Changes in glycation of fibrous type I collagen during long-term in vitro incubation with glucose

The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.     

胶原蛋白的电子活性与矽肺纤维化Ⅱ正常大鼠与实验性矽肺大鼠肺Ⅰ型胶原稳定自由基的研究

由正常大鼠肺撮取的酸溶性Ⅰ型胶原出现一个明显的ESR信号(g=2.006),而由矽肺大鼠提取的酸溶性Ⅰ型胶原较少或缺乏该ESR信号,该信号不是顺磁过渡元素产生,而是稳定自由基的反映.肺胶原可能存在两种稳定的自由基,一种易受紫外线辐照的激发并且在电场极化下自旋耦合而减弱,另一种自由基则相对稳定,对紫外线辐照和电场极化不敏感.矽肺胶原缺乏活泼性较高的稳定自由基.     

胶原蛋白的电子活性与矽肺纤维化Ⅱ正常大鼠与实验性矽肺大鼠肺Ⅰ型胶原稳定自由基的研究

由正常大鼠肺撮取的酸溶性Ⅰ型胶原出现一个明显的ESR信号(g=2.006),而由矽肺大鼠提取的酸溶性Ⅰ型胶原较少或缺乏该ESR信号,该信号不是顺磁过渡元素产生,而是稳定自由基的反映.肺胶原可能存在两种稳定的自由基,一种易受紫外线辐照的激发并且在电场极化下自旋耦合而减弱,另一种自由基则相对稳定,对紫外线辐照和电场极化不敏感.矽肺胶原缺乏活泼性较高的稳定自由基.     

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells

To elucidate the mechanism how a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0°C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1). The effects of hyperthermia on LPO and [Ca²⁺]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (ΔΨm) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca²⁺ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca²⁺]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca²⁺]i arising from increased expression of IP₃R1 and LPO. Additional increase in [Ca²⁺]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.     

Application of water-soluble radical initiator, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ek(d)) for AIPH was 2.6 x 10(-6)/s at 37 degrees C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2alpha (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.     

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride,in human histiocytic lymphoma U937 cells

To elucidate the mechanism how a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0°C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1). The effects of hyperthermia on LPO and [Ca²⁺]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (ΔΨm) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca²⁺ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca²⁺]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca²⁺]i arising from increased expression of IP₃R1 and LPO. Additional increase in [Ca²⁺]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.     

Type I collagen inhibits hydroxyl radical-induced apoptosis

The extracellular matrix (ECM) plays an important role in cell differentiation and apoptosis. Collagen is the major component of ECM. Here, an ESR signal of the hydroxyl radicals (.OH) generated via Fe2+ -mediated Fenton reaction was found to be significantly inhibited by type I collagen. Further study showed that type I collagen also inhibited cell apoptosis induced by.OH, as evidenced by morphological criteria (DAPI and annexin V staining) and quantitive assays for apoptotic cells (MTT and flow-cytometric assay for subG1 cells). By addition of type I collagen in HeLa cells, the lipid peroxidation caused by.OH was inhibited and the cellular GSH was protected. In comparison with type I collagen, BSA and the denatured collagen, gelatin, lacked such antioxidative and antiapoptotic effects. Together, the results suggest that type I collagen can uniquely prevent.OH-mediated apoptosis by scavenging free radicals.     

Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2' azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by beta-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant’s scavenging capacity against AAPH-derived free radicals

A new method is proposed for the evaluation of oxygen radical absorbance capacity (ORAC). The current fluorescence-based ORAC assay (ORAC-FL) is an indirect method that monitors the antioxidant’s ability to protect the fluorescent probe from free radical-mediated damage, and an azo-radical initiator, AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), has been used as a thermal free radical source. The new ORAC assay employs a short in situ photolysis of AAPH to generate free radicals. The electron paramagnetic resonance (EPR) spin trapping method was employed to identify and quantify AAPH radicals. In the presence of antioxidant, the level of AAPH radicals was decreased, and ORAC-EPR values were calculated following a simple kinetic formulation. Alkyl-oxy radical was identified as the sole decomposition product from AAPH; therefore, we concluded that ORAC-FL is the assay equivalent to alkyl-oxy radical scavenging capacity measurement. ORAC-EPR results for several antioxidants and human serum indicated that the overall tendency is in agreement with ORAC-FL, but absolute values showed significant discrepancies. ORAC-EPR is a rapid and simple method that is especially suitable for thermally labile biological specimens because the sample heating is not required for free radical production.     

