Experimental study on effects of acupoint application with Leima type II plaster on collagen-induced arthritis in rats / 中国针灸

<p><b>OBJECTIVE</b>To observe the therapeutic effect of acupoint application with Leima type II plaster on collagen-induced arthritis (CIA) in rats and probe its mechanism.</p><p><b>METHODS</b>Bovine type II collagen was injected intradermally into the middle line of the back to induce CIA model with 48 Wistar rats. Then the rats were randomly divided into a model control group (group A), a matrix control group (group B), acupoint application group with plaster of low concentration (group C) and high concentration plaster group (group D), 12 rats in each group. Group C and group D were treated with low and high concentration of Leima type II plaster, and "Shenzhu" (GV 12), "Zhiyang" (GV 9) and "Mingmen" (GV 4) were selected, each application for about 15 hours, once each day for 30 days. Group B was used the same method of acupoint application except using non-drug matrix plaster, and group A was not given any treatment. The morphous and the histopathological changes of affection joint were observed.</p><p><b>RESULTS</b>The paw edema volume after 30 days treatment in group C was significantly lower than that in group B (P < 0.01), and the anti-type II collagen antibody level after 15 days treatment in group C was significantly lower than that in group A (P < 0.05), and the synoviocytes proliferation of the knee joint in group C was significantly lower than that in group A and group B (both P < 0.01). The paw edema volume after 25 days treatment, arthritic index after 20 days treatment, pathological change of the paw and the synoviocytes proliferation of the knee joint in group D were significantly lower than those in group A and group B (P < 0.01, P < 0.05), and the anti-type II collagen antibody level after 15 days treatment in group D was significantly lower than that in group A (P < 0.05), and the paw edema volume and the arthritic index after 25 days treatment in group D were significantly lower than those in group C (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>Acupoint application with Leima type II plaster has a good therapeutic effect on CIA rats and the protective mechanism is related to the reduction of anti-type II collagen antibody level so as to carry out anti-inflammatory effect and immunosuppression.</p>

Overexpression of connexin 45 in rat mesenchymal stem cells improves the function as cardiac biological pacemakers / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Extensive research toward creating a biological pacemaker by enhancement of inward depolarizing current has been performed. However, studies have mainly focused on inducing spontaneous activity and have not adequately addressed ways to improve pacemaker function. In this study we attempted to improve pacemaker function by altering connexin expression in rat mesenchymal stem cells (MSCs) to a phenotype similar to native sinus node pacemaker cells.</p><p><b>METHODS</b>To generate a biological pacemaker, MSCs were transduced with a cardiac pacemaker gene-hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), via transfection with a lentiviral vector. Funny current (I(f)) in HCN4(+) MSCs was recorded by voltage-clamp. Overexpression of connexin 45 (gene Gja7) in MSCs was achieved by transfection with the plasmid pDsRED2-N1-Gja7-RFP. Double-immunolabelling with anti-connexin 43 and anti-connexin 45 antibodies were used to identify the gap junction channels. The effects of the genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Spontaneous action potentials of neonatal rat ventricular myocytes were recorded by current-clamp.</p><p><b>RESULTS</b>High level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4 mmol/L Cs(+) was detected in HCN4(+) MSCs, confirming that HCN4 acted as I(f) channels in MSCs. Connexin 43 and connexin 45 were simultaneously detected in CX45(+) MSCs. Beating frequency was (82 +/- 8) beats per minute (n = 5) in myocytes cocultured with non-transfected control MSCs, versus (129 +/- 11) beats per minute (n = 5) in myocytes cocultured with HCN4(+) MSCs. Myocytes cocultured with MSCs cotransfected with HCN4 and connexin 45 had the highest beating frequency at (147 +/- 9) beats per minute (n = 5).</p><p><b>CONCLUSION</b>These findings demonstrate that overexpression of connexin 45 and subsequent formation of heteromeric connexin 45/connexin 43 gap junction channels in HCN4 expressing MSCs can improve their function as cardiac biological pacemakers in vitro.</p>

The changes of funny currents in the ventricular myocytes of neonatal rats and adult rats / 中国应用生理学杂志

<p><b>AIM</b>To record funny currents (If) of ventricular myocytes and to analysize hyperpolarization-activated cation channel(HCN) expression in the rats of different ages.</p><p><b>METHODS</b>Fresh ventricular myocytes were isolated from 3 days rats and adult rats.HCN expressions were measured by real-time quantitative polymerase chain reaction(real-time PCR). It was recorded through whole-cell patch clamp.</p><p><b>RESULTS</b>HCN1, HCN2, HCN3, HCN4 mRNA represented 0.23% +/- 0.01%, 83.58% +/- 0.04%, 0.79% +/- 0.01%, 15.44% +/- 0.01% of total HCN mRNA in the neonatal rats, respectively. If was recorded and the threshold for activation was -75 mV. In the adult rat, HCN1, HCN2, HCN3, HCN4 mRNA represented 0.72% +/- 0.02%, 91.58% +/- 0.08%, 0.27% +/- 0.02%, 7.12% +/- 0.02% of total HCN mRNA. The ratio of HCN2 to HCN4 was approximately (13.06 +/- 0.21):1. The threshold for activation of If was approximately -115 mV in the adult rats.</p><p><b>CONCLUSION</b>With the development of rats, the value of If is smaller. The threshold for activation of If is more negative. The ratio of HCN2 to HCN4 is bigger.</p>

