Sodium nitrophenolate mediates brassinosteroids signaling to enhance cold tolerance of cucumber seedling.

Cold stress (CS) significantly limits cucumber yield. However, it remains unclear whether and how sodium nitrophenolate (CSN) regulates plant responses to cold stress. Here, H2O, CSN, 24-epibrassinolide (EBR), and CSN + EBR were sprayed on cucumber seedlings before or after CS, and on control plants. We found that CSN, EBR, or EBR + CSN pre-treatment improved seedling growth under normal conditions (control condition) and cold tolerance under CS conditions. EBR pre-treatment promoted the expression of approximately half of the genes involved in BR synthesis and signaling and CsICE-CsCBF-CsCOR under CS. However, CSN pre-treatment promoted almost all the expression of BR synthesis and signaling genes, and CsICE-CsCBF-CsCOR genes, which showed the highest expression in early CS, remarkably improving the cold tolerance of cucumber. Interestingly, EBR and CSN had a superimposed effect on the expression of BR synthesis and signaling and CsICE-CsCBF-CsCOR genes, which rapidly increased their expression under normal temperature. Spraying EBR after CS accelerated seedling recovery, whereas CSN had the opposite effect. However, spraying CSN combined with EBR accelerated the recovery of CS-injured seedlings and was better than spraying EBR alone. Although CS-injured seedlings were negatively influenced by CSN, pre-treatment with CSN accelerated seedling growth and increased cold tolerance, suggesting that the effect of CSN was related to whether the seedlings were damaged by CS. In conclusion, we firstly found that CSN enhanced cold tolerance by activating BR signaling, contributing to the gene expression of ICE-CBF-COR and that CSN + EBR contributed to cold tolerance and CS-injured seedling recovery in cucumber.

Construction of CaMKⅡγ RNA interference vector with lentivirus and its effect on osteoclast differentiation and bone resorption function / 实用医学杂志

Objective To investigate the effects of calmodulin-dependent kinase IIγ (GaMKIIγ) RNA interference on osteoclast differentiation and bone resorption. Methods Three CaMKIIγ recomninant RNA interference vectors were constructed using lentivirus. Negative vector was used to transfect RAW264.7 cells and the multiplicity of infection (MOI) with the optimal transfection efficiency was determined. Recombinant vectors were also used to transfect cells to determine the one with the best interference effect for following experiments.Then, the cells were divided into control group, negative vector group and interference vector group. Five days after virus transfection, osteoclastogenesis and bone resorption function were determined by TRAP staining and dentin resorption lacunae detection. Results Three CaMKIIγ recombinant interference vectors were constructed, and the optimal MOI was 30, under which transfection efficiency was about 81％. The #3 recombinant vector showed the best interference effect and the interference efficiency was up to 78.16％ at mRNA level and 67.02％ at protein level. When compared with control group, the number of multi-nucleated osteoclasts, the number and area of dentin resorption lacunaes in interference vector group decreased 59.99％、54.19％ and 57.94％ respectively (P < 0.01). No significant difference were observed between negative vector group and control group (P> 0.05).Conclusion CaMKIIγ RNA interference significantly inhibits osteoclastogenesis and bone resorption.

Expression profiles of CaMKⅡγ during osteoclast differentiation / 中南大学学报(医学版)