Molecular characteristics of dimethylnitrosamine induced fibrotic liver collagen

The molecular characteristics of purified pepsin solubilized collagen from rat liver was studied in control and dimethylnitrosamine administered animals. The α- and β-chains of purified pepsin solubilized liver collagen were separated by subjecting the denatured collagen to SDS-polyacrylamide gel electrophoresis. The α1(III) chains were resolved from the α1(I) chains by interrupted electrophoresis with delayed reduction of the disulfide bonds of type III collagen. The aldehyde content of the purified pepsin solubilized collagen was estimated in control and experimental samples in order to assess the extent of collagen cross-links. Fibril formation curves were studied with purified pepsin solubilized collagen to see the rate of formation of cross-links within the fibrillar mesh. The results of the unreduced electrophoretic studies revealed a significant increase in the β-subunit of type I collagen with a remarkable decrease of α/β ratio in DMN treated animals. Reduction with β-mercaptoethanol indicated the presence of type III collagen in the electrophoretic field with a proportionate increase on the 21st day. A significant increase in the aldehyde content and an increased rate of fibril formation were noticed in DMN induced fibrotic liver collagen. The data of the present investigation revealed that the DMN induced fibrotic liver collagen is more cross-linked than normal liver collagen and the deposition of type III collagen is more prominent than type I collagen in early fibrosis.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Immunohistochemical and immunoblot analyses of collagens in the developing fibrocartilage in the glans penis of the rat

The distal segment of the os penis of rats is a fibrocartilage whose development is dependent on androgens. Electrophoretic and immunoblot analysis indicated that the fibrocartilage contained both type I and type II collagens. Immunohistochemically, type I collagen was detected in the fibrous matrix of the distal segment in all the stages examined. Type II collagen was detected first at 4 weeks in the extracellular matrix surrounding the mature chondrocytes distributed sparsely as single cells or small clusters among immature chondroblasts. The clusters reactive with anti-type II collagen serum increased in size and number at 6 weeks, and almost all the region of the distal segment became reactive with anti-type II collagen serum at 8 weeks.     

Oxidative modification of rat eye lens proteins by peroxyl radicals in vitro: protection by the chain-breaking antioxidants stobadine and Trolox

In an attempt to model the processes of free radical-mediated cataractogenesis, we investigated the oxidative modification of rat eye lens proteins by peroxyl radicals generated by thermal decomposition of 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) under aerobic conditions. When incubated with AAPH, the soluble eye lens proteins precipitated in a time-dependent manner. The insolubilisation was accompanied by the accumulation of protein free carbonyls and the diminution of sulfhydryls, yet the processes were shifted in time. The SDS-PAGE analysis of the AAPH-treated proteins revealed the presence of high molecular weight cross-links and, to a lesser extent, fragments. The aggregation and cross-linking of proteins along with the generation of free carbonyls was significantly inhibited by the chain-breaking antioxidants stobadine and Trolox. On the other hand, the AAPH-initiated sulfhydryl consumption was much less sensitive to the antioxidants studied. The results point to a complex mechanism of peroxyl-radical-mediated modification of eye lens proteins with implications for cataract development and they indicate a potentially protective role of antioxidants.     

Activation of polymorphonuclear neutrophils by contact with collagen I and degradation of collagen

The contact of polymorphonuclear neutrophils with type I collagen molecules triggers the production of oxygen free radicals by these cells. This activation requires two short collagenic sequences and seems to involve a beta 2 integrin on the neutrophil membrane. Oxygen free radicals are capable to degrade collagen molecules into small peptides. This interaction takes place in inflammatory phenomena.     

Measurement of the synthesis rates of collagens and total protein in rabbit muscle.

