Effects of arsenic trioxide on activities of MMPs and expression of TIMP-1 and TGF-β1 in skin fibroblasts and polymorphonuclear neutrophils

AIM: To observe the effects of arsenic trioxide (As₂O₃) on activities of matrix metalloproteinases (MMPs), expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta1 (TGF-β₁) in human fibroblast (hFb), and to discuss weather As₂O₃ promotes the healing of chronic skin ulcer through regulating collagen metabolism. METHODS: Zymography was used for testing activity of MMP-9 deriving from rat polymorphonuclear neutrophils (PMNs) and activities of MMP-1, MMP-2 secreted by hFb. Immunocytochemical method was used to determine the expressions of TIMP-1 and TGF-β₁. RESULTS: At the concentration of 50 mg/L, As₂O₃ elevated the activity of MMP-9 (P<0.01). At the concentration of 0.8 mg/L, As₂O₃ increased the activities of MMP-1 and MMP-2 (P<0.01, respectively). After hFb was cultured with As₂O₃ for 6 h, 12 h and 18 h, the expressions of TIMP-1 and TGF-β₁ decreased continuously (P<0.01). CONCLUSION: As₂O₃ elevates the activities of MMP-1, MMP -2 and MMP-9, also inhibits the expressions of TIMP-1 and TGF-β₁, suggesting that arsenic preparation may exert positive effect on healing chronic skin ulcer through regulating collagen metabolism.     

Intervention of gossypol and relationship between cognitive function and expressions of 11β-HSD1 and GR in brain of type 2 diabetic rats

AIM: To study the effect of gossypol on the cognitive function of type 2 diabetic rats, and to explore its mechanism. METHODS: Thirty male Sprague-Dawley rats were divided into three groups randomly: normal group, type 2 diabetic group and gossypol treated group. After fed with high-fat diet for 4 weeks, the later two groups were injected with streptozotocin intraperitoneally to establish type 2 diabetic rat model. The animals in gossypol treated group were given gossypol at dosage of 15 mg/kg once per day for 4 weeks by gavage. Since 5th week, the times of gavages were changed into once per week at the same dosage and lasted to 12th week. Learning and memory abilities of rats were assayed with Morris water maze test. The concentration of blood glucose was measured by biochemical method. The levels of serum corticosterone and insulin were detected by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. The protein expressions of 11β-HSD1 and GR in cerebral cortex and hippocampus were determined by Western blotting. The morphological changes of cerebral cortex and hippocampus were observed under light microscope and transmission electronic microscope, respectively. RESULTS: Compared to normal group, the karyopyknosis, dilation of golgiosome and mitochondria swelling of neuron from cerebral cortex and hippocampus were prominent in diabetic group. The concentrations of blood glucose, serum corticosterone and insulin increased significantly (P<0.01). Protein expression of GR decreased (P<0.05), 11β-HSD1 protein tended to increase. Platform searching score was lower (P<0.01) and escape latency was longer (P<0.01) in diabetic group. After treated with gossypol, the concentrations of blood glucose, serum corticosterone and insulin declined (P<0.01). The protein expression of 11β-HSD1 was decreased (P<0.05) and GR was increased (P<0.05). Escape latency was shorter (P<0.01) and platform searching score was increased (P<0.01). CONCLUSION: Gossypol may improve the cognitive function of type 2 diabetic rats. Decreasing the level of 11β-HSD1 and increasing GR protein in the brain may be involved in the mechanism.     

Effect of caveolin-1 and ERK1/2 on 17β-estradiol-induced inhibition of VSMC proliferation

AIM: To investigate the effect of caveolin-1 and phosphorylation of ERK1/2 on 17β-estradiol (E2) induced inhibition of vascular smooth muscle cells (VSMCs). METHODS: The proliferation in cultured VSMCs was determined by using ［3H］-thymidine incorporation. The expressions of caveolin-1, MKP-1 and ERK1/2 phosphorylation were measured by Western blotting. The expression of caveolin-1 mRNA was measured by RT-PCR. RESULTS: Exposed to fetal calf serum (FCS) for 24 h, the increase in proliferation of VSMCs was detected by ［3H］-thymidine incorporation. Pretreatment with various concentrations of E2 for 24 h inhibited VSMC proliferation induced by FCS. The results of Western blotting and RT-PCR showed that pretreated with 17β-estradiol for 24 h reserved the decrease in caveolin-1 induced by FCS. Western blotting results further proved that the expression of MKP-1 was significantly increased and the expression of ERK1/2 phosphorylation was decreased after incubated with 17β-estradiol. CONCLUSION: 17β-estradiol increases caveolin-1 and MKP-1 expressions, and decreases ERK1/2 phosphorylation, leading to the inhibition of VSMC proliferation.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Effects of 1-MCP Treatment on Quality and Physiology of ‘ Whangkeunbae' Pear During Cold Storage

