Browsing by Author "Mao, Simon J. T."

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A unique tetrameric structure of deer plasma haptoglobin - an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure
(2008-03-01)
Lai, I. H.
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Lin, Kung-Yu
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Larsson, Mikael
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Yang, Ming Chi
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Shiau, Chuen-Huei
Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by alpha 2-beta subunits or (alpha 2-beta)(n) (where n >= 3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the alpha-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (alpha-beta)(4), although the locations of -SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 M urea under reducing conditions (143 mM beta-mercaptoethanol), followed by renaturation, sustained the formation of (alpha-beta)(4), suggesting that the Hp tetramers are not randomly assembled. Interestingly, an alpha-chain monoclonal antibody (W1), known to recognize both human and deer alpha-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer alpha-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage.
Abnormal Renal Resistive Index in Patients with Mild-to-moderate Chronic Obstructive Pulmonary Disease
(2013-04-01)
Chen, Cheng-Yu
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Hsu, Ta-Wei
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Mao, Simon J. T.
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Chang, Shih-Chieh
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Yang, Pan-Chyr
Background: Arterial rigidity and endothelial dysfunction are systemic manifestations of chronic obstructive pulmonary disease (COPD). The decrease in renal vascular resistance in order to adapt the increase in glomerular filtration rate after oral protein loading is known as normal renal functional reserve. We tested the hypothesis that COPD patients, even in those with mild-to-moderate airflow obstruction, are affected by systemic inflammation associated with abnormal renal functional reserve. Materials and Methods: The study enrolled 24 current smokers with a cigarette smoking history >= 25 pack-years and 8 nonsmokers with normal spirometry as control. Doppler sonography detected the renal resistive index (RRI) before and after oral protein loading to determine the renal functional reserve. Pulmonary function and serum tumor necrosis factor alpha (TNF-alpha) levels were analyzed to compare with the renal functional reserve. Results: The smokers were stratified into 3 groups (Group 1: smokers with normal spirometry, Group 2: mild COPD, Group 3: moderate COPD); nonsmokers as Group 4. The baseline RRI levels were similar in Group 1 and Group 4. After protein loading, the RRI elevated in all smoking groups; moreover, Group 3 had the highest RRI and with longer duration than other groups. The smokers with higher serum TNF-alpha levels had a longer RRI elevation. Multiple linear regression revealed forced expiratory volume in one second (FEV 1), serum TNF-alpha levels and aging were independently predictive factors of impaired renal functional reserve. Conclusions: A greater impairment in renal functional reserve of COPD patients was correlated with more severe airway obstruction and inflammation.
An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate
(2007-01-15)
Yueh, Sunny C. H.
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Lai, Yi An
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Chen, Wen Liang
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Hsu, Hsiao Han
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Mao, Simon J. T.
Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we had isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp beta-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we have developed a novel affinity column procedure using an mAb prepared against a-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl and 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then prewashed with a 0.04% sodium dodecyl sulfate (SDS)-PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS-PBS, pH 11, and collected in tubes containing 1 M Tris-HC1, pH 6.8. As a result, the isolated Hp was devoid of apoA-1 and was able to retain the biological function by forming an Hp-hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1, or 2-2 was greater than 95% with an yield greater than 50%. The procedure described here is significantly improved in time consumption, recovery, and purity. The rationale, design, and optimization for each step are described in detail. (c) 2006 Elsevier B.V. All rights reserved.
Analysis of antioxidant as a therapeutic agent for atherosclerosis
(2006-11-01)
Tseng, Chi Feng
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Mao, Simon J. T.
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生醫工程研究所
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Institute of Biomedical Engineering
Research into the oxidation of lipoprotein has yielded many insights into the underlying process of the development of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) has been suggested as an initial step in the pathogenesis of atherosclerosis. However, up until now, investigations of antioxidants have mostly focused on three main dietary antioxidant vitamins (beta-carotene, vitamin C, and vitamin E) and some synthetic compounds. Among those antioxidants, probucol, a synthetic compound, has been shown to be an extremely potent and effective antioxidant in preventing the formation of atherosclerosis in both in vitro and in vivo studies. The present review focuses on commonly used analytical methods for measuring the antioxidant potency and outlines the, critical steps on how to evaluate and design a potent antioxidant agent that can be used for the intervention of atherosclerosis. We concluded that an antioxidant should first be targeted and incorporated into human LDL. Second, the candidate compound should possess high bioavailability. The rationale and strategy for the analytical procedures are discussed in this report.
