Biomedical subjects

K M Meek

Publications and source records attributed to K M Meek.

60 records · Page 4Linked to original sources

PubMed1979

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy.

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). At pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.

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Immunolocalization of elastin, collagen type I and type III, fibronectin, and vitronectin in extracellular matrix components of normal and myxomatous mitral heart valve chordae tendineae.

The identification, distribution, and localization of matrix proteins and the proteins associated with normal and degenerated elastic fibers and collagen fibrils of myxomatous chordae tendineae were studied with immunoelectron microscopy. Ultrathin sections of L R White-embedded tissue were processed by indirect immunogold cytochemistry using primary antibodies against human alpha elastin, collagen types I and III, fibronectin, and vitronectin. In normal chordae tendineae, alpha elastin antibody heavily labeled the elastic fibers in spongiosa and fibrosa, but microfibrils around them were not labeled. Antibodies to collagen type I, collagen type III, and fibronectin all labeled the collagen fibers and microfibrils in the spongiosa. Fibronectin antibody labeling was higher than collagen type III, whereas labeling by anticollagen type I was lower. Intense labeling by vitronectin was observed on the microfibrils in the spongiosa and on electron-dense material around elastic fibers in the spongiosa and fibrosa. In myxomatous chordae tendineae, alpha elastin antibody heavily labeled degenerated elastic fibers, previously unidentified reticulated structures, and other moderately electron-dense material, both in the spongiosa and in the fibrosa, but not the electron-dense fibrous material around them. Antibodies to collagen types I, III, and fibronectin heavy labeled electron-dense aggregates of fibrous material. Vitronectin labeling was observed on electron-dense longitudinally running microfibrils and on the electron-dense microfibrils around degenerated elastic fibers.

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Ultrastructure abnormalities in proteoglycans, collagen fibrils, and elastic fibers in normal and myxomatous mitral valve chordae tendineae.

Normal and myxomatous chordae tendineae were studied using light and electron microscopy, to assess the alterations in the appearance and mutual arrangement of proteoglycans, collagen fibrils, and elastic fibers. Specific staining with ruthenium red and cuprolinic blue in a critical electrolyte concentration mode were used to localize proteoglycans. Fresh tissues were fixed in glutaraldehyde containing the cationic dyes and embedded into Spurr resin. Semithin sections of LR White (London Resin Co., Basingstoke, U.K.)-embedded tissue were used for histochemistry. In normal chordae tendineae, the fibrosa comprised close-packed collagen fibrils intermixed with elastic fibers. These were surrounded by a thin layer of elastic fibers and collagen fibrils, both of which were closely associated with proteoglycans. In myxomatous chordae tendineae, alterations were observed in the connective tissue. Proteoglycans were more abundant and were distributed throughout the tissue. The outermost layer was transformed into an undifferentiated electron-dense mass surrounding the central fibrosa, which contained degraded elastic fibers and collagen fibrils. Collagen fibrils had faint banding or lacked a banding pattern altogether. Spaces between collagen fibrils were occupied by abnormal proteoglycans or proteoglycan aggregates. Elastic fibers showed varying degrees of degeneration and were occasionally replaced by electron-lucent spaces containing microfibrils. Accumulation of abnormal proteoglycan was also observed around degenerated elastic fibres and collagen fibrils.

Aged↗

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The axial electron density in collagen fibrils from human corneal stroma.

Low angle synchrotron X-ray diffraction patterns were obtained from the demembranated human corneal stroma. These patterns showed differences in the relative intensities of the meridional reflections compared to those obtained using bovine corneas. In particular, the first order reflection, conspicuously absent in the bovine pattern, is present in the human, although it is still only one third as intense as the first order in the pattern from rat tail tendon. Using phases deduced from the electron-optical images of negatively stained corneal collagens, a difference electron density map between bovine and human corneal collagen was drawn. The features in this map are explained in terms of a different inter-relationship between collagen and proteoglycans in the two species. The function of these species differences is unknown.

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Organization of collagen fibrils in the corneal stroma in relation to mechanical properties and surgical practice.

We have used synchrotron x-ray diffraction to obtain quantitative information about the precise orientation of the collagen fibrils in the human cornea and sclera, and how these fuse at the limbus. We have shown that the human corneal stroma has preferred collagen orientation in the inferior-superior and nasal-temporal directions. At the limbus, however, the preferred orientation is tangential to the cornea. We demonstrated that these limbal fibrils are in the form of a circumcorneal annulus and quantified how this annulus varies with position. We have also started to unravel how the corneal collagen fibrils fuse with the collagen in the annulus. The results have both mechanical and surgical implications. Keratoplasty is performed without considering the preferred directions of the collagen fibrils. Surgery is increasingly used to correct astigmatism and myopia. The site and direction of an incision during, for example, cataract surgery, can influence the eventual level of astigmatism. X-ray diffraction helps our understanding of the underlying structural and hence mechanical reasons for refractive problems following surgery.

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PubMedDate not supplied

Hyaluronidase treatment, collagen fibril packing, and normal transparency in rabbit corneas.

PURPOSE: Hyaluronidase treatment is the initial step of corneaplasty, a treatment under development that induces stromal softening and involves the application of a custom designed forming lens to achieve modification of refractive error. The purpose of this investigation was to examine changes in the arrangement of stromal collagen fibrils after hyaluronidase treatment. METHODS: Rabbit corneas were evaluated by slit-lamp microscopy at 0, 2 and 7 days after treatment and haze was assessed by subjective observation. Molecular and interfibrillar Bragg spacing of corneal collagen were measured from synchrotron x-ray scattering patterns. Transmission electron microscopy and digital image analysis were used to calculate radial distribution functions from the positions of collagen fibrils. The calculated fibril sizes and positions were also used to predict the transmission of visible light through these corneas. RESULTS: Hyaluronidase-treated corneas were shown to have a decreased interfibrillar Bragg spacing of 15% to 21%. Fibril hydration did not change. Transparency of these corneas remained unaltered. CONCLUSIONS: Hyaluronidase reduced the hydration of the corneal stroma, which led to a more compacted collagen fibril arrangement. This compression was predicted to cause a small reduction in the transmission of visible light through the cornea but not to a point likely to cause visual impairment.

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