Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with "short" and "long" inverse agonists.

A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the "short" inverse agonist 6. With the increase in the size of the 6-position substituents, the "short" inverse agonist 6 first reversed its function to agonists and then to "long" inverse agonists. The cocrystal structures of RORγt complexed with the representative "short" inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the "long" inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of "short" inverse agonist 6 and "long" inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that "short" or "long" inverse agonists led to different behaviors of helixes H11, H11', and H12 of RORγt. The "short" inverse agonist 6 destabilizes H11' and dislocates H12, while the "long" inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.

αß-Dehydrocurvularin isolated from the fungus Aspergillus welwitschiae effectively inhibited the behaviour and development of the root-knot nematode Meloidogyne graminicola in rice roots.

BACKGROUND: The root-knot nematode Meloidogyne graminicola has become a serious threat to rice production as a result of the cultivation changes from transplanting to direct seeding. The nematicidal activity of Aspergillus welwitschiae have been investigated in vitro, and the disease control efficacy of the active compound has been evaluated under greenhouse and field conditions. RESULTS: The active compound αß-dehydrocurvularin (αß-DC), isolated by nematicidal assay-directed fractionation, showed significant nematicidal activity against M. graminicola, with a median lethal concentration (LC50) value of 122.2 µg mL- 1. αß-DC effectively decreased the attraction of rice roots to nematodes and the infection of nematodes and also suppressed the development of nematodes under greenhouse conditions. Moreover, αß-DC efficiently reduced the root gall index under field conditions. CONCLUSIONS: To our knowledge, this is the first report to describe the nematicidal activity of αß-DC against M. graminicola. The results obtained under greenhouse and field conditions provide a basis for developing commercial formulations from αß-DC to control M. graminicola in the future.

Evaluation of left gastric vein in Chinese healthy adults with multi-detector computed tomography.

OBJECTIVES: To evaluate the classification and diameter of left gastric vein (LGV) in healthy Chinese adults with multi-detector computed tomography (MDCT). METHODS: MDCT angiography was performed in 234 healthy adults for the portal venous system. CT cross-sectional thin-layer reconstruction combined with maximum intensity projection, volume rendering and multiplanar reconstruction were applied. The diameter of LGV was measured at the point within 2 cm from LGV origination. RESULTS: Of 234 subjects, 11 subjects (4.70%) who did not have clear images were excluded, and 223 subjects (95.30%) with excellent images were included. The LGV was originated from the portal vein in 46.15%, splenic vein in 30.77%, portal splenic angle in 14.53%, and the left branch of the portal vein in 3.85%. The maximal diameter of LGV was 4.74 ± 0.84 mm with a 95% confidence interval of 4.63-4.85 mm, and the LGV diameter was positively correlated with the weight of patients (R = 0.26, P = 0.006). No significant difference existed in the maximal diameter of LGV at different origination sites (P = 0.35). The diameter of LGV was significantly greater in males than in females (4.90 ± 0.85 vs. 4.56 ± 0.80 mm, P = 0.002), and the maximal diameter of LGV was significantly (P = 0.02) greater in the age range of 30-39 and 40-49 years than in the range of >70 years. No statistical significance (P = 0.36) was detected in the other groups. CONCLUSION: MDCT can clearly display the detailed anatomy and variation of LGV in healthy adults, providing a normal range of LGV diameter for clinical reference for diagnosing possible portal hypertension and for possible intervention.

Pretreatment with soluble ST2 reduces warm hepatic ischemia/reperfusion injury.

The interleukin-1 receptor-like protein ST2 exists in both membrane-bound (ST2L) and soluble form (sST2). ST2L has been found to play an important regulatory role in Th2-type immune response, but the function of soluble form of ST2 remains to be elucidated. In this study, we report the protective effect of soluble ST2 on warm hepatic ischemia/reperfusion injury. We constructed a eukaryotic expression plasmid, psST2-Fc, which expresses functional murine soluble ST2-human IgG1 Fc (sST2-Fc) fusion protein. The liver damage after ischemia/reperfusion was significantly attenuated by the expression of this plasmid in vivo. sST2-Fc remarkably inhibited the activation of Kupffer cells and the production of proinflammatory mediators TNF-alpha and IL-6. Furthermore, the levels of TLR4 mRNA and the nuclear translocation of NF-kappaB were also suppressed by pretreatment with sST2-Fc. These results thus identified soluble ST2 as a negative regulator in hepatic I/R injury, possibly via ST2-TLR4 pathway.

[Study on chemical constituents of volatile oil and trace elements from fruits of Clausena lansium].

OBJECTIVE: To study and determine the chemical constituents of the volatile oil and the trace elements in the fruits of the Clausena lansium. METHOD: The essential oils were extracted by supercritical fluid extraction (SFE) and separated on capillary columns with HP6890GC-5973MS. The components were quantitatively determined with normalization method, and were identified with GC-MS. And the trace elements were determined by ICP-MS and ICP-AES. RESULT: 36 Components constituting 95% of the total essential oil were separated and identified, and 11 trace elements were identified. CONCLUSION: 18 Compounds were found from the fruit of C. lansium for the first time.

