Effect of cadmium on collagen content and solubility in rat bone

The toxic action of cadmium in the bone tissue is known, but its mechanisms are still unexplained. We examined whether Cd influences collagen content and its solubility in the femoral bone of three-week-old female rats exposed to 5 or 50 mg Cd/l in drinking water. Non-cross linked collagen was extracted with 0.5 M acetic acid, and two acid-insoluble collagen fractions were extracted with pepsin and 4.0 M guanidine hydrochloride, respectively. SDS/PAGE showed the presence of two collagen types, I and V, in all three extracted fractions. Exposure of rats to Cd for 6 months increased the amount of acid-soluble collagens type I and V and decreased the level of acid-insoluble collagens. The amount of total collagen extracted from the bones of rats exposed to 50 mg Cd/l was reduced by about 14% as compared to control and those intoxicated with 5 mg Cd/l. The solubility of type I bone collagen (determined as the percentage of acetic-soluble fraction of total collagen) was increased 2.9- and 3.0-fold in rats intoxicated with 5 and 50 mg Cd/l, respectively. Similarly, the solubility of type V collagen was increased 2.3- and 2.7-fold, respectively. Our results indicate that Cd treatment affects bone collagen by decreasing its content and increasing its solubility.     

Effects of dietary xylitol on collagen content and glycosylation in healthy and diabetic rats

The effects of three-month dietary xylitol supplementation on the amounts and hexose contents of acid-soluble collagen as well as on the amounts and fluorescence of collagenase-soluble collagen were studied in healthy and streptozotocin-diabetic male rats. Collagen was extracted from skin samples. In the healthy rats, supplementation with xylitol (10%) increased the hydroxyproline content of the acid-soluble fraction and skin thickness. In diabetic rats receiving and not receiving xylitol, the acid-soluble collagen fraction was markedly lower than in healthy rats. However, its amount was significantly elevated when xylitol had been added to the diet. Supplementation with xylitol caused no changes in the amounts of collagenase-soluble fraction in either healthy or diabetic rats. Supplementation with xylitol (10%) significantly decreased the hexose content of acid-soluble collagen and the fluorescence of the collagenase-soluble fraction in both healthy and diabetic rats. The results indicate that dietary xylitol affects collagen synthesis and collagen glycosylation.     

Site variability in the solubility of collagen of human fetal membranes

The extractability of collagen was examined in different sites of amnion and chorion from term deliveries. Sequential extraction with NaCl, acetic acid and CaCl2 showed that soluble collagen accounted for 1.5% of the proteins extracted. Saline extracted more collagen from the amnion than from the chorion. Acetic acid and CaCl2 extracted decreasing and increasing amounts of collagen from the amnion and chorion respectively. The concentration of collagen decreased linearly in the chorion as the rupture site was approached. The results indicate differences in the nature of collagen between amnion and chorion as well as in various sites in the latter.     

人胎盘中胶原蛋白的提取和表征

采用碱-酶结合法以人胎盘为原料提取胶原蛋白,并使用原子力显微镜(AFM)、差示扫描量热仪(DSC)、圆二色光谱(CD)和傅立叶变换红外光谱(FTIR)对所得胶原蛋白产品进行表征。结果表明:此工艺路线的胶原蛋白得率高,达到0.102%;提取时间由48 h以上减少到24 h以内,而且所得胶原蛋白分子完整地保留了其螺旋纤维结构。此法是从人胎盘中提取胶原蛋白的一种行之有效的方法。     

The solubilization of collagen and protein–polysaccharides from the developing cartilage of lathyrytic chicks

1. The solubilization of collagen and protein-polysaccharides from the developing cartilage of normal and lathyritic chicks was studied by using mild extraction procedures. One-third of the protein-polysaccharides could be solubilized in salt solutions at neutral pH from normal cartilage, whereas 95-100% could be extracted from the cartilage of animals that were severely lathyritic. Likewise, whereas in normal animals the collagen of cartilage was essentially insoluble in salt solutions at neutral pH, in lathyritic animals it was almost completely soluble. 2. The increased solubility of the collagen of cartilage from lathyritic animals enabled sufficient material to be collected so that the pure alpha1 chains of the collagen were isolated by repeated reconstitution, precipitation and CM-cellulose column chromatography. The purified alpha1 component was characterized by its relatively high content of hydroxylysine (14 residues/1000 amino acids). 3. About 37% of the collagen from the cartilage of normal chick embryos could be extracted as the gelatin at pH7.4 in lithium chloride solution. This was accompanied by the extraction of approx. 14% of the protein-polysaccharide content. 4. The protein-polysaccharides and the collagen from normal animals could be extracted from the cartilage relatively independently of one another under mild conditions. These same components obtained from lathyritic animals easily separated from one another after solubilization. This provided evidence that the two components are probably not covalently cross-linked. 5. The collagen of cartilage extracted as a gelatin from normal animals contained a high proportion of alpha chains compared with beta dimers, similar to the lathyritic collagen of cartilage and other tissues, and similar to the gelatin extracted from normal chick bone.     

