Katharine M. Dyne

Deficient expression of the small proteoglycan decorin in a case of severe/lethal osteogenesis imperfecta.

In osteogenesis imperfecta (OI) the effects of mutations in type I collagen genes generally reflect their nature and localization. Unrelated individuals sharing identical mutations present, in general, similar clinical phenotypes. However, in some such cases the clinical phenotype differs. This variable clinical expression could be the result of abnormalities in other connective tissue proteins. Since decorin is a component of connective tissue, binds to type I collagen fibrils and plays a role in matrix assembly, we studied decorin production in skin fibroblasts from OI patients. Cultured fibroblasts from one patient with extremely severe osteogenesis imperfecta (classified as type II/III) who has an alpha 1(I)gly415ser mutation were found to secrete barely detectable amounts of decorin into culture medium. Western blotting using antibodies raised against decorin confirmed the reduction of the decorin core protein and Northern blot analysis showed decorin mRNA levels below the limit of detection. Cells from a patient, with a less severe phenotype, bearing a mutation in the same position of the triple helix (alpha 1(I)gly415) expressed decorin normally. The different clinical phenotypes could be due to the differing genetic backgrounds of the patients so it is tempting to conclude that in our most severely affected patient the absence of decorin aggravates the clinical phenotype.

Misdiagnoses in 1,250 Consecutive Patients Admitted to an Acute Stroke Unit

Admission to an acute stroke unit is decided on the basis of clinical and computed tomographic data collected in emergency, leading to a risk of misdiagnosis. The aim of this study was to evaluate the

Direct monitoring of prolidase activity in cultured skin fibroblasts using capillary electrophoresis.

Capillary electrophoresis (CE) was used as an alternative to current analysis schemes for detecting prolidase activity in erythrocytes and skin fibroblast cultures because of its unique selectivity and high resolving power. Kinetic measurement of peptide bond hydrolysis was performed using porcine kidney prolidase on different substrates (Gly-Pro, Leu-Pro and Ala-Pro) and by following the disappearance of the peptide-substrates peak. The K(m) values obtained were in agreement with those previously reported. Interestingly, in the case of Phe-Pro as the substrate, simultaneous analysis of the product and parent peptide was possible, thus showing the superiority of the capillary electrophoresis (CE) assay with respect to the standard spectrophotometric method. The application of the CE technique to the characterization of prolidase activity in control and prolidase-deficient skin cultured fibroblasts was successful. Enzyme activity was easily calculated in all controls tested and the K(m) values determined were slightly lower than those obtained with the colorimetric reaction, thus confirming our assumption that the CE assay shows higher specificity than the ninhydrin technique. Our results demonstrate the feasibility of using CE as a simple and reliable technique for determining prolidase activity.

Effect of different surfactants on the separation by micellar electrokinetic chromatography of a complex mixture of dipeptides in urine of prolidase-deficient patients.

Prolidase deficiency is a severe disorder characterized by massive excretion of metabolites with closely related structures. At present, micellar electrokinetic chromatography is the separation method which provides the highest selectivity of structurally similar solutes. However, the structure of a surfactant can greatly affect the selectivity of separation depending on factors such as the length of hydrophobic alkyl chain or the nature of the hydrophilic group. Here we investigated the effect of three non-ionic and four anionic detergents for obtaining the best separation conditions for resolving imidodipeptide mixtures. The effect on resolution of variables such as temperature, surfactant concentrations and organic solvents was also examined. The greatest resolution was obtained at the lowest temperature studied (10 degrees C) using 50 mM sodium borate, pH 9.3 containing 50 mM pentanesulfonate and 10% (v/v) methanol. Under these experimental conditions almost all excreted components were baseline separated and identified.

BIOCHEMICAL, MORPHOLOGICAL AND STEREOLOGICAL STUDY OF THE DERMIS IN THREE MEMBERS OF A LARGE FAMILY WITH TYPE IV EHLERS-DANLOS SYNDROME

Biochemical, morphological and stereological studies were carried out on dermal biopsies obtained from three members of a large family with a positive clinical history of type IV Ehlers-Danlos syndrome. Ultrastructural analysis showed that fibroblasts from two affected individuals presented abnormally dilated rough endoplasmic reticulum cisternae engorged by microfilamentous material. When cultured, fibroblasts from two affected individuals synthesized and secreted normal amounts of type I procollagen, but only a very low percentage of type III procollagen was secreted. Cellular retention of type III procollagen was confirmed by immunofluorescence. Also the secretion of fibronectin appeared delayed. Stereological analysis carried out on semithin sections of dermis by the point counting method showed that the relative volume of collagen fibers was decreased in the reticular dermis and the relative volume of elastin fibers was increased mainly in the upper layer of reticular dermis, in comparison to normal controls (P less than 0.01). Collagen fiber sizes were significantly (P less than 0.01) reduced in all dermis layers. No alterations were seen in the dermis and in cultured fibroblasts from the clinically normal individual.