Objective:To study the expression profiles and the role of Ca2+/calmodulin-dependent protein kinase Ⅱγ (CaMKⅡγ) during osteoclast differentiation.Methods:Mouse RAW264.7 cells were induced for osteoclastogenesis with 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and the cells were harvested at 0,1,3 and 5 days after induction.Tartrate-resistant acid phosphotase staining was performed to verify osteoclasts formation.RT-PCR,Western blot and immunofluorescent cytochemistry were used to detect the CaMKⅡγ gene expression during osteoclastogenesis.Results:The osteodasts were formed at day 3 under RANKL induction and more osteoclasts were observed at day 5.At day 0,1,3 and 5,the relative level of CaMKⅡγ mRNA were (1.067±0.179),(1.840±0.070),(9.493±0.453) and (30.767±0.573),respectively,and the relative protein level were (0.454±0.065),(0.613±0.021),(0.858±0.019) and (0.980±0.023),respectively.CaMKⅡγ expression was increased in a time-dependent manner except relative protein level at day 1 (P＜0.01),which showed no significant difference at day 0 (P＞0.05).Immunofluorescence assay showed that CaMKⅡγ protein was also increased with differentiation of osteoclasts.Conclusion:The CaMKⅡγ expression was increased in a time-depended manner during osteoclast differentiation and it might play a vital role during osteoclastogenesis.

Effect of zoledronate on protein interaction between Ca2+/calmodulin-dependent protein kinaseⅡ and calmodulin and expression of downstream genes during osteoclast differentiation / 中华口腔医学杂志

Objective@#To investigate the effect of zoledronate on protein interaction between Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and calmodulin and protein expression of nuclear factor of activation of T cells-1 (NFATc1) and tartrate resistant acid phosphatase (TRAP) during osteoclast differentiation. @*Methods@#Mouse RAW264.7 cells were divided into group A and B and were cultured. Group A was induced with 50 mg/L receptor activator of NF-κB ligand (RANKL) for osteoclastogenesis, and group B was treated with 1×10-6 zoledronate for two days from day 2. Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMKⅡ and calmodulin. Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc1 and TRAP in both groups. Osteoclast formation was also analyzed. @*Results@#In group B, the number of osteoclasts, number and size of dentin resorption lacunaes were 11.3±1.5, 8.7±2.1 and (5 034.4±775.4) μm2 respevtively, which were significantly lower than those (37.7±5.7, 23.0±4.0 and [15 042.7±1 906.0] μm2) in group A (P<0.01). Co-IP and reverse Co-IP examination indicated that protein-binding between CaMKⅡ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A (P<0.01). The protein level of calmodulin and CaMKⅡ in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A (P<0.01). NFATc1 and TRAP protein decreased by 52.4% and 38.9% in group B than in group A (P<0.01), respectively. @*Conclusions@#Zoledronate could significantly inhibit protein-binding between CaMKⅡ and calmodulin and down-regulate protein level of NFATc1 and TRAP.

The effects of Gegen Qinlian decoction on high sensitive C-reactive protein and interleukin-6 in serum of the rats with periodontitis / 实用口腔医学杂志

Objective:To observe the effect of Gegen Qinlian decoction on high sensitive C-reactive protein(hs-CRP)and interleu-kin-6(IL-6)in serum of the rats with periodontitis and the IL-6 expression level in periodontal tissues.Methods:45 Wistar rats were randomly divided into 2 groups:control group N(n =1 0),periodontitis model group P(n =35).After periodontitis model was identi-fied by the examination of 5 rats,the rest rats were randomly divided into 3 groups(n =1 0)and treated by gavage of saline(group P0 ), metronidazole(group P1 )and Gegen Qinlian decoction(group P2 )respectively for 4 weeks.Then all the rats were sacrificed.Serum was immediately harvested for the test of serum hs-CRP and IL-6 levels by ELISA.Attachment loss level(AL)was examined by pa-thology.IL-6 expression in periodontal tissues was examined by S-P immunohistochemistry.Results:The inflammation of periodontal tissues in P1 ,P2 were improved more than that in P0 .AL in group P2 were lower than that in other groups(P <0.05).The IL-6 expres-sion in periodontal tissues and the levels of serum hs-CRP and IL-6 of group P2 were lower than those of other groups(P <0.05).Ser-um hs-CRP level was positively correlated with IL-6 level.Conclusion:Gegen Qinlian decoction may inhibit the development of peri-odontitis by depressing the expression of serum hs-CRP and IL-6.

