AIM: To observe the effect of mouse Sim2 (mSim2) eukaryotic expression vector transfection on the cell cycle in PC12 cells in vitro and to explore the role of Sim2 in the pathogenesis of Down syndrome. METHODS: The full open reading frame of mSim2 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into the vector pcDNA3. Then the constructed pcDNA3-Sim2 vectors were transiently transfected into PC12 with Lipofectamine~ TM . The expression of mSim2 was detected by RT-PCR. The effect of mSim2 on the cell cycle was observed by flow cytometry. RESULTS: The eukaryotic expression vector mSim2 was successfully constructed. There was notable expression of mSim2 in the cells transfected with pcDNA3-Sim2. There were more cells in G_0/G_1 phase in the pcDNA3-Sim2 transfected cells than that in the control (P

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
Antibodies/*genetics ; Antibodies/immunology ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Histidine-tRNA Ligase/biosynthesis ; Histidine-tRNA Ligase/*genetics ; Histidine-tRNA Ligase/immunology ; Open Reading Frames/genetics ; Prokaryotic Cells/metabolism

To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthase isoforms (nNOS and iNOS), rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen-glucose deprivation (OGD) for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD (P < 0.05). The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase (LDH) efflux into the incubation solution. The results suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.
Animals, Newborn ; Cell Hypoxia ; Hippocampus/cytology ; Hippocampus/*metabolism ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase Type I/*metabolism ; Nitric Oxide Synthase Type II/*metabolism ; RNA, Messenger/metabolism ; Rats, Sprague-Dawley ; Reperfusion Injury/*metabolism ; Tissue Culture Techniques

Objective To study the value of high frequency ultrasound in predicting spontaneous closure of patent duct arteriosus (PDA) in premature neonates. Methods One hundred and seventy premature neonates aged less than 37 weeks of gestation were enrolled. They were all admitted in the neonatal intensive care unit of Jinhua People′ s Hospital within 24 hours after birth. Echocardiographic examinations was conducted using GE′s Vivid I Portable color Doppler ultrasonography with routine cardiac probe (frequency 1.7-3.4 MHz) and high-frequency transducer (frequency 7.5 MHz). Up to the 7th day, the echocardiography was carried out every day after birth until the closure of PDA .They were divided to two groups according to whether spontaneous closure of PDA took place with in 7 days after birth (the control group) or not (the PDA group). There were 16 premature neonates in PDA group, including 6 male and 10 female. The mean gestational week at birth was (35.3±1.4) weeks. The mean birth weight was (2 330.9±325.5) g. Thirty-two premature neonates were included incontrol group, including 13 male and 19 female. The mean gestational week at birth was (35.0±1.3) weeks. The mean birth weight was (2 337.5±287.8) g. The clinical characteristics and echocardiographic parameters were compared between the two groups. Results The detection rates of high-frequency ultrasound and routine echocardiography were all 100%. The spontaneous closure rate of PDA in preterm infants at 24 hours, 48 hours, 72 hours were 18.8%(32/170), 61.2%(104/170), 78.8%(134/170) respectively. Within 7 days after birth, the closure rate was 90.6%(154/170), therefore the incidence of PDA in premature neonates was about 9.4%(16/170) in our study. The display rate of entire duct arteriosus using high frequency ultrasound was signiifcantly higher than that using routine ultrasound [82%(39/48) vs 46%(22/48), P<0.001, 100%(48/48) vs 77.1%(37/48), P<0.01]. The diameters of duct arteriosus in PDA group were signiifcantly larger than that in control group [(2.08±0.4) mm vs (1.09±0.22) mm, P<0.001]. However, the remaining echocardiographic parameters showed no signiifcant difference between the two groups (P>0.05). To predict the spontaneous closure of PDA in preterm neonates within 7 days after birth by high frequency ultrasound, the sensitivity, specificity and accuracy were 87.5%, 90.6% and 66.5% respectively using the cutoff point of 1.55 mm. While the sensitivity, speciifcity and accuracy were 75%, 93.7%, 44.15%by routine echocardiography with the cutoff point of 2.36 mm. Conclusions High-frequency ultrasound can conifrm the diagnosis of PDA in preterm infants and it is better than routine echocardiography in depicting the structure of arterial duct. The larger PDA diameter was suggestive of the lower possibility of spontaneous close.

