The variability in type I collagen helical pitch is reflected in the D periodic fibrillar structure.

The variability in amino acid axial rise per residue of the collagen helix is a potentially important parameter that is missing in many structural models of fibrillar collagen to date. The significance of this variability has been supported by evidence from collagen axial structures determined by electron microscopy and X-ray diffraction, as well as studies of the local sequence-dependent conformation of the collagen helix. Here, sequence-dependent variation of the axial rise per residue was used to improve the fit between simulated diffraction patterns derived from model structures of the axially projected microfibrillar structure and the observed X-ray diffraction pattern from hydrated rat tail tendon. Structural models were adjusted using a genetic algorithm that allowed a wide range of structures to be tested efficiently. The results show that variation of the axial rise per residue could reduce the difference metric between model and observed data by up to 50%, indicating that such a variable is a necessary part of fibril model structure building. The variation in amino acid translation was also found to be influenced by the number of proline and hydroxyproline residues in the triple helix structure.

Nanoarchitectures of the animal extracellular matrix: opportunities for synchrotron radiation studies on collagen and fibrillin.

The extracellular matrix comprises structures that support the architectural organization of virtually all animal tissues. Within this architecture, two classes of protein assemblies found as long slender fibrils (collagen and fibrillin) characterize the bulk of the extracellular matrix. In both classes of fibrous protein, the molecular organization within a fibril ensures that the properties of the individual molecules transcend to the nanostructural and mesoscopic levels of structural organization and thence the tissue itself. The composition of the fibrils, in conjunction with other biomolecules and their suprafibrillar architecture, facilitates the formation of tissues as diverse as skin, tendon, cornea ciliary zonules and aorta. Here the relative tear resistance, strength, transparency and optical properties are paramount for proper function. Many structural investigations of fibrous protein structure have relied heavily on the use of synchrotron radiation in order to elucidate molecular packing, primarily due to the distinct benefits that X-ray diffraction provides, such as minimal sample preparation, rapid data collection and in situ mechanical testing. In this paper, an overview of the investigations that have revealed different levels of molecular architecture in fibril-based tissues is presented. Emerging future technology and how this can be matched with the pressing questions in extracellular matrix biology are also discussed.

Techniques for measurement of oxygen consumption rates of hepatocytes during attachment and post-attachment.

Three techniques for measuring oxygen consumption rate (OCR) of cultured cells relevant to the development of bioartificial liver devices are reported. In an oxystat apparatus, HepG2 cells immobilised on Cytodex 3 microcarriers at a concentration of 10(6) cells ml-1 had a mean OCR of 0.7 nmol s-1/10(6) cells. The OCR decreased with increasing cell density, a characteristic previously reported for other cell lines. Rat hepatocytes immobilised on single collagen layers in a flow cell and challenged with ammonia had a mean OCR of 0.59 nmol s-1/10(6) cells. A novel two-compartment oxystat system was used to determine the OCR of rat hepatocytes during the attachment phase. OCR declined from 1.0 nmol s-1/10(6) immediately after seeding to 0.7 nmol s-1/10(6) cells at nine hours. The low OCR for HepG2 reflects loss of certain oxygen dependent metabolic pathways. The OCR measured for rat hepatocytes during and post-attachment are significantly higher than those reported elsewhere and have major implications for the development of bioartificial liver devices.