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ABSTRACT: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been demonstrated to promote hemostasis in vivo and induce platelet aggregation in vitro. Pennogenin tetraglycoside (Tg) has been identified as one of the active ingredients in TSSPs and can induce rat platelet aggregation.
To investigate the functional role of Tg in platelets and the signaling pathway mechanisms which mediate Tg-induced platelet aggregation.
Using scanning electron microscopy, the turbidimetric method and flow cytometry, we demonstrated that Tg induces shape change and concentration-dependently induces aggregation, dense granule secretion and a-granule secretion in rat platelets. The activation characteristics were comprehensively confirmed using transmission electron microscopy. Apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, completely inhibited Tg-induced platelet aggregation, which was not sensitive to indomethacin or SQ29548 inhibition. Furthermore, ADP receptor antagonists inhibited Tg-induced a-granule secretion, and blockade of the P2Y1 receptor prevented Tg-induced platelet shape changes. Tg-induced dense granule secretion was not affected by ADP receptor antagonists or various various pharmacological inhibitors of the intracellular effectors involved in dense granule secretion signaling pathways.
We identified that Tg directly induces platelet activation and demonstrated that Tg-induced platelet activation depends on dense granule secretion of ADP, which in turn activates the P2Y1 and P2Y12 receptor signaling pathways.
Thrombosis Research 03/2012; 129(5):e209-16. · 2.44 Impact Factor
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ABSTRACT: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been widely used in China for the treatment of abnormal uterine bleeding. We previously studied the main active constituents of TSSPs and their structure-activity relationships with respect to rat myometrial contractions. Tg (pennogenin tetraglycoside) was identified as one of the active ingredients in TSSPs able to induce rat myometrial contractions. However, the mechanisms underlying the pharmacological actions on uterine activity have not been described clearly.
Here Tg was screened for effects on contractile activity in isolated uterine strips from estrogen-primed rats and on MLC20 phosphorylation and related signaling pathways in cultured rat myometrial cells as determined by Western blot. Intracellular calcium ([Ca(2+)](i)) was monitored under a confocal microscope using Fluo-4 AM-loaded myometrial cells.
Tg dose-dependently stimulated rat myometrial contractions as well as MLC20 phosphorylation in vitro, which could be completely suppressed by an inhibitor of myosin light chain kinase (MLCK). Use of Ca(2+) channel blockers and kinase inhibitors demonstrated that Tg-induced myometrial contractions are mediated by activation of the phospholipase C (PLC)-inositol triphosphate (IP3) signaling pathway, resulting in increased MLC20 phosphorylation. Furthermore, Y27632, a specific inhibitor of Rho kinase (ROK), notably suppressed Tg-stimulated myometrial contractions and decreased MLC20 phosphorylation.
These data provide evidence that rat myometrial contractility induced by Tg results from enhanced MLC20 phosphorylation, while both PLC-IP3 and RhoA/ROK signaling pathways mediate the process. These mechanisms may be responsible for the therapeutic effects of TSSPs on abnormal uterine bleeding.
PLoS ONE 01/2012; 7(12):e51536. · 4.09 Impact Factor
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YanFang Ju,
JinJu Yang,
Rong Liu, XiaoLan Liu,
XueMei Du,
Li Liu,
ZhiCheng Chen,
Jun Chi,
ShuEr Liu,
Yuan Gao,
JianEn Gao,
ShunChang Jiao,
FuChu He,
QiHong Sun
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ABSTRACT: Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning. Antibodies that recognized mitochondrial proteins were identified by screening human liver cDNA expression libraries. In this study, we identified 6 major antigens that were recognized by at least 2 different monoclonal antibodies (mAbs). The proteins that were antigenically dominant were: acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase), aldehyde dehydrogenase 1 family member A1, carbamoyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), enoyl coenzyme A hydratase 1, and hydroxysteroid (11-beta) dehydrogenase 1. We also determined the subcellular localizations of these enzymes within the mitochondria using immunohistocytochemistry. We believe that these well-characterized antibodies will provide a valuable resource for the Human Liver Proteome Project (HLPP), and will make studies aimed at investigating liver mitochondrial function far easier to perform in future. Our results provide strong evidence that, (i) depletion of dominant proteins from liver mitochondrial samples is possible and, (ii) the approaches adopted in this study can be used to explore or validate protein-protein interactions in this important organelle.
