Mammalian Connective Tissues Research Articles

Autosomal Recessive Cutis Laxa 1C Mutations Disrupt the Structure and Interactions of Latent TGFβ Binding Protein-4.

Latent TGFβ binding protein-4 (LTBP4) is a multi-domain glycoprotein, essential for regulating the extracellular bioavailability of TGFβ and assembly of elastic fibre proteins, fibrillin-1 and tropoelastin. LTBP4 mutations are linked to autosomal recessive cutis laxa type 1C (ARCL1C), a rare congenital disease characterised by high mortality and severely disrupted connective tissues. Despite the importance of LTBP4, the structure and molecular consequences of disease mutations are unknown. Therefore, we analysed the structural and functional consequences of three ARCL1C causing point mutations which effect highly conserved cysteine residues. Our structural and biophysical data show that the LTBP4 N- and C-terminal regions are monomeric in solution and adopt extended conformations with the mutations resulting in subtle changes to their conformation. Similar to LTBP1, the N-terminal region is relatively inflexible, whereas the C-terminal region is flexible. Interaction studies show that one C-terminal mutation slightly decreases binding to fibrillin-1. We also found that the LTBP4 C-terminal region directly interacts with tropoelastin which is perturbed by both C-terminal ARCL1C mutations, whereas an N-terminal mutation increased binding to fibulin-4 but did not affect the interaction with heparan sulphate. Our results suggest that LTBP4 mutations contribute to ARCL1C by disrupting the structure and interactions of LTBP4 which are essential for elastogenesis in a range of mammalian connective tissues.

Oxi-HA/ADH Hydrogels: A Novel Approach in Tissue Engineering and Regenerative Medicine

Hyaluronic acid (HA) is a natural polyelectrolyte abundant in mammalian connective tissues, such as cartilage and skin. Both endogenous and exogenous HA produced by fermentation have similar physicochemical, rheological, and biological properties, leading to medical and dermo-cosmetic products. Chemical modifications such as cross-linking or conjugation in target groups of the HA molecule improve its properties and in vivo stability, expanding its applications. Currently, HA-based scaffolds and matrices are of great interest in tissue engineering and regenerative medicine. However, the partial oxidation of the proximal hydroxyl groups in HA to electrophilic aldehydes mediated by periodate is still rarely investigated. The introduced aldehyde groups in the HA backbone allow spontaneous cross-linking with adipic dihydrazide (ADH), thermosensitivity, and noncytotoxicity to the hydrogels, which are advantageous for medical applications. This review provides an overview of the physicochemical properties of HA and its usual chemical modifications to better understand oxi-HA/ADH hydrogels, their functional properties modulated by the oxidation degree and ADH concentration, and the current clinical research. Finally, it discusses the development of biomaterials based on oxi-HA/ADH as a novel approach in tissue engineering and regenerative medicine.

Protective Effects of Collagen Peptides on the Dexamethasone-Induced Immunosuppression in Mice

Collagen peptides are collagen derived products by enzymatic proteolysis, which is widely distributed in mammalian connective tissues. They have exhibited various bioactivities, including antioxidant activity, antihypertensive activity, improvement of joint pain, etc. However, the immunomodulatory effects of collagen peptides in the condition of immune suppression have not been elucidated. The objective of this study was to investigate the effects of collagen peptides on immune function in immunosuppressed mice. The immunosuppression was induced by intraperitoneal injection of dexamethasone for three days and at same time, collagen peptides (0, 300, 600, 900 mg/kg body weight) were given by gavage for seven days. On the day 8, the mice were sacrificed by decapitation. The body weight and the weights of thymus and spleen were recorded. Thymus and spleen indexes were calculated. The proliferation and interleukin-2 production of splenic T lymphocytes and ratio of CD4+/CD8+T lymphocytes in the peripheral blood were examined. Compared with the control mice, the body weight, thymus and spleen indexes, the proliferations and interleukin-2 production in T lymphocytes and the ratios of CD4+/CD8+T cells were significantly decreased in dexamethasone induced immunosuppressed mice. However, in collagen peptides treated mice, the thymus indexes, proliferation index, secretion of interleukin-2 and the ratio of CD4+/CD8+ were significantly increased compared with the mice in experimental model group. These results indicate that collagen peptides could play a protect role in glucocorticoid-induced immunosuppression.