Type I procollagen in the severe non-lethal form of osteogenesis imperfecta.

SummaryWe have screened type I procollagen synthesized in vitro by skin fibroblasts from several patients with the severe non-lethal form of osteogenesis imperfecta. Cells from one patient synthesized and secreted both normal and a larger amount of abnormal type I procollagen. The abnormal alpha chains are larger in size due to post-translational overmodifications involving the whole triple helical domain. Abnormal collagen heterotrimers had a melting temperature 2.5°–3°C lower than normal ones or from controls. Chemical analysis of collagen in the medium showed a greater degree of both lysyl hydroxylation and hydroxylysyl glycosylation, the major increase in molecular mass of overmodified alpha chains being due to the higher hydroxylysine-bound hexose content. The probands cells modify proteoglycan metabolism and mineral probands cells modify proteoglycan metabolism and mineral crystals form in the dermis, possibly a response to abnormal collagen-proteoglycan interactions. These findings can be explained by a small defect in the product of one allele for pro-α1(I) chains: three-quarters of the synthesized type I procollagen molecules are composed of trimers containing one or two chains defective near the C-terminus of the triple helix or in the C-propeptide. The data obtained for this patient confirmed that the severity of clinical manifestations in osteogenesis imperfecta strongly depends on the location and nature of the mutations, and that the phenotype could be a consequence of a collagen defect(s) and its influence on collagen-collagen interactions and collagen interactions with other connective tissue components.

Deposition of mutant type I collagen in the extracellular matrix of cultured dermal fibroblasts in osteogenesis imperfecta.

To study how mutant type I collagen interferes with matrix deposition we investigated the extracellular matrix produced by cultured skin fibroblasts in thirteen patients affected by different forms of Osteogenesis Imperfecta. Two different approaches were used: a) the pericellular matrix produced during 24 h label was analyzed by SDS-PAGE; b) type I collagen present in the insoluble cell-layer fraction in long-term cultures was studied. Results showed that a very small amount of abnormal type I trimers were present regardless of the clinical phenotype. In only two cases mutant chains were clearly incorporated. These data indicate a selective deposition of normal collagen trimers over abnormal ones. Moreover, in long-term cultures a decreased amount of type I collagen was deposited as indicated by the relative increase in type V collagen. These data are discussed in light of results found in bone by other authors and suggest that decreased deposition of type I collagen could be a general feature in OI and not limited to null-allele OI probands.

Severe Nonlethal Osteogenesis Imperfecta: Biochemical Heterogeneity

Our studies on osteogenesis imperfecta (01) started in the middle 1970s as a consequence of the interest of our laboratory in connective tissue macromolecules and their disorders, such as mucopolysaccharidoses, lathyrism, and Larsen’s syndrome. Osteogenesis imperfecta is not a widespread disease in Italy (although quantitative data for its frequency has never been reported). It is mainly for this reason that family physicians and the great majority of clinical doctors are unaware of the disease (very few laboratories recently started their work at both the protein and nucleic-acid level). This situation recently began to improve when the Italian Association of affected families (As.It.O.1.) was founded, thanks to Professor L. Lenzi’s efforts, an orthopedic surgeon who had a great scientific and clinical interest in connective tissue diseases. Previous studies on the metabolism of collagen and proteoglycans showed alterations in several affected patients’-’ concerning differences in the relative amount of type I and I11 collagens in skin, and, more extensively, for the posttranslational modifications (hydroxylation of prolyl and lysyl residues and glycosylation of hydroxyl ysine). A step forward in the understanding of the molecular basis of the disease was made possible with the use of dermal fibroblast cultures that produce a large amount of type I and type 111 collagen, and, more importantly, that express the type I collagen defect in these in vitro conditions. While the majority of the patients whose collagen defects have been elucidated to date were affected with lethal 01, we had the opportunity to deal mainly with subjects affected with the severe nonlethal form of the disease. The knowledge of the pattern of inheritance for many of our patients is often