以“黄金梨”果实为试材,研究了1-MCP处理对冷藏“黄金梨”果实品质及生理的影响.结果表明:500 nL/L l-MCP处理能有效延缓“黄金梨”果实贮藏期间硬度下降以及可溶性固形物的增加,抑制呼吸强度、多酚氧化酶和过氧化物酶活性的变化,减轻“黄金梨”货架期间果实褐变的发生.     

Effects of diosmin on changes of TNF-α, IL-1β, IL-6, IL-8 and IL-10 in serum and kidney tissues of rats with kidney ischemia and reperfusion

AIM: To observe the effects of diosmin on the production of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, IL-8 and IL-10 in serum and kidney tissues of rats with kidney ischemia and reperfusion (I/R). METHODS: Sprague-Dawley rats (180 in total) were randomly divided into 3 groups including sham operation group (sham),I/R group and diosmin+I/R group (diosmin+I/R). At the end of the experiment, the blood and kidney tissues were obtained and TNF-α, IL-1β, IL-6, IL-8 and IL-10 were detected by ELISA. RESULTS: The levels of TNF-α, IL-1β, IL-6, IL-8 and IL-10 in serum and kidney tissues in I/R group and diosmin+I/R group were significantly higher than those in sham group (P<0.01 or P<0.05). Following the development of the pathologic process, the level of TNF-α, IL-1β, IL-6 and IL-8 was significantly increased in I/R group and diosmin+I/R groups, but the level of IL-10 was significantly decreased in I/R group and significantly increased in diosmin+I/R group. The levels of TNF-α, IL-1β, IL-6 and IL-8 in I/R group was significantly higher than those in diosmin+I/R group (except TNF-α at 1 h in diosmin+I/R group). The level of IL-10 in diosmin+I/R group was significantly higher than that in I/R group (P<0.01 or P<0.05). CONCLUSION: Diosmin not only decreases the production of TNF-α, IL-1β, IL-6 and IL-8, but also promotes the production of anti-inflammatory cytokine IL-10, suggesting that the protective effect of diosmin on kidney I/R injury was associated with anti-inflammatory mechanism.     

Expression of recombinant quadrivalent Aβ1-15 and its immunogenicity identification

AIM:To prepare the second-generation Alzheimer disease vaccine of quadrivalent tandem Aβ1-15 and to explore its immunogenicity.METHODS: The 4×Aβ15 gene was amplified by PCR with the recombinant plasmid pcDNA-4×Aβ15 as a template, which was prepared by our research group before. The 4×Aβ15 gene was then cloned to the pGEX-4T-2 plasmid. The fused GST-4×Aβ15 protein was expressed when the bacteria containing the recombinant plasmid was induced. The purified 4×Aβ15 protein was then obtained after the GST-4×Aβ15 protein was digested by thrombin. Meanwhile, BALB/c mice were used to confirm whether they could generate Aβ specific humoral immunity when immunized with the 4×Aβ15 protein.RESULTS: After identification and DNA sequence analysis, the recombinant plasmid pGEX-4×Aβ15 was constructed. Expression of recombinant 35 kD GST-4×Aβ15 was verified by SDS-PAGE and Western blotting. The 9 kD 4×Aβ15 protein was produced after thrombin digestion of GST-4×Aβ15. Immunizing mice with the 4×Aβ15 protein have induced high titre anti-Aβ antibodies.CONCLUSION:The recombinant prokaryotic vector pGEX-4×Aβ15 was constructed successfully, and the purified 4×Aβ15 protein was obtained after prokaryotic expression. It is demonstrated that the 4×Aβ15 protein is a strong immunogen, confirmed by immunization of BALB/c mice.     