The effect of Gegen Qinlian decoction on serum high sensitive C-reactive protein expression in rats with pe-riodontitis and atherosclerosis / 实用口腔医学杂志

Objective:To observe the effect of Gegen Qinlian decoction on serum high sensitive C-reactive protein(hs-CRP)expres-sion of rats with periodontitis and atherosclerosis(P-AS).Methods:40 male Wistar rats were randomly divided into 2 groups:control group(A,n=1 0),P-AS group(B,n=30).5 rats in group A and B were used for the identification of P-AS model.Then rats with P-AS in group B were randomly divided into 5 groups(n=5)and administered with saline(B1 ),atorvastatin(B2),metronidazole (B3),atorvastatin+metronidazole(B4)and Gegen Qinlian decoction(B5)respectively for 4 weeks.Serum hs-CRP level was assayed by ELISA.Periodontal attachment loss(AL)and artery change were examined by pathology.Results:Serum hs-CRP level in group B1-5 was higher than that of group A(P0.05.The inflammation of periodontal tissues in group B2-5 was improved more than that in group B1 .AL in group B4 and B5 were lower than that in other groups(P0.05.Histopathological observation of arteries re-vealed that there was no foam cell in group B4 and B5 ,the artery wall in group B4 and B5 was more even and muscle fibers were ar-ranged more regular.Conclusion:Gegen Qinlian decoction may decreas serum hs-CRP expression and depress the development of pe-riodontitis and atherosclerosis.

Clinical application of sartorius tendon transposition during radical vulvectomy: a case control study of 58 cases at a single institution / 부인종양

OBJECTIVE: The aim of this study was to investigate the clinical effects of sartorius tendon transposition versus sartorius transposition during bilateral inguinal lymphadenectomy of radical vulvectomy. METHODS: A total of 58 vulvar cancer patients who had surgery from May 2007 to October 2013, in which 30 patients received sartorius transposition and 28 patients received sartorius tendon transposition. All patients were matched by age, body mass index, stage, histology, and grade. Intraoperative variables and postoperative complications, recurrence, progression-free survival (PFS), and overall survival (OS) and postoperative life quality were compared and analyzed. RESULTS: No significant differences were found at median surgical times and amounts of bleeding (p=0.316 and p=0.249, respectively), neither at the incidences of groin cellulitis and lymphocele (p=0.673 and p=0.473, respectively), but the recovery times of the inguinal wounds were shorter (p=0.026) and the incidences of wound break and chronic lymphedema were significantly decreased in the tendon transposition group (p=0.012 and p=0.022, respectively). Postoperative quality of life in tendon transposition group was significantly improved as indicated by the EORTC QLQ-C30 questionnaire. Recurrences were similar (p=0.346) and no significant differences were found at PFS and OS (p=0.990 and p=0.683, respectively). CONCLUSION: Compared to sartorius transposition, sartorius tendon transposition during inguinal lymphadenectomy led to improved patient recovery, reduced postoperative complications, and improved life quality without compromising the outcomes.

[Effect of zoledronate on the osteoclast adhesion and gene expression of integrin α(v) and ß3].