Clinical evaluation of the eBsensor hand-held blood glucose monitoring system
(2007-02-01)
Chen, Chien-Chih
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Tai, Der-Yan
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Ho, Ping
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Lai, Yiu-Kay
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Lin, Jui-Jane
Background: Home glucose monitoring system is increasingly recognized as an important tool for glycemic control. We evaluated the clinical performance of the eBsensor glucose monitoring system. Methods: Fingertip capillary blood glucose concentrations from 282 subjects were measured using eBsensor glucose monitoring system and compared against predicate devices and the Yellow Springs Instruments (YSI) 2300 blood glucose analyzer. Accuracy and precision of the eBsensor glucose monitoring system were assessed using several methods. The comparative study between the eBsensor and 2 currently marketed monitoring systems was performed. Results: The 282 eBsensor readings covered a wide range from 2.6 to 24.4 mmol/l. Deming regression and Pearson correlation analyses showed a linear relationship between the eBsensor readings and the YSI reference method (eBsensor=0.9496 YSI+0.4127 mmol/l; r=0.98). Error Grid analysis demonstrated that 100% of the eBsensor readings in clinically acceptable zones A and B. The CVs for the 6 lots of strips were within the satisfactory interval (< 6%). The comparative study showed that the eBsensor readings correlated well with the OneTouch Ultra values (r=0.97) and the Glucocard II values (r=0.97). Conclusions: eBsensor is a reliable glucose monitoring system which provides high accurate and precise glucose readings over a wide range of glucose concentrations. (c) 2006 Elsevier B.V. All rights reserved.
Cloning and expression of human haptoglobin subunits in Escherichia coli: Delineation of a major antioxidant domain
(2007-04-01)
Lai, I. Hsiang
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Tsai, Tsung I.
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Lin, Hong Huei
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Lai, Wei Yen
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Mao, Simon J. T.
Human plasma haptoglobin (Hp) comprises alpha and beta subunits. The alpha subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that alpha 1, alpha 2, beta, and alpha 2 beta chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. Each cloned subunit retained its immunoreactivity as confirmed using antibodies specific to alpha or beta chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to the native Hp. The antioxidant activity was found to be associated with both alpha and beta chains when assessed by Cu2+-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of P chain was extremely potent and markedly greater than that of native Hp (3.5 x), alpha chain (10 x) and probucol (15 x). The latter is a clinically proved potent compound used for antioxidant therapy. The "unrestricted" structure of beta subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a "mini-Hp" in antioxidant therapy. It may also provide a new insight in understanding the mechanism involved in the antioxidant nature of Hp. (c) 2006 Elsevier Inc. All rights reserved.
Crystal structure of a secondary vitamin D(3) binding site of milk beta-lactoglobulin
(2008-05-15)
Yang, Ming-Chi
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Guan, Hong-Hsiang
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Liu, Ming-Yih
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Lin, Yih-Hung
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Yang, Jinn-Moon
beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 angstrom resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 angstrom). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an a-helix and a beta-strand I with 17.91 angstrom in length, while the span of vitamin D(3) is about 12.51 angstrom. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure Of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).
Evidence for beta-lactoglobulin involvement in vitamin D transport in vivo- role of the gamma-turn (Leu-Pro-Met) of beta-lactoglobulin in vitamin D binding
(2009-04-01)
Yang, Ming Chi
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Chen, Nai Chi
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Chen, Chun-Jung
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Wu, Chin Yun
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Mao, Simon J. T.
beta-lactoglobulin (LG) is a major bovine milk protein, containing a central calyx and a second exosite beyond the calyx to bind vitamin D; however, the biological function of LG in transporting vitamin D remains elusive. Crystallographic findings from our previous study showed the exosite to be located at the pocket between the alpha-helix and beta-strand I. In the present study, using site-directed mutagenesis, we demonstrate that residues Leu143, Pro144 and Met145 in the gamma-turn loop play a crucial role in the binding. Further evidence is provided by the ability of vitamin D(3) to block the binding of a specific mAb in the gamma-turn loop. Using the mouse (n = 95) as an animal model, we initially demonstrated that LG is a major fraction of milk proteins responsible for uptake of vitamin D. Most interestingly, dosing mice with LG supplemented with vitamin D(3) revealed that native LG containing two binding sites gave a saturated concentration of plasma 25-hydroxyvitamin D at a dose ratio of 2 : 1 (vitamin D(3)/LG), whereas heated LG containing one exosite (lacking a central calyx) gave a ratio of 1 : 1. We have demonstrated for the first time that LG has a functional advantage in the transport of vitamin D, indicating that supplementing milk with vitamin D effectively enhances its uptake.
