Abstract: The mammalian vitreous gel is a specialized type of highly hydrated extracellular matrix, which is composed of interwoven networks of uronic acid-containing polyanionic macromolecules, (i.e., hyaluronan, versican, and IX collagen) and collagen fibrils. Hyaluronan comprises the vast majority of the uronic acid-containing molecules, which contributes to structure and function of vitreous in at least two ways: its unique biophysical and hydrodynamic properties influence the vitreous homeostasis and biomechanics; it is also a template for assembly of other extracellular macromolecules, for example, versican. The other uronic acid-containing molecules namely versican and IX collagen--two chondroitin sulfate (CS) proteoglycans--occur in the vitreous without significant quantitative variations among different mammalians but with some marked variations on the molecular size and sulfation pattern of their chondroitin sulfate side chains. The contribution of versican and IX collagen (through their protein and their CS side chains) to the supramolecular organization of the vitreous gel is poorly understood. However, versican having the ability to bind hyaluronan via its N-terminal and other binding partners via its C-terminal region can play a crucial role on the structural stability and functionality of the vitreous.
Abstract: In the present study, the amounts and the fine structural characteristics of chondroitin sulphate proteoglycans (CSPGs) present in sheep and goat vitreous gels were determined. The results showed that in both examined species hyaluronan was the predominant glycosaminoglycan (GAG), whereas CSPGs were present in minor amounts. CSPGs were identified as versican and collagen IX with versican being the predominant PG type. Fine structural characterization indicated that the CS chains of versican in both mammalian species were of smaller size than those found in collagen IX. The difference in the sulphation pattern of CS chains between versican and collagen IX was also of particular interest. The results indicated that the predominant disaccharide type in CS side chains of versican and collagen IX from both sheep and goat vitreous gels was the 4-sulphated disaccharide. CS chains of versican were found to be richer in 4-sulphated disaccharide units than those in collagen IX, which also contained a significant proportion of non-sulphated disaccharides. These findings showed that, firstly, the CS content and the hydrodynamic size of the CS chain and, secondly, the sulphation pattern of CS chains from versican and collagen IX in both sheep and goat vitreous gels are PG type-dependent.
Abstract: In the present study we investigated whether the single-strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) the four most common beta-globin gene mutations in the Greek population: IVS-I-110, Cd39, IVS-I-1, and IVS-I-6. Using DNA from 50 beta-thalassemic patients and carriers, we amplified by PCR the appropriate 238-bp region of the human beta-globin gene, analyzed the reaction products by nondenaturing polyacrylamide gel electrophoresis, and visualized the bands by silver staining. Single-stranded DNA (ssDNA) fragments showed a reproducible pattern of bands that was characteristic of the mutations present. With the use of control samples containing six of the 10 possible combinations of the four most common beta-globin gene mutations, we were able to predict the mutations present in a quarter of the patients studied. Our predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. We conclude that this non-radioactive PCR-SSCP method can be used to reliably identify mutations in patients, provided that suitable controls are available. Moreover, the method is easy to apply to the identification of mutations in carriers, which makes it particularly useful for population screening.
Abstract: In the present study we investigated whether single-strand conformational polymorphism (SSCP) and polyacrylamide gel electrophoresis (PAGE) could be used for the identification of the CFTR DeltaF508 gene mutation, which is commonest in the Greek population. Using DNA from patients carrying this mutation, the appropriate 98 bp region of the CFTR gene was amplified by PCR and the reaction products were analysed by non-radioactive SSCP-electrophoresis using silver staining for band visualization and non-denaturating PAGE to confirm the results. SSCP electrophoretic analysis has been optimized for several parameters in order to achieve the best resolution. Single-strand DNA fragments gave a reproducible pattern of bands, characteristic for the particular mutation. Comparison of the obtain patterns with control samples allowed the detection of the DeltaF508 mutation in the patients studied by SSCP assay and these results were confirmed by the independent method of PAGE. Although SSCP and PAGE can be used for detection of this mutation, PAGE resulted in more distinct patterns than SSCP. It is, therefore, proposed that PAGE can be reliably used for the detection and identification of such a mutation in patients provided that suitable controls are available. The applicability of PAGE to identification of the mutation in carriers, particularly useful for population screening, is also discussed.
Abstract: The vitreous of all species is composed of essentially the same type of extracellular matrix macromolecules organized to a transparent gel. In this study, the composition and fi ne chemical structure of the glycosaminoglycans (GAGs) in the vitreous gel from sheep and goat were determined and compared with those of human and pig vitreous gels. The results showed that, in all examined species; hyaluronan (HA) was the predominant GAG, whereas chondroitin sulphate (CS) was the minor one. In the vitreous gel of the most relative species, i.e. sheep and goat, higher amounts of both of HA and CS were estimated as compared with pig and human tissues. The distribution of hydrodynamic sizes of HA and CS was significantly differed among different species. All HA preparations consisted of molecules with great variability in hydrodynamic sizes. The relative proportions of the large HA molecules (size >1.8 x 10(6) kDa) were significantly higher in sheep and goat as compared with human and pig vitreous gel. The length of CS chains was also of larger size in sheep and goat (50 and 58 kDa, respectively) than the respective chains in human and pig vitreous gel (38 and 28 kDa, respectively). The sulphation patterns of CS preparations were determined following enzymic treatments, HPLC and capillary electrophoretic analyses. The human vitreous-derived CS chains showed quite different sulphation profile than that of CS isolated from other species, since 4-sulphated disaccharides were identified as the dominant moiety. In conclusion, significant compositional and structural variations between the vitreous matrixes of different species at the GAG level were identified. The functional significance of these species-dependent variations is discussed.
Abstract: The aim of this study was to examine the macromolecular composition of pig vitreous body with particular emphasis on hyaluronan-binding proteoglycans. The whole pig vitreous gel was found to contain 76 microg of hyaluronan-derived uronic acid, 700 microg of total protein and 150 microg of collagen per ml of gel. The contents of neutral hexoses and sialic acids were 80 and 22 microg/ml of vitreous gel, but only a minor proportion of them were found to be associated with the proteoglycan fraction. As estimated by gel chromatography on Sepharose CL-2B, hyaluronan presents a polydisperse hydrodynamic behavior with a lower molecular mass (M(r)) value of 220 kDa. The existence of low amounts of a hyaluronan-binding proteoglycan population with structural and immunological characteristics similar to a member of the hyalectan family, versican, has also been demonstrated. The concentration of this versican-like proteoglycan in whole vitreous accounts for 50 microg proteoglycan protein per ml of vitreous gel and represents a minor proportion (about 7%) of the total protein content. The proteoglycan has an average M(r) of 360 kDa and is substituted by chondroitin sulphate (CS) side chains. Study of the CS sulphation pattern showed that the chains were composed of both type 4- and 6-sulphated disaccharide units.