Isolation and properties of a preparation of arterial collagen solubilized by pretreatment with alkali (author's transl)]

The acid-soluble, highly cross-linked aorta collagen, of which about 30% can be converted into a soluble form by alkali treatment, followed by extraction with aetic acid, was obtained predominantly in the form of monomeric, helical molecules, as indicated by the value for the intrinsic viscosity and its behaviour in sodium dodecylsulphate disc electrophoresis. Apart from decreased values for tyrosine (0.26%), arginine (4.4%) and aspartic acid (3.9%), the amino acid composition of the aorta collagen fraction was similar to that of the acid-soluble calf skin collagen. This finding, together with the cyanogen bromide peptide pattern, shows that the collagen extracted from the artery is predominantly type I. Treatment with alkali probably shortens the alpha1-CB6-peptide by about 45 amino acids. The collagen extracted from artery was compared with acid soluble skin collagen by sodium dodecylsulphate polyacrylamide electrophoresis. The arterial collagen showed a marked increase in the rations alpha1 to alpha2 (4:1), alpha to beta (3:1) and beta11 to beta12 (2.5:1). Compared with acid soluble skin collagen, the aorta collagen contained twice as much galactose and glucose (13.5 and 9.6 nmol/mg protein respectively), which are bound to hydroxylysine. 50% of the hydroxylysine residues are unsubstituted, 15% are present as galactosyl hydroxylysine, and 35% as glucosyl-galactosyl hydroxylysine. On the basis of its reported properties, arterial collagen obtained by the method of Fujii appears to be a suitable substrate for the study of the enzymic synthesis and enzymic degradation of hydroxylysine glycosides of native arterial collagen.     

Reconstituted basement-membrane matrix modulates fibroblast activities in vitro

Cellular growth and collagen biosynthesis were compared in dermal calf fibroblasts cultured on plastic or on a reconstituted basement membrane gel, termed matrigel. This matrix, extracted from Engelbreth-Holm-Swarm tumors, consists mainly of laminin, entactin, type IV collagen, and heparan sulfate proteoglycan. The multiplication rate of fibroblasts grown on matrigel was stimulated compared to that of monolayered cells cultured on plastic, and these cells formed multilayers after 4 days. Protein and collagen biosynthesis was reduced in fibroblasts cultured on matrigel. A higher proportion of the newly synthesized collagen (40%) was incorporated to the extracellular matrix in cultures grown on matrigel than in those grown on plastic (14%). Type III collagen was the preferential collagen type deposited on matrigel, and the ratio of type III:type I collagens secreted in the medium was also slightly higher in cultures grown on matrigel. Partially processed collagen was more abundant in fibroblasts grown on matrigel than in cells cultured on plastic. Finally, cells grown on matrigel exhibited a higher catabolic activity than cells grown on plastic. In this experimental model, the reconstituted basement-membrane matrix seems to influence the activities of fibroblasts significantly.     

乌贼皮胶原蛋白的提取及结构表征

为了充分利用乌贼加工废弃物,分析了乌贼皮的基本组成成分,优化了从乌贼皮中提取胶原蛋白的工艺条件,并利用SDS-PAGE垂直电泳、紫外扫描和傅里叶变换红外光谱对所提取的胶原蛋白进行了结构表征.结果表明,乌贼皮中含有大量胶原蛋白,可作为胶原蛋白来源的补充.采用酸酶复合提取胶原蛋白的最佳条件为:酒石酸浓度为0.1mol/L,胃蛋白酶添加量为1400U/g,料液比为1:20(m:V,原料),4℃提取18h,提取率为12.08％.SDS-PAGE垂直电泳、紫外扫描和傅里叶变换红外光谱的结果表明,采用酸酶复合法从乌贼皮中提取的胶原蛋白为I型胶原蛋白,保持了完整的三螺旋结构.     