1. Collagen- and total-protein-synthesis rates were determined in rabbit muscle by continuous infusion of radioactive proline. 2. The precursor pool of free proline used for collagen synthesis was defined by measuring the specific radioactivity of hydroxy-proline in isolated type I procollagen. The specific radioactivities of type I procollagen were about 40% of those for free proline in the homogenate. 3. The mean ratio (+/- S.E.M.) between the fractional synthesis rates of muscle collagen and total protein was 0.99 +/- 0.10, where the total protein values were based on specific radioactivities of the homogenate free proline pools. 4. Types I, III and V collagen were solubilized by pepsin and isolated by fractional precipitation with NaCl. The fractional synthesis rates of types I and III collagens were very similar. Type V collagen samples had higher specific radioactivities than the other collagens, but this was not necessarily indicative of a higher rate of synthesis because of uncertainty about the cellular origin of this collagen and, hence, the specific radioactivity of its precursor proline pool.     

Solubilization of Peripheral-Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.     

The interaction of collagen and acid mucopolysaccarides. A model for connective tissue

1. The interaction of acid mucopolysaccharides of connective tissue with solubilized collagen of native, or near-native, structure was investigated by free solution electrophoresis at pH7.0. 2. Complex-formation was detected by the appearance of a third peak in the ascending limb only, indicating reversible association. 3. Complex-formation was destroyed by prior heating of solubilized collagen, indicating a probable requirement for high molecular weight or internal structure of the protein. 4. Hyaluronate and chondroitin sulphate of mol.wt. 50000 gave complexes with soluble collagen at I 0.4, whereas heparin and chondroitin sulphate of mol.wt. 15000-18000 did not. All mucopolysaccharides yielded complexes at I 0.1. The stability of the complex appears mainly dependent on electrostatic forces and is increased with increase in chain length of the polysaccharide. 5. Solubilized collagen interacted to yield gels with the ;native' chondroitin sulphate-protein macro-molecule from cartilage. 6. A schematic model for the interaction of collagen and chondroitin sulphate-protein macromolecules shows parallel-ordered interaction of collagen fibrils with chondroitin sulphate side chains of the chondroitin sulphate-protein macromolecule. The biological implications of this model are discussed, particularly in relation to the ordered structures and the ionic-network properties of the intercellular components of connective tissue.     

Red wine polyphenols affect the collagen composition in the aorta after oxidative damage induced by chronic administration of CCl(4)

Increased amount of collagen type I and decreased amount of type III is described in various pathological processes in the vascular wall. Polyphenols were shown to have protective effect on endothelium, decrease blood pressure and prevent oxidative damage induced by various stimuli. Tetrachlormethane (CCl(4)) is a toxic substance with known negative systemic effects induced by free radicals. Chronic administration of CCl(4) for 12 weeks led to an increase of collagen type I and a decrease of type III in the wall of aorta. Parallel administration of red wine polyphenols significantly reduced the increase of collagen type I, at the same time the content of type III rose to the level above controls. After 4 weeks of spontaneous recovery no changes were observed. If polyphenols were administered during the recovery period, there was a decrease of type I and an increase of type III collagen content in the aorta. It can be concluded that polyphenols have a tendency to lower the amount of type I and to increase the proportion of type III collagen in the wall of the aorta. These changes are significant in prevention or in regression of changes induced by chronic oxidative stress. This effect of polyphenols is most likely the result of their influence on MMP-1 and TIMP activities through which they positively influence the collagen types I and III content ratio in the vascular wall in favor of the type III collagen.     

A binding assay for the solubilized receptors of type beta transforming growth factor: adsorption and removal of free ligand by dextran-coated charcoal

A binding assay was developed for the measurement of solubilized receptors for transforming growth factor type beta (TGF-beta). Solubilized receptors were incubated with 125I-TGF-beta, then the unbound ligand was removed by adsorption to dextran-coated charcoal. The binding of TGF-beta to solubilized receptors was saturable and specific, and increased in a linear manner with respect to the amount of membrane protein present. Crosslinking of radioactive complexes after adsorptive removal of unbound TGF-beta yielded complexes similar to affinity-labeled TGF-beta receptors from whole cells. Treatment of a 20% charcoal suspension with 0.2-0.4% dextran was optimal for the protection of receptors from adsorption to charcoal while allowing free TGF-beta to be removed; Mr approximately 250,000 dextran was most effective. This method can assay receptors from purified membranes and crude extracts of cells and tissues, and was used to demonstrate that TGF-beta receptors are glycosylated and retain a high affinity (Kd approximately 530 pM) for ligand after solubilization.     

Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis

A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase. When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability. Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei. Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.