The hierarchical response of human corneal collagen to load.

Fibrillar collagen in the human cornea is integral to its function as a transparent lens of precise curvature, and its arrangement is now well-characterised in the literature. While there has been considerable effort to incorporate fibrillar architecture into mechanical models of the cornea, the mechanical response of corneal collagen to small applied loads is not well understood. In this study the fibrillar and molecular response to tensile load was quantified using small and wide angle X-ray scattering (SAXS/WAXS), and digital image correlation (DIC) photography was used to calculate the local strain field that gave rise to the hierarchical changes. A molecular scattering model was used to calculate the tropocollagen tilt relative to the fibril axis and changes associated with applied strain. Changes were measured in the D-period, molecular tilt and the orientation and spacing of the fibrillar and molecular networks. These measurements were summarised into hierarchical deformation mechanisms, which were found to contribute at varying strains. The change in molecular tilt is indicative of a sub-fibrillar "spring-like" deformation mechanism, which was found to account for most of the applied strain under physiological and near-physiological loads. This deformation mechanism may play an important functional role in tissues rich in fibrils of high helical tilt, such as skin and cartilage. STATEMENT OF SIGNIFICANCE: Collagen is the primary mediator of soft tissue biomechanics, and variations in its hierarchical structure convey the varying amounts of structural support necessary for organs to function normally. Here we have examined the structural response of corneal collagen to tensile load using X-rays to probe hierarchies ranging from molecular to fibrillar. We found a previously unreported deformation mechanism whereby molecules, which are helically arranged relative to the axis of their fibril, change in tilt akin to the manner in which a spring stretches. This "spring-like" mechanism accounts for a significant portion of the applied deformation at low strains (<3%). These findings will inform the future design of collagen-based artificial corneas being developed to address world-wide shortages of corneal donor tissue.

Regional distribution of muscarinic and nicotinic cholinergic receptor binding activities in the human brain.

The distribution of nicotinic and muscarinic receptor subtypes was examined in human brain tissue obtained at autopsy from neurologically normal adult (50-60 years) individuals. Membrane preparations from 15 brain regions were examined for nicotinic (L-[3H]nicotine) binding, both M1 and M2 muscarinic receptor binding (distinguished on the basis of pirenzepine affinity) and high (H) and low (L) affinity muscarinic agonist binding (distinguished on the basis of carbachol displacement). Total muscarinic receptor binding sites were relatively high in striatal and cortical areas, where both M1 subtype and agonist binding type L predominated, compared with thalamic, nigral and cerebellar regions and spinal cord, where the M2 subtype and agonist binding type H predominated. Nicotinic receptor binding sites (predominantly high affinity, measured at low ligand concentrations) did not parallel any of the muscarinic subtypes, being concentrated in thalamic, neocortical and striatal regions. Scatchard analysis indicated the presence of both high and low affinity nicotinic sites, the numbers of the latter generally exceeding the former by over one order of magnitude. Neither muscarinic nor nicotinic receptor binding sites were closely related to the distribution of the cholinergic neuronal marker, choline acetyltransferase, suggesting that individual cholinergic pathways may be distinguished by the relative proportion of the different types of cholinergic receptors present.

Cortical somatostatinergic system not affected in Alzheimer's and Parkinson's diseases.

A reduction in the levels of somatostatin-like immunoreactivity (SLI) and somatostatin binding sites in the cerebral cortex has been previously reported to occur in Alzheimer's disease (AD) and Parkinson's disease with associated dementia. In the present study, the levels of both SLI and high affinity [3H]somatostatin binding sites have been measured in the frontal (Brodmann area 9) and temporal (Brodmann area 21) cortices in patients with presenile and senile Alzheimer's disease, and in mentally normal and cognitively impaired cases of Parkinson's disease. The results were compared with those obtained from a group of normal patients matched for age and postmortem delay. No significant changes in SLI or somatostatin binding in the frontal and temporal cortex were found between any of the disease groups. These results suggest that involvement of the somatostatinergic system in AD or Parkinson's disease is not a consistent or primary neurochemical feature of these diseases.

Autoradiographic visualization of binding sites for [3H]somatostatin in the rat brain.