Collagen Extract from Marine Finfish Scales as a Potential Mosquito Larvicide

Collagen is a peptide being utilized in medical, health care, nutrient and decorative industry. Marine fish scales are one of the good sources of collagen, which is extracted using the advanced enzymatic digestion method. Scales of Sardinella longiceps (Oil Sardine) have a high proportion of collagen. This product is well absorbed with broad adaptive values that encourage the inclusion of nutriments. In this paper, we have performed the isolation and characterization of collagen from S. longiceps fish scales. The unnecessary proteins on the surface of fish scales was removed by demineralization process. The fish scale collagen was extracted in two different methods: acid (acetic acid) and enzymatic (pepsin) technique. The molecular mass of the extracted collagen was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The absorption spectra of the extracted collagen was measured to estimate its amino acid (tyrosine) content. Fourier transform infrared (FTIR) spectrum showed the existence of bands corresponding to the collagen extracted from S. longiceps fish scale and the crystallinity of extracted collagen was obtained using X-ray diffraction (XRD) analysis. The morphological micrograph was also analyzed by scanning electron microscope (SEM). The anti-larval effect of the collagen extract was determined using mosquito larvae of Aedes aegypti (Ae. aegypti) and the activity was statistically significant.     

The removal of non-collagen components from newborn calf dermis with magnesium chloride solution.

1. Non-collagenous substances in newborn calf dermis were extracted with solutions of various concentrations of MgCl2. The total protein and hydroxyproline contents in MgCl2 extracts increased with increase in the concentration of MgCl2 in the solutions. In particular, steep increases of their contents were observed at concentrations of MgCl2 from 0.5 to 1.0 M. Total amounts of hydroxyproline in 1.0, 2.0, and 3.0 M MgCl2 extracts were equivalent to 40-50% of the hydroxyproline content in the whole connective tissue. Hexose and hexosamine contents of MgCl2 extracts increased with increase of the MgCl2 concentration. Hexuronic acid was hardly present in the residues after extractions with 0.5, 1.0, 2.0, and 3.0 M MgCl2. 2. Plasma proteins, hyaluronic acid, and dermatan sulfate were extracted at low concentrations of MgCl2. A non-collagenous protein and MgCl2-soluble collagen were extracted with 1.0, 2.0, and 3.0 M MgCl2 solutions. The disperson of collagen fibrils was observed in the residue extracted with 1.0 M MgCl2 solution by electron microscopy; the fibril structure of collagen was disordered by extraction with 2.0 and 3.0 M MgCl2. The results suggest that the dispersion and disorder of collagen fibrils lead to the release of a non-collagenous protein. Furthermore, it is suggested that the removal of hyaluronic acid and dermatan sulfate was not very effective for the solubilization of a large amount of collagen, but was suitable as a pretreatment to the extraction of a non-collagenous protein accompanied by the solubilization of a large amount of collagen. 3. The non-collagenous protein was purified by DEAE-cellulose column chromatography. Polyacrylamide gel electrophoresis of this protein at pH 8.5 showed a single band moving to the cathode. The non-collagenous protein contained 3.7% hexose, 1.8% hexosamine, and no hexuronic acid. This protein is rich in glycine, glutamic acid, and alanine, and contains neither hydroxyproline nor hydroxylysine. Sedimentation analysis showed a single peak with 1.8 S and the molecular weight was approx. 43,000 as determided by SDS polyacrylamide gel electrophoresis.     

Programmed synthesis of collagen during postembryonic development of the nematode Panagrellus silusiae.