Effects of immune-activated platelets and LDL on expression and activity of COX-2 and PPAR-α in HUVECs

AIM: To observe the effect of immune-activated platelets and low-density lipoprotein cholesterol (LDL) on the expression and activity of cyclooxygenase-2 (COX-2) and peroxisome proliferator activated receptor α (PPAR-α) in human umbilical vein endothelial cells (HUVECs) treated with activated platelets and LDL. METHODS: The platelets were activated by ADP. The co-culture system of HUVECs with immune activated platelets and/or LDL were established. The activity of COX-2 and expression of PPAR-α at mRNA and protein levels in HUVECs were detected by RT-PCR and Western blotting. The concentration of PGE2 was measured by ELISA for representing the COX-2 activity. The PPAR-α activity was determined by a nuclear factor assay kit. RESULTS: The COX-2 activity and mRNA expression of PPAR-α, the protein levels of COX-2 and PPAR-α and PGE2 concentration in activated platelets group were significant higher than those in un-activated platelets group (all P<0.01). No difference of PPAR-α binding activity was observed between two groups. LDL didnt affect the COX-2 activity and PPAR-α expression, but significantly promoted the stimulating effect of immune-activated platelets. CONCLUSION: Immune-activated platelets significantly promote COX-2 activity and PPAR-α expression in HUVECs, but dont change the PPAR-α binding activity. LDL at general concentration does not affect the expression and activity of COX-2 and PPAR-α, but promote the effect of activated platelets on HUVECs.     

Effects of TNF-α-induced changes of expression and activity of 11-β HSD-1 on the insulin sensitivity in 3T3-L1 adipocytes

AIM: To observe the effects of tumor necrosis factor-α（TNF-α）-induced changes of expression and activity of 11-beta-hydroxysteroid dehydrogenase type1(11-β HSD-1) on the insulin sensitivity in 3T3-L1 adipocytes.METHODS: 3T3-L1 adipocytes were treated with TNF-α and TNF-α combined with aspirin, 2’-hydroxyflavanone or RU486, then mRNA expression and activity of 11-β HSD-1 and insulin-stimulated glucose uptake were examined.RESULTS: TNF-α increased expression and activity of 11-β HSD-1 in 3T3-L1 adipocytes, and decreased insulin-stimulated glucose uptake. Aspirin decreased expression and activity of 11-β HSD-1 induced by TNF-α, and alleviated the inhibiting effect of TNF-α on insulin-stimulated glucose uptake. 11-β HSD-1 specific inhibitor 2’-hydroxyflavanone and cortisol-receptor antagonist RU486 also alleviated the inhibiting effect of TNF-α on insulin-stimulated glucose uptake. CONCLUSION: TNF-α may decrease the insulin sensitivity in 3T3-L1 adipocytes through increasing expression and activity of 11-β HSD-1.     

Effect of miR-206 over-expression on proliferation and migration of pap?illary thyroid carcinoma K1 cells

AIMTo determine the effect of microRNA-206 (miR-206) on proliferation and migration of human papillary thyroid carcinoma K1 cells and to explore its possible mechanism. METHODSThe expression of miR-206 in the K1 cells was detected by RT-qPCR. The cell viability was detected by CCK-8 assay. The number of viable K1 cells was counted by the method of Trypan blue exclusion. The migration ability of K1 cells was detected by Transwell chamber migration assay. Bioinformatics software was used to predict the target gene of miR-206. The targeting relationship between miR-206 and c-Met was verified by dual-luciferase reporter assay. The protein levels of c-Met, p-Met, AKT, p-AKT, mTOR and p-mTOR were determined by Western blot. RESULTSAfter the K1 cells were transfected with miR-206 mimic transiently, the relative expression of miR-206 in treatment group was significantly higher than that in blank group and negative control group (P<0.01). The results of CCK-8 assay and Trypan blue exclusion assay showed that the proliferation ability of K1 cells in treatment group transfected with miR-206 mimic was significantly inhibited compared with other groups (P<0.01). The results of Transwell assay showed that the number of migratory K1 cells in treatment group was lower than that in blank group and negative control group (P<0.01). Moreover, our results demonstrated that miR-206 directly targeted c-Met and repressed the activation of downstream AKT/mTOR signaling pathway. CONCLUSION miR-206 over-expression inhibits the proliferation and migration abilities of papillary thyroid carcinoma K1 cells, and its mechanism may be related to the inhibition of c-Met/AKT/mTOR signaling pathway.     