Evidence of tandem repeat and extra thiol-groups resulted in the polymeric formation of bovine haptoglobin: A unique structure of Hp 2-2 phenotype
(2007-11-30)
Lai, Yi An
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Lai, I. Hsiang
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Tseng, Chi Feng
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Lee, James
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Mao, Simon J. T.
Human plasma Hp is classified as 1-1, 2-1, and 2-2. They are inherited from two alleles Hp 1 and Hp 2, but there is only Hp 1 in almost all the animal species. Hp 2-2 molecule is extremely large and heterogeneous associated with the development of inflammatory-related diseases. In this study, we expressed entire bovine Hp in E. coli as a alpha beta linear form. Interestingly, the antibodies prepared against this form could recognize the subunit of native Hp. In stead of a complicated column method, the antibody was able to isolate bovine Hp via immunoaffinity and gel-filtration columns. The isolated Hp is polymeric containing two major molecular forms (660 and 730 kDa). Their size and hemoglobin binding complex are significantly larger than that of human Hp 2-2. The amino-acid sequence deducted from the nucleotide sequence is similar to human Hp 2 containing a tandem repeat over the cc chain. Thus, the Hp 2 allele is not unique in human. We also found that there is one additional -SH group (Cys-97) in bovine alpha chain with a total of 8 -SH groups, which may be responsible for the overall polymeric structure that is markedly different from human Hp 2-2. The significance of the finding and its relationship to structural evolution are also discussed.
Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting
(2007-04-15)
Cheng, Tsai-Mu
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Huang, Pei-Chen
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Pan, Ju-Pin
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Lin, Kuan-Yu
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Mao, Simon J. T.
Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3 mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SIDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within I min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed. (c) 2006 Elsevier B.V. All rights reserved.
Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H(2)O(2) generation from polyunsaturated fatty acids
(2011-03-01)
Cheng, Tsai-Mu
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Mao, Simon J. T.
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Lai, Shiau-Ting
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Chang, Chia-Ching
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Yang, Ming-Chi
Patients with increased haemolytic haemoglobin (Hb) have 10-20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H(2)O(2) generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN(3), a peroxidase inhibitor, catalase, a H(2)O(2) decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H(2)O(2) production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN(3) and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.
Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids
(2011-03-01)
Cheng, Tsai-Mu
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Mao, Simon J. T.
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Lai, Shiau-Ting
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Chang, Chia-Ching
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Yang, Ming-Chi
Patients with increased haemolytic haemoglobin (Hb) have 10-20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H2O2 generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN3, a peroxidase inhibitor, catalase, a H2O2 decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H2O2 production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN3 and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.
Haptoglobin phenotypes and plasma haptoglobin levels in patients with abdominal aortic aneurysm
(2011-05-01)
Pan, Ju-Pin
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Cheng, Tsai-Mu
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Shih, Chun-Che
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Chiang, Shu-Chiung
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Chou, Shiu-Chin
Objective: Inflammation is associated with the disruption of the aortic media and appears to play a fundamental role in the progression and development of abdominal aortic aneurysm (AAA). Haptoglobin (Hp) is a genetically determined acute phase protein, the synthesis of which is increased during inflammation. This study was designed to investigate both phenotype and plasma levels of Hp in patients with AAA. Methods: Patients with documented AAA who were admitted for elective open repair operation or endograft stem implantation, and non-AAA subjects admitted for coronary arteriography, but found to have normal or insignificant coronary artery disease, were included in the study. Plasma Hp levels were determined using a standard specific enzyme-linked immunosorbent assay, while Hp phenotype was determined by native polyacrylamide gel electrophoresis. Total cholesterol, high density lipoprotein, low density lipoprotein, and triglyceride levels were analyzed enzymatically, and C-reactive protein was analyzed by immunochemistry. Results: Forty-five patients with AAA and 49 non-AAA subjects were included. The Hp 2-2 phenotype was more predominant in AAA patients compared with non-AAA subjects, but this difference was not significant (67% vs 47%; P = .141), while plasma Hp concentrations were significantly higher in AAA patients (237 +/- 144 vs 163 +/- 86 ng/mL; P = .024). Further analysis revealed that plasma Hp concentrations were significantly higher in AAA patients with the 2-2 phenotype compared with corresponding non-AAA subjects (238 +/- 144 vs 163 +/- 86 ng/mL;P = .024). Conclusions: Our findings suggest that plasma Hp concentrations are elevated in patients with AAA, particularly those with the Hp 2-2 phenotype. (J Vasc Surg 2011;53:1189-94.)