OBJECTIVE: To explore the effect of zoledronate (ZOL) on the osteoclast adhesion and expression of integrin α(v) and ß3 in vitro. METHODS: Mice RAW264.7 cells were used for osteoclast differentiation in vitro, and osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining and dentin resorption lacunae examination. The cells were then divided into 2 groups, the control group and ZOL treatment group (treated with 1 x 10(-6) mol · L(-1) ZOL for 2 d). The adhesion ability of osteoclasts and mRNA and the protein expressions of integrin α(v) and ß3 were examined by crystal violet staining, real-time fluorescence quantitative polymerase chain reaction, Western blot analysis, and immunofluorescent chemistry. RESULTS: TRAP staining and dentin resorption lacunae examination revealed the formation of multi-nuclear osteoclasts. ZOL treatment significantly decreased the adhesion ability of osteoclasts (P < 0.01). In the ZOL-treated group, the mRNA levels of integrin α(v) and ß3 were 0.66 ± 0.05 and 0.59 ± 0.08, respectively. In the control group, the mRNA levels of integrin α(v) and ß3, were 1.01 ± 0.01 and 1.01 ± 0.02, respectively; these values were higher than those in the ZOL-treated group (P < 0.01). The protein level of integrin α(v) and ß3 in the ZOL-treated group (31,934.84 ± 112.91 and 18,812.79 ± 194.13) was downregulated by approximately 39.19% and 40.17%, respectively, compared with those in the control group (52,517.81 ± 211.72 and 31,441.93 ± 456.87) (P < 0.01). Immunofluorescent examination showed that the fluorescent intensities of integrin α(v) and ß3 in the ZOL-treated group (9.491 ± 0.748 and 4.744 ± 0.759) were also significantly decreased compared with those in the control group (15.159 ± 1.143 and 11.418 ± 1.095) (P < 0.01). CONCLUSION: ZOL significantly inhibits osteoclast adhesion and downregulates integrin α(v) and ß3, expression, thus contributing to the ZOL-induced inhibition of osteoclast- mediated bone resorption.

Multiple enamel pearls on left maxillary third molar: a case report / 华西口腔医学杂志

Enamel pearl is an ectopic enamel, which usually occurs in the root bifurcate or approaching enamel-cementum site of the first maxillary molar. A case of multiple enamel pearls on the left maxillary third molar is reported in this paper, and relevant literature was reviewed.

Effect of zoledronate on the osteoclast adhesion and gene expression of integrin α(v) and β3 / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the effect of zoledronate (ZOL) on the osteoclast adhesion and expression of integrin α(v) and β3 in vitro.</p><p><b>METHODS</b>Mice RAW264.7 cells were used for osteoclast differentiation in vitro, and osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining and dentin resorption lacunae examination. The cells were then divided into 2 groups, the control group and ZOL treatment group (treated with 1 x 10(-6) mol · L(-1) ZOL for 2 d). The adhesion ability of osteoclasts and mRNA and the protein expressions of integrin α(v) and β3 were examined by crystal violet staining, real-time fluorescence quantitative polymerase chain reaction, Western blot analysis, and immunofluorescent chemistry.</p><p><b>RESULTS</b>TRAP staining and dentin resorption lacunae examination revealed the formation of multi-nuclear osteoclasts. ZOL treatment significantly decreased the adhesion ability of osteoclasts (P < 0.01). In the ZOL-treated group, the mRNA levels of integrin α(v) and β3 were 0.66 ± 0.05 and 0.59 ± 0.08, respectively. In the control group, the mRNA levels of integrin α(v) and β3, were 1.01 ± 0.01 and 1.01 ± 0.02, respectively; these values were higher than those in the ZOL-treated group (P < 0.01). The protein level of integrin α(v) and β3 in the ZOL-treated group (31,934.84 ± 112.91 and 18,812.79 ± 194.13) was downregulated by approximately 39.19% and 40.17%, respectively, compared with those in the control group (52,517.81 ± 211.72 and 31,441.93 ± 456.87) (P < 0.01). Immunofluorescent examination showed that the fluorescent intensities of integrin α(v) and β3 in the ZOL-treated group (9.491 ± 0.748 and 4.744 ± 0.759) were also significantly decreased compared with those in the control group (15.159 ± 1.143 and 11.418 ± 1.095) (P < 0.01).</p><p><b>CONCLUSION</b>ZOL significantly inhibits osteoclast adhesion and downregulates integrin α(v) and β3, expression, thus contributing to the ZOL-induced inhibition of osteoclast- mediated bone resorption.</p>

Effect of bisphosphonate on osteoclast differentiation and tartrate-resistant acid phosphatase / 中国组织工程研究

