The effect of food restriction and low protein diet upon collagen type I and III ratio in rat skin

It has been demonstrated that the content of the collagen type I is more affected by both chronic low protein diet feeding and chronic food deprivation (50% food intake) than the content of collagen type III. By introducing these dietary regimes the proportion of collagen type I to collagen type III ratio drops from 2.1 to 1.3 indicating the higher proportion of collagen type III in the skin at the end of the experiment (after 18 months of chronic feeding). It was also observed that the total concentration of hydroxyproline (hyp) in the skin decreases considerably in both food restricted animals and those fed a low protein diet. It is suggested that, under the present experimental conditions, the balance between collagen break-down and synthesis is shifted and, furthermore, that this shift is different for collagen type I and III and results in an altered ratio of these two collagen species in the skin. Refeeding of animals leads to a higher than normal collagen type I to III ratio indicating thus a relatively higher proportion of collagen type I in this tissue.     

Type IX collagen: a possible function in articular cartilage

The effect of type IX on in vitro fibrillogenesis of type II collagen indicated that, while not preventing fibrillogenesis, the presence of type IX collagen reduced the size of the type II fibre aggregates. This observation is consistent with the in vivo localisation studies of type IX collagen. Using the immunogold labelling technique, type IX collagen was shown to be located evenly on small fibrils which occur at higher concentration closer to the cell. Therefore type IX collagen may function as a regulator of fibre diameter in articular cartilage.     

Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen

The functional integrity of articular cartilage is dependent on the maintenance of the extracellular matrix (ECM), a process which is controlled by chondrocytes. The regulation of ECM biosynthesis is complex and a variety of substances have been found to influence chondrocyte metabolism. In the present study we have investigated the effect of degraded collagen on the formation of type II collagen by mature bovine chondrocytes in a cell culture model. The culture medium was supplemented with collagen hydrolysate (CH) and biosynthesis of type II collagen by chondrocytes was compared to control cells treated with native type I and type II collagen and a collagen-free protein hydrolysate. The quantification of type II collagen by means of an ELISA technique was confirmed by immunocytochemical detection as well as by the incorporation of (14)C-proline in the ECM after a 48 h incubation. Chondrocytes in the control group were maintained in the basal medium for 11 days. The presence of extracellular CH led to a dose-dependent increase in type II collagen secretion. However, native collagens as well as a collagen-free hydrolysate of wheat proteins failed to stimulate the production of type II collagen in chondrocytes. These results clearly indicate a stimulatory effect of degraded collagen on the type II collagen biosynthesis of chondrocytes and suggest a possible feedback mechanism for the regulation of collagen turnover in cartilage tissue.     

A nonradioactive assay for type IV collagen degradation

A sensitive assay for type IV collagen degradation using an avidin-biotin sandwich technique is described. Biotinylated type IV collagen is allowed to bind to an avidin-coated microtiter plate. The solution to be assayed is incubated with the biotinylated collagen bound to the avidin plate. Collagen degraded by the solution is released into the supernatant and transferred to a second plate coated with avidin. By addition of biotinylated horseradish peroxidase to this second plate, the amount of collagen degraded is determined. Our assay requires only 0.5 microgram of type IV collagen per microtiter plate and detects nanogram quantities of bacterial collagenase activity.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

Fibroblast behavior on gels of type I, III, and IV human placental collagens

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.     

Immunohistochemical localization of type II and type I collagens in articular cartilage of the femoral head of dexamethasone-treated rats

The immunohistochemical localization of type II and type I collagens was examined in the articular cartilage of the femoral head of growing rats injected systemically with 5 mg kg−1 dexamethasone for 2 weeks every other day. The intensities of immunostaining for type II collagen, measured by microphotometry, was highest in the flattened cell layer and high in the hypertrophic cell layer, moderate in the proliferative cell and transitional cell layers and low in the superficial layer. After dexamethasone administration, the intensities decreased markedly in the flattened cell layer and slightly in the hypertrophic cell layer, although the decreases in other layers were negligible. The staining intensities for type I collagen were highest in the flattened cell layer, low in the superficial and transitional cell layers and very low in the proliferative and hypertrophic cell layers. After dexamethasone administration, the intensities increased markedly in the flattened cell layer and slightly in the superficial and proliferative cell layers, but did not change in the transitional and hypertrophic cell layers. Thus, dexamethasone administration caused a decrease in type II collagen and an increase in type I collagen in the matrix of the surface portion of articular cartilage. The composition of isoforms of collagen in the matrix changed after the steroid administration. The results strongly suggest that the shift in collagen composition from type II to type I predominance is a cause of the degeneration of the articular cartilage after glucocorticoid administration. This revised version was published online in November 2006 with corrections to the Cover Date.     

