AIM: To investigate the activity and distribution of calcineurin (CaN) in different tissues of rat. METHODS: Using western blot and immunohistochemical staining methods to measure the amount and location of CnA?, isoform of catalytic subunit of CaN in different tissues of rat. CaN activity was measured by labelled substrate peptide. RESULTS: 1. Western blot showed that CnA? expression was detected in brain, heart, skeletal muscle and lung tissues. There was no detectable CnA? expression in kidney and aorta. 2. In immunohistochemical staining study, there was strong immunostaining of CnA? in brain. CnA? was located in cytoplasm of cardiac cell, macrophage and connective tissue of peribronchiolar in lung tissue, aorta adventitia, connective tissue around small vessels and outer wall of renal tube. 3. CaN activity was highest in brain, the following was skeletal muscle, myocardium and lung tissue. CaN activity was lowest in aorta and kidney tissue. CONCLUSION: CaN is widely distributed in rats and might be involved in functional regulation of various organs and tissues.

Objective: To determine the role of cross-talk between calcineurin-dependent signal transduction pathway and protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and protein kinase A (PKA) in airway remodeling in asthma. Methods: Male guinea pigs were sensitized with intraperitoneal injections of ovabumin (OVA), then treated with cyclosporin A (CsA,5 mg/kg), an inhibitor of calcineurin, then inhaled OVA for 2 weeks 14 days later. Activities of calcineurin, PKC, MAPK, and PKA were was analyzed by phosphorylation and dephosphorylation. In primary cultures of rat airway smooth muscle cells (ASMC), activities of calcineurin, PKC, MAPK, and cross-talk induced by urotensin Ⅱ (UⅡ), a recently identified strong mitogen, were measured. Results: (1) The activities of calcineurin, MAPK and PKC increased by 19% (P0.05). (4) CsA 10 -6 mol/L inhibited UⅡ-stimulated PKC activity by 14% (P0.05). Conclusion:The signal transduction pathways between calcineurin and other protein kinases such as PKC, MAPK and PKA have cross-talk in airway remodeling in asthma.

Objective To analyze the patterns of amino acid changes in liver failure patients treated with non-bioartificial liver support system (ALSS), and to explore the efficacy of ALSS in liver failure treatment. Methods A total of 146 liver failure patients treated with ALSS from June 2009 to August 2010 were recruited in this study. Paired blood samples were collected from every patient and serum amino acids and ammonia were tested by automatic amino acid analyzer. The changes of amino acids in patients with different prognoses, different types/phases of liver failure were evaluated.Measurement data were compared by paired t test. Results After ALSS treatment, liver failure patients experienced a significant decrease in serum glutamic acid and lysine [(395.62±200.24)μmol/Lvs (260. 05±169.56) μmol/L and (436. 73±326. 18)μmol/L vs (407. 12±292.01) μmol/L,respectively; t= 8. 611 and 2. 659, respectively; both P＜0.01)], while experienced greatly increases in threonine and branched-chain amino acids/aromatic amino acid ratio [( 1302. 90 ±1288.70) μmol/L vs (1406.70 ±1272. 34) μmol/L and 1. 23 ± 0. 53 vs 1. 36 ± 0.57, respectively; t = 2. 895 and 1. 061,respectively; both P＜0. 01)]. The changes of glutamic acid, tyrosine, arginine and methionine before and after ALSS treatment in patients with different prognoses, different types/phases of liver failure were all significantly different. Conclusions ALSS treatment could improve the serum amino acid disorder in liver failure patients. The amino acids in patients with different types/phases or different prognoses of liver failure change significantly after ALSS treatment.

AIM:To study lipopolysaccharide (LPS)-stimulated secretion of endothelin-1 (ET-1) and adrenomedullin (Adm) from human vascular endothelial cells (HVEC) and its mechanism. METHODS:In cultured HVEC, LPS was used to stimulate ET-1 and Adm secretion from HVEC. The contents of ET-1 and Adm in medium were determined by radioimmunoassay. RESULTS:LPS stimulated secretion of ET-1 and Adm from HVEC in time-dependent and concentration-dependent manner. The ratio of secreted ET-1 to Adm was not changed compared with the control group. The increase of ET-1 could be inhibited by inhibitor of extracellular signal-regulated protein kinases (PD098059) and inhibitor of P38 kinase (SB202190)（P＜0.01）, while the increase of Adm could only be inhibited by SB202190（P＜0.05）, both had no response to inhibitor of protein kinase C (H7), inhibitor of calmodulin (W7), inhibitor of calcineurin (cyclosporin A) and inhibitor of Ca2+ (nicardipine)（P＞0.05）.CONCLUSION:ERKs and P38 signal pathways may play an important role in the secretion of ET-1 from LPS -stimulated HVEC, while only P38 kinase signal pathway is invovled in the secretion of Adm.

AIM: To investigate the effect of endothelin (ET), angiotensin II (AngII) and homocysteine (Hcy) on C-type natriuretic peptide (CNP) synthesis and release. METHODS: Human endothelial cell was cultured; CNP was measured by radioimmunoassay method. RESULTS: ET and AngII could augment CNP synthesis in human endothelial cells. Compared with control group, 10-9,10-8,10-7 mol/L ET and Ang II increased CNP content of endothelial cells by 1%(P＞0.05), 49%(P＜0.05),117%(P＜0.01) and 137% (P＜0.01),165%(P＜0.01),201%(P＜0.01),respectively. A great dose of ET and Ang II also stimulated CNP release from cultured human endothelial cells. Hcy had no effect on CNP synthesis, but 10-9，10-8,10-7 mol/L Hcy enhanced CNP release from cultured human endothelial cells by 17%(P＞0.05),84%(P＜0.01) and 555%(P＜0.01), respectively. CONCLUSION: ET, AngII and Hcy might be involved in the synthesis and release of human endothelial cell CNP.

