This Article Abstract Full Text (PDF) Services Download to citation manager Citing Articles Citing Articles via HighWire PubMed PubMed Citation

Proteomic Analysis of Nitrated and 4-Hydroxy-2-Nonenal–Modified Serum Proteins During Aging

¹ Research Institute of Genetic Engineering, ² College of Pharmacy, and ³ Longevity Life Science and Technology Institute, Aging Tissue Bank, Pusan National University, Busan, Korea.
⁴ University of Texas Health Science Center at San Antonio.

Address correspondence to Hae Young Chung, PhD, College of Pharmacy, Pusan National University, 30 Jangjeon-dong, Geumjeong-gu, Busan 609-735, Korea. E-mail: hyjung{at}pusan.ac.kr

	Abstract

Top Abstract Methods Results Discussion References

 
Using proteomic techniques, we investigated peroxynitrite (ONOO^–) and 4-hydroxy-2-nonenal (HNE) modified serum proteins from young and old Fischer 344 rats. Two-dimensional gel electrophoresis/western blot analysis of nitrotyrosine and HNE–histidine revealed that serum proteins were differentially modified by ONOO^– and HNE. Among them, 16 of the modified proteins, identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS), are involved in blood coagulation, lipid transport, blood pressure regulation, and protease inhibition. Furthermore, nitration and HNE adduction were found to increase with age, lending support to the oxidative stress hypothesis of aging. Our data showed that proteomic techniques can be valuable tools in the study of protein profiling modifications during aging.

Peroxynitrite (ONOO^–), formed from nitric oxide reacting with superoxide, and 4-hydroxy-2-nonenal (HNE) from lipid peroxidation, are highly reactive molecules that can modify proteins. ONOO^– oxidizes proteins by the preferential nitration of tyrosine to form nitrotyrosine (3,4). HNE, in contrast, forms adducts with proteins by interacting with redox-sensitive histidine, cysteine, and lysine residues of proteins (5,6). The proteins oxidatively modified by nitration or HNE adduction have been detected in vascular and neurodegenerative diseases (3,5–7). In addition, HNE-modified low density lipoprotein (LDL) is known to promote the generation of foam cells (8) and to interact with tyrosine kinase receptors, which contribute to the development of atherogenesis (9).

Previously, we and others reported that elevated oxidative stress occurs in old animals (10–12), and leads to enhanced formation of reactive modifiers and damaged proteins in serum during aging (13,14). The lack of precise data on oxidatively modified proteins in serum during aging warrants the documented profile analysis conducted in the present study.

Proteomic techniques for analyzing posttranslational modifications (PTMs) were introduced recently into biological research. Two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) are core techniques in the application of qualitative, quantitative, and functional characterizations of the proteome (15), as our laboratory has reported (16). These techniques, combined with molecular and biochemical approaches, can provide distinct advantages for PTM profile analysis of complex biological changes over conventional biochemical techniques (17–23).

In the present study, we investigated protein modification by ONOO^– and HNE in the serum from young (age 7 months) and old (age 25 months) male Fischer 344 rats using 2DE/western blot analysis. Further, MALDI-TOF MS techniques allowed us to identify 16 serum proteins that were oxidatively modified during aging.

	METHODS

Top Abstract Methods Results Discussion References

 
Chemicals and Materials
Mouse antinitrotyrosine and horseradish peroxidase-conjugated goat antimouse immunoglobulin G (IgG) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-HNE–histidine monoclonal antibody was obtained from the Japan Institute for the Control of Aging (Shizuoka, Japan). Alpha-cyano-4-hydroxycinnamic acid (CHCA) and goat anti-serotransferrin antibody were purchased from Sigma-Aldrich (St. Louis, MO). The IPGphor Isoelectric Focusing (IEF) System, Linear Immobiline pH gradient (IPG) dry strips pH 3–10 (3 mm wide and 130 mm long), dithiothreitol (DTT), Coomassie Brilliant Blue (CBB) G-250, enhanced chemiluminescence western blotting detection reagents, and low-molecular-weight calibration kit were obtained from Amersham Biosciences (Piscataway, NJ).

