Mechanism of protein phosphatase-2Aregulating phosphorylation of amyloid precursor proteosome and Aß generation.

OBJECTIVE: To discuss whether PP-2A influences the phosphorylation level at APP threonine 668 locus thus regulating Aß secretion. METHOD: In the experiment, 24 hours after N2a cells of stably transfected human APP (N2a/APP) were treated with okadaic acid (OA) or DES (C6-ceramide) (N2a/APP), an injection of OA cerebral stereotactic was administered to a SD rat in the hippocampal region, or PP-2A overexpressed plasmids was transfected transiently, the aggregate levels of APP phosphorylated APP, and APP-CTF were detected through immunoblotting and the activity of PP-2A and secretase was also detected using a reagent kit. RESULT: The phosphorylation level of APP was significantly increased after the PP-2A activity was inhibited by OA. DES activated PP-2A or over-expressed PP-2A was able to reduce the phosphorylation level of APP. Either can inhibit PP and reduce the phosphorylation level of APP. The level of phosphorylated APP was increased significantly after the SD rat was injected with OA through the hippocampal region. The activity of ß- and γ-secretases in N2a/APP cells significantly increased after OA treatment whereas the α-secretase activity had no significant changes; the Aß level increased. CONCLUSION: We discovered that PP-2A was capable of regulating the Aß level by regulating APP phosphorylation level and ß and γ-secretase activity(Fig. 5, Ref. 30).

[Inhibition of CD4(+)T cell infiltration by interleukin-10 competent B cells in periodontitis tissues].

Objective: To study the immune regulation function of high expressing interleukin-10 (IL-10) in B cells on CD4(+)T-cells in periodontitis mouse model. Methods: Twenty-four 7-weeks-old female C57BL/6 mice were randomly and equally assigned into 4 groups: the healthy control group (HC group, n=6), the ligation combined Porphyromonasgingivalis (Pg) infection group (P group, n=6), the ligation combined Pg infection with non-stimulated B cell transfer group (P+NSB group, n=6) and the ligation combined Pg infection with stimulated B cell transfer group (P+SB group, n=6). Ligation combined Pg infection of the maxillary second molar was used to induce periodontitis of mice. The exogenous non-stimulated B cells or stimulated B cells were injected into the palate gingivalat the 5th day after ligation, and all mice were sacrificed at the 14th day. HE stain was used to detect the histological of periodontal tissues, toluidine blue stain was used to analysis the alveolar bone loss, immunofluorescence stain was used to detect the expression of CD4(+)T-cell and IL-10, immunohistochemical was used to detect the expression of receptor activator of NF-κB ligand (RANKL) and IL-1ß. Results: Results of HE staining showed that more hyperplasia of gingival epithelium and the alveolar bone resorption in P group, P+NSB group and P+SB group compared with HC group. Results of toluidine blue staining showed that the alveolar bone losses in P group [(0.668±0.041) mm(2)], P+NSB group [(0.750±0.039) mm(2)] and P+SB group [(0.517±0.038) mm(2)] were significantly increased compared with that in HC group [(0.336±0.029) mm(2)](F=146.051, P<0.01), and the alveolar bone resorption was significantly increased in P+NSB group compared with that in P group (F=146.051, P<0.01). However, compared with P+NSB group and P group, the alveolar bone loss in P+SB groupwas significantly decreased (F=146.051, P<0.01). Results of immunofluorescence staining showed that CD4(+)T-cells expressed in P group [(287.5±37.9) cell/mm(2)], P+NSB group [(314.6±53.3) cell/mm(2)] and P+SB group [(185.4±42.9) cell/mm(2)] were higher than that in HC group [(12.5±13.7) cell/mm(2))(F=71.284, P<0.01). Compared with P group and P+NSB group, CD4(+)T-cells expression in group P+SB was decreased (F=71.284, P<0.01). IL-10 levels were increased in P group [(111.7±20.4) cell/mm(2)], P+NSB group [(126.7±15.1) cell/mm(2)] and P+SB group [(191.0±22.6) cell/mm(2)] compared with that in HC group [(22.7±4.3) cell/mm(2)] (F=98.516, P<0.01), and the IL-10 expressed in P+SB group was significantly higher than those in P+NSB group and P group. Results of immunohistochemical tests showed that RANKL expressions in gingival tissues among P group [(674.0±71.5) cell/mm(2)], P+NSB group [(831.0±97.5) cell/mm(2)] and P+SB group [(420.1±40.8) cell/mm(2)] were significantly higher than that in HC group [(69.3±29.1) cell/mm(2)] (F=154.886, P<0.01). However, it dramatically decreased in P+SB group compared with those in P group and P+NSB group.The IL-1ßexpression in P group [(447.8±40.8) cell/mm(2)], P+NSB group [(512.5±38.2) cell/mm(2)] and P+SB group [(281.6±32.4) cell/mm(2)] were significantly higher than that in HC group [(50.8±20.9) cell/mm(2)], and it also higher in P+NSB group compared with in P group. However, it decreased in P+SB group compared with those in P group and P+NSB group (F=221.185, P<0.01). Conclusions: High expression IL-10 in B cell smight inhibit alveolar bone loss, RANKL and IL-1ß expressions and CD4(+)T-cell infiltration through IL-10.

