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Background: Diabetes mellitus can occur after acute pancreatitis (AP), but the accurate quantitative methods to predict post-acute pancreatitis diabetes mellitus (PPDM-A) are lacking. This retrospective study aimed to establish a radiomics model based on contrast-enhanced computed tomography (CECT) for predicting PPDM-A., Methods: A total of 374 patients with first-episode AP were retrospectively enrolled from two tertiary referral centers. There were 224 patients in the training cohort, 56 in the internal validation cohort, and 94 in the external validation cohort, and there were 86, 22, and 27 patients with PPDM-A in these cohorts, respectively. The clinical characteristics were collected from the hospital information system. A total of 2,398 radiomics features, including shape-based features, first-order histogram features, high order textural features, and transformed features, were extracted from the arterial- and venous-phase CECT images. Intraclass correlation coefficients were used to assess the intraobserver reliability and interobserver agreement. Random forest-based recursive feature elimination, collinearity analysis, and least absolute shrinkage and selection operator (LASSO) were used for selecting the final features. Three classification methods [eXtreme Gradient Boosting (XGBoost), Adaptive Boosting, and Decision Tree] were used to build three models and performances of the three models were compared. Each of the three classification methods were used to establish the clinical model, radiomics model, and combined model for predicting PPDM-A, resulting in a total of nine classifiers. The predictive performances of the models were evaluated by the area under the receiver operating characteristic curve (AUC), accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and F1-score., Results: Eleven radiomics features were selected after a reproducibility test and dimensionality reduction. Among the three classification methods, the XGBoost classifier showed better and more consistent performances. The AUC of the XGBoost's radiomics model to predict PPDM-A in the training, internal, and external cohorts was good (0.964, 0.901, and 0.857, respectively). The AUC of the XGBoost's combined model to predict PPDM-A in the training, internal, and external cohorts was good (0.980, 0.901, and 0.882, respectively). The AUC of the XGBoost's clinical model to predict PPDM-A in the training, internal, and external cohorts did not perform well (0.685, 0.733, and 0.619, respectively). In the external validation cohort, the AUC of the XGBoost's radiomics model was significantly higher than that of the clinical model (0.857 vs. 0.619, P<0.001), but there was no significant difference between the combined and radiomics models (0.882 vs. 0.857, P=0.317)., Conclusions: The radiomics model based on CECT performs well and can be used as an early quantitative method to predict the occurrence of PPDM-A., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-23-1232/coif). R.H. reports that this research was funded by the Chengdu University of Traditional Chinese Medicine “Xinglin Scholars” Discipline Talents Research Promotion Plan (grant No. YYZX2021059). D.Z. reports that this research was sponsored by Natural Science Foundation of Chongqing, China (grant No. CSTB2023NSCQ-MSX0154) and the First Affiliated Hospital of Chongqing Medical University (grant No. CYYY-BSHPYXM-202305). The other authors have no conflicts of interest to declare., (2024 Quantitative Imaging in Medicine and Surgery. All rights reserved.)

Purpose: To assess the characteristics of pancreatic perfusion in normal pancreas versus cirrhotic patients using intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI)., Methods: A total of 67 cirrhotic patients and 33 healthy subjects underwent IVIM on a 3.0 T MRI scanner. Diffusion coefficient (ADC slow ), pseudo-diffusion coefficient (ADC fast ), and perfusion fraction (f) were calculated based on the bi-exponential model. The pancreatic IVIM-derived parameters were then compared. In the cirrhotic group, the relationship was analyzed between IVIM-derived pancreatic parameters and different classes of hepatic function as determined by the Child-Pugh classification. Also, the pancreatic IVIM-derived parameters were compared among different classes of cirrhosis as determined by the Child-Pugh classification., Results: The f value of the pancreas in cirrhotic patients was significantly lower than that in normal subjects (p = 0.01). In the cirrhotic group, the f value of the pancreas decreased with the increase of the Child-Pugh classification (R = - 0.49, p = 0.00). The f value of the pancreas was significantly higher in Child-Pugh class A patients than in class B and C patients (p = 0.02, 0.00, respectively), whereas there was no significant difference between class B and C patients (p = 0.16)., Conclusion: The IVIM-derived perfusion-related parameter (f value) could be helpful for the evaluation of pancreatic perfusion in liver cirrhosis. Our data also suggest that the blood perfusion decrease in the pancreas is present in liver cirrhosis, and the pancreatic perfusion tends to decrease with the increasing severity of hepatic function., Trial Registration: Trial registration number is 2021-ky-68 and date of registration for prospectively registered trials is February 23, 2022., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acupuncture points have a positive effect on the auxiliary prevention and treatment of diseases, so medical devices such as acupuncture robots often need to combine acupuncture points to improve the treatment effect when working, however, intelligent acupoint selection technology is not yet mature, the automatic rapid and accurate positioning of acupoints is still challenging. Therefore, this paper proposes a method of back acupoint location and an evaluation index of acupoint location. First, we propose an improved Keypoint RCNN network for the preliminary location of back acupoints and introduce a channel and spatial attention mechanism module (CBAM) to improve the performance of the model. Then, we set up a posterior median line positioning method to improve the accuracy of acupoint positioning. Finally, expand and locate other acupoints according to the prior information of acupoints. According to the experimental results, the accuracy of acupoint positioning was 87.32%. After the correction of acupoint positioning, the accuracy was increased by 2.8%, which was 90.12%. In this paper, the application of depth learning in automatic location of back acupoints is realized for the first time. Only one image can be used to locate the back acupoints, with an accuracy of 90.12%., (© 2023 John Wiley & Sons Ltd.)