Animals and Serum Preparation
Male, young (age 7 months, n = 7) and old (age 25 months, n = 8), specific pathogen-free Fischer 344 rats were obtained from Samtaco BioKorea (Osan, Korea). The body weights of the young and old rats were 378.9 ± 4.8 g and 457.3 ± 6.7 g, respectively. To obtain serum samples, rats were killed by decapitation under anesthesia, and blood was collected into a conical tube and allowed to clot at 4°C for 30 minutes before being centrifuged at 3000 rpm for 20 minutes. The supernatant was collected as serum, frozen, and stored at –80°C until analyses were performed. Protein amounts were measured with a Bio-Rad Protein Assay Kit (Hercules, CA). Three sera in each group were randomly selected and used for 2DE/western blot and MALDI-TOF MS. All protocols used in animal maintenance are approved by the Institutional Animal Care and Use Committee at Pusan National University, Busan, Korea.

2DE
The 2DE was carried out as described previously (16). Briefly, serum protein was supplemented with lysis buffer (9 M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], 50 mM DTT, 1% IPG buffer, and a trace of bromophenol blue [BPB]); vortexing was performed for 1 hour at room temperature (RT) and then the serum and lysis buffer mixture was utilized for the 2DE. For IEF, protein samples of 500 µg for analytical gels and 1400 µg for preparative gels were mixed with lysis buffer to obtain a volume of 250 µl. The samples were then loaded onto individual strip holders. The complete sample uptake onto the IPG strip was achieved after 14 hours at 20°C. Then, damp electrode pads were positioned under the rehydrated strip over the electrode at anodic and cathodic ends. IEF was carried out at 20°C under the current limit of 50 µA per strip under conditions described by the user's manual with a few modifications: 500 V for 1 hour, 1000 V for 1 hour, 8000 V for 2.5 hours (a total of 21,500 Vh). Immediately after IEF, IPG strips were equilibrated twice at 15 minutes each with gentle shaking in 6 M urea, 50 mM Tris–HCl (pH 8.6), 1% sodium dodecyl sulfate (SDS), 100 mg/10 ml DTT, 30% glycerol, and a trace of BPB. For the second dimension, homemade vertical SDS slab gels (11%, dimensions 140 x 170 x 1.5 mm) were used. The equilibrated IPG strips were placed on top of gels and covered with 2 ml of sealing solution (0.5% agarose, 25 mM Tris, 192 mM glycine, 0.1% SDS, and a trace of BPB). Molecular weight markers were applied at one end of the IPG strip. Electrophoresis was carried out at 30 V for 1 hour, and then continued at 230 V until the BPB line reached the bottom of the gel. The gels were stained with CBB G-250 for MALDI-TOF MS or transferred to polyvinylidene fluoride (PVDF) membrane for western blotting.

Peptide Mass Mapping by MALDI-TOF MS and Database Search
Protein spots were excised from 2DE gels, rehydrated with water for 30 minutes, washed 3 times by 50% acetonitrile (ACN) in 25 mM ammonium bicarbonate pH 8.0, then dehydrated with 100% ACN. For in-gel digestion, 10 µl of trypsin solution (2 ng/µl in 25 mM ammonium bicarbonate pH 8.0) was added and digested at 37°C overnight. Peptides were extracted with 50% ACN containing 0.2% trifluoreacetic acid and dried under vacuum, then reconstructed with 3 µl of CHCA solution (8 mg CHCA in 1 ml of 50% ACN containing 0.2% trifluoreacetic acid) (24). Peptide mass mapping was performed as previously reported (16). One microliter of sample mixture was loaded on a 96 x 2 MALDI plate. Peptide mass was acquired with Voyager DE-PRO (Applied Biosystems, Framingham, MA) at reflector mode under the following conditions: 20,000 V of accelerating voltage, 76% of grid voltage, and 0.02% of guide wire voltage. For calibration, a CalMix 2 calibration kit (Applied Biosystems) was used as external standard, and autolysis fragments of trypsin were used as internal standard. The experimental peptide masses were matched with theoretical peptide masses of all proteins from mammals of the SwissProt and NCBInr protein databases, with 50 ppm of mass error tolerance using local Protein Prospector software (Applied Biosystems).

Image Analysis
ImageMaster 2D Elite version 4.01 and ImageQuant TL software (Amersham Biosciences, Uppsala, Sweden) were used for 2DE/ and 1DE/western blot images, respectively.