Biochemical characterization of a thermophilic hyaluronate lyase TcHly8C from Thermasporomyces composti DSM22891

Hyaluronic acid (HA) is an anionic linear polysaccharide abundantly distributed in the extracellular matrix of mammalian connective, growing, and tumor tissues. Hyaluronidase is used as an important drug diffusion promoter and a tool enzyme to produce HA oligosaccharides. However, there is no thermostable hyaluronidase suitable for application to date. In this study, a thermophilic hyaluronate lyase, TcHly8C, from Thermasporomyces composti DSM22891 was expressed in Escherichia coli. The recombinant TcHly8C was most active at 70 °C, and it retained about 30% of initial activity after incubation at 60 °C for 28 days. The half-lives of TcHly8C at 60 °C and 70 °C were 16.1 d and 2.3 h, respectively. The optimum pH of TcHly8C is 5.93, and it was stable at pH 6.15–10.90. The presence of Mg2+ could enhance its enzymatic activity significantly. Km, kcat, and kcat/Km of TcHly8C towards HA were 3.69 mg∙ml−1, 17.82 s−1, and 4.82 ml∙mg−1∙s−1, respectively. TcHly8C degraded HA in an exolytic mode, and the end product was unsaturated HA disaccharide (ΔUA-GlcNAc). Overall, our results show that TcHly8C is the first reported PL8 exo-type hyaluronate lyase with high thermostability, which provides a potential enzyme used in medicine and production of HA oligosaccharides.

Collagen extract obtained from Nile tilapia (Oreochromis niloticus L.) skin accelerates wound healing in rat model via up regulating VEGF, bFGF, and \u03b1-SMA genes expression

BackgroundCollagen is the most abundant structural protein in the mammalian connective tissue and represents approximately 30% of animal protein. The current study evaluated the potential capacity of collagen extract derived from Nile tilapia skin in improving the cutaneous wound healing in rats and investigated the underlying possible mechanisms. A rat model was used, and the experimental design included a control group (CG) and the tilapia collagen treated group (TCG). Full-thickness wounds were conducted on the back of all the rats under general anesthesia, then the tilapia collagen extract was applied topically on the wound area of TCG. Wound areas of the two experimental groups were measured on days 0, 3, 6, 9, 12, and 15 post-wounding. The stages of the wound granulation tissues were detected by histopathologic examination and the expression of vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-ß1) were investigated using immunohistochemistry. Moreover, relative gene expression analysis of transforming growth factor-beta (TGF-ß1), basic fibroblast growth factor (bFGF), and alpha-smooth muscle actin (α-SMA) were quantified by real-time qPCR.ResultsThe histopathological assessment showed noticeable signs of skin healing in TCG compared to CG. Immunohistochemistry results revealed remarkable enhancement in the expression levels of VEGF and TGF-β1 in TCG. Furthermore, TCG exhibited marked upregulation in the VEGF, bFGF, and α-SMA genes expression. These findings suggested that the topical application of Nile tilapia collagen extract can promote the cutaneous wound healing process in rats, which could be attributed to its stimulating effect on recruiting and activating macrophages to produce chemotactic growth factors, fibroblast proliferation, and angiogenesis.ConclusionsThe collagen extract could, therefore, be a potential biomaterial for cutaneous wound healing therapeutics.

Sub-Ångstrom structure of collagen model peptide (GPO)10 shows a hydrated triple helix with pitch variation and two proline ring conformations

Collagens are large structural proteins that are prevalent in mammalian connective tissue. Peptides designed to include a glycine-proline-hydroxyproline (GPO) amino acid triad are biomimetic analogs of the collagen triple helix, a fold that is a hallmark of collagen-like sequences. To inform the rational engineering of collagen-like peptides and proteins for food systems, we report the crystal structure of the (GPO)10 peptide at 0.89-Å resolution, solved using direct methods. We determined that a single chain in the asymmetric unit forms a pseudo-hexagonal network of triple helices that have a pitch variation consistent with the model 7/2 helix (3.5 residues per turn). The proline rings occupied one of two states, while the helix was found to have a well-defined hydration shell involved in the stabilization of the inter-helix crystal network. This structure offers a new high-resolution basis for understanding the hierarchical assembly of native collagens, which will aid the food industry in engineering new sustainable food systems.

