Junctophilin-2 allosterically interacts with ryanodine receptor type 2 to regulate calcium release units in mouse cardiomyocytes.

The interaction between junctophilin-2 (JPH2) and ryanodine receptor type 2 (RyR2) regulated Ca2+ signaling in mouse cardiomyocytes. However, their exact interaction remains unclear. This study elucidates the interaction between JPH2 with RyR2 using co-immunoprecipitation of cardiac sarcoplasmic reticulum vesicles. Additionally, a glutathione S-transferase (GST) pull-down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. JPH2 interacted with RyR2 and the C terminus of the JPH2 protein can pull-down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines in isolated mouse cardiomyocytes. Knockdown of JPH2 reduced the amplitude of Ca2+ transients and disrupted its interaction with RyR2. Therefore, the C-terminus domain of JPH2 is required for interactions with RyR2 in mouse cardiomyocytes, which provides a molecular mechanism for seeking Ca2+-related disease prevention strategies.

[Identification of yougui and jisheng shenqi pills with FTIR and EDS fingerprint spectra by new visual comparison].

FTIR combined with EDS fingerprint spectra was first applied to the identification of two kind of traditional Chinese compound formulae-Yougui pill and Jisheng shenqi pills, which have the similar composition The IR FPS of extraction of two kinds of pills extracted with chloroform were measured by liquid membrane method. The exclusively characteristic peak groups of these two kinds of formulae were theoretically established based on the Shapiro-Wilk W testing method,and the characteristic radicals and compound species corresponding to each characteristic peak were determined. Meanwhile, EDS fingerprint spectra of the two kinds of original powders were also measured which can reflect the element species and content information. Based on the three kinds of information (characteristic peak groups, radicals and compound species, different elements), Yougui and Jisheng shenqi pills were identified quickly, precisely and reliably. In this method, infrared fingerprint spectra possess the similar ability with chromatograph fingerprint spectra in identification of traditional Chinese compound formulae. The results show that the new visual comparison method is suitable for identifying traditional Chinese compound formulae with the same dosage-form and similar composition.

[Influence of infection of murine chemokine receptor-7 recombinant lentivirus on the immunogenicity and migration of DC 2.4 cells].

OBJECTIVE: To observe the influence of infection of murine chemokine receptor-7 recombinant lentivirus on the immunogenicity and migration of dendritic cell strain DC 2.4 cells. METHODS: DC 2.4 cells were routinely cultured. Lentiviruses carrying GFP and those with up-regulated CCR7 were constructed. DC 2.4 cells were divided into DC 2.4 group (without any treatment), GFP-DC 2.4 group (infected with GFP-carrying lentivirus), and CCR7-DC 2.4 group (infected with CCR7-carrying lentivirus labeled by GFP) according to the random number table. The expressions of surface molecules MHCII, CD80, CD86, and CCR7 were detected by flow cytometry, Western blotting, and confocal laser scanning microscope. The migration of cells was detected by chemotaxis assay in vitro. The immunogenicity of cells was detected with mixed lymphocyte reaction. LPS-DC 2.4 group was set up as positive control. Data were processed with one-way analysis of variance and t test. RESULTS: Lentiviruses carrying stably-expressing CCR7 were constructed, and the transfection rate of which into DC 2.4 cells was 87.4%. There was no statistically significant difference among DC 2.4, GFP-DC 2.4, and CCR7-DC 2.4 groups in the expressions of MHC II, CD80, and CD86 as showed by flow cytometry (with F values from 0.17 to 1.19, P values all above 0.05). The protein expression of CCR7 of cells in CCR7-DC 2.4 group (45.1 ± 2.1) was obviously higher than that in DC 2.4 and GFP-DC 2.4 groups (25.3 ± 1.4, 28.6 ± 0.9, F = 162.90, P < 0.01), while the difference of which between DC 2.4 group and GFP-DC 2.4 group was not statistically significant (t = 2.20,P > 0.05). The fluorescence intensity of CCR7 in CCR7-DC 2.4 group was obviously increased compared with that of DC 2.4 group. The chemotaxis migration rate of cells in CCR7-DC 2.4 group with the influence of CCL19 was (41.0 ± 2.0)%, which was significantly higher than that of DC 2.4 and GFP-DC 2.4 groups [(6.0 ± 0.5)%, (6.8 ± 0.3)%, F = 84.21, P < 0.01]. There was no statistically significant difference between DC 2.4 group and GFP-DC 2.4 group in the migration rate (t = 0.45, P > 0.05). The absorbance values in DC 2.4, GFP-DC 2.4, CCR7-DC 2.4, and LPS-DC 2.4 groups were respectively 1.6 ± 0.4, 1.9 ± 0.4, 1.7 ± 0.4, 3.8 ± 0.4, and the differences among the former three groups were not obvious (F = 1.56, P > 0.05). The absorbance value in LPS-DC 2.4 group was obviously higher than that of the other three groups (with t values from 1.53 to 1.82, P values all below 0.01). CONCLUSIONS: DC 2.4 cells infected with efficiently CCR7-expressing lentivirus showed high chemotaxis to CCL19, but without obvious change in immunogenicity.