Gene therapy using adenoviral vector encoding 4-1BBIg gene significantly prolonged murine cardiac allograft survival.

4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, interacts with 4-1BBL expressed on APC and delivers a costimulatory signal for T cell activation and growth. In this study, we investigated the efficacy of an adenoviral vector encoding murine 4-1BB extracellular domain and human IgG1 Fc (Ad4-1BBIg) fusion gene on murine cardiac allograft survival. Abdomen heterotopical heart graft model was performed from Balb/c to C57BL/6 mice. The adenoviral vectors, Ad4-1BBIg or an adenoviral vector containing EGFP gene (AdEGFP), were administered intravenously to recipient animals after cardiac grafting. The cardiac allograft survival was monitored by daily palpation. The serum level of 4-1BBIg and graft histology was assessed. Cytokine profiles in the grafts were detected by RT-PCR. IFN-gamma producing cells in recipient spleen were examined by flow cytometry. 4-1BBIg gene expression was achieved highly level at 72 h after vector injection. The proportion of IFN-gamma producing cells in recipient spleen was significantly reduced after administration of Ad4-1BBIg, compared to the group given AdEGFP or to the untreated control group. Unlike in controls, cardiac allograft expression of mRNA coding for IL-2 and IFN-gamma remained low in the Ad4-1BBIg group. Ad4-1BBIg therapy markedly reduced T cell infiltration into the graft and significantly prolonged recipient survival time (13.5 days), compared to the untreated group (7.5 days) and the AdEGFP-treated group (8.0 days) (P < 0.05). These results indicate that blockade of 4-1BB/4-1BB ligand interactions by Ad4-1BBIg inhibited alloreactive T-cell activation and attenuated T-cell infiltration into the graft, resulting in significant prolongation of murine cardiac allograft survival. Therefore, Ad4-1BBIg may be useful for preventing allograft rejection.

[Study on amino acids from the fruit of Clausena lansium].

OBJECTIVE: To investigate the amino acids from the fruit of Clausenae lansium. METHODS: To determine the content of hydrolyzed and free amino acids from fresh fruit and dried fruit of Clausenae lansium by HPLC, 10 mmol/L Na2HPO4 (PB) was used as mobile phase (A) and PB-methanol-acetic acid (50:35:15) was used as mobile phase (B) on a Hypersil ODS column. RESULTS: 16 kinds of amino acids were assayed, 10 of all was necessary or half-necessary amino acids. The total content of free and hydrolyzed amino was 4.8 mg/g and 15.0 mg/g in fresh fruit; 4.8 mg/g and 15.0 mg/g in dried fruit. CONCLUSION: There are abundant amino acids in the fruit of Clausenae lansium.

[HLA-G1 molecule expressed by ECV304 cells inhibit cytotoxic activity of allogeneic NK cells].

AIM: To study the effect of HLA-G1 molecule expressed by an endothelial cell line (ECV304) on the cytotoxic activity of allogeneic NK cells. METHODS: ECV304 cells were transfected with recombinant plasmid pcDNA3-HLA-G1 by the liposome transfection, and the expressed HLA-G1 on the cell surface was detected by indirect immunofluorescent assay and flow cytometry. The cytotoxic activity of allogeneic NK cells against ECV304 cells was analyzed by the MTT method. RESULTS: HLA-G1 was expressed on the surface of the transfected ECV304 cells. The specific lysis of NK cells against plasmid pcDNA3 transfected ECV304 was (50.6+/-18.1)%, while the specific lysis against pcDNA3-HLA-G1 transfected ECV304 was (29.7+/-11.4)%, which was significantly lower than the former (P<0.001). CONCLUSION: HLA-G1 expressed by the ECV304 cells can inhibit cytotoxicity of allogeneic NK cells.

[In vitro refolding and functional study of soluble HLA-G1-peptide complex].

AIM: To form soluble HLA-G1-peptide complex by refolding in vitro, and to study its immune function. METHODS: The heavy chain and beta(2m) of sHLA-G1 were expressed as insoluble aggregates in E. coli, and then the two subunits were refolded to form HLA-G1-peptide complex by dilution method in the presence of specific peptide. The refolded product was purified through Sephadex G-75 gel filtration. The purified product was identified by Western blot with mAb W6/32. The function of soluble HLA-G1 was explored from following three aspects, namely, the influences on cytotoxicity of NK cells, on proliferation of T cells in mixed lymphocyte culture and apoptosis of activated T cells. RESULTS: The refolded complex was recognized by mAb W6/32. It effectively inhibited cytotoxicity of NK cells and proliferation of T cells, and induced apoptosis of activated T cells. CONCLUSION: The refolding of soluble HLA-G1-peptide complex has been successfully realized in vitro. The complex can inhibit the functions of NK cells and T cells.