Protein Analysis by Western Blot
Western blot was carried out as described previously (25). For 1DE, serum samples diluted 10 times were boiled for 5 minutes with a sample-loading buffer at a ratio of 1:1. Total protein equivalents for each sample were separated by SDS–polyacrylamide gel electophoresis (PAGE) minigel as described by Laemmli (26). After 1DE or 2DE, protein was transferred to PVDF at 100 V for 1.5 hours (1DE gel) or 30 V for 12 hours (2DE gel) in a Trans-Blot Electrophoretic Transfer Cell (BioRad). The membrane was immediately placed into a blocking solution (3% skim milk powder in Tris-buffered saline (TBS)–Tween buffer containing 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, and 0.1 mM Tween-20) at RT for 30 minutes. The membrane was incubated with a primary antibody at a 1:200 dilution in Tris-buffered saline–Tween buffer containing 0.5% skim milk at RT for 2 hours, then incubated with a horseradish peroxidase–conjugated secondary antibody at RT for 1 hour. The secondary antibody was detected by enhanced chemiluminescence western blotting detection reagents following the manufacturer's instructions.

Determination of Free HNE
Free HNE in serum was measured with the LPO-586 kit (Oxis International Inc., Portland, OR) based on colorimetric methods. All measurement procedures were performed following the user's manual.

Statistical Methods
Numeric data are presented as mean ± standard error of the mean. Student's t test was used to test for differences in nitrotyrosine, HNE adducts, free HNE, and total cholesterol levels between young and old rat sera. Values of p <.05 were considered statistically significant.

	RESULTS

Top Abstract Methods Results Discussion References

 
Proteomic Profiling of Nitrated and HNE-Adducted Serum Proteins
To understand the physiological consequences and potential pathogenic capacity of nitrated and HNE-adducted serum proteins it is important to recognize and identify damaged proteins. Serum proteins from young and old rats were separated by 2DE (Figure 1), followed by immunodetection of nitrated and HNE-adducted proteins (Figure 2). In this analysis, proteins that showed cross-reactivity against secondary antibodies were not included for identification.

View larger version (41K): [in this window] [in a new window]   Figure 1. Two-dimensional electrophoresis (2DE) images of serum proteome from young and old rats. Serum proteins from young and old rats were separated by 2DE and visualized by Coomassie Brilliant Blue G-250 as described in Methods. Arrows indicate modified proteins by peroxynitrite and 4-hydroxy-2-nonenal (HNE) shown in Table 1. *Indicates fragment of a given protein number

View larger version (54K): [in this window] [in a new window]   Figure 2. Two-dimensional electrophoresis (2DE)/western blot images of young and old rat sera. Nitrotyrosine, 4-hydroxy-2-nonenal (HNE)–histidine, and rat immunoglobulin Gs (IgGs) of serum proteins from young and old rats were detected by using specific antibodies as described in Methods

We realize that some modified proteins could not be identified from databases even though their reliable peptide masses were obtained from MALDI-TOF MS. In our experiments, 16 of 93 spots analyzed were not identified, probably because of the lack of entries in protein databases, or due to higher molecular weight protein fragments, thereby exceeding the search limits of peptide mass fingerprint (under 200 kd).

Proteomic Quantitation of Nitrated and HNE-Adducted Proteins
The effect of aging on nitrated and HNE-adducted proteins was quantitatively assessed by image analysis, and the quantitation of three representative proteins is shown in Table 2. We quantified and compared the extent of modification, namely serotransferrin (No. 4), A1M (No. 2) and VDBP (No. 5), between young and old groups using 2DE/western blot data. Both nitrotyrosine and HNE–histidine residues on serotransferrin (No. 4) were significantly increased in old rats (Table 2). A1M (No. 2) fragments from old rats showed an increased HNE–histidine content and only a slightly enhanced nitrotyrosine level. However, VDBP (No. 5) showed increased HNE–histidine levels but decreased nitrotyrosine with age. Therefore, although both nitration and HNE adduction levels in old rat serum proteins showed a pattern of increase with 1DE/western blot analysis, neither of these modifications showed the same pattern using 2DE/western blot analysis.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effect of aging on nitration and 4-hydroxy-2-nonenal (HNE) adduction of serotransferrin. Nitrotyrosine and HNE–histidine on serotransferrin in sera from young (7 months) and old (25 months) rats were detected by western blot analysis using specific antibodies as described in Methods