Immunochemical property of human haptoglobin phenotypes: Determination of plasma haptoglobin using type-matched standards
(2007-09-01)
Cheng, Tsai-Mu
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Pan, Ju-Pin
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Lai, Shiau-Ting
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Kao, Li-Pin
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Lin, Hong-Huel
Objectives: Haptoglobin (Hp) phenotypes 1-1, 2-1, and 2-2 are associated with inflammatory diseases. Since their biochemical structures are rather heterogeneous, it is necessary to accurately determine the plasma Hp levels. Design and methods: Immunodiffusion, immunoturbidimetric, and noncompetitive ELISA were conducted to determine the differences in immunoreactivity among Hp phenotypes and to verify that such difference may significantly affect the outcome of Hp determinations. A novel ELISA using phenotype-matched calibrators was performed to compared with a commercial GenWay ELISA kit using a single calibrator in normal healthy males. Results: In immunodiffusion and immunoturbidimetric assays, the immumoreactivity of Hp 1-1 was markedly higher than 2-1 and 2-2, while an opposite result was observed using an ELISA. The latter was primarily due to the repeated antigenic epitopes in polymeric 2-1 and 2-2. Thus, Hp levels could be significantly over- or underestimated depending on the method. An accurate ELISA could be achieved when using each type-specific Hp calibrator matched to each type subject. We show the mean levels of Hp 1-1 subjects (n= 16; 184 +/- 42 mg/dL) to be significantly and differentially greater than 2-1 (n=28; 153 +/- 55 mg/dL) (p < 0.05) and 2-2 (n=24; 93 +/- 54 mg/dL) (p < 0.01) subjects. Conclusions: Due to the diverse immunochemical structure among the Hp types, phenotyping should be performed in all the patients and a type-matched Hp calibrator should be used in clinical Hp determination. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Immunopathogenic behaviors of canine transmissible venereal tumor in dogs following an immunotherapy using dendritic/tumor cell hybrid
(2011-02-15)
Pai, Chien-Chun
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Kuo, Tzong-Fu
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Mao, Simon J. T.
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Chuang, Tien-Fu
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Lin, Chen-Si
Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor that can be transmitted between dogs via live tumor cell inoculation. It is also a spontaneous self-regression tumor and its behavior is closely related to host immune responses. Since CTVT had been widely used for tumor models in canine cancers, whether this self-regression may overtake the immunity elicited from an exogenous tumor vaccine remains unclear and certainly worthwhile to be investigated. In this study, we used DCs/tumor hybrids as a tumor vaccine to evaluate the CTVT model. We prepared mature allogeneic dendritic cells from bone marrow and then assessed their phenotype (CD80, CD83, CD86, CD1a, CD11c, CD40 and MHC II), antigen uptake and presenting abilities. Fused dendritic cell/CTVT hybrids were then used as a vaccine, administered three times at two-week intervals via subcutaneous injection near the bilateral auxiliary and inguinal lymph nodes. In comparison with unvaccinated dogs (spontaneous regressed group), within a period of 2.5 months, the vaccinations substantially inhibited tumor progression (p<0.05) and accelerated the rate of regression by a mechanism involving amplification of the host tumor-specific adaptive immune responses and NK cytotoxicity (p<0.001). Pathologic examination revealed early massive lymphocyte infiltration resulting in final tumor necrosis. In addition, there are not any detectable effects on routine physical, body temperature or blood chemistry examinations. In conclusion, our data furnishes a reference value showing that CTVT is a model of potential use for the study of immunity elicited by vaccines against tumors, and also enable early-phase evaluation of the dendritic cell/tumor vaccine in terms of raising host immunity. (C) 2010 Elsevier B.V. All rights reserved.