Objective: To explore and design a novel bi-specific chimeric antigen receptor (CAR) structure. To obtain the corresponding CAR-T cells and verify killing effects on tumor cells in vitro and in vivo., Methods: Five kinds of bi-specific CAR structures including humanized CD19 scFv and CD79b scFv, CD8 hinge & TM-4-1BB-CD3ζ and/or CD3ε chain intracellular regions were constructed and prepared. CAR-19-79b cells were obtained. Five kinds of CAR-T cells were co-incubated with the 3M-CD19-CD79b-Luc target cells. Luciferase assay and ELISA were used to detecte the killing ability of these five groups of CAR-T cells and the secretion of cytokines and compared. The optimal structure of CAR-T cells was used to treat the leukemia mouse model constructed by Daudi-Luc cells. And the treatment efficacy was evaluated. At the same time, other targets were used in this structure. With the same methods, the stability and effectiveness of the structure were verified., Results: CAR-19-79b-T cells were cultured for 7 days, the expression rates of CAR-19 and CAR-79b were 21.6%-36.3% and 21.7%-37.8%, respectively. The killing rates of 5 kinds of CAR-19-79b-T cells prepared by T cells from 3 healthy donors on 3M-CD19-CD79b-Luc cells were significantly higher than those of the T cell control group at the effect-target ratio of 10∶1. Among them, the killing rates of CAR-19-79b-T cells with No. III and No. IV structures were the strongest. After co-incubation with 3M-CD19-CD79b-Luc target cells, the amount of IFN-γ and TNF-α secreted by CAR-T cells with CAR IV and CARV structures was the lowest. And there was no significance between the two groups ( P >0.05). CAR IV cells with remarkable killing effect and low secretion factor had obvious therapeutic effect on Daudi-Luc leukemia mice, extending the survival period of mice to 64 days. And all mice in the T cell control group died at 41.0±2.4 days. The CAR-19-BCMA-T and CAR-19-22-T with the same structure showed significant killing ability and low cytokine expression levels., Conclusion: A novel bi-specific CAR structures was successfully designed, which could efficiently kill the corresponding tumor cells and secrete less cytokines (such as TNF-α, IFN-γ). Moreover, it shows obvious therapeutic effect on Daudi lymphoma mouse model. The bi-specific CAR structure shows good killing specificity and safety.

Background: This study aimed to investigate the impact of the COVID-19 pandemic on ST-segment elevation myocardial infarction (STEMI) care in China., Methods: We conducted a multicenter, retrospective cohort study in Hunan province (adjacent to the epidemic center), China. Consecutive patients presenting with STEMI within 12 h of symptom onset and receiving primary percutaneous coronary intervention, pharmaco-invasive strategy and only thrombolytic treatment, were enrolled from January 23, 2020 to April 8, 2020 (COVID-19 era group). The same data were also collected for the equivalent period of 2019 (pre-COVID-19 era group)., Results: A total of 610 patients with STEMI (COVID-19 era group n = 286, pre-COVID-19 era group n = 324) were included. There was a decline in the number of STEMI admissions by 10.5% and STEMI-related PCI procedures by 12.7% in 2020 compared with the equivalent period of 2019. The key time intervals including time from symptom onset to first medical contact, symptom onset to door, door-to-balloon, symptom onset to balloon and symptom onset to thrombolysis showed no significant difference between these two groups. There were no significant differences for in-hospital death and major adverse cardiovascular events between these two groups., Conclusion: During the COVID-19 pandemic outbreak in China, we observed a decline in the number of STEMI admissions and STEMI-related PCI procedures. However, the key quality indicators of STEMI care were not significantly affected. Restructuring health services during the COVID-19 pandemic has not significantly adversely influenced the in-hospital outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tang, Wang, Hu, Fang, Zheng, Zeng, Jiang, Ouyang, Liu, Zeng, Guo and Zhou.)

Aims: Prostate cancer is a well-known aggressive malignant tumor in men with a high metastasis rate and poor prognosis. Adapalene (ADA) is a third-generation synthetic retinoid with anticancer properties. We investigated the anti-tumor activity and molecular mechanisms of ADA in the RM-1 prostate cancer cell line in vivo and in vitro . Methods: The effects of ADA on cell proliferation were estimated using the CCK-8 and colony formation assays. The wound-healing assay and the Transwell assay were employed to examine the migratory capacity and invasiveness of the cells. Flow cytometry was utilized to evaluate the cell cycle and apoptosis, and Western blotting analysis was used to assess the expression of the associated proteins. Micro-CT, histomorphological, and immunohistochemical staining were used to assess the effects of ADA on bone tissue structure and tumor growth in a mouse model of prostate cancer bone metastasis. Result: ADA dramatically inhibited cell proliferation, migration, invasiveness, and induced S-phase arrest and apoptosis. ADA also regulated the expression of S-phase associated proteins and elevated the levels of DNA damage markers, p53, and p21 after ADA treatment, suggesting that the anti-tumor effect of ADA manifests through the DNA damage/p53 pathway. Furthermore, we observed that ADA could effectively inhibited tumor growth and bone destruction in mice. Conclusion: ADA inhibited prostate cancer cell proliferation, elicited apoptosis, and arrested the cell cycle in the S-phase. ADA also slowed the rate of tumor growth and bone destruction in vitro . Overall, our results suggest that ADA may be a potential treatment against prostate cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nong, Zhang, Bai, Zhang, Liu, Zhou, Shi, Zeng and Zong.)