Effect of erythromycin on transforming growth factor-β1 and γ-glutamylcysteine synthetase in lung of smoking rats

AIM: To investigate the effect of erythromycin on the level of transforming growth factor-β₁ (TGF-β₁) and γ-glutaglutamylcysteine synthetase (γ-GCS) in smoking rats,and to explore the antioxidate therapeutic role of erythromycin in chronic obstructive pulmonary disease.METHODS: Wistar rats were exposed to cigarettes smoking to establish the model.After passive smoking for 4 weeks,erythromycin intragastric intervention was administered continuously for 8 weeks.The expiratory airway resistance and lung compliance were assessed and the expression levels of TGF-β₁ and γ-GCS proteins (and the mRNA) in airway endothelial cells and alveolar macrophages were observed respectively by immunohistochemical,immunocytochemical and (in situ) hybridization.RESULTS: The expiratory airway resistance was increased and the lung compliance was degraded significantly in smoking group and erythromycin group,compared to control group.In erythromycin group,the airway resistance was lower and the lung compliance was higher than that in smoking group (P<0.05).The levels of TGF-β₁ and γ-GCS in smoking group and erythromycin group was obviously increased in airway endothelial cells and alveolar macrophages in comparison with control group (P<0.05).The levels of TGF-β₁ and γ-GCS were inhibited by erythromycin (P<0.05).TGF-β₁ was obviously positive correlated with γ-GCS in smoking group,but this was not found in erythromycin group.CONCLUSION: Erythromycin therapy improves pulmonary function and relieves emphysema change induced by smoking in rats,and decreases the expression of TGF-β₁ and γ-GCS in alveolar macrophages and airway endothelial cells,suggesting that erythromycin may play a role in the antioxidate therapeutic in COPD.     

Effect of the acupotome therapy and electro-acupuncture on the serum contents of IL-1β, IL-6 and TNF-α in rabbits with knee osteoarthritis

AIM: IL-1β, IL-6 and TNF-α play important roles in emergence of osteoarthritis. This study aims at observing the effect of the acupotome therapy and electro-acupuncture on the cytokines in serum of rabbits with osteoarthritis. METHODS: 52 New Zealand rabbits were divided randomly into the normal group, model group, acupotome group and electro-acupuncture group. Knee osteoarthritis of the model rabbits was made with the straightened immobilization method. The acupotome and electro-acupuncture therapies were applied for three weeks. One week after the treatment the serum was collected, and the changes of IL-1β, IL-6 and TNF-α in serum were detected with RIA. RESULTS: In comparison with the control group, the contents of IL-1β, IL-6 and TNF-α elevated significantly in the knee osteoarthritis model group (P<0.05). Compared to the knee osteoarthritis model group, the contents of the cytokines in acupotome treatment group and electro-acupuncture treatment group decreased significantly (P<0.05). No significant difference between the content of cytokines in the acupotome treatment or electro-acupuncture treatment groups (P<0.05) was observed, also no statistical difference between the acupotome treatment or electro-cupuncture treatment groups and the control group was found. However, the contents of the three cytokines in the acupotome treatment and electro-acupuncture treatment groups were still higher than those in control group. CONCLUSION: The acupotome and electro-acupuncture treatment can decrease the release of IL-1β, IL-6 and TNF-α, indicating that the two therapies play an important role in improvement of the articular cartilage cell injury and function through inhibiting the generation of matrix protease and alleviation of degradation of the cartilage matrix.     

Effect of Pruning and Partial Disbudding on the Cropping of Comice and Beurré Hardy Pears

The effects of three intensities of pruning on fruit set, fruit bud development, cropping and vigour of intensively grown Cornice and Beurré Hardy pears were compared over a three-year period. Severe pruning, in which extension shoots were cut back to fruit buds on the two-year-old wood, greatly increased the set of fruits per 100 flower clusters and reduced vigour, in comparison with a renewal type of pruning. Severe pruning of Comice caused a relative reduction in the number of fruit buds in subsequent years, so the improvement in fruit set did not lead to an increase in crop per tree, but with Beurré Hardy fruit bud formation was unaffected and the crop per tree was increased by hard pruning. There was an improvement in yield in relation to tree size with both varieties. Pruning to fruit buds provides a means of increasing yields per acre by ‘containing’ pear trees at close spacings without reducing the yield per tree.

