A novel method of isolation of a bone-morphogenetic-protein-like protein from ossein.

A new bone-morphogenetic-protein (BMP)-like protein has been isolated through a new protocol from a novel source, ossein. The BMP-like protein was hydrophilic and characterized through Fourier-transform IR studies, SDS/PAGE and coupled with a neutral binder, hydroxypropylmethylcellulose (HPMC) for control release. The IR spectrum of the protein showed peaks in tandem with BMP from bone matrix, and its molecular mass was in the range 18-21 kDa. Sustained release from the surface of HPMC was achieved for a period of 3 days.

Dimethylnitrosamine-induced liver injury in rats: the early deposition of collagen.

Dimethylnitrosamine (DMN) is a potent hepatotoxin that can cause fibrosis of the liver. It's ability to provide a suitable rapid experimental murine model for early human cirrhosis was examined. The drug was administered to adult male albino rats in order to document sequential pathological and biochemical alterations. Injury was produced by intraperitoneal injections of DMN on three consecutive days of each week over a 3-week period. A rapid increase in collagen content was documented, with linear increases occurring from days 7 to 21. Livers were examined for histopathological changes on days 7, 14 and 21 following the beginning of exposure. Severe centrilobular congestion and haemorrhagic necrosis could be observed on day 7. Centrilobular necrosis and intense neutrophilic infiltration were observed on day 14. By day 21, collagen fiber deposition could be observed, together with severe centrilobular necrosis, with focal fatty changes, bile duct proliferation, bridging necrosis and fibrosis surrounding the central veins. A decrease in total protein and increase in DNA were also documented. DMN-induced liver injury in rats appears to be a potential animal model for early human cirrhosis and the rapid deposition of collagen, and may serve as a convenient procedure for screening antifibrotic agents.

Biochemical abnormalities during the progression of hepatic fibrosis induced by dimethylnitrosamine.

OBJECTIVES: The pathogenesis of hepatic fibrosis is accompanied with several biochemical and metabolic abnormalities. To obtain more information about the alteration of biochemical and metabolic parameters during alcoholic liver fibrosis, we have monitored the changes of certain important biochemical compounds in experimentally induced hepatic fibrosis. DESIGN AND METHODS: The liver injury was induced in adult male albino rats by using dimethylnitrosamine (DMN) in doses of 1 mg/100 g body weight. Total collagen, total protein, cholesterol, lipid peroxides, glucose, urea, and inorganic phosphorus were monitored in liver and blood/serum samples on Days 0, 7, 14, and 21 after the start of DMN administration. Serum insulin levels were assayed by radioimmunoassay. The serum and urinary levels of hydroxyproline, uric acid, and creatinine were also monitored. RESULTS: The total collagen content in the liver was increased about 4-fold by Day 21 after the start of DMN administration. A significant increase was observed in lipid peroxide levels in both liver and blood samples. Although inorganic phosphorus level decreased in both serum and liver tissue, cholesterol was lowered only in the serum. Increased serum insulin level with impaired glucose tolerance was observed after 21 days. Serum hydroxyproline level increased throughout after the start of DMN administration. The urinary excretion of hydroxyproline was also significantly increased with a striking elevation on Day 7. Elevated uric acid levels were recorded in serum and urine samples during the latter periods of DMN treatment. No alteration was observed in blood urea and creatinine levels. CONCLUSIONS: The results of the present investigation demonstrated important alterations in metabolic parameters and biochemical abnormalities during experimentally induced liver damage. All alterations are compatible with the deterioration of liver functions during the pathogenesis of hepatic fibrosis.

Molecular species of collagen in the intramuscular connective tissues of the marine crab, Scylla serrata.

Type V like collagens are widely distributed in marine invertebrates, particularly crustaceans and molluscs. We have been investigating the nature of collagens in the muscular tissues of crustaceans. The presence of type V like homotrimeric collagen in prawn muscle was noted before. We report here a comparative analysis of collagens purified from the pepsin digest of abdominal and pereiopod muscle tissues of the crab, Scylla serrata. The major collagen in either muscle precipitated at 1.2 M NaCl at acid pH, suggestive of a type V like property. The homotrimeric collagen was then purified to near homogeneity by precipitation with 20% ammonium sulphate. Solubility characteristics and biochemical studies indicated the leg muscle collagens to be highly crosslinked and stabilised by more bound carbohydrates, as compared to the abdominal muscle collagen. Analysis of amino acid composition revealed a close similarity to known type V collagens and the leg muscle collagen was characterised by more lysine hydroxylation and slightly reduced glycine content. The leg muscle collagen had a higher denaturation temperature and intrinsic viscosity than the abdominal muscle collagen. Our results confirm the similarity of major crustacean muscle collagens to vertebrate type V collagen. Further, the relative complexity of leg muscle collagen, unlike the abdominal muscle collagen, correlates to the specific functional requirements, where the former is involved in locomotion and preying and the latter in normal growth and development.

