8 +/- 64.3 in 100 mumol/L CCh group. KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchange, did not change the baseline level of cell shortening and Ca(2+) transient, while completely abolished CCh-induced increments of both Ca(2+) transient and cell shortening. CCh increased cell shortening and Ca(2+) transient in the presence of nicardipine, indicating that the positive inotropic effect of CCh was through activation of Na(+)/Ca(2+) exchange. Calcium sensitivity was not changed by CCh. Both atropine and methoctramine abolished the positive inotropic effects of CCh, demonstrating that CCh induced positive inotropism via the M(2) muscarinic receptor. The results suggest that CCh increases cell contraction and Ca(2+) transient in rat ventricular myocytes. This positive inotropic effect of CCh is through activation of reverse mode Na(+)/Ca(2+) exchange, and M(2) receptors are involved in mediating CCh-induced contraction.
Animals ; Calcium ; Carbachol ; pharmacology ; Heart Ventricles ; Male ; Myocardial Contraction ; Myocytes, Cardiac ; drug effects ; Rats ; Receptor, Muscarinic M2 ; Receptors, Muscarinic ; drug effects ; Sodium ; Sodium-Calcium Exchanger ; Thiourea ; analogs & derivatives

Objective To evaluate the practicability of using CRISPR/Cas9 genome editing tech-nology for inhibition of hepatitis B virus ( HBV) replication. Methods Two sgRNA targeting sites were de-signed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were con-structed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen ( HBsAg ) were determined by using chemiluminescent immunoassay ( CLIA ) . The expression of HBV at mRNA level was analyzed by quantitative real-time PCR ( qRT-PCR) . The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing ( NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were de-tected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24. 2% (P<0. 05) and 16. 9% (P>0. 05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16. 4% (P>0. 05) and 32. 1% (P>0. 05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indica-ted by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0. 05) and 35% (P<0. 05), and the levels of intracellular HBV DNA were respectively sup-pressed by 7. 2% (P>0. 05) and 18% (P>0. 05). Different types of insertion/deletion mutation were de-tected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection.

OBJECTIVETo provide new germplasm materials for breeding new varieties of Pseudostellaria heterophylla.

METHODThe method of single plant selection was adopted, with the comparative experiments being carried out under the same conditions in Shibing county. The 8 plants of Shibing SB-4 were compared respectively with factor analysis for 27 phenotypic traits and 8 yield traits, and single factor variance analysis for the contents of polysaccharides.

RESULTUsing factor analysis, 27 phenotypic traits were classified into 7 principal divisors and 8 yield traits were simplified into 3 principal divisors. The 4 strains of P. heterophylla, ZT-01, ZT-02, ZT-06 and ZT-07, performed better than others in the phenotypic traits, and ZT-01, ZT-02, ZT-03 and ZT-07 in the yield traits. The contents of polysaccharides of ZT-01, ZT-02, ZT-05 and ZT-08 showed significantly higher value.

CONCLUSIONThere is significant difference among the 8 strains of P. heterophylla in phenotypic traits, yield traits and quality traits, making it possible to select certain strains for different purposes. ZT-01 and ZT-02 can be breaded further. ZT-06 and ZT-07 were used as ornamental cultivars for its great phenotypic traits. ZT-03 with good resistance and high yield was taken as resistant variety, and ZT-05 would face next selection on the basis of its high content of polysaccharide.

Breeding ; Caryophyllaceae ; chemistry ; growth & development ; China ; Phenotype ; Polysaccharides ; analysis

OBJECTIVETo investigate the effect of moxibustion at Sanyinjiao (SP 6) for uterine contraction pain in labor, and evaluate the safety of the parturient and newborn.

METHODSOne hundred and seventy-four cases of singleton pregnancy and cephalic presentation primipara were single blinded and randomly divided into three groups: observation group (59 cases), placebo treated group (57 cases) and blank group (58 cases). The observation group was treated with moxibustion at Sanyinjiao (SP 6) for 30 min when the uterus cervix openning at 3 cm, the placebo treated group was treated with moxibustion at no acupoint for 30 min and the blank group was treated with routine labor nursing, the uterine contraction pain and the safety of the mother and infant were compared among three groups.