Single molecule mechanical properties of type II collagen and hyaluronan measured by optical tweezers

Type II collagen and hyaluronan are the two major components of extracellular molecules in cartilage and play an important role in mechanical functions of extracellular matrix. Currently, their mechanical properties have been investigated only at the gross-level. In this study, the mechanical properties of single type II collagen and hyaluronan molecules were directly measured using optical tweezers technique. The persistence length was found to be 11.2+/-8.4 nm in type II collagen and 4.5+/-1.2 nm in hyaluronan. This result suggested that type II collagen is stiffer than hyaluronan at the individual molecule level, which supports the general concept that collagen is responsible for resisting tensile force. The experimental system developed here also provides a powerful tool for quantifying mechanical properties of extracellular matrix at the single molecule level.     

Deficiency of tenascin-X causes a decrease in the level of expression of type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX-null fibroblasts exhibit decreased cell-matrix and cell-cell adhesion. In this study, we used a differential display technique to determine the genes involved in this process. Differential display analysis of wild-type and TNX-null fibroblasts revealed that mRNA expression level of type VI collagen alpha3 is predominantly decreased in TNX-null fibroblasts. Expression levels of mRNAs of other subunits of type VI collagen, alpha2 and alpha3 chains, were also remarkably decreased in TNX-null fibroblasts. The protein level of alpha3 chain of type VI collagen was also reduced in TNX-null fibroblasts. However, the organization of type VI collagen in the extracellular matrix of TNX-null fibroblasts was similar to that of wild-type fibroblasts. Transient expression of TNX in Balb3T3 cells caused an increase in the level of mRNA of type VI collagen compared with that in vector control and increased the promoter activity of type VI collagen alpha1 subunit gene. In addition, the expression levels of type I collagen and other collagen fibril-associated molecules such as type XII and type XIV collagens, decorin, lumican and fibromodulin in wild-type and TNX-null fibroblasts were compared. It was found that the mRNA expression levels of type I collagen and collagen fibril-associated molecules other than decorin were decreased and that the expression level of decorin was increased in TNX-null fibroblasts. The results suggest the possibility that TNX mediates not only cell-cell and cell-matrix interactions but also fibrillogenesis via collagen fibril-associated molecules.     

Heterogeneity of collagen isolated from methylcholanthrene-induced sarcoma

Rat fibrosarcoma induced by subcutaneous injection of methylcholanthrene was found to contain at least three different types of collagen. Two of them were identified as type I and type III collagens, the third (fraction B) seems to be specific for this tumour. The ratio of type I to type III collagen is lower in fibrosarcoma than in normal rat skin. The number of hydroxyproline residues in alpha 1 (I), alpha 2 (I) and alpha 1 (III) chains of tumour collagen appears to be higher than in the corresponding chains of rat skin collagen. Fraction B is composed of three identical alpha chains connected with disulphide bonds. It contains a relatively low amount of glycine: 234 molecules per 1000 residues. The amount of hydroxyproline and cysteine is similar to that found in the type III collagen.     

Analysis of type II collagen RNA localization in chick wing buds by in situ hybridization

Type II collagen is a major component of cartilage extracellular matrix. Differentiation of mesenchyme into cartilage involves the cessation of type I collagen synthesis and the onset of type II collagen synthesis. Solution hybridization of mRNA isolated from chick limb buds with a cDNA probe to type II collagen mRNA showed the presence of small amounts of type II collagen message in mesenchymal chick limbs. We have examined the localization of type II collagen mRNA in mesenchymal chick wing buds by in situ hybridization using single stranded RNA probes. Our results show a small but detectable amount of type II collagen RNA distributed uniformly in early limbs until the first precartilage condensations form at stage 22. This is interesting because it is known that mesenchyme isolated from chick wing buds has the capacity to undergo chondrogenesis in culture, even if taken from nonchondrogenic areas of the limb. At stage 23, type II collagen mRNA is found at significantly increased levels in the cells of the precartilage condensation when compared to the other limb cells. As chondrogenesis proceeds, the amount of type II collagen RNA increases even more in cells of the cartilage elements. The signal in the peripheral tissue is indistinguishable from background. These results show that type II collagen message exists at low levels in cells throughout the mesenchymal chick wing bud, until the formation of the condensation results in an elevation of type II mRNA in the prechondrogenic cells found in the core of the limb.     