Partial disbudding just before blossoming increased fruit set but not enough to offset the reduction in number of fruit buds. Supplementary pollination did not improve the yields of Comice and increased the crop of Hardy in one year only.     

Effect of Yangjing-Zhongyu decoction on in vitro maturation and expression of PDGFA and PDGFRα of mouse oocytes

AIMTo investigate the effects of Yangjing-Zhongyu decoction on the in vitro maturation and expression of platelet-derived growth factor subunit A (PDGFA) and platelet-derived growth factor receptor α (PDGFRα) of mouse oocytes. METHODSThe SPF female KM mice were given Yangjing-Zhongyu decoction, and the blood was collected to prepare serum. The serum containing Yangjing-Zhongyu decoction was used to culture immature oocyte-granulosa cell complexes. After the in vitro culture, the oocyte maturation rate, fertilization rate and cleavage rate were observed and calculated, and the expression of PDGFA and PDGFRα in the oocytes at protein and mRNA levels was determined by West?ern blot and real-time PCR. RESULTSYangjing-Zhongyu decoction increased the in vitro maturation rate and fertil?ization rate of the oocytes (P<0.05 or P<0.01), and down-regulated the protein and mRNA expression of PDGFA and PDGFRα (P<0.01). CONCLUSION Yangjing-Zhongyu decoction may promote the in vitro maturation of mouse oocytes by down-regulating the expression of PDGFA and PDGFRα.     

Analysis the efficiency of inter and intra-specific hybridization of new cultivars of sweet cherry and chinese cherry北大核心CSCD

【Objective】To explore the rules of intra-specional and inters-pecific cross breeding of some new sweet cherry cultivars and improve the efficiency of cross breeding of sweet cherry in the area where winter is warm and summer is hot and moist.【Methods】The intraspecial and interspecifichybridization test of sweet cherry was carried out by using sweet cherry as the mother,using sweet cherry and chinese cherry as the father. The rate of fruit set,embryo,embryo abort of the cross and quality of the F0 fruit were investigated in order to analysis the efficiency of the cross.【Results】The four cultivars of sweet cherry‘Jiangnanhong’‘Chaoyang-1’‘Changfeng-1’and‘Van’were taken as the mother. Five sweet cherry cultivars or selections‘04-8’‘04-11’‘Chaoyang-1’‘Van’‘Lapins’and five cultivars of chinese cherry‘Chaozaohong’‘Duanbing’‘Gejiawu’‘Heizhenzhu’‘Zijing’were taken as the male parent. Among them‘Jiangnanhong’and‘Zijing’are newly identified cultivars in Zhejiang province,and‘Chaoyang-1’and‘Changfeng-1’are new selections. After pollination for 1 week,the fruit setting rate was relatively high and significant decrease in the 4 weeks and mature stages in intra and inter- species with the development of fruits. In the mature stage,all the fruit setting of the other cross of intra-species were higher than 5%,except that of‘Jiangnanhong’ב04-11’was 1.85% lower than 3%,and that of‘Changfeng-1’בLapins’was 4.35% lower than 5%,which indicates the basic affinity between the cross of intra-specific sweet cherry. The fruit setting rate of intraspecific cross was higher than that of interspecific cross. The fruit setting rate of‘Chaoyang-1’was the highest in both intraspecific and interspecific hybridization. The interspecific hybrid combination of‘Changfeng-1’had the lowest fruiting rate. The embryo rate and abortion rate of the intraspecific cross were higher than those of interspecific cross when the female parent is same. The abortion rate is high when hybrid embryos mature,and only 4 crosses group can obtain non-abortion embryos,which are the hybrid cross of‘Jiangnanhong’with‘04-8’‘Lapins’‘Chaozaohong’,and the cross of‘Changfeng-1’with‘Van’. The abortion is lower when the hybrid embryos immature. The embryo rate was lowest and the abortion degree was the highest of the cross of‘Jiangnanghong’in immature embryo stage. The cross with the mother of‘Chaoyang-1’was the best with high setting rate,high embryo rate and low abortion rate in immature embryo stage,so‘Chaoyang-1’is the best female parent in this test.‘Chaoyang-1’and‘Zijing’were the better fathers in the ten test male parents for the cross with them had a high embryo rate (65.84% and 75.21%,respectively),and most of the embryos were bright white and full with good development in the immature embryos stage. ‘Chaoyang-1’is the best female and male parent in this test. The cross between‘Chaoyang-1’and‘Van’shows‘Chaoyang-1’is the better female than male parent. The ripening stage of F0 fruit was delayed,and the color and shape of F0 fruit showed the same col- or and shape of open pollination of parent fruit. The soluble solid of interspecific hybrid fruit was lower than that the intra-specific hybrid,and they all lower than that of the open pollinated female fruit. In order to obtain hybrid offspring,the author suggests three methods could be used in the area where is warm in winter,hot and moist in summer: one is selecting hybrid offspring which comes from the main producing areas,which means making cross and getting hybrid offspring in the main producing areas but select it in future planting area. The other is using embryos rescue technology after getting immature fruit; the third one is using mixed pollen for open pollination.【Conclusion】The fruit set and embryo development have significant differences among the different cross group in the area where winter is warm and summer is hot and moist. The fruit set is higher and the embryo abort is lower of intraspecific cross than that of interspecific cross when the mother is the same sweet cherry.‘Chaoyang-1’is the best female and male parent in this test when making cross. © 2019 Journal of Fruit Science     

