[Cloning and expression analysis of 8 bHLH transcription factors in Panax quinquefolius].

A total of 8 bHLH transcription factors were cloned from Panax quinquefolius and the response of them to methyl jasmonate(MeJA) was studied.To be specific, based on the preliminary transcriptome screening, 8 bHLH transcription factors were cloned with seedlings which had been cultured for 3 weeks.The content of ginsenosides Rg_1, Re, and Rb_1, and total saponins in the adventitious roots of P.quinquefolius was determined at different time of MeJA treatment by high performance liquid chromatography(HPLC) and spectrophotometry.Real-time quantitative polymerase chain reaction(PCR) was used to detect the relative expression of 8 transcription factors after MeJA treatment.The correlation between the relative expression of the 8 transcription factors and the saponin content after MeJA treatment was checked by Pearson's correlation analysis.The results showed that the PCR products(Pq-bHLH21-Pq-bHLH28) of the 8 bHLH transcription factors were 762-2 013 bp in length.They were submitted to NCBI to obtain the Genbank access numbers.The proteins yielded from Pq-bHLH21-Pq-bHLH28 showed amino acid sequence identity of 24.90%, and each amino acid sequence had the bHLH(Basic Helix-loop-helix) conserved domain and belonged to the bHLH family.The 5 amino acid sequences of Pq-bHLH22 and Pq-bHLH24-Pq-bHLH27 contained the bHLH-MYC N domain, which belonged to the MYC transcription factors.Pq-bHLH21-Pq-bHLH28 responded to MeJA within 48 h of treatment.At 72 h, the expression of Pq-bHLH24 reached 106.53 folds the highest in the treatment group.Pq-bHLH25, Pq-bHLH27, and Pq-bHLH28 showed synergic expression.Pq-bHLH21 may re-gulate the biosynthetic pathway of ginsenoside Rb_1, while Pq-bHLH22, Pq-bHLH25, and Pq-bHLH28 were in significantly positive correlation with the biosynthetic pathway of ginsenoside Re.The result lays a foundation for further verifying the regulation of ginsenoside biosynthesis by bHLH transcription factors.

Molecular modeling and biological effects of peptidomimetic inhibitors of TACE activity.

We investigated the molecular basis of specificity for the interaction between tumor necrosis factor-alpha converting enzyme (TACE) and peptidomimetic inhibitors. Four novel peptidomimetic TACE inhibitors (8a-d) were designed and synthesized by introducing a substituted sulfur group and a hydrophobic group to a novel matrix metalloprotease (MMP) inhibitor. Inhibition was determined by in vitro lipopolysaccharide (LPS) cytotoxicity tests in HL-60 cell lines and by measuring the expression of mTNF-alpha using FCM techniques and immunohistochemistry in vivo. We simulated the interaction of the inhibitors with the 3D structure of the TACE active site in the Brookhaven Protein Database (PDB). The four inhibitors (8a-d) inhibited activity by 9.1%, 54.5%, 27.3%, and 54.5%, respectively. 8b and 8d showed significant in vitro inhibition in cytotoxicity tests, which corresponded to the molecular docking results. 8d also showed inhibitory activity in vivo. We explored the interface between enzyme and substrate by combining bioinformatics with experimental observations to further the development of specific TACE inhibitors to reduce inflammatory responses.

Noggin Inhibits IL-1ß and BMP-2 Expression, and Attenuates Cartilage Degeneration and Subchondral Bone Destruction in Experimental Osteoarthritis.

Osteoarthritis (OA) is a chronic inflammatory and progressive joint disease that results in cartilage degradation and subchondral bone remodeling. The proinflammatory cytokine interleukin 1 beta (IL-1ß) is abundantly expressed in OA and plays a crucial role in cartilage remodeling, although its role in the activity of chondrocytes in cartilage and subchondral remodeling remains unclear. In this study, stimulating chondrogenic ATDC5 cells with IL-1ß increased the levels of bone morphogenetic protein 2 (BMP-2), promoted articular cartilage degradation, and enhanced structural remodeling. Immunohistochemistry staining and microcomputed tomography imaging of the subchondral trabecular bone region in the experimental OA rat model revealed that the OA disease promotes levels of IL-1ß, BMP-2, and matrix metalloproteinase 13 (MMP-13) expression in the articular cartilage and enhances subchondral bone remodeling. The intra-articular injection of Noggin protein (a BMP-2 inhibitor) attenuated subchondral bone remodeling and disease progression in OA rats. We also found that IL-1ß increased BMP-2 expression by activating the mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK), and specificity protein 1 (Sp1) signaling pathways. We conclude that IL-1ß promotes BMP-2 expression in chondrocytes via the MEK/ERK/Sp1 signaling pathways. The administration of Noggin protein reduces the expression of IL-1ß and BMP-2, which prevents cartilage degeneration and OA development.