Flavonoids have been reported to have therapeutic potential for spinal cord injury. Hawthorn leaves have abundant content and species of total flavonoids, and studies of the effects of the total flavonoids of hawthorn leaves on spinal cord injury have not been published in or outside China. Therefore, Sprague-Dawley rats were used to establish a spinal cord injury model by Allen's method. Rats were intraperitoneally injected with 0.2 mL of different concentrations of total flavonoids of hawthorn leaves (5, 10, and 20 mg/kg) after spinal cord injury. Injections were administered once every 6 hours, three times a day, for 14 days. After treatment with various concentrations of total flavonoids of hawthorn leaves, the Basso, Beattie, and Bresnahan scores and histological staining indicated decreases in the lesion cavity and number of apoptotic cells of the injured spinal cord tissue; the morphological arrangement of the myelin sheath and nerve cells tended to be regular; and the Nissl bodies in neurons increased. The Basso, Beattie, and Bresnahan scores of treated spinal cord injury rats were increased. Western blot assays showed that the expression levels of pro-apoptotic Bax and cleaved caspase-3 were decreased, but the expression level of the anti-apoptotic Bcl-2 protein was increased. The improvement of the above physiological indicators showed a dose-dependent relationship with the concentration of total flavonoids of hawthorn leaves. The above findings confirm that total flavonoids of hawthorn leaves can reduce apoptosis and exert neuroprotective effects to promote the recovery of the motor function of rats with spinal cord injury. This study was approved by the Ethics Committee of the Guangxi Medical University of China (approval No. 201810042) in October 2018., Competing Interests: None

Objective: To explore the effects of direct antiviral agent (DAAs) on the frequency of peripheral blood mononuclear cells and their activating factors sCD14s and CD163 in patients with chronic hepatitis C. Methods: Data of 15 treatment-naive chronic hepatitis C patients and 10 healthy controls were collected. Patients with chronic hepatitis C were treated with DAAs for 12 weeks. Blood samples were collected at 0, 4 and 12 weeks respectively, and blood samples of healthy controls were used as controls. Flow cytometry was used to detect the frequency of classical CD14(++)CD16(-) mononuclear cells and pro-inflammatory CD14(+)CD16(+) mononuclear cells in peripheral blood. Serum sCD14s and sCD163 were detected by enzyme-linked immunosorbent assay. The comparison between the two groups was performed by t-test. The comparison between multiple groups was performed by analysis of variance, and further pairwise comparison was performed by LSD-t test. Results: Prior DAAs treatment, peripheral blood CD14(+)CD16(+) mononuclear cell frequency (18.49% ± 1.54% vs. 10.65% ± 0.83%), serum sCD14s [(64 407.38 ± 5778.49) pg/ml vs. (28 370.76 ± 2 357.68 ) pg/ml] and sCD163 [(22 853.80 ± 4 137.61) pg/ml vs. (2 934.41 ± 223.31) pg/ml] were all higher than healthy controls ( P < 0.05), while the frequency of CD14(++)CD16(-) mononuclear cells in peripheral blood was lower than healthy controls (59.14%±0.54% vs. 72.75%±1.31%, P < 0.01). During DAAs treatment, CD14(+)CD16(+) mononuclear cells frequency, serum sCD14 and sCD163 were all decreased significantly. After 12 weeks of treatment, CD14(+)CD16(+) mononuclear cells had decreased to nearly normal level (12.42% ± 1.60% vs. 10.65% ± 0.83%, P > 0.05), and serum sCD14 and scd163 were still higher than those of healthy controls [sCD14: (44 390.06 ± 3 330.17) pg / ml vs. (28 370.76 ± 2 357.68) pg/ml, Scd163: (11 494.79 ± 1 836.97) pg / ml vs. (2 934.41 ± 223.31) pg / ml, P < 0.01], while the frequency of CD14(++)CD16(-)mononuclear cells had gradually increased during the course of treatment and neared healthy control level after 12 weeks of treatment. There was no statistically significant difference between the two groups (71.54) % ± 2.99% vs. 72.75% ± 1.31%, P > 0.05). Conclusion: DAAs therapy can reduce the activation of peripheral blood mononuclear cells in patients with chronic hepatitis C.

Objective: To assess the clinical characteristics and identify the risk factors in the acute myocardial infarction (AMI) patients complicating with ventricular septal rupture (VSR). Methods: A retrospective study was performed on 96 AMI patients complicating with VSR, who were hospitalized in the Second Xiangya Hospital of Central South University, Hunan Provincial Peoples' Hospital, the First Affiliated Hospital of University of South China, the Second Affiliated hospital of University of south China, Xiangtan Central Hospital from December 2007 to May 2017. There were 46 females and the age was (66.2±10.7) years (from 43 to 90 years). Patients were divided into in-hospital survival group ( n =64) and in-hospital death group ( n =32). The 96 patients were also divided into the early death group (survived ≤2 weeks after admission, n= 50) and non-early death group (survived>2 weeks after admission, n= 46). Multivariate logistic regression was used to analyze the independent risk factors of the early death. Results: Location of VSR was available in 71 patients, VSR was located at the apical or anterior septum near the apical region in 64.0% (32/50) patients with the anterior AMI, VSR was located at the posterior wall and basal inferior segment in 57.1% (12/21) patients with non-anterior AMI. Compared to the in-hospital survival group, patients in the in-hospital death group were older ((69.6±11.3) years vs. (64.6±10.1) years, P= 0.031), incidence of non-ventricular aneurysm (71.9% (23/32) vs. 37.5% (24/64), P= 0.001) and anterior AMI (84.4%(27/32) vs. 62.5%(40/64), P= 0.028) was significantly higher in the in-hospital death group than in the in-hospital survival group. The comparison between the early death group and non-early death group showed that older age, female, no history of angina or myocardial infarction, Killip grade>Ⅲ, and non-ventricular aneurysm were related to increased risk of the early mortality in this patient cohort. Logistic regression analysis revealed that female ( OR= 5.109,95 %CI 1.19-22.00, P= 0.012), no history of angina or myocardial infarction ( OR= 23.34, 95 %CI 3.44-158.37, P= 0.001), Killip grade>Ⅲ( OR= 5.35, 95 %CI 1.26-22.66, P= 0.019) and non-ventricular aneurysm ( OR= 6.30,95 %CI 1.67-23.73, P= 0.005) were independent risk factors for early death in this patient cohort. Conclusion: The risk factors of in-hospital death include older age, non-ventricular aneurysm and anterior AMI. Female, no history of angina or myocardial infarction, Killip grade>Ⅲ and non-ventricular aneurysm are independent risk factors for the early death of AMI patients complicating VSR.

