[Preparation and preliminary application of rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha].

OBJECTIVE: To prepare rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha. METHODS: Two peptides corresponding to human platelet GPIbalpha C-terminus were synthesized and purified by high-performance liquid chromatography (HPLC). The peptides were cross-linked with keyhole limpet hemocyanin (KLH). Two New Zealand white rabbits were immunized with conjugated peptides for 3 times. The polyclonal antibodies were purified by Ammonium Sulfate Precipitation and identified by dot blotting and ELISA. GPIbalpha intracellular peptides phosphorylation was tested with these polyclonal antibodies by ELISA. RESULTS: The titers of the two polyclonal antibodies against the GPIbalpha C-terminus peptides were 1:32 000 and 1:64 000 respectively and both of these antibodies reacted with purified GPIbalpha. CONCLUSIONS: Two rabbit polyclonal antibodies against C-terminal peptides of human platelet GPIbalpha have been prepared successfully, providing a way for the preparation of these kinds of antibody. Both phosphorylation and dephosphorylation states exist in the intracellular peptide of human platelets.

Effect of Kaiyu Qingwei Jianji on the morphometry and residual strain distribution of small intestine in experimental diabetic rats.

AIM: To investigate the effect of a Chinese medicine, Kaiyu Qingwei Jianji (KYQWJJ) used for diabetic treatment, on the morphometry and residual strain distribution of the small intestine in streptozotocin (STZ) -induced diabetic rats. Correlation analysis was also performed between the opening angle and residual strain with the blood glucose level. METHODS: Forty-two male Wistar rats weighing 220-240 g were included in this study. Thirty-two STZ-induced diabetic rats were subdivided into four groups (n = 8 in each group), i.e. diabetic control group (DM); high dose of KYQWJJ (T1, 36 g/kg per day); low dose of KYQWJJ (T2, 17 g/kg per day) and Gliclazide (T3, 50 mg/kg per day). Another ten rats were used as non-diabetic control (CON). The medicines were poured directly into stomach lumen by gastric lavage twice daily. The rats of CON and DM groups were only poured the physiological saline. Blood glucose and plasma insulin levels were measured. Experimental period was 35 d. At the end of experiment, three 5-cm long segments were harvested from the duodenum, jejunum and ileum. Three rings of 1-2 mm in length for no-load and zero-stress state tests were cut from the middle of different segments. The morphometric data, such as the circumferential length, the wall thickness and the opening angle were measured from the digitized images of intestinal segments in the no-load state and zero-stress state. The residual strain was computed from the morphometry data. Furthermore, the linear regression analysis was performed between blood glucose level with morphometric and biomechanical data in the different intestinal segments. RESULTS: The blood glucose level of DM group was consistent 4-fold to 5-fold higher than those in CON group during the experiment (16.89+/-1.11 vs 3.44+/-0.15 mmol/L, P < 0.001). The blood glucose level in the T1 (16.89+/-1.11 vs 11.08+/-2.67 mmol/L, P < 0.01) and T3 groups (16.89+/-1.11 vs 13.54+/-1.73 mmol/L, P < 0.05), but not in T2 group (P > 0.05) was significantly lower than those in DM group. The plasma insulin levels of DM, T1, T2 and T3 groups were significantly lower than those in CON group (10.98+/-1.02, 12.52+/-1.42,13.54+/-1.56,10.96+/-0.96 vs 17.84+/-2.34 pmol/L respectively, P < 0.05), but no significantly difference among the groups with exception of CON group. The wet weight/cm and total wall thickness of duodenum, jejunum and ileum in DM group were significantly higher than those in CON group (wet weight (g/cm): duodenum 0.209+/-0.012 vs 0.166+/-0.010, jejunum 0.149+/-0.008 vs 0.121+/-0.004, ileum 0.134+/-0.013 vs 0.112+/-0.007; Wall thickness (mm): duodenum 0.849+/-0.027 vs 0.710+/-0.026, jejunum 0.7259+/-0.034 vs 0.627+/-0.025, ileum 0.532+/-0.023 vs 0.470+/-0.010, all P < 0.05), T1 and T3 treatment could partly restore change of wall thickness, but T2 could not. The opening angle and absolute value of inner and outer residual stain were significantly smaller in duodenal segment (188+/-11 degrees, -0.31+/-0.02 and 0.35+/-0.03 vs 259+/-15 degrees, -0.40+/-0.02 and 0.43+/-0.05) and larger in jejunal (215+/-20 degrees, -0.30+/-0.03 and 0.36+/-0.06 vs 172+/-19 degrees, -0.25+/-0.02 and 0.27+/-0.02) and ileal segments (183+/-20 degrees, -0.28+/-0.01 and 0.34+/-0.05 vs 153+/-14 degrees, -0.23+/-0.03 and 0.29+/-0.04) in DM group than in CON group (P < 0.01). T1 and T3 treatment could partly restore this biomechanical alteration, but strong effect was found in T1 treatment (duodenum 243+/-14 degrees, -0.36+/-0.02 and 0.42+/-0.06, jejunum 180+/-15 degrees, -0.26+/-0.03 and 0.30+/-0.06 and ileum 163+/-17 degrees, -0.23+/-0.03 and 0.30+/-0.05, compared with DM, P < 0.05). The linear association was found between the glucose level with most morphometric and biomechanical data. CONCLUSION: KYQWJJ (high dose) treatment could partly restore the changes of blood glucose level and the remodeling of morphometry and residual strain of small intestine in diabetic rats. The linear regression analysis demonstrated that the effect of KYQWJJ on intestinal opening angle and residual strain is partially through its effect on the blood glucose level.