View larger version (15K): [in this window] [in a new window]   Figure 4. Nitrotyrosine levels of serum proteins from young and old rats. Nitrotyrosine levels of serum proteins were detected by western blot analysis as described in Methods. Data are expressed as relative band intensity (%), i.e., level of nitrotyrosine in old rat (n = 8) serum compared with that in young rats (n = 7). *p <.05

View larger version (17K): [in this window] [in a new window]   Figure 5. 4-hydroxy-2-nonenal (HNE)–histidine levels of proteins and free HNE concentrations in young and old rat sera. A, HNE–histidine levels of serum proteins from young and old rats were detected by western blot analysis as described in Methods. Data are expressed as relative band intensity (%), i.e., level of nitrotyrosine in old rat (n = 8) serum compared with that in young rats (n = 7). *p <.01. B, Free HNE concentrations in serum were detected as described in Methods. Data are expressed as µM. **p <.001; young = serum from young rats (n = 7); old = serum from old rats (n = 8)

	DISCUSSION

Top Abstract Methods Results Discussion References

Recently, proteomic techniques have been applied in aging and biomedical research to profile protein expressions or modifications. In a previous study, we documented the protein expression profiles of postmitochondrial fraction of aged kidney, and showed that proteomic techniques can be a powerful analytical tool for detecting age-related alterations in protein expression and for a better understanding of the aging status of a given tissue (16). Moreover, these techniques, combined with western blotting, proved to be valuable in analyzing the PTMs of proteins. Similar applications have been used in a number of other studies in this area. For example, analyses of nitrated mitochondrial proteins during inflammation (17), nitrated skeletal muscle proteins in aged rat (18), and carbonyl and nitrated proteins in brain affected by Alzheimer's disease (19,20) are reported. Proteins modified by malondialdehyde, HNE, and advanced glycation end-product (AGE) in lipofusin of human pigment epithelium also have been analyzed (21). More recently, HNE-modified proteins were identified in G93A-SOD1 transgenic mice (22). Although these studies clearly show the advantage and efficiency of proteomic techniques as a high throughput method in biomedical research, such a technique has not been used to study oxidatively modified serum proteins, except for a technical report showing the detection of carbonylated serum proteins (23).

In the present study, we identified 16 modified serum proteins as listed in Table 1. One important finding in our study is that the oxidative modifications are selective, depending on the protein. As shown in Table 1, five proteins (No. 7–9, 11, 13) were preferentially modified by nitration; in contrast, three proteins (No. 14–16) were modified by only HNE. This single modification by either ONOO^– or HNE suggests the possibility that proteins differ in their susceptibility to modification by a particular oxidative modifier. However, other proteins (No. 1–6, 10, 12) were modified by both ONOO^– and HNE (Table 1). At present, whether these double modifications have any biological relevance remains unclear.

For nitrated proteins, comparisons of young rats with old rats showed that only the old rats had modifications to A1AT (No. 12) and T-kininogen I (No. 13). The A1AT protein, a member of the class of protease inhibitor proteins known as serpins (serine protease inhibitors), functions to inhibit neutrophil elastase (27). T-kininogen I is a protein functionally involved in the regulation of blood pressure and acute phase of immune response (28). Regarding HNE-adducted proteins, four proteins (haptoglobin [No. 6], A1AT [No. 12], ApoE [No. 15], and T-kininogen II [No. 16]) were found to form HNE adducts only in old animals. Haptoglobin prevents loss of iron through the kidneys and protects the kidneys from damage by hemoglobin by combining with free plasma hemoglobin (29). ApoE (No. 15) acts as lipid transporter of LDL (30) and T-kininogen II (No. 16), an alternatively spliced form of T-kininogen I that regulates blood pressure (31). As discussed earlier, modification of these serum proteins by nitration and/or HNE may have dysfunctional physiological consequences (32,33). It is reported that HNE-modified LDL contributes to the generation of foam cells (8) and the development of atherogenesis (9). In addition, HNE-modified A1AT (No. 12) is suggested to enhance tissue destruction not only by the loss of its proteinase inhibitory activity, but also by its ability to recruit and activate monocytes in the atherosclerotic lesion (34). Therefore, the nitrated and HNE-adducted serum proteins (such as A1AT, T-kininogen I and II, ApoE, and haptoglobin) that were observed only in aged rats, suggest that these modified proteins likely participate in the age-related vascular alterations and dysfunctions that lead to vascular diseases.