Atherosclerosis is a dyslipidemia disease characterized by foam cell formation driven by the accumulation of lipids. Visceral adipose tissue-derived serine protease inhibitor (vaspin) is known to suppress the development of atherosclerosis via its anti-inflammatory properties, but it is not yet known whether vaspin affects cholesterol efflux in THP-1 macrophage-derived foam cells. Here, we investigated the effects of vaspin on ABCA1 expression and cholesterol efflux, and further explored the underlying mechanism. We found that vaspin decreased miR-33a levels, which in turn increased ABCA1 expression and cholesteorl efflux. We also found that inhibition of NF-κB reduced miR-33a expression and vaspin suppressed LPS-mediated NF-κB phosphorylation. Our findings suggest that vaspin is not only a regular of inflammasion but also a promoter of cholesterol efflux., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Postinfarction ventricular septal rupture (PI-VSR) is a rare but devastating complication of acute myocardial infarction (AMI). Risk stratification in the acute phase is crucial for decision-making, and this study analyzed the risk factors for early mortality and the effects of various management options on the outcome of PI-VSR patients in the era of percutaneous intervention., Methods: A total of 96 patients with PI-VSR were identified and divided into an acute-phase survivor group (n = 46, survived ≥2 weeks after admission) and a nonsurvivor group (n = 50, died within 2 weeks after admission). Percutaneous closure was considered in acute-phase survivors. Patients were followed up for a mean 47 (quartiles 15-71) months by clinical visit or telephone interview., Results: The overall acute-phase (i.e., < 2 weeks after the diagnosis of PI-VSR) mortality rate was 52%. Female sex and Killip Class III-IV at admission were associated with an increased risk of acute-phase death. Of the 46 patients who survived ≥2 weeks, 20 underwent interventional occlusion and the procedure was successful in 19. Percutaneous closure in the acute-phase survivor group improved the immediate (21% in-hospital mortality rate) and long-term (53% mortality) outcomes., Conclusions: Patients with PI-VSR are at a high risk of acute-phase mortality. Female sex and severe cardiac dysfunction at admission are linked with a high rate of acute-phase deaths. Percutaneous closure in acute-phase survivors results in favorable short- and long-term benefits for PI-VSR patients., (© 2019 S. Karger AG, Basel.)

Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Previous studies have shown that miR-467b plays a central role in the progression of atherosclerosis via regulating LPL expression. However, the regulatory mechanism of miR-467b in regulateing the CE and FC formation is still unclear. Interestingly, computational analysis demonstrated that ACAT1 which converts intracellular FC into the storage form of CE, and ABCA1 which promotes cellular FC efflux may be target gene of miR-467b. Here, we examined whether miR-467b could target ACAT1 and ABCA1, thereby affecting the CE and FC formation in oxLDL-treatment RAW 264.7 cells. We found that miR-467b regulates the CE:FC ratio in oxLDL-treatment RAW 264.7 macrophages, and the luciferase activity of ACAT1 is regulated by the miR-467b, but the luciferase activity of ABCA1 has no effect. Furthermore, our data suggested that miR-467b highly regulates the endogenous levels of ACAT1 expression, thereby affecting the CE formation in oxLDL-treatment RAW 264.7 macrophages. Taken together, our findings demonstrate that ACAT1 is a target gene of miR-467b, and miR-467b regulated the CE and FC formation via directly target the ACAT1 3'UTR., (Copyright © 2016. Published by Elsevier B.V.)

Bone homeostasis is maintained by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoporosis occurs when osteoclast activity surpasses osteoblast activity. Our previous studies showed the plant-derived natural polysaccharide (Polygonatum sibiricum polysaccharide or PSP) had significant anti-ovariectomy (OVX)-induced osteoporosis effects in vivo, but the mechanisms of PSP's anti-osteoporosis effect remains unclear. In this study, we assessed PSP's effect on the generation of osteoblast and osteoclast in vitro. This study showed that PSP promoted the osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) without affecting BMPs signaling pathway. This effect was due to the increased nuclear accumulation of β-catenin, resulting in a higher expression of osteoblast-related genes. Furthermore, the study showed PSP could inhibit the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and exert prophylatic protection against LPS-induced osteolysis in vivo. This effect was also related to the increased nuclear accumulation of β-catenin, resulting in the decreased expression of osteoclast-related genes. In conclusion, our results showed that PSP effectively promoted the osteogenic differentiation of mouse BMSCs and suppressed osteoclastogenesis; therefore, it could be used to treat osteoporosis.

To investigate the effect and mechanism of serum amyloid A (SAA) on the expression of scavenger receptor class B type I (SR-BI) and inflammatory response in THP-1 macrophages, the human THP-1 cells were treated with SAA and p38-MAPK agonist (anisomycin) or p38-MAPK inhibitor (SB203580). Then, the expressions of SR-BI, phosphorylated p38-MAPK and inflammatory factors (MCP-1, TNF-α, IL-1β) were examined by real-time quantitative PCR, Western blotting and ELISA, respectively. The results showed that, compared with control group, SAA increased the levels of inflammatory factors (MCP-1, TNF-α, IL-1β), down-regulated the expressions of SR-BI, and up-regulated the expression of phosphorylated p38-MAPK protein in a concentration- and time-dependent manner in THP-1 cells (P < 0.05). After treatment with SAA and p38-MAPK agonist (anisomycin) in THP-1 cells, the expression of SR-BI was down-regulated, and the levels of inflammatory factors and phosphorylated p38-MAPK protein expression were increased, compared with the group only treated by SAA (P < 0.05). In contrast, the SR-BI expression was up-regulated, whereas inflammatory factors and phosphorylated p38-MAPK protein expressions were decreased after the cells were treated with SAA and p38-MAPK inhibitor (SB203580) (P < 0.05). The results suggest that SAA-promoted inflammatory response in THP-1 macrophages may be through the phosphorylation of p38-MAPK and inhibition of SR-BI expression.