Science China. Life sciences 01/2011; 54(1):16-24. · 2.02 Impact Factor
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ABSTRACT: Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10-120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.
Journal of Biomedicine and Biotechnology 02/2009; 2009:973754. · 2.44 Impact Factor
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Jianen Gao,
Yuan Gao,
Yanfang Ju,
Jinju Yang,
Qiong Wu,
Jinchao Zhang,
Xuemei Du,
Zhaoqing Wang,
Yingbo Song,
Hui Li, [......],
Han Xu, Xiaolan Liu,
Jinlan Wang,
Yangjun Zhang,
Yun Cai,
Yufang Cui,
Xiaohong Qian,
Fuchu He,
Ming Li,
Qi-Hong Sun
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ABSTRACT: Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.
PROTEOMICS 02/2006; 6(2):427-37. · 4.51 Impact Factor
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Yunshan Ning,
Yundan Wang,
Yan Li,
Yanhua Hong,
Dandan Peng,
Yichu Liu,
Junxiang Wang,
Wenbo Hao,
Xuemei Tian,
Fenfang Wu,
Wenqi Dong,
Lihong Wang,
Qiong Wu, Xiaolan Liu,
Jianen Gao,
Fuchu He,
Xiaohong Qian,
Qi-Hong Sun,
Ming Li
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ABSTRACT: Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.
PROTEOMICS 02/2006; 6(2):438-48. · 4.51 Impact Factor
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ABSTRACT: The aim of the present study was to examine the serum lipid profile in obese Chinese children, their serum lipid and apolipoprotein A-I (apoA-I) and B (apoB) levels were examined.
The subjects were 153 patients (109 male and 44 female) aged 4-16 years with obesity, who attended the outpatient clinic of Beijing Children's Hospital. Percentage bodyweight (%BW) ([(bodyweight - standard weight)/standard weight]x 100) were obtained. Skinfold thickness and hip and waist circumference were measured. Percentage body fat (%BF) was estimated by bioelectrical impedance analyzer. Serum total cholesterol (TC), high density lipoprotein cholesterol (HDLC), triglyceride (TG), apoA-I and apoB levels were also measured.
TC showed an acceptable level in 86.8% of obese children. The prevalence of high TC levels (3.3%) or high LDLC levels (6.0%) was rather low. The HDLC level was reduced in 31.3% of obese children. Anthropometric variables had no linear relationship to TC, HDLC, TG, LDLC, apoA-I or apoB, but in the older age group (over 10 years old) %BW and %BF showed a weak correlation with HDLC (r = -0.202, r = -0.211, respectively).
In obese Chinese children, HDLC as well as TC levels should be examined in order to assess coronary risk.
Pediatrics International 09/2004; 46(4):425-8. · 0.63 Impact Factor
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Jing Hao,
Liansheng Sun,
Haixiao Huang,
Guolin Xiong, Xiaolan Liu,
Liling Qiu,
Guozhi Chen,
Bo Dong,
Yuanmin Li,
Wangqiu Chen,
Yingji Buechler,
Jim Sun,
Chun Shen,
Qingliang Luo
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ABSTRACT: The effects of recombinant human interleukin 11 (rhIL11) on thrombocytopenia and neutropenia in irradiated rhesus monkeys were evaluated after administration different doses at different times. Twenty-three rhesus monkeys were exposed to a total-body irradiation (TBI) with a single dose of 3 Gy 60Co gamma rays. Either placebo, rhIL11 at a dose of 30, 60 or 120 microg/kg day(-1) on days 0-13, or rhIL11 at a dose of 60 microg/kg day(-1) on days 13-26 after TBI was administered to the animals. The results showed that the immediate treatment with rhIL11 but not treatment on days 13-26 resulted in much higher platelet nadirs than in the placebo-treated group. The accelerated recovery of platelets to normal levels after TBI was demonstrated in all groups treated with rhIL11, but the effects of rhIL11 were independent of dose. However, rhIL11 treatment could also accelerate the recovery of leukocytes to normal levels. The numbers of colony-forming bone marrow cells (CFU-E, CFU-Mix, CFU-MK and CFU-GM) in all groups treated with rhIL11 were increased 4- to 14-fold relative to those of the placebo group on day 30. We conclude that rhIL11 may directly promote megakaryocyte development and ameliorate myelosuppression in irradiated monkeys.
Radiation Research 09/2004; 162(2):157-63. · 2.68 Impact Factor