Tellimagrandin I enhances gap junctional communication and attenuates the tumor phenotype of human cervical carcinoma HeLa cells in vitro.

Tellimagrandin I and chebulinic acid, two hydrolysable tannins, have been shown to exert anti-tumor properties. Dysfunctional gap junctional communication (GJIC) has been recognized as being involved in carcinogenesis. The human cervical carcinoma HeLa cells have been reported to be deficient in functional GJIC. In present study, we investigated whether tellimagrandin I and chebulinic acid might restore functional GJIC in HeLa cells. Both compounds could inhibit the growth of HeLa cells. Either Lucifer yellow transfer assay or calcein transfer assay demonstrated that tellimagrandin I improved GJIC in HeLa cells while chebulinic acid showed no effect on GJIC. The GJIC enhancement by tellimagrandin I occurred along with an increase of Cx43 gene expression at mRNA and protein levels. Exposure to tellimagrandin I also led to inhibition of proliferation and anchorage-independent growth of HeLa cells. In addition, tellimagrandin I decreased the percentage of cells in the G0/G1 and G2/M phases coinciding with an increase in the percentage of cells in the S phase. The accumulation of cells in S phase was coupled with a decreased expression of cyclin A that was critical to the progression of S phase. These results suggested that restoring GJIC might be one explanation for tellimagrandin I antitumor effects, whereas chebulinic acid exerted antitumor action through other pathways.

Morphological properties and residual strain along the small intestine in rats.

AIM: Residual stress and strain are important for gastrointestinal function and relate to the geometric configuration, the loading conditions and the zero-stress state of the gastrointestinal tract. The purpose of this project is to provide morphometric data and residual strains for the rat small intestine ( n =11). METHODS: To approach the no-load state, the intestine was surgically excised, transferred to an organ bath and cut transversely into short ring-shaped segments. Each ring was cut radially for obtaining the zero-stress state. The residual stress can be characterised by an opening angle. The strain difference between the zero-stress state and the no-load state is called residual strain. RESULTS: Large morphometric variations were found along the small intestine. The wall thickness was highest in the proximal duodenum and decreased in distal direction along the axis of the small intestine (P<0.001). The circumferential length of the inner and outer surfaces decreased rapidly along the length of duodenum by 30-50% (P<0.001). The wall area and lumen area showed a similar pattern (P<0.001). In zero-stress state the rings always opened up after making the cut. The experiments resulted in larger inner circumferential length and smaller outer circumferential length when compared to the no-load state. The wall thickness and wall area did not differ between the no-load and zero-stress state. The opening angle and tangent rotation angle increased along the length of the duodenum and had its highest value 30% down the intestine. Further down the intestine it decreased again (P<0.001). The serosal residual strain was tensile with the highest value close to the ligament of Treitz (P<0.001). The mucosal residual strain was compressive in all segments of the small intestine with average values between -0.25 and -0.4 and with the lowest values close to the ligament of Treitz (P<0.001). CONCLUSION: Axial variation in morphometric properties and residual strains were found in the small intestine. Existence of large residual strains indicates that the zero-stress state must be considered in future biomechanical studies in the gastrointestinal tract.