A study on grafting and characterization of HMDI-modified calcium hydrogenphosphate.

It is known that the organic molecules can provide an effective means to manipulate the surface properties of the biodegradable ceramic. There are two ways to modify the surface of the biodegradable ceramic by organic molecules. The first one is through surface adsorption but organic molecules will easily be washed out in the physiological environment. The second approach is to graft organic molecules through covalent bond to the hydroxyl groups that are available on the surface of the ceramics. Isocyanate group has been reported as a coupling agent for hydroxyapatite and organic molecule. The studies showed that the isocyanate could react with hydroxyl groups of hydroxyapatite and form a covalent bond between isocyanate and hydroxyapatite. In the study, hexamethylene diisocyanate (HMDI) was used as coupling agent and calcium hydrogenphosphate (CaHPO4, CHP) was the candidate ceramic. CHP will react with HMDI at the temperature of 20 degrees C, 30 degrees C, 40 degrees C, 50 degrees C, 60 degrees C, and 70 degrees C for 4h. Dibutyltin dilaurate and hydroquinone were used as catalyst and inhibitor, respectively. The effect of reaction temperature on the grafted yield will be described. The linkage between CHP and HMDI will be characterized by DTA, TGA, FTIR, XRD, and 31P, 13C liquid state NMR. From the results, we successfully modified the surface of CHP with coupling agent of HMDI. The grafted yield of HMDI on CHP was increasing with the reaction temperature. The best temperature for CHP modified by HMDI is around 50 degrees C. The linkage between HMDI and the surface of CHP is a urethane linkage as CHP-O-CO-NH-(CH2)6-N=C=O. After further treatment, the terminal group of CHP treated with HMDI (MCHP) will be converted into a primary amine group as the formula of CHP-O-CO-NH-(CH2)6-NH2. If reaction temperature is 60 degrees C, long extension chain will occur with a urea linkage between the isocyanate groups as the formula of CHP-O-CO-NH-(CH2)6-(NH-CO-NH-(CH2)6)n-NH2. At reaction temperature higher than 60 degrees C, the HMDI will become prepolymerized forms in solution. The prepolymerized forms such as allophanate, biuret, uretidione and urea linkage will turn the solution into gel type mixture, which will lead to low grafted yield of HMDI on CHP. When MCHP prepared at the temperature 20 degrees C, there is no evidence of long extension but the grafted yield is the lowest only 0.9 wt% around.

Evaluation of adhesion force and binding affinity of phytohemagglutinin erythroagglutinating to EGF receptor on human lung cancer cells.

PHA-E is a natural product extracted from red kidney beans, and it has been reported to induce cell apoptosis by blocking EGFR in lung cancer cells. Because EGF is the major in vivo competitor to PHA-E in clinical application, PHA-E must be proved that has better affinity to EGFR than EGF. This study would focus on how PHA-E tightly bind to EGFR and the results would compare with EGF. The adhesion force, measured by AFM, between EGFR and PHA-E was 207.14±74.42 pN that was higher than EGF (183.65±86.93 pN). The equilibrium dissociation constant of PHA-E and EGF to EGFR was 2.4 10(-9)±1.4 10(-9) and 7.3 10(-8)±2.7 10(-8), respectively, that could evaluate binding affinity. The result showed that binding affinity of PHA-E to EGFR was one order higher than EGF to EGFR. In the results of flow cytometer and confocal microscope, we found binding efficiency of EGF to EGFR was decrease as the concentration of PHA-E increased. In the analysis of Western blot, treatment of A-549 cells with PHA-E resulted in a dose-dependent decrease in EGFR phosphorylation. In conclusion, we found that PHA-E had better adhesion force and binding affinity to EGFR than that of the EGF. The interaction between PHA-E and EGFR could block EGF binding and then inhibit EGFR phosphorylation. PHA-E could be developed into a new target molecule for lung cancer treatment that could be immobilized on the drug carrier to guide therapeutic particles to the tumor site.

Preparation and characterization of surface-modified calcium hydrogenphosphate by hexamethylene diisocyanates.

Isocyanate group has been reported as a coupling agent of hydroxyapatite and polymers. The studies showed that the isocyanate would react with hydroxyl groups of hydroxyapatite and form a covalent bond between isocyanate and hydroxyapatite. In the study, hexamethlene diisocyanate (HMDI) was used as coupling agent. Calcium hydrogen-phosphate (CaHPO4, CHP) powders was the candidate ceramic due to higher content of hydroxyl group, which would react with HMDI at the temperature of 30, 40, 50, 60, and 70 degrees C for 4 hours. Dibutyltin dilaurate and hydroquinone were used as catalyst and inhibitor, respectively. The product was analyzed by DTA, TGA, FTIR, XRD, 13C solid state NMR and 31P, 13C liquid state NMR. From the results, we could prove the surface of calcium hydrogen-phosphate has been successfully modified. The largest amount (5.6 wt%) of HMDI could be grafted on the surface of CHP when reacted at 50 degrees C for 4 hours. Some chain extension could be observed and their structure would also be described in the research.