An in vitro study on the role of metal catalyzed oxidation in glycation and crosslinking of collagen.

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 microM) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentration-dependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 microM copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.

Influence of Aloe vera on collagen turnover in healing of dermal wounds in rats.

Treatment of full-thickness wounds with A. vera, on rats resulted in increased biosynthesis of collagen and its degradation. A corresponding increase in the urinary excretion of hydroxyproline was also observed. Elevated levels of lysyl oxidase also indicated increased crosslinking of newly synthesised collagen. The results suggest that A. vera influences the wound healing process by enhancing collagen turnover in the wound tissue.

Biochemical and connective tissue changes in cyclophosphamide-induced lung fibrosis in rats.

The present investigation was designed to characterize the biochemical and connective tissue components and to correlate the significance of morphological and biochemical perturbations in cyclophosphamide (CP)-induced lung fibrosis in rats. Lung fibrosis was induced in male Wistar rats by intraperitoneal injection of 20 mg/100 g body weight of CP, and their pneumotoxic derangements were characterized during an early destructive phase followed by a proliferative and synthetic phase. Serum angiotensin-converting enzyme (ACE) activity was higher in CP-treated rats at days 2, 3, 5, 7, and 11, but there was a significant decrease in lung ACE activity during the same time period. Elevated levels of beta-glucuronidase activity were observed in the lung lavage fluid of CP-administered rats days 2, 3, 5, and 7. Lung myeloperoxidase activity was higher in CP rats. Of significance was the presence of collagenase and collagenolytic cathepsin in the lavage fluid of CP rats, when compared with the barely detectable levels in controls. A similar increase in these enzyme activities was also noticed in the lung tissue of CP rats during the same experimental period. Lavage fluid hydroxyproline content was higher in CP rats when compared with controls. Similarly, lung protein and DNA levels were elevated significantly after treatment with CP. The pulmonary histamine and serotonin contents were significantly higher in CP rats. The incorporation of [3H]thymidine into lung total DNA, [3H]proline into lung hydroxyproline, and [35S]sulphate into lung glycosaminoglycan, measured as indicators of lung DNA, collagen, and glycosaminoglycan synthesis, respectively, was also higher in CP groups. Increased levels of hydroxyproline, elastin, hexosamine, total hexose, fucose, sialic acid, and uronic acid in the lungs of rats 14, 28, and 42 days after CP insult were characterized as biomarkers of CP-induced interstitial changes. These findings indicate that CP-induced lung fibrosis results in alterations not only in collagen synthesis and accumulation, but also in glycosaminoglycan and glycoprotein content.

Advanced glycation end products induce crosslinking of collagen in vitro.

We have investigated the effect of advanced glycation end products (AGEs) on the crosslinking of collagen. The potential pathological significance of AGEs and the altered metabolism of ascorbic acid (ASA) in diabetes have prompted us to investigate the role of ASA in the crosslinking and advanced glycation of collagen. Rat tail tendons were incubated with ASA and dehydroascorbic acid (DHA) under physiological conditions of temperature and pH, and the crosslinking and the level of AGEs were analyzed. Analysis of crosslinking was conducted by pepsin solubility and cyanogen bromide digestion. Level of AGEs was estimated by enzyme-linked immunosorbent assay (ELISA) using antibodies raised against AGE-ribonuclease. It was noted that ASA and DHA induced crosslinking of collagen and stimulated the formation of AGEs. It was also noted that these pathways were dependent on oxidative conditions. Similarly incubation of collagen with AGEs, prepared by the in vitro incubation of bovine serum albumin (BSA) with glucose, also resulted in increased crosslinking. The extent of crosslinking was dependent on the duration of incubation. The novel finding of this study, which is in contrast to the earlier reports on glucose-induced crosslinking of collagen, was that AGEs-induced crosslinking of collagen was not inhibited by radical scavengers and the metal chelator. EDTA, whereas glucose-induced crosslinking of collagen was almost completely prevented by free radical scavengers. The increased fluorescence intensity observed in collagen incubated with AGEs was also not prevented by radical scavengers. Estimation of AGEs by ELISA revealed an increased accumulation of AGEs in collagen incubated with AGE-BSA. The inhibitory effect of aminoguanidine and aspirin on AGEs-induced modification of collagen, strongly suggests that the amino-carbonyl interaction between AGEs and collagen may play a key role in the crosslinking process. The results obtained in this study indicate that soluble AGEs can directly induce crosslinking of collagen and this process is independent of oxidative conditions. From these results it may be hypothesized that glucose, under oxidative conditions, reacts with proteins to form potentially reactive end products called AGEs. These AGEs, once formed, could induce crosslinking of collagen even in the absence of both glucose and oxygen.