Effects of amiodarone on funny current I(f) channel gene expression in neonatal rat ventricular myocytes / 中华心血管病杂志

<p><b>OBJECTIVE</b>To analysis the effect of amiodarone on funny current (I(f)) and hyperpolarization-activated cation channel (HCN) gene expressions of the neonatal rat ventricular myocytes.</p><p><b>METHODS</b>Ventricular myocytes of 1 - 3 days-old rats were isolated and cultured. The cardiomyocytes were treated by amiodarone (0.01, 0.1, 1, 10, 100 micromol/L) for 3 hours or amiodaron (10 micromol/L) for 0, 0.5, 1, 3, 6 hours. The I(f) and HCN 1 - 4 gene expressions were measured through the whole-cell configuration of the patch-clamp technique and real-time quantitative polymerase chain reaction (real-time PCR) using SYBR Green PCR kit.</p><p><b>RESULTS</b>(1) HCN1, HCN2, HCN3 and HCN4 represented (0.23 +/- 0.01)%, (83.58 +/- 0.04)%, (0.79 +/- 0.01)% and (15.44 +/- 0.01)% of total HCN mRNA, respectively. (2) Amiodaron resulted in a dose-dependent I(f) [(3.1 +/- 0.9)%, (9.7 +/- 2.4)%, (36.7 +/- 5.8)%, (80.3 +/- 1.8)% and (85.9 +/- 3.1)%, respectively at -145 mV, IC(50) (1.32 +/- 0.28) micromol/L], HCN2 [(2.1 +/- 0.8)%, (8.9 +/- 3.6)%, (30.1 +/- 4.2)%, (78.3 +/- 3.6)% and (81.1 +/- 1.9)%, respectively] and HCN4 decrease [(0.5 +/- 0.2)%, (2.1 +/- 2.6)%, (8.8 +/- 3.2)%, (60.1 +/- 4.6)% and (59.6 +/- 6.5)%, respectively]. (3) Amiodaron (10 micromol/L) also induced a time-dependent I(f) [(1.1 +/- 0.1)%, (12.6 +/- 2.3)%, (80.6 +/- 2.2)% and (80.1 +/- 2.1)%, respectively], HCN2 [(1.0 +/- 0.1)%, (9.8 +/- 3.9)%, (82.9 +/- 4.6)% and (83.9 +/- 1.7)%, respectively] and HCN4 decrease [(0.1 +/- 0.1)%, (1.9 +/- 1.1)%, (59.4 +/- 7.8)% and (60.9 +/- 3.1)%, respectively]. However, HCN1 and HCN3 expressions were not affected by amiodaron treatment.</p><p><b>CONCLUSION</b>Current density of I(f) and the expression of HCN2 and HCN4 were decreased by amiodaron which might be the possible antiarrhythmic working mechanisms of amiodaron.</p>

Current researches on molecular enzyme engineering / 生物工程学报

The developments of recombinant DNA technology and structural biology make it possible to modify enzyme in molecular level. Scientists show growing interests in the evolution or functional fusion of enzymes. Recent advances and applications of the molecular enzyme engineering are reviewed and discussed in this article.

High expression and identification of DNA mismatch repair gene mutS in Escherichia coli / 生物工程学报

DNA mismatch repair gene mutS (2.56 kb) was PCR modified and cloned into a secretive prokaryotic expression vector pET32a (+) which carries a N-terminal His.tag + and thioredoxin sequence. MutS protein was expressed with high level after IPTG induction using the strain E. coli AD494(DE3). SDS-PAGE revealed that the expected protein with a molecular weight of 108 kD which is about 35% of the total bacterial proteins is almost soluble. The expected protein was purified directly by immobilized metal (Ni2+) chelation affinity chromatography and the purity is over 90%. MutS protein activity verified using mismatch DNA showed that the expression product can recognize and bind to base-pair mismatch specifically.

Heterogenous Gene Expression of Methyl Parathion Hydrolase and Analysis of the Enzyme Activity / 微生物学通报

Methyl parathion hydrolase (MPH, E.C.3.1.8.1) coding gene mph from Pseudomonas sp. WBC-3, isolated and identified by our lab, was successfully expressed in E. coli AD494 (DE3)/ pET32a(+) system as soluble fusion form at high level. The recombinant MPH showed nearly 4～5 fold higher specific activity to parathion than the enzyme from Pseudomonas sp. WBC-3. In addition, the thermal stability of the recombinant enzyme was improved comparing with the wild type enzyme.