正常椎间盘胶原的研究

腰椎间盘突出症是引起腰腿痛常见的原因。胶原作为椎间盘结构的主要成分,构成椎间盘的纤维框架,其类型与分布直接决定着椎间盘结构的强度和功能的稳定。本文利用溴化氰消化椎间盘胶原产生多肽,借助于梯度层析。SDS-PAGE及光密度定量扫描等对正常人椎间盘胶原进行了研究。结果表明:正常人椎间盘含Ⅰ型及Ⅱ型两种胶原,它们的分布呈明显而特征性的移行性变化:纤维环外层边缘以Ⅰ型胶原为主(83％),由外向内Ⅰ型胶原逐渐移行为Ⅱ型胶原,靠近髓核处以Ⅱ型胶原为主(72％);髓核中心含有Ⅱ型胶原。此为椎间盘的一个结构特性,以满足椎间盘的特殊功能的需要。     

Different behavior of type I and type I-trimer collagen in neutral sodium chloride solutions

Type I and type I-trimer collagen, isolated from ductal infiltrating carcinoma of the human breast, have been tested for their behavior in neutral NaCl solutions. Evident diversities in their rate of precipitation at different saline concentrations have been found, since type I-trimer collagen precipitates at low NaCl molarity while type I collagen is mostly recovered in 2.6-3.6 M NaCl solutions. The native conformation of homotrimer collagen is proved by its ability to produce segment long-spacing crystallites and native-type fibrils.     

Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)₇ with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)₇-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.     

Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro

Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils.     

The distribution of type VI collagen in the developing tissues of the bovine femoral head

Type VI collagen appears central to the maintenance of tissue integrity. In adult articular cartilage, type VI collagen is preferentially localised in the chondron where it may be involved in cell attachment. In actively remodelling developing cartilage, the distribution is less certain. We have used confocal immunohistochemistry and in situ hybridisation to investigate type VI collagen distribution in third trimester bovine proximal femoral epiphyses. In general, type VI collagen immunofluorescence was concentrated in the chondrocyte pericellular matrix, with staining intensity strongest in regions which persist to maturity and weakest in regions that remodel during development. Type VI collagen was also present in cartilage canals. In the growth plate and around the secondary centre of ossification, the intensity of type VI collagen stain rapidly decreased with chondrocyte maturation and was absent at hypertrophy, except where canal branches penetrated the growth plate and stain was retained around the adjacent chondrocytes. In situ hybridisation confirmed the presence of type VI collagen mRNA in cartilage canal mesenchymal cells but the signal was low in chondrocytes, suggesting minimal levels of synthesis and turnover. The results are consistent with a role for type VI collagen in stabilising the extracellular matrix during development.     

原子力显微镜研究UV-B对I型胶原结构的影响

利用原子力显微镜(AFM)成像技术观察胶原蛋白溶液在UV-B照射前后形态的变化,发现UV-B引起胶原纤维交联度的增加,当交联达一定程度后,照射时间的增加对交联度增加的影响不明显。AFM作为一种高分辨的表面分析仪器,为分子生物学领域的研究提供了一种新的手段。是探讨胶原光作用机理直观、有效的方法。     

The use of Fourier‐transform infrared spectroscopy to characterize connective tissue components in skeletal muscle of Atlantic cod (Gadus morhua L.)

In the present study, Fourier‐transform infrared spectroscopy (FTIR) is investigated as a method to measure connective tissue components that are important for the quality of Atlantic cod filets (Gadus morhua L.). The Atlantic cod used in this study originated from a feeding trial, which found that fish fed a high starch diet contained relative more collagen type I, while fish fed a low starch (LS) diet contained relative more glycosaminoglycans (GAGs) in the connective tissue. FTIR spectra of pure commercial collagen type I and GAGs were acquired to identify spectral markers and compare them with FTIR spectra and images from connective tissue. Using principal component analysis, high and LS diets were separated due to collagen type I in the spectral region 1800 to 800 cm^?1. The spatial distribution of collagen type I and GAGs were further investigated by FTIR imaging in combination with immunohistochemistry. Pixel‐wise correlation images were calculated between preprocessed connective tissue images and preprocessed pure components spectra of collagen type I and GAGs, respectively. For collagen, the FTIR images reveal a collagen distribution that closely resembles the collagen distribution as imaged by immunohistochemistry. For GAGs, the concentration is very low. Still, the FTIR images detect the most GAGs rich regions.      

Differential effect of hypertonic initiation block on the synthesis of collagen chains by cultured chick embryo cells

The major type of collagen synthesized by fibroblasts or bone cells, type I collagen, consists of two chains normally found in a 2:1 ratio designated alpha 1(I)2 alpha 2(I) or more simply alpha 1(I)2 alpha 2. I have analyzed the relative synthesis of type I chains in these cells under conditions which reduce the initiation of protein synthesis. It was found that in bone cells, which make a large amount of collagen, the alpha 1(I):alpha 2 ratio is unaltered whereas in fibroblasts, which make smaller amounts of collagen, the alpha 2 chain is particularly sensitive to these same conditions. Examination of the collagen secreted into the medium, under these same conditions, also revealed an altered chain ratio from cells making low amounts of collagen.