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Biomedical subjects

K M Meek

Publications and source records attributed to K M Meek.

At least 55 records · Page 3Linked to original sources
PubMed1991

Alteration of the stromal architecture and depletion of keratan sulphate proteoglycans in oedematous human corneas: histological, immunochemical and X-ray diffraction evidence.

The structure and content of the extracellular stromal matrix of several oedematous human corneas was investigated using electron microscopy, X-ray diffraction and biochemical techniques. Electron microscopy revealed the presence of wavy lamellae and various sized collagen-free 'lakes' within the stroma of the oedematous corneas, with their posterior sections containing by far the largest 'lakes'. The existence of 'lakes' was supported by the equatorial X-ray diffraction evidence. Staining the oedematous corneas with Cuprolinic blue prior to electron microscopical and meridional X-ray diffraction studies demonstrated a loss of stromal proteoglycans normally associated with collagen. Immunochemical evidence demonstrated reduced levels of antigenic keratan sulphate in the oedematous corneas while biochemical techniques revealed constant chondroitin sulphate levels in the same corneas.

Chondroitin Sulfate Proteoglycans↗
PubMed1991

Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration.

The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern.

Animals↗
PubMed1990

The collagen fibril--a model system for studying the staining and fixation of a protein.

A collagen fibril is made up of long rod-like molecules regularly D-staggered with respect to one another. This means that (i) its axially projected fine structure, resolvable to approximately 2 nm in electron micrographs, repeats D-periodically (D = 67 nm), and (ii) the amino acid residues contributing to each element of the fine structure can be inferred from sequence data. Electron-optical data from a fibril D-period can can therefore be correlated directly with chemical data. Such correlations confirm the electrostatic nature of the staining reaction when a fibril is positively stained. After negative staining, the principal factor determining the small-scale distribution of stain is local exclusion by 'bulky' amino acid side-chains. ('Bulkiness' is the average cross-sectional area, or 'plumpness', of a side-chain.) A small superimposed positive staining contribution can also be detected. Fixation of collagen by aldehydes and diimidoesters occurs via an initial reaction with lysyl (and hydroxylsyl) side-chains and alpha-amino groups, followed by secondary cross-linking reactions that differ from fixative to fixative. These secondary reactions determine the nature and abundance of the cross-links and the extent to which they influence subsequent staining behaviour.

Amino Acid Sequence↗
PubMed1990

Macular corneal dystrophy: reduction in both corneal thickness and collagen interfibrillar spacing.

The interfibrillar spacing of collagen fibrils was measured at twenty different positions across a macular dystrophy cornea using synchrotron X-ray diffraction. Unlike previous work of this type the cornea had not been frozen for storage. The spacings were all significantly lower than the spacings which existed at similar positions across a normal adult human cornea. This close-packing of collagen fibrils seems to be responsible for the reduced thickness of the central cornea in macular dystrophy. Neither the patient's serum or corneal tissue contained appreciable amounts of sulfated keratan sulfate, this classifies the disease as Type I macular corneal dystrophy.

Adult↗
PubMed1989

Macular corneal dystrophy: the macromolecular structure of the stroma observed using electron microscopy and synchrotron X-ray diffraction.

The distribution of sulphated proteoglycans within the stromas of three patients (A,B,C) suffering from macular corneal dystrophy was studied using the specific dye Cuprolinic Blue in a 'critical electrolyte concentration' method. The corneas were examined using transmission electron microscopy and A and C were further studied by low-angle synchroton X-ray diffraction. Sera from all three patients were analyzed for the presence of keratan sulphate using a monoclonal antibody in an enzyme-linked immunosorbent assay. The serum from Patient A contained keratan sulphate, but the chains were thought to be shorter or less sulphate in their sera. Electron microscopy showed many electron-transparent lacunae randomly distributed throughout the specimens. The average collagen fibril diameter was normal but there were differences in packing between the specimens. Specimen A was closely-packed with most collagen fibrils in contact with their neighbours. Specimens B and C showed fewer regions of close packing; in most of the tissue the interfibrillar spacing appeared normal. Staining with Cuprolinic Blue revealed an unusual distribution of proteoglycans in some parts of the interfibrillar matrix, particularly in A, with 'small' proteoglycans running exclusively parallel to the collagen fibrils. Furthermore in A, and to a lesser extent in B and C, some lacunae were filled with clusters of abnormal sulphated proteoglycan filaments (of various sizes) which were chondroitinase ABC susceptible. Clearly defined regions, both within the lacunae and elsewhere, failed to stain with Cuprolinic Blue; this suggests an absence of sulphated proteoglycans within these areas. Equatorial X-ray diffraction of the wet tissues (A and C) gave values for the mean interfibrillar centre-to-centre separation of 43 +/- 2 nm in Specimen A and 52 +/- 3 nm in Specimen C. The differences observed in the serum keratan sulphate levels, the packing of the collagen fibrils and the distribution of chondroitin/dermatan sulphate proteoglycans confirm the heterogeneity that exists within the macular corneal dystrophies.