The Relationship of Collagen Structural and Compositional Heterogeneity to Tissue Mechanical Properties: A Chemical Perspective.

Collagen is the primary protein component in mammalian connective tissues. Over the last 20 years, evidence has mounted that collagen matrices exhibit substantial heterogeneity in their hierarchical structures and that this heterogeneity plays important roles in both structure and function. Herein, an overview of studies addressing the nanoscale compositional and structural heterogeneity is provided and connected to work exploring the mechanical implications for a number of tissues.

Alteration in immune responses toward N-deacetylation of hyaluronic acid.

Hyaluronic acid (HA) is an ubiquitous nonsulfated glycosaminoglycan of the extracellular matrix in all mammalian connective tissues. Along with the age growth, HA will lose its N-acetyl groups in vivo; however, the significance of this physiological process remains largely unknown. Herein, three highly N-deacetylated HAs (dHAs), dHA-5 kDa (Mw: 5 kDa, DD: 100%), dHA-16 kDa (Mw: 16 kDa, DD: 94%) and dHA-110 kDa (Mw: 110 kDa, DD: 72%), were generated after hydrazinolysis. Their capability in the activation of antigen-presenting cells (APCs) was compared with that of their respective HAs. Our results demonstrated that both HAs and dHAs could activate the nuclear factor-kappa B (NF-κB) transcription factor in APCs and induced cytokine production through the Toll-like receptor (TLR)/MyD88 pathway. Notably, the capacity of dHAs in cytokine induction was much lower than that of HAs. In addition, the TLR-2 pathway was much involved following the appearance of zwitterionic motifs in dHAs. Thus, our findings highlight that N-deacetylation renders HA divergences in immune response, which might be implicated in age-induced functional change in endogenous glycosaminoglycans due to the structural modification in vivo.

Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices

The collagen matrix constitutes the primary extracellular matrix (ECM) portion of mammalian connective tissues in which the interaction of the cell and the surrounding collagen fibers has a significant impact on cell and tissue physiology, including morphogenesis, development and motility. Discoidin domain receptors (DDR1 and DDR2) have been identified as the receptor tyrosine kinases that are activated upon collagen binding. However, there is a lack of evidence regarding the effect of DDRs on the mechanical interaction between fibroblasts and ECM. In this study, we demonstrated that one of the major phosphotyrosine proteins in human fibroblasts during 3D collagen matrix polymerization is DDR2. Treatment of fibroblasts in 3D collagen matrices with platelet-derived growth factor (PDFG) has been shown to increase DDR2 phosphorylation. Silencing of DDR2 with siRNA in fibroblasts significantly reduced the number of dendritic extensions regardless of whether cells were cultured in the collagen or fibronectin 3D matrices. Decreasing dendritic extensions in DDR2-silenced cells has also been shown to decrease the ability of fibroblast entanglement to collagen fibrils in 3D collagen matrices. Finally, we also showed that the silencing of DDR2 decreased the cell migration in 3D nested collagen matrices but had no effect on 3D floating matrix contraction. Collectively, these results suggest that DDR2 functioning is required for the membrane dynamics to control the mechanical attachment of fibroblasts to the 3D collagen matrices in an integrin-independent manner.

A tissue‐engineered human dermal construct utilizing fibroblasts and transforming growth factor β1 to promote elastogenesis

Numerous studies have shown that extracellular matrix (ECM)-based scaffolds are suitable for dermal constructs for the differentiation of various cell types in vitro and for constructive tissue remodeling after implantation in vivo. However, a shortcoming of these ECM materials is its limited elastogenesis. Elastic fibers constitute an essential component of mammalian connective tissue and the presence of elastic fibers is crucial for the proper function of the cardiovascular, pulmonary, and intestinal systems. Since it is still largely unknown how cells coordinate the molecular events of elastic-fiber assembly, understanding the ability to regenerate elastic fibers in tissues remains a significant challenge. For this reason, human neonatal dermal fibroblasts (HDFneo) were analyzed for their potential to serve as a cell culture model for elastic fiber assembly. Using optical technologies such as multiphoton laser-scanning microscopy (MPSLM) we demonstrate that HDFneo stimulated with transforming growth factor β1 (TGF-β1) are able to produce a distinct and complex elastic fiber system in vitro. As shown by the desmosine and isodesmosine content, crosslinked elastic fibers were formed within the 3D ECM-based scaffold. This tissue-engineered dermal construct may prove to be an effective template for the development of medicinal approaches in regenerative soft skin tissue reconstruction through TGF-β1 induction.