Multiple shRNA-mediated knockdown of TACE reduces the malignancy of HeLa cells.

Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) is a key enzyme involved in the proteolytic shedding of the ectodomain of several membrane-bound growth factors, cytokines and receptors. Here, we constructed a multiple short hairpin RNA (shRNA) expression vector containing four shRNAs against TACE. We found that in HeLa cells our multiple shRNAs vector produced a higher level of TACE knockdown than any single shRNA vector containing only one TACE shRNA. Silencing TACE expression in HeLa cells decreased their malignancy by decreasing the proliferation, adhesion and migration, as well as inducing apoptosis in these cells. Furthermore, our data suggest that the effects of TACE on the malignancy of HeLa cells may be mediated via activation of the EGFR (epidermal growth factor receptor) signaling pathway. Our findings suggest that using a combination of shRNAs within one vector to silence the expression of TACE might be a potential therapeutic strategy for tumors.

[A comparative study of the diagnostic value of endoscopic ultrasonography with pathological features of upper gastrointestinal mesenchymal tumors].

OBJECTIVE: To study the pathological and immunohistochemical features of upper gastrointestinal mesenchymal tumors (GIMTs) and compare them with endoscopic ultrasonographic (EUS) characteristics so as to evaluate the diagnostic value of EUS in upper digestive tract GIMTs. METHODS: Seventy-two pathological specimens of upper digestive tract GIMTs (34 surgical specimens and 38 endoscopic mucosal resection (EMR) specimens) were collected. The pathological features and the expression of CD(117), CD(34), SMA and S-100 were observed by immunohistochemical method with light microscope. The pathological types and characteristics were determined by pathologists and compared with the layer of origin and sonographic characteristics determined with preoperative EUS. RESULTS: In the 72 cases of upper digestive tract GIMTs, 37 cases were diagnosed as stromal tumor with pathological and immunohistochemical methods (51.4%); 21 of them were malignant, accounting for 56.7% of the stromal tumors. Thirty-four cases were diagnosed as leiomyoma (47.2%) and 1 case as schwannoma (1.4%). In the 72 GIMTs cases, 40 were esophageal GIMTs. EUS showed that 38 cases were originated from the muscularis mucosae layer; 33 of them were leiomyoma and 5 stromal tumor. The 2 cases originating from the muscularis propria layer which were both stromal tumors. Thirty-two cases were gastric GIMTs, EUS showed that 2 cases originating from the muscularis mucosae layer were gastric stromal tumor. Of the 30 cases originating from the muscularis propria layer, 28 cases were stromal tumor, 1 case was leiomyoma and 1 case was schwannoma. The sensitivity and the specificity of EUS in distinguishing benign and malignant stromal tumors according to sonographic characteristics were 81.0% and 93.8% respectively. CONCLUSION: Stromal tumor is more common in stomach mesenchymal neoplasms and is more often originated from the muscularis propria layer in EUS; leiomyoma is more common in esophagus and is more often originated from the muscularis mucosae layer. The diagnostic sensitivity and specificity of EUS are high in distinguishing benign and malignant character of upper digestive tract GIMTs. EUS plays an important role in guiding the clinical management of upper digestive tract GIMTs.

[The effects of sympathetic neurotransmitters and adrenergic receptors on liver fibrosis in murine schistosomiasis].

OBJECTIVE: To investigate the effects of sympathetic neurotransmitters and adrenergic receptors on liver fibrosis in murine schistosomiasis. METHODS: Mice were infestated with schistosoma by means of pasting cercariae on their abdomens. Thirty mice were randomly divided into a control group and a model group. Hematoxylin eosin and Van Gieson staining were used to view the histopathology of their livers. Immunofluorescence histochemistry and laser scanning confocal fluorescence microscopy were used to measure the a1A and beta2 adrenergic receptors in livers of the two groups of mice. High performance liquid chromatography-electrochemical detector (HPLC-ECD) was used to determine the concentration of norepinephrine (NE) and dopamine (DA) in the plasma of the mice. RESULTS: Immunofluorescence histochemistry showed that a1A and beta2 receptors were present in hepatocytes and hepatic sinusoids of the livers of the mice of the two groups, but there were many more in the livers of the schistosoma infected mice (t=-2.888; t=-6.648) (P<0.05). The results of HPLC-ECD showed that the levels of NE and DA in the model group were higher than those of the control group (t=-3.372; t=-4.428) (P<0.05). CONCLUSION: Sympathetic neurotransmitters and adrenergic receptors may participate in liver fibrogenesis in mice infected with schistosoma.