Chondroitin Sulfates↗
PubMed1988

Bovine corneal stroma contains a structural glycoprotein located in the gap region of the collagen fibrils.

Treatment of bovine corneal stroma using SDS-containing extracting solutions removes a 135,000 MW glycoprotein from the main collagen framework of the tissue. Low-angle synchrotron X-ray diffraction patterns obtained from corneas extracted in this way indicate that the glycoprotein has been removed from the gap regions of the collagen fibrils and is thus an important structural component of the corneal stroma. The glycoprotein (GP 135) shares a number of properties with one of the subunits of type VI collagen, but tests have so far failed to establish their identity.

Amino Acids↗
PubMed1988

Axial electron density of human scleral collagen. Location of proteoglycans by x-ray diffraction.

The low angle meridional x-ray diffraction pattern from fresh human sclera was analyzed to ascertain if collagen-bound proteoglycans affect the axially-projected electron density distribution to the same extent as appears to occur in the cornea. The results showed that, unlike cornea, the electron density of the sclera is similar to that seen in rat tail tendon collagen. The proteoglycans were specifically stained using either Cuprolinic blue or Cupromeronic blue, both under critical electrolyte conditions. The tissue was then examined by electron microscopy and by low angle x-ray diffraction. The electron-optical observations suggested that proteoglycans associate with collagen near the d/e staining bands in the gap zone. A difference Fourier analysis from the x-ray results confirmed that these observations were not e.m. preparative artefacts and allowed a quantitative estimate to be made of the axial extent of the proteglycans in the wet tissue.

Animals↗
PubMed1987

The organisation of collagen fibrils in the human corneal stroma: a synchrotron X-ray diffraction study.

The low angle equatorial diffraction pattern from the human corneal stroma shows that the collagen fibrils have two preferred orientations: inferior-superior and medial-lateral. We have not observed this effect in any other animal species. This arrangement, which was found to be more pronounced in the posterior than in the anterior stroma, was maintained until the last 1 to 2 millimetres before the limbus at which point uniaxial orientation was observed along the circumference. Our interpretation of this result is that most collagen fibrils wrap around the circumference of the cornea and relatively few continue radially into the limbus where uniformity of collagen fibril diameters is lost.

Aged↗
PubMed1986

A synchrotron X-ray diffraction study of bovine cornea stained with cupromeronic blue.

Bovine corneal stroma was stained with the dye Cupromeronic Blue under "critical electrolyte" conditions in order to locate the proteoglycans specifically. Electron microscopy revealed that most of the stained proteoglycan filaments are randomly distributed in the tissue although there are many regions where a close proteoglycan-collagen relationship is evident. On counterstaining the collagen in order to identify the intra-periodic banding pattern, the proteoglycan filaments were seen to predominate at three axial positions within the collagen D-period corresponding to electron-optical staining bands a, c and d/e. X-ray diffraction patterns were obtained from the stained and unstained corneae using a high intensity synchrotron source. Integrated intensities from the meridional patterns were used to calculate a difference electron density distribution which revealed where the collagen was axially modified by the stain. This distribution indicated the presence of the dye periodically associated with the collagen at three major sites, one adjacent to each gap/overlap junction (at electron-optical staining bands a and c) and one in the gap zone (including staining bands d and e). However, the Cupromeronic Blue treatment is estimated to have contributed only about 12% to the variation in electron density along the collagen. The staining also gave rise to a greatly increased level of diffuse background scatter, which, in agreement with the electron-optical observations, indicates that much of the stained material is not ordered with respect to the collagen but is randomly distributed in the tissue.

Animals↗
PubMed1985

Glutaraldehyde-induced changes in the axially projected fine structure of collagen fibrils.