The relative rate of collagen synthesis in the free-living nematode Panagrellus silusiae during postembryonic development was found to be discontinuous by measuring either the incorporation of tritium into material extracted as collagen or the amount of collagen-bound tritiated proline and hydroxyproline after 2-hr incubations of whole worms with [³H]proline. A peak of collagen production preceded each of the three molts that were examined. Moreover, protocollagen prolyl hydroxylase activity during each intermolt period paralleled the pattern of collagen synthesis. On the other hand, a triphasic pattern was not observed when noncollagenous proteins were labeled with either [³H]tryptophan or [³H]leucine. In addition, the level of soluble radioactive proline that accumulates in whole organisms after 2-hr incubation periods did not fluctuate appreciably during postembryonic development. The mean ratio of hydroxy-proline to proline in a number of collagen samples extracted at various times during the maturation phase was 0.113 ± 0.040. Pulse and chase experiments with [³H]proline indicated that most of the collagen synthesized during a peak period is lost after the second ecdysis following the labeling interval. In contrast, a considerable proportion of the collagen synthesized during nonpeak periods is retained throughout the postembryonic period. It is postulated that the modulated pattern of collagen biosynthesis in Panagrellus reflects, for the most part, a quantitative regulation of the production of cuticular collagen during postembryonic development.     

Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen

The different collagen types were extracted sequentially, by 4 M guanidinium chloride and pepsin, from human foetal and normal and osteoarthritic adult articular cartilage. They were characterized by electrophoresis and immunoblotting. Most of the collagenous proteins present in articular cartilage from young human foetuses were solubilized: almost 40% of the total collagen was extracted in the native form with 4 M guanidinium chloride. Type VI collagen was detected in this fraction as high-molecular-mass chains (185-220 kDa) and a low-molecular-mass chain (140 kDa). Type II, IX and XI collagens were also present, but were extracted more extensively by pepsin digestion. Comparative analysis of normal and osteoarthritic cartilage from adults reveals some major differences: an increase in the solubility of the collagen and modifications of soluble collagen types in osteoarthritic cartilage. Furthermore, type VI collagen was present at a higher concentration in guanidinium chloride extracts of osteoarthritic cartilage than those of normal tissue. This finding was corroborated by electron microscopic observations of the same samples: abundant (100 nm) periodic fibrils were observed in the disorganized pericellular capsule of cloned cells in osteoarthritic cartilage. In normal tissues the pericellular zone was more compact and contained only a few such banded fibrils. The differences in the collagen types solubilized from normal and osteoarthritic cartilage, although corresponding to a minor proportion of the total collagen, demonstrate that important modifications in chondrocyte metabolism and in the collagenous network do occur in degenerated cartilage.     

Viscoelastic and Thermal Properties of Collagen–Xanthan Gum and Collagen–Maltodextrin Suspensions During Heating and Cooling

The purpose of this work was to investigate the viscoelastic properties of aqueous suspensions of crude collagen powder extracted from bovine hides and nonsubmitted to the hydrolysis reaction that leads to gelatin. The studied variables included the collagen concentration and the addition of xanthan gum or maltodextrin at varied concentrations during heating/cooling of the mixtures. Differential scanning calorimetry thermograms showed that the addition of polysaccharides decreased the endothermic peak areas observed at the denaturation temperature of collagen. The rheological properties of the pure collagen suspensions were highly dependent on concentration: 4% and 6% collagen suspensions presented a great increase in the storage modulus after heating/cooling, whereas for concentrations of 8% and 10% G′ decreased during heating and did not recover its original value after heating/cooling. The frequency sweeps showed that the thermal treatment was responsible by the strengthening of the interactions that formed the polymer network. Addition of 0.1% xanthan gum to collagen suspensions increased the gel strength, especially after heating/cooling of the system, whereas increasing gum concentration to 0.3% resulted in a weaker gel, which could indicate thermodynamic incompatibility between the biopolymers. Mixtures of collagen and maltodextrin resulted in more fluid structures than those obtained with pure collagen at the same collagen concentration and the range of temperatures in which these mixtures behaved as a gel decreased with increasing concentrations of both collagen and maltodextrin, suggesting incompatibilities between the biopolymers.     

Tissue form of type VII collagen from human skin and dermal fibroblasts in culture

The triple-helical domain of type VII collagen was isolated from human placental membranes by mild digestion with pepsin, and polyclonal antibodies were raised in rabbits against this protein. After affinity purification the antibodies specifically recognized type VII collagen in both the triple-helical and the unfolded state. They also reacted with the fragments P1 and P2, derived from the triple-helical domain by further proteolysis with pepsin, but did not crossreact with other biochemical components of the dermal connective tissue. In skin the presence of a fragment of type VII collagen, similar to that isolated from placenta, was demonstrated by SDS-PAGE and immunoblotting. Type VII collagen represented less than 0.001% of the total collagen extracted by pepsin digestion from newborn or adult skin. The tissue form of type VII collagen was obtained from dermis after artificial epidermolysis with strongly denaturing buffers under conditions reducing disulfide bonds. The protein was identified by immunoblotting with the antibodies. The molecule was composed of three polypeptides with an apparent molecular mass of about 250 kDa, each. Similar large-molecular-mass chains could be identified by immunoblotting in extracts of human fibroblasts in culture.     