Background: Increased expression of METCAM/MUC18, a trans-membrane cell adhesion molecule in the Ig-like gene superfamily, has been associated with the malignant progression of epithelial ovarian carcinomas. To investigate if this is a fortuitous correlation or if METCAM/MUC18 actually plays a role in the progression of the cancer, we tested effects of enforced expression of METCAM/MUC18 on in vitro behaviors, in vivo tumorigenesis, and in vivo malignant progression of human ovarian cancer SK-OV-3 cells, which minimally expressed this protein., Methods: For in vitro and in vivo tests, we transfected human METCAM/MUC18 cDNA gene into SK-OV-3 cells in a mammalian expression vector pcDNA3.1+ and obtained G418-resistant (G418(R)) clones, which expressed various levels of human METCAM/MUC18. To mimic physiological situations, we used pooled METCAM/MUC18-expressing and control (vector) clones for testing effects of human METCAM/MUC18 over-expression on in vitro motility and invasiveness, and on in vivo tumor formation and metastasis in female athymic nude mice. Effects of METCAM/MUC18 on the expression of various downstream key factors related to tumorigenesis were also evaluated by Western blot analyses., Results: The over-expression of METCAM/MUC18 inhibited in vitro motility and invasiveness of SK-OV-3 cells. SK-OV-3 cells of the control (vector) clone (3D), which did not express human METCAM/MUC18, supported the formation of a solid tumor after SC injection of the cells at dorsal or ventral sites and also formation of solid tumor and ascites after IP injection in the intraperitoneal cavity of nude mice. In contrast, SK-OV-3 cells from the METCAM/MUC18-expressing clone (2D), which expressed a high level of METCAM/MUC18, did not support the formation of a solid tumor at SC sites, or formation of ascites in the intraperitoneal cavity of nude mice. Expression levels of downstream key factors, which may affect tumor proliferation and angiogenesis, were reduced in tumors induced by the METCAM/MUC18-expressing clone (2D)., Conclusions: We conclude that increased human METCAM/MUC18 expression in ovarian cancer SK-OV-3 cells suppressed tumorigenesis and ascites formation in nude mice, suggesting that human METCAM/MUC18 plays a suppressor role in the progression of ovarian cancer, perhaps by reducing proliferation and angiogenesis.

Our previous study suggests that ginger root extract can reverse behavioral dysfunction and prevent Alzheimer's disease (AD)-like symptoms induced by the amyloid-β protein (Aβ) in a rat model. 6-Gingerol is the major gingerol in ginger rhizomes, but its effect on the treatment of AD remains unclear. In this study, we aimed to determine if 6-gingerol had a protective effect on Aβ1-42-induced damage and apoptotic death in rat pheochromocytoma cells (PC12 cells) and to investigate the underlying mechanisms by which 6-gingerol may exert its neuroprotective effects. Our results indicated that pre-treatment with 6-gingerol significantly increased cell viability and reduced cell apoptosis in Aβ1-42-treated cells. Moreover, 6-gingerol pretreatment markedly reduced the level of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), the production of nitric oxide (NO), and the leakage of lactate dehydrogenase (LDH) and increased superoxide dismutase (SOD) activity compared with the Aβ1-42 treatment group. In addition, 6-gingerol pretreatment also significantly enhanced the protein levels of phosphorylated Akt (p-Akt) and glycogen synthase kinase-3β (p-GSK-3β). Overall, these results indicate that 6-gingerol exhibited protective effects on apoptosis induced by Aβ1-42 in cultured PC12 cells by reducing oxidative stress and inflammatory responses, suppressing the activation of GSK-3β and enhancing the activation of Akt, thereby exerting neuroprotective effects. Therefore, 6-gingerol may be useful in the prevention and/or treatment of AD.

Objective: Foam cell formation in the arterial wall plays a key role in the development of atherosclerosis. Recent studies showed that Urotensin II (U II) is involved in the pathogenesis of atherosclerosis. Here we examined the effects of human U II on ATP-binding cassette transporter A1 (ABCA1) expression and the underlying mechanism in THP-1 macrophages., Methods and Results: Cultured THP-1 macrophages were treated with U II, followed by measuring the intracellular lipid contents, cholesterol efflux and ABCA1 levels. The results showed that U II dramatically decreased ABCA1 levels and impaired cholesterol efflux. However, the effects of U II on ABCA1 protein expression and cellular cholesterol efflux were partially reversed by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa B (NF-κB) activity, suggesting the potential roles of ERK1/2 and NF-κB in ABCA1 expression, respectively., Conclusion: Our current data indicate that U II may have promoting effects on the progression of atherosclerosis, likely through suppressing ABCA1 expression via activation of the ERK/NF-κB pathway and reducing cholesterol efflux to promote macrophage foam cell formation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aim: To investigate the effects of dihydromyricetin (DHM) on the migration and invasion of human hepatic cancer cells., Methods: The hepatoma cell lines SK-Hep-1 and MHCC97L were used in this study. The cells were cultured in RPIM-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. DHM was dissolved in dimethyl sulfoxide and diluted to various concentrations in medium before applying to cells. MTT assays were performed to measure the viability of the cells after DHM treatment. Wound healing and Boyden transwell assays were used to assess cancer cell motility. The invasive capacity of cancer cells was measured using Matrigel-coated transwell chambers. Matrix metalloproteinase (MMP)-2/9 activity was examined by fluorescence analysis. Western blot was carried out to analyze the expression of MMP-2, MMP-9, p-38, JNK, ERK1/2 and PKC-δ proteins. All data were analyzed by Student's t tests in GraphPad prism 5.0 software and are presented as mean ± SD., Results: DHM was found to strongly inhibit the migration of the hepatoma cell lines SK-Hep-1 (without DHM, 24 h: 120 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 65 ± 10 μmol/L, P < 0.001) and MHCC97L (without DHM, 24 h: 126 ± 7 μmol/L vs 100 μmol/L DHM, 24 h: 74 ± 6 μmol/L, P < 0.001). The invasive capacity of the cells was reduced by DHM treatment (SK-Hep-1 cells without DHM, 24 h: 67 ± 4 μmol/L vs 100 μmol/L DHM, 24 h: 9 ± 3 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 117 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 45 ± 2 μmol/L, P < 0.001). MMP2/9 activity was also inhibited by DHM exposure (SK-Hep-1 cells without DHM, 24 h: 600 ± 26 μmol/L vs 100 μmol/L DHM, 24 h: 100 ± 6 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 504 ± 32 μmol/L vs 100 μmol/L DHM 24 h: 156 ± 10 μmol/L, P < 0.001). Western blot analysis showed that DHM decreased the expression level of MMP-9 but had little effect on MMP-2. Further investigation indicated that DHM markedly reduced the phosphorylation levels of p38, ERK1/2 and JNK in a concentration-dependent manner but had no impact on the total protein levels. In addition, PKC-δ protein, a key protein in the regulation of MMP family protein expression, was up-regulated with DHM treatment., Conclusion: These findings demonstrate that DHM inhibits the migration and invasion of hepatoma cells and may serve as a potential candidate agent for the prevention of HCC metastasis.