Another interesting finding from our study is that aging is associated with differential oxidative modifications to serum proteins. In the case of nitration, the total modification (1DE/western blot) was significantly higher in the old rats (Figure 4), which might be the result of increased generation of reactive nitrogen species (13), possibly due to an imbalanced redox status during aging (10,14). Previously, we reported that increased lipid peroxidation caused by redox imbalance occurred during aging in rat heart and brain (12,35). Paralleling these findings, total levels of HNE–histidine (Figure 5A) and free HNE (Figure 5B) in old rat serum were significantly higher than those found in young rat serum. As shown in Table 2, some of the nitrated proteins showed similar modifications in both young and old rat groups (e.g., A1M) or a reduced modification with age (e.g., VDBP). At present, the biological basis for this phenomenon is not known. However, several possibilities can be offered, including the susceptibility of proteins to modification, the turnover rate of unaltered and modified proteins, and the circulating level of a given protein.

It should be pointed out that proteomic techniques still have some inadequacies that interfere with the analysis procedure, as we experienced in the current work, including the detection limits of the CBB staining method, variances in chemiluminescence sensitivities on spots, and the lack of entries in protein databases needed for peptide mass fingerprints. However, we believe that the most significant endeavor of the current study is the application of advanced proteomic techniques used in the analysis of the complex biological changes that occur during aging.

Conclusion
By proteomic analysis, we identified 16 proteins modified by nitration and/or HNE adduction during aging. As far as we know, this is the first time an investigation of serum protein modification profiling using proteomic techniques has been applied to an aging study. We hypothesize through our data that during aging, increased oxidative stress, including enhanced ONOO^– and free HNE generation, will enhance serum protein modifications. As a result, oxidatively modified serum proteins may lose their biological integrity and functional capabilities, which could lead to age-related vascular diseases. Our data showed the advantage of using proteomic techniques in profiling age-related modifications of serum proteins. With the information provided by this new biological technique, we can thereby gain a better understanding of the mechanisms associated with serum protein damage and vascular aging.

	Acknowledgments

Top Abstract Methods Results Discussion References

 
This study was supported by a grant from the Korea Health 21 R&D project, Ministry of Health and Welfare, Republic of Korea (A050166-AA0718O5N100010A).

We are grateful to the Aging Tissue Bank for supplying the tissues used in this study.

Chul Hong Kim and Yani Zou contributed equally to this work.

	Footnotes

Top Abstract Methods Results Discussion References

 
Decision Editor: James R. Smith, PhD

Received May 18, 2005

Accepted September 7, 2005

	References

Top Abstract Methods Results Discussion References

 