AIM: To study the role and regulation of calcineurin(CaN) in angiotensin II(AngⅡ)-stimulated cardiacmyocyte hypertrophy of rats. METHODS: Using AngⅡ to induce the cultured cardiac myocyte hypertrophy of rats, and investigating the effect of CaN inhibitor on [ 3H]-leucine incorporation of AngⅡ-stimulated cardiomyocytes and the regulation of various factors on CaN activity in cardiomyocytes.RESULTS: AngⅡ can stimulate the CaN activity in cultured neonatal rat cardiomyocytes in a dose- and time-dependent manner. In cardiac myocytes incubated with 10, 100, 1000 nmol?L -1 of AngⅡ for 12h, the CaN activities increased respectively by 13%,57%( P

AIM: To study lipopolysaccharide (LPS)-stimulated secretion of endothelin-1 (ET-1) and adrenomedullin (Adm) from human vascular endothelial cells (HVEC) and its mechanism. METHODS: In cultured HVEC, LPS was used to stimulate ET-1 and Adm secretion from HVEC. The contents of ET-1 and Adm in medium were determined by radioimmunoassay. RESULTS: LPS stimulated secretion of ET-1 and Adm from HVEC in time-dependent and concentration-dependent manner. The ratio of secreted ET-1 to Adm was not changed compared with the control group. The increase of ET-1 could be inhibited by inhibitor of extracellular signal-regulated protein kinases (PD 098059 ) and inhibitor of P38 kinase (SB 202190 )( P 0.05).CONCLUSION: ERKs and P38 signal pathways may play an important role in the secretion of ET-1 from LPS -stimulated HVEC, while only P38 kinase signal pathway is invovled in the secretion of Adm.

AIM: To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS: DNA synthesis of cultured rat aortic VSMC was measured by -TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [?- 32 P]-ATP. RESULTS: UⅡ(10 -8 mol/L) significantly increased -TdR incorporation of VSMC and MAPK activities by 38%( P0.05 ), 32%( P0.05 ), 32%( P

According to the super-position principle of the reinforcement of biological activities, a series of novel E-substituted 2, 3-diaryl propenoic acyloxy phosphonate derivatives were designed and synthesized. And the structures of the target compounds were confirmed by IR, 1H NMR, 13C NMR and elemental analysis. Furthermore, the cytotoxicities of all compounds on A-549, SGC-7901 and EC-109 in vitro were evaluated by MTT assay, and some of them showed good antitumor activity. Among the active compounds, especially, the IC50 value of compound 3e was (12.7 ± 1.9) μmol x L(-1) against A-549 cells, similar to cisplatin [IC50 = (8.0 ± 1.5) μmol x L(-1)], compounds 3g and 3k had better inhibition effect on EC-109 cells growth, with the IC50 values of (9.5 ± 1.8) μmol x L(-1) and (11.5 ± 0.9) μmol x L(-1) respectively, and compounds 3i and 3k exhibited good cytotoxic property on A-549, SGC-7901 and EC-109, which were worth further investigation.

Objective To establish a model for preparation of tissue-engineered skin grafts with hTERT cells carrying human papillomavirus type 6 (HPV 6) genome in vitro, so as to lay a foundation for studying HPV life cycle. Methods The full-length linear HPV6 genome and plasmid pEGFP-▲EGFP were electrophoretically cotransferred into hTERT cells. After selection using G418 resistance, Southern blotting was performed to determine the viral load of HPV6 in transfected cells. 3T3 J2 trophoblastic cells, type I rat-tail collagen and hTERT cells containing the full-length HPV6 genes (HPV6.hTERT cells)were mixed and cocultured on metal meshes to form skin graft-like structures. Hematoxylin and eosin (HE)staining was performed to observe the structure of formed skin grafts, an immunohistochemical assay to measure the expression of HPV6 L1 protein, and electron microscopy to observe virus particles in the skin grafts. Results The linear HPV6 gene was successfully transferred into hTERT cells, and Southern blotting showed the presence of HPV6 DNA in the transferred hTERT cells. The HPV6.hTERT cells, which were cocultured with 3T3 J2 trophoblastic cells and type I rat-tail collagen, proliferated and differentiated over time, and gradually formed skin grafts giving the appearance of verrucous hyperplasia. HE staining showed that the cocultured HPV6.hTERT cells could form typical stratified structure of skin after 7 days of cultivation, and histopathologic features of HPV infection, including obvious papillomatous hyperplasia, presence of vesicular cells, hyperkeratosis and parakeratosis, could be observed after 21 days. The immunohistochemical assay showed the expression of HPV6 L1 protein in the upper portion of skin grafts, and electron microscopy revealed the presence of HPV6 virus particles in skin grafts. Conclusions The established model for preparation of tissue-engineered skin grafts using HPV 6 genome-carrying cells provides a basis for biological studies of HPV, but its application is limited to some degree.