Phytoestrogens are candidate drugs for the treatment of osteoporosis. Many experiments have been designed to investigate the preventive effects of phytoestrogens for osteoporosis; however, it is easy for a single dissenting result from animal experiments to mislead clinical investigations. Herein, we use meta-analysis to assess the evidence for a protective effect of phytoestrogens on ovariectomized rat models of osteopenia. With respect to osteoporosis, PubMed and Web of Science were searched from January 2000 to March 2013 for relevant studies of phytoestrogens in ovariectomized rats. Two reviewers independently selected and assessed the studies. Data were aggregated using a random effects model. Meta-analysis revealed that the phytoestrogen treatment group demonstrated a significantly higher femur bone mineral density and trabecular bone and lower bone turnover markers (serum alkaline phosphatase and serum osteocalcin) compared with the control ovariectomized group, thus showing a bone protective effect of phytoestrogens in ovariectomized rats. Subsequent sensitivity analyses indicated that the effect of phytoestrogens on serum alkaline phosphatase and serum osteocalcin are not robust. Despite the high heterogeneity in the systematic review of animal experiments, the present results indicated that phytoestrogens may offer the most potential for the prevention of bone loss by reducing the expected loss of trabecular bone and bone mineral density. Their effects are likely due to inhibition of bone resorption, but their benefits on bone formation are still unclear. Further studies are needed to assess the effect of phytoestrogens on bone formation and the efficacy and safety of individual phytoestrogens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Apolipoprotein AI (apoA-I) is the major protein component of high-density lipoprotein (HDL), and apoA-I can stabilize the structure of HDL. Whereas the amphipathic alpha-helix is the structural motif for apoA-I to complete the corresponding functionality. In the lipid-free state, the N-terminal of apoA-I molecule is a dynamic four-helix bundle structure, most amino acid residues of the C-terminal domain is the formation of a disordered structure, which is the initial domain that mediates bingding to phospholipid surface. Two molecules of apoA-I are arranged in an anti-parallel, double-belt conformation around the surface of the discoidal HDL particles. However, the apoA-I molecule forms a trefoil scaffold structure, which can adapt to the surface of spherical HDL particles. ATP-binding cassette transporter A1 (ABCA1) can mediate phospholipid and cholesterol efflux from intracellular and interact with apoA-I to generate nascent HDL particles. Overall, apoA-I and HDL as an anti-atherosclerotic effect of primary target, we focus on the molecular structure of apoA-I, which determines the structure and function of different size of HDL particles, as well as the conformational changes after interaction with lipids, in order to learn more about the relationship of apolipoprotein and lipids metabolism against atherosclerosis.

Objective: The aim of this study was to determine whether ATP-binding cassette transporter A1 (ABCA1) was up-regulated by growth differentiation factor-15 (GDF-15) via the phosphoinositide 3-kinase (PI3K)/protein kinase Cζ (PKCζ)/specificity protein 1 (SP1) pathway in THP-1 macrophages., Methods and Results: We investigated the effects of different concentrations of GDF-15 on ABCA1 expression in THP-1 macrophages. The results showed that GDF-15 dramatically increased cholesterol efflux and decreased cellular cholesterol levels. In addition, GDF15 increased ABCA1 mRNA and protein levels. The effects of GDF-15 on ABCA1 protein expression and cellular cholesterol efflux were abolished by wither inhibition or depletion of PI3K, PKCζ and SP1, respectively, suggesting the potential roles of PI3K, PKCζ and SP1 in ABCA1 expression. Taken together, GDF-15 appears to activate PI3K, PKCζ and SP1 cascade, and then increase ABCA1 expression, thereby promoting cholesterol efflux and reducing foam cell formation., Conclusion: Our results suggest that GDF-15 has an overall protective effect on the progression of atherosclerosis, likely through inducing ABCA1 expression via the PI3K/PKCζ/SP1 signaling pathway and enhancing cholesterol efflux., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of this study was to assess the ability of a traditional Chinese medicinal ginger root extract (GRE) to prevent behavioral dysfunction in the Alzheimer disease (AD) rat model. Rat AD models were established by an operation (OP) in which rats were treated with a one-time intra-cerebroventricuIar injection of amyloid β-protein (Aβ) and continuous gavage of aluminum chloride every day for 4 weeks. GRE was administered intra-gastrically to rats. After 35 days, learning and memory were assessed in all of the rats. Brain sections were processed for immunohistochemistry and Hematoxylin & Eosin (H&E) and Nissl staining. The latency to show significant memory deficits was shorter in the group that received OP with a high dose of GRE (HG)(OP+HG) than in the groups that received OP with a low or moderate dose of GRE (LG, MG)(OP+LG, OP+MG) (p<0.05). The expression of superoxide dismutase (SOD) and catalase (CAT) in the OP+MG and OP+LG groups was up-regulated compared to the OP+HG groups (p<0.05). The rats in the OP+HG groups had lower levels of nuclear factor-κB (NF-κB), interleukin-1β (IL-1β), and malondialdehyde (MDA) expression than the rats in the OP+MG and OP+LG groups (p<0.05). This experiment demonstrates that the administration of GRE reverses behavioral dysfunction and prevents AD-like symptoms in our rat model.

