Browsing by Author "Pan, JP"

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Epidemiology of haptoglobin phenotypes in chronic diseases
(2003-03-17)
Cheng, TM
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Pan, JP
;
Wang, JS
;
Mao, SJT
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生物科技學系
Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography
(2003-06-25)
Liau, CY
;
Chang, TM
;
Pan, JP
;
Chen, WL
;
Mao, SJT
Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Up is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 mul of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 nil of human plasma. Affinity purified Up 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2. (C) 2003 Elsevier Science B.V. All rights reserved.
Vitamin C protects against lysophosphatidylcholine-induced expression of monocyte chemoattractant protein-1 in cultured human umbilical vein endothelial cells
(2003-03-01)
Pan, JP
;
Cheng, TM
;
Chou, SC
;
Lai, ST
;
生物科技學系
Background and Purpose: It is well documented that oxidized low-density lipoproteins (LDLs) can stimulate human vascular endothelial cells to produce monocyte chemoattractant protein-I (MCP-I). Vitamin c is known to be an important antioxidant for vasodilation. The purpose of this study was to determine whether pretreatment with vitamin could protect against oxidized LDL induced expression of MCP-1 in cultured human umbilical vein endothelial cells (HUVECs). Methods: Cultured HUVECs were used for desired experiments before passage 4. Lysophosphatidylcholine (lyso PC), an oxidized components of LDL, was designated as the stimulator for MPC-1 synthesis from cultured HUVECs MCP-1 concentration in the cultured media were determined by enzyme-linked immunosorbent assay MCP-1 RNA was evaluated by a semi quantitative reverse transcriptase-polymerase chain reaction. Results: HUVECs secreted MCP-1 within 30 minutes after exposure to 50 muM lysoPC. Compared with samples treated with lyso PC alone, pretreatment with vitamin C in concentration of 50, 100, 150 and 200 muM. reduced levels of MCP-1 in the culture medium by 44%, 51%, 60% and 67%, respectively, while levels of MCP-1 mRNA decreased by 15%, 18%, 80% and 82%, respectively. Conclusions: Our findings imply that pretreatment with vitamin C can suppress lysoPC induced expression and secretion of MCP-1 in cultured HUVECs. Therefore, vitamin C is protective against lyso mediated inflammatory insults to the vascular endothelium in vitro.
Voice over wireless local area network
(2006-02-01)
Shen, XM
;
Lin, YB
;
Pang, AC
;
Pan, JP
;
資訊工程學系