Mast cells are present in the choroid of the normal eye in most vertebrate classes

Mast cells are bone marrow-derived tissue-homing leukocytes, which have traditionally been regarded as effector cells in allergic disorders, responses against parasites, and regulation of blood flow, but a broader perspective of their functional heterogeneity, such as immunomodulation, angiogenesis, tissue repair, and remodeling after injury, is now emerging. The persistence of mast cells in connective tissues throughout the evolution of vertebrates is evidence of strong selective pressure suggesting that these cells must have multiple beneficial and important roles in normal homeostasis. While mast cells are present within the uveal tract of eutherian mammals, there is little known about their presence in the choroid of other vertebrate classes. Eye tissues from a range of vertebrate species (fish, amphibian, reptiles, birds, marsupials, monotreme, and eutherian mammals) were investigated. Tissues were fixed in either 2% glutaraldehyde, 2% paraformaldehyde or a mixture of both and processed for resin embedding. Semi-thin sections of the retina and choroid were cut and stained with toluidine blue. Mast cells were identified in the choroid of all classes of vertebrates investigated except sharks. Their morphology, location, and staining characteristics were remarkably similar from teleost fish through to eutherian mammals and bore close morphological resemblance to mammalian connective tissue mast cells. The similar morphology and distribution of mast cells in the choroid of all vertebrate classes studied suggest a basic physiological function that has been retained since the evolution of the vertebrate eye.

Molecular and structural mapping of collagen fibril interactions

The fibrous collagens form the structural basis of all mammalian connective tissues, including the vasculature, dermis, bones, tendons, cartilage, and those tissues that support organs such as the heart, kindneys, liver, and lungs. The helical structure of collagen has been extensively studied but in addition to its helical character, its molecular packing arrangement (in its aggregated or fibrillar form) and the presence of specific amino acid sequences govern collagen's in vivo functions. Collagen's molecular packing arrangement helps control cellular communication, attachment and movement, and conveys its tissue-specific biomechanical properties. Recent progress in understanding collagen's molecular packing, fibrillar structure, domain organization, and extracellular matrix (ECM) interactions in light of X-ray fiber diffraction data provides significant new insights into how the ECM is organized and functions. In this review, the hierarchy of fibrillar collagen structure is discussed in the context of how this organization affects ECM–“ligand” interactions, with specific attention to collagenolysis, integrins, fibronection, glycoprotein VI receptor (GPVI), and proteoglycans (PG). Understanding the complex structure of collagen and its attached ligands should provide new insights into tissue growth, development, regeneration, and disease.

Molecular Architecture of the Collagen Based Extra-Cellular Matrix

The fibrous collagens are the fundamental constituents of the Extracellular Matrix (ECM) of animals, forming the structural basis of all known mammalian connective tissues and organ systems (1). Yet, despite the fundamental biological importance of collagen, many of us are perplexed by the complexity of the assemblies that the collagens form. This is particularly true at what may be the most significant aspect of collagen structure from a cellular point of view, at the intermediate sub-fibrillar and at fibril surface levels (i.e. collagens molecular packing) where many important biological processes occur in growth, development and disease (1, 2). These include but are not limited to: fibrillogenesis, tissue remolding and in forming the scaffolding upon which organ systems, bones, cartilage, etc., i.e. the animal body, are built upon. Clearly, obtaining an unambiguous and contextualized visualization of collagen molecules would be of significant value to the scientific community. We have recently determined the structure of the type I collagen microfibril (3) and fibril (2) at the molecular level from whole intact rat-tail tendons and produced an initial one dimensional structure for type II collagen from lamprey notochord. Using these data, it is possible to map the amino acid chemistry, ligand binding data and other observations onto the defining shape modality of the fibrillar collagen ECM. In so doing, we have been able to propose the first fibrillar based mechanism of collagenolysis and provide a number of illuminating observations regarding other collagen fibril - ligand interactions involving cell adhesion and matrix organization.1. Sweeney S et al. (2008) “Candidate cell and matrix interaction domains…” J Biol Chem 283:21187-97.2. Perumal et al., (2008) “Collagen fibril architecture,… govern its proteolysis.” PNAS 105:2824-9.3. Orgel J et al (2006) Microfibrillar structure of type I collagen in situ. PNAS 103:9001-5.