Ethnopharmacological Relevance: To assess the ability of traditional Chinese medicine Polygonatum sibiricum polysaccharide to prevent bone loss in the ovariectomized rat., Materials and Methods: PSP was administered intragastrically to the rats. After 35 days, the total body bone mineral density (BMD) was assessed in all of the rats. All sections were processed for immunohistochemistry and hematoxylin-eosin staining (H.E.)., Results: BMD was lower in the ovariectomized group (OVX, 0.163 g/cm(2)), the group that received a moderate dose of PSP on OVX animals (OVX+MP, 0.163 g/cm(2)) and the group that received a low dose of PSP on OVX animals (OVX+LP, 0.162 g/cm(2)) than in the sham-operated group (SHAM, 0.180 g/cm(2)), the OVX+E(2) group (OVX+E(2), 0.176 g/cm(2)) and the group that received a high dose of PSP on OVX animals (OVX+HP, 0.174 g/cm(2)) (P<0.05). Clear arrangements of bone trabeculae were observed in the OVX+E(2) and OVX+HP. The expression of bone morphogenetic proteins (BMP) and basic fibroblast growth factor (bFGF) in the OVX, OVX+MP and OVX+LP was down regulated compared to the SHAM, OVX+E(2) and OVX+HP (P<0.05). The rats in the OVX+E(2) and OVX+HP had lower levels of bone Gla protein (BGP), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP) and tumor necrosis factor α(TNF-α) expression than the rats in the OVX, OVX+MP and OVX+LP (P<0.05)., Conclusion: This experiment demonstrates that the administration of PSP to ovariectomized rats reverses bone loss and prevents osteoporosis., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Conflicting research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as both a tumor promoter and a tumor suppressor in the development of breast cancer. To resolve this, we have re-investigated the role of this CAM in the progression of human breast cancer cells., Methods: Three breast cancer cell lines were used for the tests: one luminal-like breast cancer cell line, MCF7, which did not express any METCAM/MUC18, and two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which expressed moderate levels of the protein.MCF7 cells were transfected with the human METCAM/MUC18 cDNA to obtain G418-resistant clones which expressed the protein and were used for testing effects of human METCAM/MUC18 expression on in vitro motility and invasiveness, and in vitro and in vivo tumorigenesis. Both MDA-MB-231 and MDA-MB-468 cells already expressed METCAM/MUC18. They were directly used for in vitro tests in the presence and absence of an anti-METCAM/MUC18 antibody., Results: In MCF7 cells, enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, anchorage-independent colony formation (in vitro tumorigenesis), and in vivo tumorigenesis. In both MDA-MB-231 and MDA-MB-468 cells, the anti-METCAM/MUC18 antibody inhibited both motility and invasiveness. Though both MDA-MB-231 and MDA-MB-468 cells established a disorganized growth in 3D basement membrane culture assay, the introduction of the anti-METCAM/MUC18 antibody completely destroyed their growth in the 3D culture., Conclusion: These findings support the notion that human METCAM/MUC18 expression promotes the progression of human breast cancer cells by increasing their motility, invasiveness and tumorigenesis.

Background: Inflammation is involved in the atherogenesis and pathogenesis of acute coronary syndrome (ACS). As the acute-phase reaction proteins in ACS, myeloperoxidase (MPO) and C-reactive protein (CRP) may play critical roles. Anti-inflammation may be one of benefits of statin drugs in ACS. Studies have showed that statins can suppress serum CRP concentrations. However, whether statins also reduce serum MPO concentrations in patients with ACS is unknown., Methods: Seventy-eight patients with ACS were randomly separated into Group A and Group B, the patients in Group A receiving conventional therapy, which include no cholesterol-lowering drugs, +atorvastatin (10 mg/day, n=40), the patients in Group B receiving conventional therapy (n=38). The serum concentrations of MPO were measured by enzyme-linked immunosorbent assay (ELISA) and CRP were measured by turbidimetric immunoassay., Results: Serum concentrations of MPO were significantly lower after 1-week therapy in both groups of patients [Group A from 590+/-168 to 496+/-154 microg/l, Group B from 570+/-165 to 521+/-153 microg/l; P<0.01, respectively]. Serum concentrations of CRP also were markedly lower than pretreatment [Group A from 6.56+/-1.87 to 5.14+/-2.07 mg/l; Group B from 6.36+/-1.94 to 5.45+/-1.90 mg/l, P<0.05, respectively]. Compared with conventional therapy alone, atorvastatin significantly further reduced serum MPO [P=0.014] and CRP concentrations [P=0.032]. There were no correlations detected between the reduction of MPO and CRP (r=0.124, P=0.068)., Conclusions: Atorvastatin reduced serum MPO and CRP concentrations in patients with ACS. These effects may explain some clinical benefits of statins in the treatment of these patients.

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.

Infrared spectra of 2-alkyl-7,7,8,8-tetracyanoquinodimethane (C12H25 TCNQ, C15H31 TCNQ, C18H37 TCNQ) were measured with the resolution of 1 and 4 cm(-1). In order to identify the peak number correctly in the CH2 stretching region, second derivative and Fourier self-deconvolution were applied to the infrared spectra, respectively. The overlapping bands in the CH2 stretching region could be identified when the infrared spectra, which were measured with the resolution of 4 cm(-1), were dealt with by Fourier self-deconvolution. However, the bands overlapped in the CH2 symmetric stretching region could not be observed when these infrared spectra were dealt with by second derivative. The above results reveal that Fourier self-deconvolution method is more powerful than second derivative in identifying bands that are involved in an overlapping band feature.