Increased expression of extracellular matrix proteins in rapid atrial pacing-induced atrial fibrillation
(2007-07-01)
Lin, Chih-Sheng
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Lai, Ling-Ping
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Lin, Jiunn-Lee
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Sun, Yu-Ling
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Hsu, Chih-Wei
BACKGROUND: Atria[ fibrillation (AF) is characterized by structural remodeling of the extracellular matrix (ECM) in cardiac atrium. OBJECTIVE: The purpose of this study was to gain further insight into atrial ECM remodeling at the molecular Level and to test whether altered expression of ECM proteins was associated with the disease. METHODS: Sustained AF was induced in nine adult pigs after 3-4 weeks of continuous rapid atrial pacing at a rate of 600 bpm. Histologic studies and immunohistochemical stain were performed to identify the potential pathologic substrate underlying abnormalities in atrial tissues with sustained AF. RESULTS: In the pathologic findings, the fraction of myocardial ECM (ECM%) was measured, with a significantly greater ECM% found in the AF group compared with the sham operated group (n = 6; i.e., pigs with normal sinus rhythm [SR]). A set of 9,182 genes was screened with cDNA microarray analysis. In AF animals, expression of 121 genes increased and 24 genes decreased by >= 1.75-fold compared with SR animals. Significant up-regulation of fibronectin-1 (4.9-fold), fibrillin-1 (3.1-fold), and fibromodulin (1.9-fold) in the fibrillating atria was confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction. Western blot analysis revealed significantly increased atrial fibronectin-1, fibrillin-1, and fibromodulin in the AF group compared with the SR group (1.5-, 2.7-, and 2.1-fold, respectively). Immunohistochemical staining of AF tissue displayed increased accumulation of fibronectin-1 and fibrillin-1 in the atrial interstitial space. CONCLUSION: Increased expression of ECM proteins in fibrillating atria supports the hypothesis that ECM metabolism contributes to the development of AF.
Low levels of haptoglobin and putative amino acid sequence in Taiwanese Lanyu miniature pigs
(2008-04-01)
Yueh, Sunny C. H.
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Wang, Yao Horng
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Lin, Kuan Yu
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Tseng, Chi Feng
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Chu, Hsien Pin
Porcine haptoglobin (Hp) is an acute phase protein. Its plasma level increases significantly during inflammation and infection. One of the main functions of Hp is to bind free hemoglobin (Hb) and inhibit its oxidative activity. In the present report, we studied the Hp phenotype of Taiwanese Lanyu miniature pigs (TLY minipigs; n=43) and found their Hp structure to be a homodimer (beta-alpha-alpha-beta) similar to human Hp 1-1. Interestingly, Western blot and high performance liquid chromatographic (HPLC) analysis showed that 25% of the TLY minipigs possessed low or no plasma Hp level (<0.05 mg/ml). The Hp cDNA of these TLY minipigs was then cloned, and the translated amino acid sequence was analyzed. No sequences were found to be deficient; they showed a 99.7% identity with domestic pigs (NP_999165). The mean overall Hp level of the TLY minipigs (0.21 +/- 0.25 mg/ml; n=43) determined by enzyme-linked immunosorbent assay (ELISA) was markedly lower than that of domestic pigs (0.78 +/- 0.45 mg/ml; p<0.001), while 25% of the TLY minipigs had an Hp level that was extremely low (<0.05 mg/ml). In addition, the initial recovery rate (first 40 min) in the circulation of infused fluorescein isothiocyanate (FITC)-Hb was significantly higher in the TLY minipigs with extremely low Hp levels than those with high levels. This data suggests that the low concentration of Hp-Hb complex is responsible for the higher recovery rate of Hb in the circulation. TLY minipigs have been used as an experimental model for cardiovascular diseases; whether they can be used as a model for inflammatory diseases, with Hp as a marker, remains a topic of interest. However, since the Hp level varies significantly among individual TLY minipigs, it is necessary to prescreen the Hp levels of the animals to minimize variation in the experimental baseline. The present study may provide a reference value for future use of the TLY minipig as an animal model for inflammation-associated diseases.
Neutrophils as one of the major haptoglobin sources in mastitis affected milk
(2009-05-01)
Lai, I-Hsiang
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Tsao, Jung Hsu
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Lu, Yi Ping
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Lee, Jai Wei
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Zhao, Xin
The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to de. ne the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: < 100 (n = 19); B: 100-200 (n = 10); C: 201-500 (n = 10); and D: > 500 x 10(3) (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were similar to 10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immuno fluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation.
Rational Design for Crystallization of beta-Lactoglobulin and Vitamin D(3) Complex: Revealing a Secondary Binding Site
(2008-12-01)
Yang, Ming Chi
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Guan, Hong-Hsiang
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Yang, Jirin-Moon
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Ko, Cheng-Neng
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Liu, Ming-Yih
beta-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D(3) binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D3 in the complex by altering the pH and initial ratios of vitamin D(3)/LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D(3)/LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D(3) in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.
Structure of a Secondary Vitamin D-3 Binding Site of Milk beta-Lactoglobulin for Vitamin D Transport.
(2009-01-01)
Chen, Chun-Jung
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Yang, Ming-Chi
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Guan, Hong-Hsiang
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Yang, Jinn-Moon
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Chen, Wen-Liang