Development of hypo-allergenic apples: silencing of the major allergen Mal d 1 gene in ‘Elstar’ apple and the effect of grafting

Summary

Many people who are allergic to birch pollen are also allergic to apple fruit, due to cross- allergenicity. Since apples are the most extensively consumed fruit in Europe, it is highly relevant to develop a hypo-allergenic apple.Apples with significantly reduced levels of the allergen, Mal d 1, may allow many apple allergics to eat them without an allergic reaction. We are currently collaborating to develop a hypo-allergenic apple within the European Integrated Research Project, ISAFRUIT (www.isafruit.org). Hypo-allergenic apple plants (Malus × domestica Borkh., ‘Elstar’) with decreased levels of Mal d 1 mRNA were produced by RNA interference (RNAi) technology. Ten genetically modified (GM) apple lines were selected. In vitro plantlets were first transferred to a greenhouse, then grafted onto wild-type M.9 rootstock to promote the development of fruit-producing trees. Levels of Mal d 1 gene silencing were measured repeatedly by quantitative real-time PCR. Compared to leaf samples from wild-type ‘Elstar’, two GM lines showed modest levels of gene silencing (up to 250-fold), whereas the other eight GM lines were significantly silenced (up to10,000-fold) in Mal d 1 gene expression. These levels of silencing were unaffected by grafting, and have been stable over more than 3 years, and throughout all developmental stages.     

Effect of breviscapin on Ito and IK1 channel current in isolated ventricular myocytes of rats

AIM: The effects of breviscapin on transient outward potassium current (I_to) and inward rectifier potassium current (I_K1) channel in isolated ventricular myocytes of rats were determined. The mechanism of breviscapin at the ionic channel level was explored. METHODS: Single ventricular myocyte of rats was isolated by enzymatic dissociation. The whole-cell patch-clamp recording technique was used to record the change of I_to and I_K1 channel current influenced by breviscapin. RESULTS: Breviscapin decreased the I_to channel current in a dose-dependent and voltage-dependent manner in ventricular myocytes of rats. (1) The current-voltage curve was significantly decreased. Breviscapin at concentrations of 0.02, 0.05, 0.08, 0.10 g·L^-1 respectively decreased I_to current density (10.07±0.50)%, (27.47±2.36)%, (42.72%±2.50)% and (56.09±5.60)%. I_to was inhibited in a voltage-dependent manner, showing a significant attenuating effect at test potentials from 0 to + 50 mV. (2) The I_to activation curve, the activation curve and the recovery curve were not altered. (3) Breviscapin did not affect I_K1. CONCLUSION: Breviscapin concentration-dependently decreases I_to channel current in ventricular myocytes of rat.