The fine structure of the collagen fibril, as seen in axial projection, is changed by treatment with glutaraldehyde. The changes are detectable in electron-optical staining patterns and in the intensities of the low-angle meridional X-ray diffraction maxima. Current knowledge of the amino acid sequence of collagen and of the axial arrangement of molecules in fibrils permits interpretation in terms of specific alterations to the axial distribution of electron density along the fibril. Analysis of fibril staining patterns from glutaraldehyde-treated calf skin collagen shows that uptake of staining ions in positive staining patterns is inhibited at residues known to interact with glutaraldehyde (lysyl, hydroxylysyl and probably histidyl side-chains) and on other charged residues in the immediate neighbourhood of the glutaraldehyde-reactive residues. This can be seen as a "stain-exclusion effect" due to the presence of bulky polymeric complexes of glutaraldehyde molecules at cross-linking sites. Such stain exclusion accounts for the drastic changes in the negative staining pattern following treatment with glutaraldehyde. The intensity changes observed in the low-angle meridional X-ray reflections from rat tail tendon, similarly treated, also can be explained by the presence of these bulky complexes. Existing data have been used to predict a model of the altered electron density profile indicating the axial distribution of glutaraldehyde along a D-period of moist tendon collagen.

Aldehydes↗
PubMed1985

An X-ray diffraction analysis of rat tail tendons treated with Cupromeronic Blue.

Cupromeronic Blue was used to stain selectively the proteoglycans in rat tail tendons under 'critical electrolyte' conditions. Earlier electron microscopical observations indicated that at least one type of proteoglycan filament is associated with tendon collagen fibrils at the positive staining band 'd'. To ensure that this was not an artefact caused by specimen preparation or the subsequent positive staining of the collagen fibrils, we have analysed low angle meridional diffraction patterns from stained but not dehydrated, embedded or counterstained tissues. Axial electron density profiles of Cupromeronic Blue-stained compared with unstained rat tail tendons revealed the axial locations and relative amounts of dye in both mature and young wet specimens. In mature tendons, the difference electron density profile contained a broad peak centred near residue 180 along the 234-residue D-period. This corresponds to the electron-optical staining band 'd'. In young tendons a similar distribution of stain was observed although in this case there was evidence of a doublet of peaks, one centred near residue 182 (band 'd') and the other near residue 165 (midway between bands d and e1). The wet proteoglycan--Cupromeronic Blue complexes distribute over about 30 nm along the collagen fibril axis. Comparison with the images of filaments seen in the electron microscope suggests that the dye complexes collapse significantly on dehydration and embedding.

Aging↗
PubMed1981

The use of glutaraldehyde and tannic acid to preserve reconstituted collagen for electron microscopy.

The effects of glutaraldehyde and tannic acid on the axial periodicity of collagen have measured. Both fixatives produce axial shrinkage of the collagen but whereas glutaraldehyde produces 7% shrinkage, tannic acid produces only 2% shrinkage. The technique of carbon/platinum shadowing was used to estimate the extent to which the collagen fibrils flatten down when they are dried onto grids for electron microscopy without prior embedding and sectioning. The influence of fixation was studied and it was found that minimum distortion occurred when both tannic acid and glutaraldehyde were used to preserve the protein structure.

Aldehydes↗
PubMed1981

The distribution of electron density in corneal collagen fibrils.

Using high intensity synchrotron X-ray diffraction data, an electron density profile has been obtained for the collagen fibrils in corneal stroma at higher resolution than has been possible before. The details within this electron density distribution are discussed in terms of the collagen and its interactions with other components present in the tissue.

Collagen↗
PubMed1979

The staining pattern of collagen fibrils. Improved correlation with sequence data.

The periodic banding pattern of stained collagen fibrils observed in the electron microscopic can be correlated with the charge distribution deduced from the amino acid sequence. Earlier work used alpha 1 chain sequence data only. The present study incorporates alpha 2 as well as alpha 1 sequence data, so that the complete distribution of charged residues is used. Correlation is improved if it is supposed that the extrahelical terminal regions are contracted. The optimal value of the periodicity, D, (previously 232.3 +/- 0.5 residues using alpha 1 data only), is now 234.2 +/- 0.5 residues, assuming uniform spacing of residues in the helical body of the molecule. This value agrees better with values obtained by others from analyses of interactions between molecules, using sequence data alone. Using the improved value of D, the relative axial locations of the charged residues in the fibril are displayed. In this way, the charged residues contributing to each band in the fibril staining pattern can be identified.

Amino Acid Sequence↗