BACKGROUND:Tartrate-resistant acid phosphatase is a specific marker for osteoclast differentiation and bone resorption, which is a sign of osteoclast maturity. OBJECTIVE:To study the effect of alendronate on tartrate-resistant acid phosphatase related to osteoclast differentiation and bone resorption. METHODOsteoclasts were cultured by mouse monocyte-macrophage cellline-RAW264.7. The cells were divided into two groupcontrol group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor;alendronate group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor+10-7 mol/L alendronate. Osteoclastogenesis and resorption function of osteoclasts were examined at 7 days of culture. Gene expression of tartrate-resistant acid phosphatase was detected by immunofluorescence method. Western blot assay was used to detect protein expression of tartrate-resistant acid phosphatase. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase positive multinuclear cells were observed and resorption lacunae formed in two groups. Control group showed the higher number of tartrate-resistant acid phosphatase positive multinuclear cells and larger size of resorption lacunae than the alendronate group (P<0.01). Immunofluorescence showed expression of tartrate-resistant acid phosphatase was higher in the control group than the alendronate group (P<0.01);furthermore, the protein expression of tartrate-resistant acid phosphatase was also lower in the alendronate group than the control group (P<0.01). These findings indicate that bisphosphonates could strongly inhibit osteoclastogenesis and its resorption function by inhibiting protein expression of tartrate-resistant acid phosphatase.

Bisphosphonate effects on capthesin K and bone resorption function during osteoclast differentiation / 中国组织工程研究

BACKGROUND:Studies have shown that bisphosphonates inhibit osteoclast resorption, but whether cathepsin K, a key cytokine of bone resorption, plays an effect has rarely been reported. OBJECTIVE:To study the effect of bisphosphonate on capthesin K and bone resorption function during osteoclast differentiation. METHODS:Osteoclasts were cultured by mouse monocyte-macrophage cellline-RAW264.7. The cells were divided into two groups:control group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor;alendronate group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor+10-7 mol/L alendronate. Osteoclastogenesis and resorption function of osteoclasts were examined at 7 days of culture and gene expression of capthesin K was detected by immunofluorescence method at 72 hours of culture. Western blot assay was used to detect capthesin K protein expression at 72 hours of culture. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase positive multinuclear cells were observed and resorption lacunae formed in two groups. Control group showed the higher number of tartrate-resistant acid phosphatase positive multinuclear cells and larger size of resorption lacunae than the alendronate group (P<0.01). Immunofluorescence showed expression of capthesin K was higher in the control group than the alendronate group (P<0.01);furthermore, the protein expression of capthesin K was also lower in the alendronate group than the control group (P<0.01). These findings indicate that bisphosphonates could strongly inhibit osteoclastogenesis and its resorption function by inhibiting gene expression of capthesin K.

Effect of alendronate on expressions of osteoprotegerin and receptor activator of nuclear factor κB ligand in mouse osteoblasts / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of alendronate on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in mouse osteoblasts.</p><p><b>METHODS</b>Mouse calvarial osteoblasts cultured in vitro were identified by alkaline phosphatase (ALP) staining and immunofluorescence assay of OPG and RANKL expressions. The second passage of the osteoblasts were treated with different concentrations of alendronate (10(-4) to 10(-7) mol/L) for 48 h, and the changes in OPG and RANKL mRNA and protein expressions were examined using real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>The isolated osteoblasts were positive for ALP and expressed OPG and RANKL. Real-time PCR and Western blotting showed that at the concentration of 1×10(-4) mol/L, alendronate caused an obvious down-regulation of OPG and RANKL expressions in the cells, whereas at lower concentrations, alendronate increased the expressions of both genes with the highest expressions occurring after treatment with 1×10(-5) mol/L.</p><p><b>CONCLUSION</b>High concentrations of alendronate (>1×10(-4) mol/L) decrease the expressions of OPG and RANKL, whereas low concentrations (1×10(-5) to 1×10(-7) mol/L) increase their expressions in mouse osteoblasts cultured in vitro.</p>