Short-chain basement membrane collagen. Further characterization and its biosynthesis by F-9 embryonal carcinoma cells

The paper describes further characterization of the 55-kDa short-chain collagen from lens capsule. Lens capsules were extracted with 5.5 M guanidine.HCl and the extracted material was fractionated on agarose A-5M followed by high-pressure liquid chromatography (HPLC). By amino acid composition, the major fraction obtained from HPLC was found to be different than type-IV collagen fragments. The 55-kDa short-chain collagen on pepsin digestion produced a 45-kDa pepsin-resistant fragment. The undifferentiated embryonal carcinoma (F-9) cells were found to synthesize increased amounts of 55-kDa short-chain collagen. The identity of this biosynthesized molecule with 55-kDa short-chain collagen from lens capsules was established by immunoprecipitation experiments. The results indicated a close similarity or identical nature of the short-chain collagens from these two sources.     

Isolation and characterization of collagen from brown backed toadfish (Lagocephalus gloveri) skin

Collagen was isolated and characterized from the skin of brown backed toadfish (Lagocephalus gloveri), processing wastes. The total collagen yield extracted was 54.3% on the basis of lyophilized dry weight compared to other vertebrates. According to the electrophoretic pattern, the skin collagen of the fish consisted of heterotrimer alpha1alpha2alpha3. Moreover, the denaturation temperature was 28 degrees C, which was about 9 degrees C lower than that of collagen from porcine skin. Therefore, there is a possibility to use brown backed toadfish skin collagen as an alternative source of collagen for industrial purposes and subsequently it may maximize the economical value of the fish.     

Effect of the triterpenoid fraction of Centella asiatica on macromolecules of the connective matrix in human skin fibroblast cultures

The mechanism of action of the total triterpenoid fraction extracted from Centella Asiatica (TTFCA) was evaluated using human skin fibroblasts cultures as the experimental system. In particular its influence on the biosynthesis of collagen, fibronectin and proteoglycans was considered. The presence of TTFCA (25 micrograms/ml) does not seem to affect cell proliferation, total protein synthesis or the biosynthesis of proteoglycans in a significant way. A statistically important increase was observed in the percentage of collagen and, as revealed by immunofluorescence measurements, in cell layer fibronectin. This effect on collagen and fibronectin may help to explain the action of TTFCA in promoting wound healing, and suggests an interesting working hypothesis for its action on basal endothelia.     

Identification, content, and distribution of type VI collagen in bovine tendons

Tendon composition changes according to differentiation, mechanical load, and aging. In this study, we attempted to identify, localize, and quantify type VI collagen in bovine tendons. Type VI collagen was identified by the electrophoretic behavior of the alpha chains and Western blotting, and by rotary shadowing. Type VI collagen was extracted from powdered tendon with three sequential 24-h extractions with 4 M guanidine-HCl. The amount of type VI collagen was determined by enzyme-linked immunosorbent assay for purely tensional areas and for the compressive fibrocartilage regions of the deep flexor tendon of the digits, for the corresponding fetal and calf tendons, and for the extensor digital tendon. The distal fibrocartilaginous region of the adult tendon was richer in type VI collagen than the tensional area, reaching as much as 3.3 mg/g (0.33%) of the wet weight. Calf tendons showed an accumulation of type VI at the fibrocartilage site. Immunocytochemistry demonstrated that type VI collagen was evenly distributed in the tensional areas of tendons but was highly concentrated around the fibrochondrocytes in the fibrocartilages. The results demonstrate that tendons are variable with regard to the presence and distribution of type VI collagen. The early accumulation of type VI collagen in the region of calf tendon that will become fibrocartilage in the adult suggests that it is a good marker of fibrocartilage differentiation. Furthermore, the distribution of type VI collagen in tendon fibrocartilage indicates that it organizes the pericellular environment and may represent a survival factor for these cells.