[4-3H][Phe6]somatostatin-14 was used to localize somatostatin binding sites in the rat brain by tritium-film autoradiography. The distribution of binding sites using 0.7 nM [3H]somatostatin confirmed that previously described for iodinated tyrosyl analogues of somatostatin, with highest densities of sites in the cerebral cortex (particularly in laminae III-V), amygdala, lateral septal nucleus, hippocampus and claustrum. Investigation of the pharmacological specificity of the binding sites showed that somatostatin-28, but not its N-terminal dodecapeptide, somatostatin-28 (1-12) or des-Ala1[Gly2,Lys4,Asn5,Thr12,Ser13]somatostatin displaced [3H]somatostatin. Further examination of the binding inhibition characteristics, using a homogenate assay, suggested the presence of two classes of binding sites in the cerebral cortex, hippocampus, midbrain and striatum. The existence of sub-populations of somatostatin binding sites in the rat brain has implications for future studies on the physiological and pharmacological significance of somatostatin receptors in the central nervous system.

Cortical neuropathological and neurochemical substrates of Alzheimer's and Parkinson's diseases.

Whilst the neuropathological correlates of Alzheimer type dementia--cortical neurofibrillary tangles and senile plaques--are well defined, the prevalence of these cortical abnormalities in Parkinson's disease and their relation to dementia is unclear. In a series of 46 consecutive cases of clinically and pathologically established Parkinson's disease the prevalence of mild Alzheimer-type pathology (exceeding the normal but not as extensive as in Alzheimer's disease) was increased 2 to 3 fold compared with an age-matched control group, although there was no obvious relation to the presence or severity of dementia. In a subgroup of Parkinsonian cases (both demented and non-demented), examined neurochemically, there were both similarities (decreased choline acetyltransferase, nicotinic and serotonergic S 1 receptor activities) and distinctions (increased muscarinic receptor binding--particularly to the "L" subtype, and normal serotonergic S 2, somatostatin, and D-aspartate binding together with normal levels of an endogenous nicotine binding inhibitor) compared with a group of cases with Alzheimer's disease. Amongst the various pathological and chemical indices examined, only presynaptic cholinergic markers (including the number of Meynert neurons) and S 1 receptor binding were related to dementia in Parkinson's disease. It is suggested that whilst coincidental classical Alzheimer's disease is infrequent in Parkinson's disease (5% in the present series) Alzheimer's disease itself is distinguished from Parkinson's disease by the formation of numerous neocortical neurofibrillary tangles and a reduction in glutamate uptake, serotonergic S 2 receptors and possibly in endogenous nicotine binding inhibitor.

Regional distribution of high-affinity [3H]somatostatin binding sites in the human brain.

The distribution of high-affinity binding sites for [3H]somatostatin has been studied in membrane preparations from a number of regions of normal human brain. The highest densities of binding sites (greater than 48 fmol/mg protein) were found in the cerebral and cerebellar cortices and the hippocampus, with intermediate binding densities (30-46 fmol/mg protein) being present in the basal ganglia, amygdala, septum and claustrum. The lowest densities of binding sites (less than 14 fmol/mg protein) were observed in the hypothalamus, thalamus and substantia nigra. The binding of [3H]somatostatin in both the frontal cortex and cerebellar cortex demonstrated pharmacological specificity, since somatostatin-28, but not somatostatin-28(1-12) or Des AA1,2,4,5,12,13, D-Trp8-somatostatin, competed for the binding sites. Scatchard analysis of the binding in both frontal cortex and cerebellar cortex revealed the presence of two classes of high-affinity binding sites.

Human cerebellar cortex possesses high affinity binding sites for [3H]somatostatin.

Somatostatin binding sites have been identified in the human brain using [4-3H-(Phe6)]-somatostatin-14. In contrast to that of the rat, the human cerebellar cortex possesses a high density of somatostatin binding sites, comparable to that found in either the rat or human cerebral cortex. Autoradiographic localisation of somatostatin binding sites in the human cerebellum reveals that the highest density is associated with the granule cell layer.

Rat brain membranes possess two high-affinity binding sites for [3H]somatostatin.

This report described the first use of [4-3H-Phe6]somatostatin-14 to characterize binding sites on rat brain membranes for somatostatin-14. This ligand is superior to previously used iodinated analogues and is chemically and biologically identical to the natural ligand. Two high-affinity binding sites were found, from Scatchard analysis of competitive displacement experiments, with Kd SS1 = 0.41 and Kd SS2 = 22.9 nM. Specific binding was reversible, and kinetic analysis of the dissociation and association time-course gave an apparent Kd of 0.44 nM, in good agreement with the Kd of the higher-affinity site. Specific binding of the ligand was enriched in cerebral cortex and hippocampus, with intermediate levels in the striatum, hypothalamus and midbrain, and low levels in the pons/medulla and cerebellum. This ligand should prove to be valuable for elucidating the physiological and pharmacological significance of the two subtypes of somatostatin binding sites we have demonstrated.

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa.

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.