Glycoprotein composition in cyclophosphamide-induced lung fibrosis.

The present study investigated the glycosylation state of proteins in lung tissue of a cyclophosphamide-induced model of pulmonary fibrosis in rats. In fibrotic lung, the carbohydrate constituents (total hexose, fucose, sialic acid and hexosamine) of salt-soluble, collagenase, elastase and papain digested glycoproteins were significantly higher compared to normal lungs. Interestingly, fibrotic lung tissues had higher activities of mannosyl, glucosyl, galactosyl, sialyl and fucosyl transferases than normal lung tissues. Similarly, mannosyl, glucosyl, galactosyl, sialyl and fucosyl transferases were higher in serum from rats with fibrosis than in that from normals. These data indicate that glycoprotein metabolism is significantly altered from normal in animals with interstitial lung fibrosis.

Occurrence of a novel collagen with three distinct chains in the cranial cartilage of the squid Sepia officinalis: comparison with shark cartilage collagen.

A unique collagen with three distinct chains, was purified from the cranial cartilage of the squid Sepia officinalis, by pepsinisation and salt precipitation and compared with shark cartilage collagen. These chains, which were different from the known cartilage collagen chains, were referred as C1, C2 and C3, had approximate molecular weights of 105 kDa, 115 kDa and 130 kDa, respectively, and were present in a ratio of 3:2:1, suggestive of two molecules of composition, [(C1)2C2] and [C1C2C3]. These collagens were purified by fractionation at acid and neutral pH, and by ammonium sulfate precipitation. Solubility data indicated that this collagen was more crosslinked than the type I collagen isolated from cartilage of shark, Carcharius acutus. In vitro fibrillogenesis revealed that the sepia collagen formed denser aggregates, as compared to shark collagen, and was stabilised by a higher degree of carbohydrate association. Polyclonal antisera raised against shark collagen was also reactive against the sepia collagens, while the converse was not true, indicating the high immunospecificity of the latter. These results demonstrate collagen polymorphism in an invertebrate cartilage and may hold significance in understanding tissue calcification and molecular evolution. Further, these collagens may represent ancestral forms of vertebrate minor collagens like typeV/XI.

The role of metal-catalyzed oxidation in the formation of advanced glycation end products: an in vitro study on collagen.

The present study investigates the role of metal catalysed oxidation in the formation of Advanced Glycation End products (AGEs). Rat tail tendon collagen was incubated with glucose (250 mM) and increasing concentrations of copper ions (5-500 microM) under physiological conditions of temperature and pH. After 1 and 3 weeks of incubation the level of AGEs in collagen samples were estimated by enzyme linked immunoassay, using antibodies raised against AGE ribonuclease. It was observed that the presence of metal ions significantly increased the rate of accumulation of AGEs. The increase was dependent on the concentration of metal ions present in the incubation medium. Free radical scavengers such as mannitol, benzoate, catalase, and the antiglycating agent aminoguanidine almost completely inhibited the formation of AGEs. Incubation of collagen with copper ions alone did not show any increase in crosslinking, as detected by cyanogen bromide digestion, and AGEs formation. Further it was also noted that glycoxidation, i.e., oxidation of glycated collagen, was the major pathway that leads to increased formation of AGEs. These results indicate that metal-catalyzed oxidation and free radicals play a major role in the formation of AGEs. This work also strongly suggests that increased oxidative stress in diabetes may accelerate the formation of AGEs and thus contribute to the pathogenesis of diabetic complications.

Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats.

Wound healing is a fundamental response to tissue injury that results in restoration of tissue integrity. This end is achieved mainly by the synthesis of the connective tissue matrix. Collagen is the major protein of the extracellular matrix, and is the component which ultimately contributes to wound strength. In this work, we report the influence of Aloe vera on the collagen content and its characteristics in a healing wound. It was observed that Aloe vera increased the collagen content of the granulation tissue as well as its degree of crosslinking as seen by increased aldehyde content and decreased acid solubility. The type I/type III collagen ratio of treated groups were lower than that of the untreated controls, indicating enhanced levels of type III collagen. Wounds were treated either by topical application or oral administration of Aloe vera to rats and both treatments were found to result in similar effects.

Physiological and nutritional factors affecting biosynthesis of extracellular protease by Streptomyces spp. G157.

The aim of the present study was to evaluate the effect of culture conditions on extracellular protease biosynthesis by Streptomyces spp. G157. It was found that there was optimum expression of extracellular proteases in a medium of soyabean meal (2%), mannitol (2%) at a pH of 7.2 and temperature of 28 degrees C. Among the sugars glucose and starch had a greater repressive effect than the others. Phosphate was found to have an inhibitory effect on protease biosynthesis.

Influence of Aloe vera on the glycosaminoglycans in the matrix of healing dermal wounds in rats.

The influence of Aloe vera (L.) Burman f. on the glycosaminoglycan (GAG) components of the matrix in a healing wound was studied. Wound healing is a dynamic and complex sequence of events of which the major one is the synthesis of extracellular matrix components. The early stage of wound healing is characterized by the laying down of a provisional matrix, which is then followed by the formation of granulation tissue and synthesis of collagen and elastin. The provisional matrix or the ground substance consists of GAGs and proteoglycans (PGs), which are protein GAG conjugates. In the present work, we have studied the influence of Aloe vera on the content of GAG and its types in the granulation tissue of healing wounds. We have also reported the levels of a few enzymes involved in matrix metabolism. The amount of ground substance synthesized was found to be higher in the treated wounds, and in particular, hyaluronic acid and dermatan sulphate levels were increased. The levels of the reported glycohydrolases were elevated on treatment with Aloe vera, indicating increased turnover of the matrix. Both topical and oral treatments with Aloe vera were found to have a positive influence on the synthesis of GAGs and thereby beneficially modulate wound healing.

Influence of aloe vera on the healing of dermal wounds in diabetic rats.

The positive influence of Aloe vera, a tropical cactus, on the healing of full-thickness wounds in diabetic rats is reported. Full-thickness excision/incision wounds were created on the back of rats, and treated either by topical application on the wound surface or by oral administration of the Aloe vera gel to the rat. Wound granulation tissues were removed on various days and the collagen, hexosamine, total protein and DNA contents were determined, in addition to the rates of wound contraction and period of epithelialization. Measurements of tensile strength were made on treated/untreated incision wounds. The results indicated that Aloe vera treatment of wounds in diabetic rats may enhance the process of wound healing by influencing phases such as inflammation, fibroplasia, collagen synthesis and maturation, and wound contraction. These effects may be due to the reported hypoglycemic effects of the aloe gel.

Effect of curcumin on the advanced glycation and cross-linking of collagen in diabetic rats.

A close association between increased oxidative stress and hyperglycemia has been postulated to contribute significantly to the accelerated accumulation of advanced glycation end products (AGEs) and the cross-linking of collagen in diabetes mellitus. In the present work, we report the influence of curcumin, an efficient antioxidant, on the level of AGEs and the cross-linking of collagen in diabetic rats. Diabetic rats were given curcumin (200 mg/kg body wt) orally for a duration of 8 weeks. The antioxidant status in serum and the level of AGEs, cross-linking and browning of collagen in tail tendons and skin were investigated. The oxidative stress observed in diabetic rats was reduced significantly by curcumin administration. Nonenzymic antioxidants such as vitamin C, vitamin E, and glutathione were maintained at near normal values in curcumin-treated diabetic animals. Similarly, the accumulation of lipid peroxidation products in diabetic serum was reduced significantly by curcumin. Accelerated accumulation of AGE-collagen in diabetic animals, as detected by ELISA, was prevented by curcumin. Extensive cross-linking of collagen in the tail tendon and skin of diabetic animals was also prevented to a greater extent by curcumin treatment. A correlation between the level of AGEs and collagen cross-linking was noted, suggesting the involvement of advanced glycation in cross-linking. It was also noted that the preventive effect of curcumin on the advanced glycation and cross-linking of collagen was more pronounced than its therapeutic effect. However, the Maillard reaction fluorescence in both tail and skin collagen remained unaltered by curcumin. This study confirms the significance of free radicals in the accumulation of AGEs and cross-linking of collagen in diabetes. It supports curcumin administration for the prevention of AGE-induced complications of diabetes mellitus.