Genetic and orthopedic aspects of collagen disorders

'Collagens' are a family of structurally related proteins that play a wide variety of roles in the extracellular matrix. To date, there are at least 29 known types of collagen. Accordingly, abnormality in the various collagens produces a large category of diseases with heterogeneous symptoms. This review presents genetic and orthopedic aspects of type II, IX, and XI collagen disorders. Although a diverse group of conditions, mutation of collagens affecting the articular cartilage typically produces an epiphyseal skeletal dysplasia phenotype. Often, the ocular or auditory systems or both are also involved. Treatment of these collagenopathies is symptomatic and individualized. Study of tissue from animal models allows examination of mutation effects on the abnormal protein structure and function. The collagen superfamily comprises an important structural protein in mammalian connective tissue. Mutation of collagens produces a wide variety of genetic disorders, and those mutations affecting types II, IX, and XI collagens produce an overlapping spectrum of skeletal dysplasias. Findings range from lethal to mild, depending on the mutation of the collagen gene and its subsequent effect on the structure and/or metabolism of the resultant procollagen and/or collagen protein and its function in the body.

Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis

We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

Microfibrillar structure of type I collagen in situ

The fibrous collagens are ubiquitous in animals and form the structural basis of all mammalian connective tissues, including those of the heart, vasculature, skin, cornea, bones, and tendons. However, in comparison with what is known of their production, turnover and physiological structure, very little is understood regarding the three-dimensional arrangement of collagen molecules in naturally occurring fibrils. This knowledge may provide insight into key biological processes such as fibrillo-genesis and tissue remodeling and into diseases such as heart disease and cancer. Here we present a crystallographic determination of the collagen type I supermolecular structure, where the molecular conformation of each collagen segment found within the naturally occurring crystallographic unit cell has been defined (P1, a approximately 40.0 A, b approximately 27.0 A, c approximately 678 A, alpha approximately 89.2 degrees , beta approximately 94.6 degrees , gamma approximately 105.6 degrees ; reflections: 414, overlapping, 232, and nonoverlapping, 182; resolution, 5.16 A axial and 11.1 A equatorial). This structure shows that the molecular packing topology of the collagen molecule is such that packing neighbors are arranged to form a supertwisted (discontinuous) right-handed microfibril that interdigitates with neighboring microfibrils. This interdigitation establishes the crystallographic superlattice, which is formed of quasihexagonally packed collagen molecules. In addition, the molecular packing structure of collagen shown here provides information concerning the potential modes of action of two prominent molecules involved in human health and disease: decorin and the Matrix Metallo-Proteinase (MMP) collagenase.

Tissue specific differences in fibrillin microfibrils analysed using single particle image analysis

Fibrillin microfibrils endow mammalian connective tissues with elasticity and play a fundamental role in the deposition of elastin. The microfibrils are 57 nm periodic supramolecular protein polymers with a mass of 2.5 MDa per repeat. The organisation of molecules within a microfibril is still open to debate and structural studies are only just starting to unravel this issue. The contribution of microfibril associated proteins to microfibril ultrastructure and whether there are any tissue specific differences in microfibril structure is still unknown. Therefore, we have used low dose electron microscopy, single particle image analysis and atomic force microscopy to study the structure of fibrillin microfibrils from different tissues. EM images of microfibrils from aorta, ciliary zonules and vitreous humor were collected and more than 500 microfibril repeats from each sample were subjected to averaging. Averages from each sample were analysed using axial stain exclusion patterns and difference images to detect any variations between them. The overall morphology of fibrillin microfibrils was conserved between tissues and there were only very minor differences in the bead and shoulder region of microfibrils. These data suggest that the structure of isolated microfibrils represents the fibrillin scaffold, and either microfibril associated molecules are lost on purification or play only a minor role in microfibril structure.