TRK family proteins, which mediate the concentrative uptake of potassium by plant cells, fungi, and bacteria, resemble primitive potassium channels in sequence and have recently been proposed actually to fold like potassium channels in a 4-MPM motif (Durell, S. R., and Guy, H. R. (1999) Biophys. J. 77, 789 - 807), instead of like conventional substrate porters in the 12-TM motif (Gaber, R. F., Styles, C. A., and Fink, G. R. (1988) Mol. Cell. Biol. 8, 2848-2859). The known fungal members of this family possess a very long hydrophilic loop, positioned intracellularly in the K(+)-channel model and extracellularly in the substrate porter model. This and two shorter hydrophilic segments have been tested as topological markers for the true folding pattern of TRK proteins using Saccharomyces cerevisiae Trk2p. Hemagglutinin epitope tags were inserted into all three segments, and the enhanced green fluorescent protein (EGFP) was fused to the C terminus of Trk2p. The gene constructs were expressed from a high copy plasmid, and sidedness of the tags was determined by native fluorescence (EGFP), indirect immunofluorescence, and immunoelectron microscopy. Both the long-loop tag and the C-terminal EGFP fusion allowed abundant protein to reach the plasma membrane and support normal yeast growth. In all determinations, the long-loop tag was localized to the inner surface of the yeast cell plasma membrane, thus strongly supporting the channel-like folding model. Additional observations showed (i). membrane-associated Trk2p to lie in proteolipid rafts; (ii). significant tagged protein, expressed from the plasmid, to be sequestered in cytoplasmic vesicular-tubular clusters; and (iii). suppression of such clusters by yeast growth in 5-10% glycerol. This chaperone-like effect may assist other membrane proteins (overexpressed or heterologously expressed) to function within the yeast plasma membrane.

Objective: To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. pastroris, and to purify the expressed product and detect its biological activity., Methods: By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His+ Muts phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol., Results: SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF165 and inhibit the proliferation of HUVEC stimulated by hVEGF165., Conclusion: Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

New vessel formation plays a key role in tumor growth and transforming, and the Vascular endothelial growth factor (VEGF) is the most important factor inducing tumor vessel formation. The cDNA of extracellular domain of VEGF receptor KDR was cloned from primary cultured HUVEC by RT-PCR and subcloned into AAV vector pSNAV. Recombinant AAV was obtained from BHK cells transformed with pSNAV/KDR after adding helper virus. The recombinant AAV expressing soluble KDR that can bind to VEGF. In vivo experiment demonstrated that the recombinant virus can inhibit the new vessel formation of melanoma in C57 mice model.

The cDNA fragment of the first 3 loops of VEGF receptor, KDR, was cloned by PCR and inserted into a baculovirus expression plasmid pFASTBACI. The competent E. coli DH10BAC cell, which contain another plasmid with baculovirus genome in it, was transformed with pFASTBACI-KDRn3. Homologous recombination in the prokaryotic cells resulted in a recombinant plasmid containing KDRn3 in baculovirus genome. Transfection of the insect cell SF-9 with above plasmid generated a recombinant baculorvirus contain target gene fragment. SDS-PAGE and Western blot analysis of the supernatant of the infected SF-9 cell showed that KDRn3 was secreted in the medium. The recombinant protein was verified with Western blot and tested for their binding activity with VEGF. Its anti-angiogenic activity was assayed on chorionic allantoic membrane(CAM) of fertilized egg. The results showed that the recombinant protein could inhibit new vessel formation on CAM of fertilized eggs.

The human VEGF(165) cDNA was amplified by PCR, and was inserted, after confirming by DNA sequence analysis, into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and lead sequence of alpha factor gene to form a recombinant expression plasmids pPIC9K/hVEGF(165). This recombinant plasmid was transformed into KM71. Transformants were screened, cultured inflasks and induced by the addition of 1% methanol. After 4 days of methanol induction, the expressed hVEGF(165) came up to 60% of total proteins in medium supernatant as shown by SDS-PAGE. Western blot assay proved that the expressed hVEGF(165) had good antigenicity and high specificity. The recombinant protein was further purified by using Heparin-Sepharose CL6B affinity chromatography, and was proved that it had good biological activity to stimulate HUVEC proliferation and to promote collateral blood vessel formation in an acquired limb artery occlusion animal model.

By inserting VEGF receptor Flt-1(1-3 loop) cDNA into Pichia pastoris expression vector pPIC9K containing AOX1 promotor and the sequences of alpha secreting signal peptides, the expression plasmid pPIC9K/Flt-1(1-3) was constructed and transformed into GS115. The multi-copy insert transformants were selected and cultivated in flasks. After 4 days of 1% methanol induction, the expressed Flt-1(1-3) accumulated up to 30% of total proteins in supernatant. The expressed Flt-1(1-3) was further proved with good antigenicity and high specificity by ELISA and Western blot. They can bind to VEGF and inhibit HUVEC proliferation stimulated by VEGF.

Four cDNA clones, encoding truncated Flt-1 mutants consisting of loop 2, 1-2, 2-3 and 1-3, were amplified by PCR from human placenta cDNA library and the corresponding gene fragments were named Flt-1(2) Flt-1(1-2) Flt-1(2-3) and Flt-1(1-3). In order to detect which part of the extracellular domain was involved in ligand binding, the interaction between Flt-1 mutants and hVEGF(165) was studied with yeast two-hybrid system. The data presented here suggest that both Flt-1(2-3) and Flt-1(1-3) were able to bind hVEGF(165). Two recombinant expression plasmids, pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1(2-3), were constructed and transformed into Pichia pastoris strain GS115, respectively. After 4 days of methanol induction, the amount of the expressed Flt-1s reached 60% of total proteins in supernatant by SDS-PAGE. Recombinant proteins were purified with CM-Sepharose Fast Flow and Sephacryl S-100 chromatography. Biological activities analysis proved that 1-3 loop had slightly better biological activity than 2-3 loop in VEGF(165) binding and in inhibition of HUVEC proliferation stimulated by hVEGF(165).