Curcumin protects bleomycin-induced lung injury in rats.

The present study was designed to determine the protective effects of curcumin against bleomycin (BLM)-induced inflammatory and oxidant lung injury. The data indicate that BLM-mediated lung injury resulted in increases in lung lavage fluid biomarkers such as total protein, angiotensin-converting enzyme (ACE), lactate dehydrogenase (LDH), N-acetyl-beta-D-glucosaminidase (NAG), lipid peroxidation (LPO) products, superoxide dismutase (SOD) and catalase. Bleomycin administration also resulted in increased levels of malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage (BAL) cells and greater amounts of alveolar macrophage (AM) superoxide dismutase activity. By contrast, lower levels of reduced glutathione (GSH) were observed in lung lavage fluid, BAL cells and AM. Stimulated superoxide anion and hydrogen peroxide release by AM from BLM rats were found to be higher. Curcumin treatment resulted in a significant reduction in lavage fluid biomarkers. In addition, curcumin treatment resulted in the restoration of antioxidant status in BLM rats. These data suggest that curcumin treatment reduces the development of BLM-induced inflammatory and oxidant activity. Therefore, curcumin offers the potential for a novel pharmacological approach in the suppression of drug or chemical-induced lung injury.

Effects of Centella asiatica extract on dermal wound healing in rats.

Effects of oral and topical administration of an alcoholic extract of C. asiatica on rat dermal wound healing was studied. The extract increased cellular proliferation and collagen synthesis at the wound site, as evidenced by increase in DNA, protein and collagen content of granulation tissues. Quicker and better maturation and crosslinking of collagen was observed in the extract-treated rats, as indicated by the high stability of acid-soluble collagen and increase in aldehyde content and tensile strength. The extract treated wounds were found to epithelialise faster and the rate of wound contraction was higher, as compared to control wounds. The results show that C. asiatica produced different actions on the various phases of wound repair.

Glycoprotein metabolism in dimethylnitrosamine induced hepatic fibrosis in rats.

Glycoproteins play a major role in the pathogenesis of hepatic fibrosis by accumulating in the sinusoids and the space of Disse. In order to obtain more information about the altered metabolism of glycoproteins during the development of human hepatic fibrosis, the carbohydrate moieties of the glycoproteins were monitored in experimentally induced hepatic fibrosis. The liver injury was induced by injecting dimethylnitrosamine intraperitoneally in male albino rats. The injections were given on the first 3 consecutive days of each week over a period of 21 days. Glycoprotein moieties such as total hexose, hexosamine, fucose and sialic acid were estimated in liver, serum and urine samples on days 7, 14 and 21 of the experiment. The results indicated a significant decrease in total hexose and an increase in fucose levels in the liver tissue during dimethylnitrosamine administration. While protein bound hexose in the serum showed a significant decrease, sialic acid levels were notably increased. The other glycoprotein moieties both in liver and serum also showed an increase in the later periods of study, especially on day 21. All glycoprotein moieties exhibited a significant increase in the rate of urinary excretion on the 14th and 21st days, indicating an increased rate of metabolic degradation in the acute phase of hepatic fibrosis. The results suggest that glycoproteins undergo changes in both synthesis and the degradation during hepatic fibrosis. The relative alterations in these processes will play a vital role in determining the progression of hepatic fibrosis.

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 alpha- and beta-chains of purified pepsin solubilized liver collagen were separated by subjecting the denatured collagen to SDS-polyacrylamide gel electrophoresis. The alpha 1(III) chains were resolved from the alpha 1(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 beta-subunit of type I collagen with a remarkable decrease of alpha/beta ratio in DMN treated animals. Reduction with beta-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 in more prominent than type I collagen in early fibrosis.