1. Yu BP, Yang R. Critical evaluation of the free radical theory of aging. A proposal for the oxidative stress hypothesis. Ann N Y Acad Sci. 1996;786:1-11.[Medline]
2. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408:239-247.[Medline]
3. Stadtman ER. Protein oxidation in aging and age-related diseases. Ann N Y Acad Sci. 2001;928:22-38.[Medline]
4. Spiteller G. Lipid peroxidation in aging and age-dependent diseases. Exp Gerontol. 2001;36:1425-1457.[Medline]
5. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991;11:81-128.[Medline]
6. Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res. 2003;42:318-343.[Medline]
7. Shishehbor MH, Aviles RJ, Brennan ML, et al. Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy. JAMA. 2003;289:1675-1680.[Abstract/Free Full Text]
8. Hoff HF, O'Neil J, Chisolm GM, 3rd, et al. Modification of low density lipoprotein with 4-hydroxynonenal induces uptake by macrophages. Arteriosclerosis. 1989;9:538-549.[Abstract/Free Full Text]
9. Negre-Salvayre A, Vieira O, Escargueil-Blanc I, Salvayre R. Oxidized LDL and 4-hydroxynonenal modulate tyrosine kinase receptor activity. Mol Aspects Med. 2003;24:251-261.[Medline]
10. Zou Y, Kim AR, Kim JE, Choi JS, Chung HY. Peroxynitrite scavenging activity of sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) isolated from Brassica juncea. J Agric Food Chem. 2002;50:5884-5890.[Medline]
11. Baek BS, Kim JW, Lee JH, et al. Age-related increase of brain cyclooxygenase activity and dietary modulation of oxidative status. J Gerontol Biol Sci. 2001;56A:B426-B431.[Abstract/Free Full Text]
12. Kim JW, Baek BS, Kim YK, et al. Gene expression of cyclooxygenase in the aging heart. J Gerontol A Biol Sci Med Sci. 2001;56A:B350-355.[Abstract/Free Full Text]
13. Van der Loo B, Labugger R, Skepper JN, et al. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med. 2000;192:1731-1744.[Abstract/Free Full Text]
14. Kim JW, No JK, Ikeno Y, et al. Age-related changes in redox status of rat serum. Arch Gerontol Geriatr. 2002;34:9-17.[Medline]
15. Schoneich C. Proteomics in gerontological research. Exp Gerontol. 2003;38:473-481.[Medline]
16. Kim CH, Park DU, Chung AS, et al. Proteomic analysis of post-mitochondrial fractions of young and old rat kidney. Exp Gerontol. 2004;39:1155-1168.[Medline]
17. Aulak KS, Miyagi M, Yan L, et al. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc Natl Acad Sci U S A. 2001;98:12056-12061.[Abstract/Free Full Text]
18. Kanski J, Alterman MA, Schoneich C. Proteomic identification of age-dependent protein nitration in rat skeletal muscle. Free Radic Biol Med. 2003;35:1229-1239.[Medline]
19. Castegna A, Aksenov M, Thongboonkerd V, et al. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J Neurochem. 2002;82:1524-1532.[Medline]
20. Castegna A, Thongboonkerd V, Klein JB, Lynn B, Markesbery WR, Butterfield DA. Proteomic identification of nitrated proteins in Alzheimer's disease brain. J Neurochem. 2003;85:1394-1401.[Medline]
21. Schutt F, Bergmann M, Holz FG, Kopitz J. Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2003;44:3663-3668.[Abstract/Free Full Text]
22. Perluigi M, Fai Poon H, Hensley K, et al. Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice-A model of familial amyotrophic lateral sclerosis. Free Radic Biol Med. 2005;38:960-968.[Medline]
23. Conrad CC, Choi J, Malakowsky CA, et al. Identification of protein carbonyls after two-dimensional electrophoresis. Proteomics. 2001;1:829-834.[Medline]
24. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis. 1999;20:601-605.[Medline]
25. Habib A, Creminon C, Frobert Y, Grassi J, Pradelles P, Maclouf J. Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2. J Biol Chem. 1993;268:23448-23454.[Abstract/Free Full Text]
26. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.[Medline]
27. Brantly M, Nukiwa T, Crystal RG. Molecular basis of alpha-1-antitrypsin deficiency. Am J Med. 1988;84:13-31.[Medline]
28. Santiago JA, Champion HC, Kadowitz PJ. T-kinin has endothelium-dependent vasodilator activity in the cat. Am J Physiol. 1997;272:H1491-1498.
29. Murray RK, Connell GE, Pert JH. The role of haptoglobin in the clearance and distribution of extracorpuscular hemoglobin. Blood. 1961;17:45-53.[Abstract/Free Full Text]
30. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 1988;240:622-630.[Abstract/Free Full Text]
31. Furuto-Kato S, Matsumoto A, Kitamura N, Nakanishi S. Primary structures of the mRNAs encoding the rat precursors for bradykinin and T-kinin. Structural relationship of kininogens with major acute phase protein and alpha 1-cysteine proteinase inhibitor. J Biol Chem. 1985;260:12054-12059.[Abstract/Free Full Text]
32. Kashiba-Iwatsuki M, Miyamoto M, Inoue M. Effect of nitric oxide on the ligand-binding activity of albumin. Arch Biochem Biophys. 1997;345:237-242.[Medline]
33. Ji C, Kozak KR, Marnett LJ. IkappaB kinase, a molecular target for inhibition by 4-hydroxy-2-nonenal. J Biol Chem. 2001;276:18223-18228.[Abstract/Free Full Text]
34. Mashiba S, Wada Y, Takeya M, et al. In vivo complex formation of oxidized alpha(1)-antitrypsin and LDL. Arterioscler Thromb Vasc Biol. 2001;21:1801-1808.[Abstract/Free Full Text]
35. Baek BS, Kwon HJ, Lee KH, et al. Regional difference of ROS generation, lipid peroxidation, and antioxidant enzyme activity in rat brain and their dietary modulation. Arch Pharm Res. 1999;22:361-366.[Medline]

This article has been cited by other articles:

				P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Download to citation manager Citing Articles Citing Articles via HighWire PubMed PubMed Citation