Evidence for the Intramolecular Pleating Model of Fibrillin Microfibril Organisation from Single Particle Image Analysis

Fibrillin microfibrils endow mammalian connective tissues with elasticity and are fundamental for the deposition of elastin. The microfibrils are 57nm periodic supramolecular protein polymers with a mass of 2.4MDa per repeat. The detailed structure and organisation of most matrix assemblies is poorly understood due to their large size and complexity and it has proved a major challenge to define their structural organisation. Therefore, we have used low dose electron microscopy and single particle image analysis to study the structure of fibrillin microfibrils. Three novel features were detected: a globular feature that bridges the "arm" region, a double band of density crossing the microfibril and stain penetrating holes present in the interbead region, possibly produced by the removal of microfibril associated proteins in the purification procedure. Fine filaments of approximately 2.4nm diameter are resolved in the interbead region, which correspond to the reported diameter of the fibrillin molecule. Comparison of the stain exclusion pattern of microfibrils with the theoretical stain exclusion pattern of fibrillin packing models indicates that the intramolecular pleating model, where each fibrillin molecule is pleated within one microfibril period allowing extensibility by unpleating, has the best fit to the data.

Mechanical forces and signaling in connective tissue cells: cellular mechanisms of detection, transduction, and responses to mechanical deformation

The environment provides mechanical input to tissues through ground reaction forces, gravity, barometric pressure, vibration, and contact with bodies. When tissues are deformed, resident cells are deformed in complex modes. Cells detect “outside-in” mechanical signals by diverse means, including stretch-activated ion channels and other ion channels as the plasma membrane is deformed, through integrin linkages from the matrix to the cytoskeleton, and through cadherins or desmosomes in cell-cell connections. Ion channels, particularly sodium, chloride, potassium, and calcium channels, play an important role in the signal transduced from the mechanical stimulus to a chemical signal. This signal often results in an increase in the concentration of intracellular calcium. This part of the signaling pathway is often blocked by removal of extracellular calcium. Once an initial step in signal transduction has occurred, activation of intracellular pathways initiates phosphorylation events and protein-protein associations including association of integrin β subunits with the cytoskeleton and associated proteins, such as FAK, paxillin, filamin, integrin-linked kinase (ILK), and talin. PLA, PLC, adenyl cyclase, guanyl cyclase, iNOS, and other enzymes are activated producing the second messengers: cAMP, cGMP, ATP, GTP, NO, PGE 2 , IP 3 , and DAG that act, not only in the target cell but also may have paracrine effects. Furthermore, specific pathways may be activated that drive mitogenesis (MEK/MAPK), a stress response (JAK/STAT and JNK), or other responses that result in specific gene expression through activation of transcription factors, cell division, and contraction. The chemical signal can be passed from cell to cell by mediator secretion or through gap junctions. A cell must be able to respond, then desensitize to a mechanical stimulus, just as a receptor may desensitize to a ligand. Therefore, “stop,” or inhibitory signals, are perhaps as important as “go,” or stimulatory signals with respect to a mechanical load response. A cell must be able to attenuate its response to load. This review discusses the recent concepts in mechanical signal detection, transduction, and response pathway in mammalian connective tissue cells.

Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit

Hydroxyl radicals (•OH) may cause non-enzymic scission of polysaccharides in vivo, e.g. in plant cell walls and mammalian connective tissues. To provide a method for detecting the action of endogenous •OH in vivo, we investigated the products formed when polysaccharides were treated with •OH (generated in situ by ascorbate-H2O2-Cu2+ mixtures) followed by NaB3H4. Treatment with •OH increased the number of NaB3H4-reacting groups present in citrus pectin, homogalacturonan and tamarind xyloglucan. This increase is attributed partly to the formation of glycosulose and glycosulosuronic acid residues, which are then reduced back to the original (but radioactive) sugar residues and their epimers by NaB3H4. The glycosulose and glycosulosuronic acid residues were stable for > 16h at 20°C in ethanol or buffer (pH4.7), but were destroyed in alkali. Driselase-digestion of the radiolabelled polysaccharides yielded characteristic patterns of 3H-products, which included galactose and galacturonate from pectin, and isoprimeverose, galactose, glucose and arabinose from xyloglucan. Pectin yielded at least eight 3H-labelled anionic products, separable by electrophoresis at pH3.5. The patterns of radioactive products form useful ‘fingerprints’ by which •OH-attacked polysaccharides may be recognized. Applied to the cell walls of ripening pear (Pyrus communis) fruit, the method gave evidence for progressive •OH radical attack on polysaccharides during the softening process.