RESULTS1) The uterine contraction pain was tested by Visual Analogue Scale (VAS): the scores of VAS in the observation group were obviously decreased after 15 min and 30 min of moxibustion (both P<0.05), there were no obvious changes of the VAS scores in placebo treated group and the blank group, the scores of VAS in observation group decreased much more obviously than those in the other two groups (all P<0.05); 2) Midwife rating of the uterine contraction pain: after 30 min of moxibustion, the effective rate of labor analgesia was 69.5% (41/59) in observation group, which was higher than that of 45.6% (26/57) in placebo treated group and 43.1% (25/58) in blank group, with significant differences between them (both P<0.05); 3) The postpartum hemorrhage amount of the observation group was obviously lower than those of placebo treated group and blank group (both P<0.05); 4) The Apgar score of newborn was higher in observation group and placebo treated group than that of blank group (both P<0.05).

CONCLUSIONMoxibustion at Sanyinjiao (SP 6) can relieve the uterine contraction pain, and has no side effect to mother and infant, it is one of the safe, effective and simple non-drug analgesia methods.

Acupuncture Points ; Adult ; Female ; Humans ; Labor Pain ; physiopathology ; therapy ; Labor, Obstetric ; Moxibustion ; Pregnancy ; Treatment Outcome ; Uterine Contraction ; Young Adult

OBJECTIVETo investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells.

METHODSUnder the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity.

RESULTSThe Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01).

CONCLUSIONSOverexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Mitochondrial Proteins ; biosynthesis ; genetics ; Mitomycin ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Neoplasms ; metabolism ; pathology ; Transfection

The effects of 5-(N,N-dimethyl)amiloride (DMA) (a blocker of Na(+)/H(+) exchanger or Na(+)/Ca(2+) exchanger) on calcium transient and cell contraction in isolated ventricular myocytes in normal rats and rats with myocardial hypertrophy were examined using ion imaging system with a charge coupled digital camera (CCD camera). Loading myocytes with Fura-2, electrically triggered Ca(2+) transients and cell shortening were measured simultaneously. The results showed that 10 micromol/L DMA increased Ca(2+) transient and cell shortening from 209.60+/-54.96 and 3.07+/-0.97 micrometer to 238.50+/-80.41 and 4.07+/-1.02 micrometer, respectively (P<0.05), which was completely abolished by application of KB-R7943, a specific reverse mode Na(+)/Ca(2+) exchanger blocker. After blocking L-type Ca(2+) channels by nicardipine, DMA also enhanced Ca(2+) transient and cell shortening. In rats with myocardial hypertrophy, DMA showed the common pharmacologic profile as in normal rats but more intense stimulating effects on Ca(2+) transient and cell contraction. The results suggest that DMA increase Ca(2+) transient and cell contraction via stimulating reverse mode Na(+)/ Ca(2+) exchange, and the stimulating effect is more pronounced in rats with myocardial hypertrophy than in normal ones.
Amiloride ; analogs & derivatives ; pharmacology ; Animals ; Calcium ; metabolism ; Cardiomyopathy, Hypertrophic ; drug therapy ; Heart Ventricles ; cytology ; Myocardial Contraction ; drug effects ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Wistar ; Sodium-Calcium Exchanger ; antagonists & inhibitors ; pharmacology

Objective:To observe whether the effect of electroacupuncture (EA) on improving sex hormone disorders and follicle development is by decreasing the expression of anti-Mullerian hormone (AMH) in rats with experimental polycystic ovarian syndrome (PCOS).Methods:Forty rats were randomly divided into four groups,a normal group (NG),a model group (MG),an EA at acupoints group (EAAG),and an EA at non-acupoints group (EANAG),with 10 rats in each group.The rats in the EAAG and EANAG were intervened by EA treatment for consecutive 14 d.Zhongji (CV 3) and Guanyuan (CV 4) were selected as the acupoints in the EAAG,and the tip of the tail and 1 cm up from the tail tip were selected as the non-acupoints in the EANAG.After treatment,the histomorphological changes of the ovary,the levels of aromatase P450 (P450arom),testosterone and estradiol in the ovarian tissues,and the expressions of follicle stimulating hormone (FSH) and AMH were observed.Results:After treatment,compared with the MG and EANAG,the expression of AMH decreased (P＜0.05),the levels of P450arom and estradiol increased significantly,and the level of testosterone decreased significantly (all P＜0.01) in the EAAG.Additionally,several normal follicles were present and the number of cystically dilated follicles decreased in the EAAG.Compared with the MG and EANAG,the EAAG obviously had more follicular granulosa cells.Conclusion:EA can down-regulate the abnormally increased expression of AMH to improve sex hormone disorders and follicle development in PCOS rats.