This study was aimed to observe the analgesia of curing-injury Cataplasma and discuss the Nav1 . 7 expression in dorsal root ganglion ( DRG ) in model rats with formaldehyde-induced inflammatory pain . A total of 36 Sprague-Dawley rats were divided into three groups, which were the blank control group (n = 12), model group ( n = 12 ) , and treatment group ( n = 12 ) . The blank control group was without any treatment . The model group was injected with 0 . 1 mL 5% saline formalin on the left rear foot . The treatment group was applied with curing-injury Cataplasma on the left rear foot 24 h before the injection of 0 . 1 mL 5% saline formalin in the establishment of animal model . The behavior reactions to pain of model rats were observed . Dubuisson score was recorded and compared . Meanwhile , L3-6 DRG was collected from rats in each group . The expres-sion of Nav1 . 7 was detected by real-time quantitative PCR and western blot . The results showed that the pain reaction integral in the treatment group was lower than the model group ( P < 0 . 05 ) . Results from the real-time quantitative PCR showed that the relative expression of Nav1 . 7 mRNA in the model group was more than the treatment group . And the relative expression of Nav1 . 7 mRNA in the treatment group was more than the blank control group . There was significant difference among three groups ( P < 0 . 05 ) . There was no statistical difference at the three time points within three groups. Results from the western blot showed that the relative expression of Nav1 . 7 in the model group was more than the treatment group . And the expression of Nav1 . 7 in the treatment group was more than the blank control group . There was significant difference among three groups (P < 0.05). There was no statistical difference at the three time points within three groups. It was concluded that the curing-injury Cataplasma can alleviate inflammatory pain response in rats, and have certain analgesia effect . Meanwhile , it can influence Nav1 . 7 expression in DRG in model rats with formaldehyde-induced inflam-matory pain .

To investigate the protective effects of eplerenone on adriamycin-induced renal injury and the possible mechanisms involved, 36 male Sprague-Dawley rats were randomly divided into control group, adriamycin nephropathy (AN) group and eplerenone-treated group (100 mg·kg(-1)·d(-1) eplerenone). Blood pressure, 24-h urinary protein, serum potassium, sodium and creatinine were measured 28 days after adriamycin injection (a single tail intravenous injection of 6.5 mg/kg adriamycin). The morphological changes of renal tissues were observed by light and electron microscopy. Immunohistochemistry and Western blotting were performed to examine the expression of TGF-β(1) and desmin in renal cortex. The results showed that 28 days after adriamycin injection, there were no significant changes in the level of serum potassium, sodium, creatinine concentrations and blood pressure values in the rats of the three groups. Meanwhile, the 24-h proteinuria excretion in the AN group was significantly higher than that in the control group (P<0.01), but that in the eplerenone-treated group was substantially reduced when compared with that in the AN group (P<0.05). Mild mesangial cell proliferation and matrix expansion, diffuse deformation and confluence of foot processes in podocytes were found in the AN group. By contrast, rats in the eplerenone-treated group exhibited obvious attenuation of these morphological lesions. The protein expression of TGF-β(1) and desmin in the AN group was markedly up-regulated in contrast to that in the control group (P<0.01), whereas that in the eplerenone-treated group was much lower than in the AN group (P<0.05). It was concluded that eplerenone may ameliorate the proteinuria and the development of pathological alteration in adriamycin-induced nephropathy presumably via the inhibition of cytokine release, and restore the morphology of podocytes independent of its blood pressure-lowing effects.

The effect of electroacupuncture (EA) on TRPM7 mRNA expression of focal cerebral ischemia in rats and further the role of EA in the relationship between TRPM7 and trkA pathway was investigated. Thirty SD rats were randomly divided into 5 groups : normal group, ischemia/reperfusion group, EA treated group (ischemic rats with EA treatment), TE infusion group (ischemic rats with EA treatment and TE buffer infusion), AS-ODN group (ischemic rats with EA treatment and antisense trkA oligonucleotide infusion). The stroke animal model was established by the modified method of middle cerebral artery occlusion. Antisense trkA oligonucleotide that blocked NGFs effects was injected into cerebroventricle before EA. The TRPM7 mRNA was detected by RT-PCR method. The results showed that there were low TRPM7 mRNA levels in cortex and hippocampus in normal group. Compared with normal group, TRPM7 mRNA expression was increased significantly in ischemia/reperfusion group (P<0.05). A significant reduction in the expression of TR-PM7 mRNA was found in EA treated group in contrast to ischemia/reperfusion group (P<0.05). The expression of TRPM7 mRNA in AS-ODN group was remarkably increased compared with EA treated group and TE infusion group (P<0.05). The results indicated that TRPM7 channels in the ischemic cortex and hippocampus in rats might play a key role in ischemic brain injury. EA could reverse the overexpression of TRPM7 in cerebral ischemia/reperfusion rats. And the inhibitory effect of EA on TRPM7 channels might be through trkA pathway.

As a most effective monomer composition from bark of Pacific Yew,paclitaxel and its derivatives are used in clinical practice as broad spectrum anticancer drugs.Since its discovery in the 1970 s,many researches had been carried out,mainly focusing on the modification,structure-activity relationship and pharmacological activity.The great successes pressed ahead the development of a series of taxol-like drugs,including taxol,docetaxel,cabazitaxel,larotaxel.Nowadays,studies of taxol are still the hotpots,which concentrated on the new source such as cultivation of tissue,fungus culture and new dosage forms.As the representative of drugs research from natural source,taxol is worth to be summarized of its history and ongoing development for looking forward to bring new innovation mentality in new drugs.

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
Antibodies ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Histidine-tRNA Ligase ; biosynthesis ; genetics ; immunology ; Humans ; Open Reading Frames ; genetics ; Prokaryotic Cells ; metabolism