Decreased levels of nitric oxide production and nitric oxide synthase-2 expression are associated with the development and metastasis of hepatocellular carcinoma.

Many studies have demonstrated the function of nitric oxide (NO) or nitric oxide synthase-2 (NOS-2) in cancer as pro-neoplastic or anti-neoplastic effectors, but the role of NO and NOS-2 in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the levels of NO production and NOS-2 expression in HCC and adjacent non-tumor liver tissues and to clarify whether the levels of NO/NOS-2 are related to the clinicopathological features of HCC. The levels of NO production were examined in tumor and adjacent non-tumor liver tissues of 30 patients with HCC. The expression of NOS-2 was detected by real-time polymerase chain reaction (RT-PCR) and immunohistochemical analysis in HCC and/or adjacent non-tumor liver tissues. Mutant p53 and proliferating cell nuclear antigen (PCNA) were also immunohistochemically investigated in liver tissues. The levels of NO in HCC were significantly lower compared to adjacent non-tumor liver tissues (P<0.001). The relative mRNA and protein expression levels of NOS-2 in HCC were also significantly lower compared to adjacent non-tumor liver tissues (P<0.01 for both). We found that the levels of NO in patients suffering from HCC metastasis were lower compared to those without metastasis (P<0.05) and NOS-2 expression was correlated with tumor diameter (P<0.05) and metastasis (P<0.05). In addition, mutant p53 protein was expressed in the majority of HCC samples and the proliferation rate of HCC was significantly higher than that of adjacent non-tumor liver tissues. These data indicate that decreased levels of NO/NOS-2 may partially contribute to overexpression of the mutant p53 protein and excessive proliferation; this may be a potential mechanism in the development and progression of HCC.

[Effect of glucose-6-phosphate dehydrogenase on intracellular gsh level in Raji cells during oxidative stress].

AIM: To explore a role of G6PD in replenishment of intracellular GSH during oxidative stress. METHODS: In vitro Raji cell was cultured, intracellular GSH levels and G6PD, GR, GPX activities were determined at different time points after PMS treatment when G6PD activity was inhibited or not by DHEA. RESULTS: Intracellular GR, GPX, G6PD activities elevated significantly combined with GSH level decreased dramatically before 30 minutes, replenished gradually after 30 minutes and restore normal levels about 6 h after PMS treatment when G6PD was not inhibited. No change in GR and significant increase in GPX activity were shown following depleted GSH after PMS treatment when G6PD was inhibited by DHEA. CONCLUSION: G6PD contributes to replenish intracellular GSH and is a critical factor regulating GSH levels during oxidative stress.

[Inhibition of proliferation, adhesion and invasion ability of human lung carcinoma cell A549 by tumor necrosis factor-alpha converting enzyme (TACE)].

We constructed prokaryotic expression vectors for different domains of TACE gene and expressed the fusion proteins, so as to explore their effects on the proliferation, adhesion and invasion potential of tumor cells in vitro. The total RNA was isolated from THP1 cell. TACE cDNA was amplified by RT-PCR and subcloned into pMD18-T vector to construct pMD-18T-TACE vector. The different cDNA fragment of TACE were amplified from plasmid pMD-18T-TACE and then cloned into pET-28a( + ) to construct expression vector pET28a( + )- 300, pET28a( + )-T800, and pET28a( + )-T1300, which respectively transformed into E. coli BL21 (DE3). The expression of His-tagged fusion proteins were induced with IPTG and purified through BBST NTA resin. The proliferation ability was examined by MTT assay. The adhesive and invasive ability were examined by plated adhesion model and Transwell assay. The protein pET28a( + )-T300 and pET28a( + )-T1300 can reduce the proliferation, adhesion and invasion ability of human lung carcinoma cell A549 in vitro, but otherwise the protein pET28a( + )-T800 had not shown the inhibitive function. The fusion protein of disintegrin domain of TACE have the similar biological function to other disintegrins, which can be used for further research on function of TACE in inflammation and tumor.

[Screening of TACE peptide inhibitors from a phage display random 15-peptide library by recombinant TACE ecotodomain].

Tumour necrosis factor-alpha converting enzyme (TACE) is the major protease responsible for processing proTNF from membrane-anchored precursor into secreted TNF-alpha. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET-28a/pET-28c and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni2+ -NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE-binding peptides from the phage display random 15-peptide library. After four rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence-TRWLVYFSRPYLVAT was found and synthesized. The synthetic peptide was shown to bind to TACE and inhibit the TNF-alpha release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-alpha on LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti-inflammation drugs.