Establishment of a Huntington’s Disease in vitro Drug Screening Cell Model / 中国生物工程杂志

To develop a Huntington’s disease(HD) cell model in vitro to screen drugs targeting the aggregation of polyQ,different length of CAG repeat fragments were amplified by random primer PCR, identified by DNA sequencing and were fused to the N-terminus of CAT in the pCAR system respectively which had been constructed and identified before. Recombinant plasmids were transformed into and induced to express in the host E.coli. SDS-PAGE and chloramphenicol resistance test were done to determine the solubility of the polyQ and chloramphenicol resistance levels of the fusions. With different length of CAG repeat fragments cloned and expressed in the CAT-fusion protein reporting system, it is found that when the length of the fragments increased over 40, their encoding polyQ expressed as insoluble protein and chloramphenicol resistance levels are lower, while under 40, the polyQ expressed as soluble ones and chloramphenicol resistance levels are higher. A in vitro HD model that could minimize the pathological process of the HD thus has been developed. With which by measure the recombinant bacteria’s resistance to chloramphenicol, the polyQ’ solubility and folding state in vitro by quality and quantity could be determined. Thus this model can be used to screen drugs or bioactivity materials that can inhibit aggregation of the polyQ, which thereby shedding new light on the prevent, diagnosis and therapy of HD.

Effect of antisense oligonucleotides of ryanodine receptor on proliferation and Ca2+i of airway smooth muscle cells / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of antisense oligonucleotides (ASON) of ryanodine receptor on proliferation and [Ca2+]i concentration of airway smooth muscle cells (ASMCs).</p><p><b>METHODS</b>ASMCs were cultivated with collagen enzyme digestion method. Different concentrations of ASON were added to the cultures with Lipofectamine 2000 to observe the ASMCs proliferation using MTS/PES method. The changes of ASMCs [Ca2+]i were also observed by flow cytometry. The expression of mRNA of subtypes of RyR was assayed by RT-PCR.</p><p><b>RESULTS</b>RyR ASON restrained the proliferation of ASMCs, decreased the expression of RyR and reduced the concentration of [Ca2+]i.</p><p><b>CONCLUSION</b>RyR ASON could inhibit the proliferation of ASMCs by influencing the concentration of [Ca2+]i after excited.</p>

Adaptation of myofibrilla, MHC and metabolic enzyme of rabbit diaphragm muscle to different frequency chronic electrical stimulation / 中国应用生理学杂志

<p><b>AIM</b>To detect effect of the different frequency of chronic electrical stimulation (CES) on myofibrillar isoform, myosin heavy chain (MHC) and metabolic enzyme activities.</p><p><b>METHODS</b>The histochemical method and SDS-polyacrylamide gel electrophoresis were respectively employed.</p><p><b>RESULTS</b>(1)There were a significant increase in I myo-fibrillar isoform and I MHC isoform and decrease in II B myofibrillar isoform and II B MHC isoforms in the chronic low frequency electrical stimulation (CLFES) 10 Hz and 20 Hz groups, but opposite results were found in the chronic high frequency electrical stimulation (CHFES) 50 Hz and 100 Hz groups. (2) There were a significant increase in the aerobic-oxidative enzyme activities and capacity, and a concomitant significant drop in glycolysis enzyme activities in CLFES groups, but opposite results were found in CHFES 50 Hz and 100 Hz groups.</p><p><b>CONCLUSION</b>It was suggested that there was a significant dependent relation between chronic electrical stimulation frequency and myofibrilla isoforms, myosin heavy chain (MHC) and metabolic enzyme activities.</p>

Expression of ryanodine receptor isoforms in airway smooth muscle cells of rats / 中国应用生理学杂志

<p><b>AIM</b>To detect the expression of ryanodine receptor (RyR) subtypes in normol rat airway smooth muscle cells(ASMCs) and changes during chronic asthma formation.</p><p><b>METHODS</b>ASMCs were cultured with collagen enzyme digestion method. The expression of subtypes of RyR were detected by RT-PCR. Purified PCR product linked with pGEM-T vector to make DNA sequence assay. Chronic asthma model was made with OVA, the changes of RyRs detected by RT-PCR.</p><p><b>RESULTS</b>All subtypes of RyR were expressed in airway smooth muscle cells of normol rat. The expression of RyR1 increased obviously compared with control group (P < 0.05) on chronic asthma.</p><p><b>CONCLUSION</b>Co-expression of three subtypes of RyR in ASMCs of normal rat, indicate that there are complicated intercellular Ca2+ regulation mechanism in ASM, moreover RyR1 might play a role during asthma development.</p>

Effect of chronic electrical stimulation of phrenic nerve at different frequencies on mRNA and protein expression of skeletal DHPR(alpha1) and RyRs in the diaphragm muscle of rabbits / 生理学报

The mRNA and protein expression of skeletal dihydropyridine receptor isoform alpha1 subunit (DHPR(alpha1)) and ryanodine receptor(1-3) (RyR(1-3)) during chronic electrical stimulation (CES) of phrenic nerve have rarely been explored. In the present study, we explored the signal translation mode of calcium release unit in diaphragm muscle of rabbits after CES. Thirty rabbits were used and randomly divided into the normal, 10, 20, 50 and 100 Hz groups. Phrenic nerve was continuously (5 weeks, 2x 2 h/d) stimulated at 10, 20, 50 and 100 Hz respectively (impulse width 0.2 ms, 3~6 waves/time, 45 times/min, 10~20 V). Reverse transcription PCR and immunohistochemical methods were employed. The results showed that mRNA and protein expressions of DHPR(alpha1) and RyR(1) in 10 and 20 Hz groups were more significantly lower than those in the control group (P<0.01), but mRNA and protein expressions of DHPR(alpha1) and RyR(1) were significantly higher in 50 and 100 Hz groups than those in the control group (P<0.01); a lower level of mRNA expression of RyR(2) was found in 10 and 20 Hz groups. It is suggested that the calcium release unit and the signal transduction mode between DHPR and RyRs were altered from conformational changes of linked proteins to Ca(2+)-induced Ca(2+) release (CICR) in the diaphragmatic muscle of rabbits after chronic low-frequency electrical stimulation of phrenic nerve for 5 weeks.