Proliferation characteristics and polyploidization of cultured myofibroblasts from a patient with fibroblastic rheumatism.

Fibroblast-like cells were obtained from a nodule of a patient with fibroblastic rheumatism, and grown in culture for different times (from passage 3 to 21). These cells as well as the fibroblasts taken from an unaffected skin area (controls) of the same patient, have been investigated by fluorescence microscopy, cytochemical methods and cytometry, to evaluate their cytodifferentiation features and cytokinetic characteristics. In addition, in low-passage cultures, the secretion of collagen and of non-collagenic proteins was evaluated using electrophoretic techniques.

Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts.

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 microM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.

Human cells unable to express decoron produced disorganized extracellular matrix lacking "shape modules" (interfibrillar proteoglycan bridges).

The shapes of extracellular matrices are determined by positioning collagen fibrils in the right places, oriented and maintained viv-à-vis each other. The fibrils are linked orthogonally by dermatan/chondroitin sulfates or keratan sulfate (in small proteoglycans) attached every approximately 65 nm via their protein moieties to collagen fibrils at specific binding sites. These regular repeating structures are the "shape modules.

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.

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-substrate's peak. The K(m) values obtained were in agreement with those previously reported.

Complete resolution of imidodipeptide mixtures in urine of prolidase-deficient patients using micellar electrokinetic chromatography.

The use of capillary zone electrophoresis as an efficient method for the identification of urinary imidodipeptides of prolidase-deficient patients has already been reported. However, owing to the complexity of the components excreted, the resolution of electrophoretic patterns obtained was poor. Here we examine the use of micellar electrokinetic chromatography to enhance peak resolution in order to obtain better insight into the electropherograms of patients' urine.

Use of capillary zone electrophoresis for analysis of imidodipeptides in urine of prolidase-deficient patients.

Prolidase deficiency (PD) is characterized by massive urinary excretion of imidodipeptides X-Pro and X-Hyp. We report the applicability of capillary zone electrophoresis to urinary imidodipeptide determination. The protocol is fast, simple, reliable, only small amounts of sample are required and there is minimal sample preparation.

Type I collagen CNBr peptides: species and behavior in solution.

The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides).

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.

Prolidase deficiency: biochemical study of erythrocyte and skin fibroblast prolidase activity in Italian patients.

Background And Methods: Prolidase deficiency (PD), a rare, autosomally inherited disorder causing iminodipeptiduria is associated with a number of clinical manifestations, the principle feature being chronic skin ulceration. The enzyme prolidase cleaves iminodipeptides containing C-terminal prolyl or hydroxyprolyl residues and is important in the final stages of protein catabolism. We report clinical and biochemical findings in 8 Italian patients with proven prolidase deficiency.

Ehlers-Danlos syndrome type VIII: biochemical, stereological and immunocytochemical studies on dermis from a child with clinical signs of Ehlers-Danlos syndrome and a family history of premature loss of permanent teeth.

We studied collagen expressed by skin fibroblasts in culture, and performed immunocytochemical, ultrastructural and stereological analysis of dermis from a child with signs reminiscent of a mild Ehlers-Danlos syndrome (EDS) type IV and severe periodontitis. No alterations in types I and III [corrected] collagen synthesis and secretion, or serum levels of type III procollagen aminoterminal propeptide were found, and morphological studies revealed non-specific alterations of collagen and elastic components observed in many connective tissue disorders.

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.

[Ehlers-Danlos syndrome. Description of 2 clinical cases].

The paper describes the clinical symptoms and biochemical tests carried out in two girls suffering from Ehlers-Danlos syndrome. The most typical clinical feature, which is common to both cases, was the presence of skin alterations at the extensor surface level of elbows and, above all, knees. These alterations appeared to be delimited areas with irregular margins where the skin was thin, dry, hyperpigmented, wrinkled, with scanty or absent subcutaneous tissue.

Blood transfusions in the therapy of a case of prolidase deficiency.

A case is reported of a 15-year-old boy with prolidase deficiency and marked urinary excretion of the iminodipeptide gly-pro. Prolidase activity of erythrocytes against substrate glycyl-proline was deficient, but after blood transfusions this was increased to 15.7% of donor activity and declined to 12% and 3.

Ultrastructural studies on dermis from prolidase deficient subjects.

Ultrastructural analyses were performed on clinically normal skin from forearm and/or femoral regions of five subjects, all excreting high levels of gly-pro dipeptides into the urine and exhibiting very low prolidase activity on hemolyzed erythrocytes. In both regions, the overall organization of the dermis was normal. Stereological analysis, however, showed that collagen volume density was reduced when compared to that of age-matched controls.

Phenotypic variability and abnormal type I collagen unstable at body temperature in a family with mild dominant osteogenesis imperfecta.

Autosomal dominant inheritance of a mild form of osteogenesis imperfecta (osteogenesis imperfecta type I) with different phenotypic expression was found in a family. Phenotypic expression was different for the affected mother and son, in the presence of the same biochemical results. Dermal fibroblast cultures synthesized normal and mutant type I collagen alpha chains.

Some properties of the alkaline proteinase from Aspergillus melleus.

Seaprose is a semi-alkaline proteinase produced by Aspergillus melleus. The aim of our study was to further characterize the properties of this enzyme, particularly looking at its interaction with alpha 1-proteinase inhibitor, the major human plasma proteinase inhibitor. We studied the cleavage of three synthetic peptide substrates induced by seaprose and the inhibitory profile of the enzyme by means of a panel of inhibitors, including alpha 1-proteinase inhibitor.

Low levels of serum type III procollagen aminoterminal propeptide confirmed type III collagen deficiency in patients without typical clinical symptoms of Ehlers-Danlos type IV.

Biochemical analysis of skin samples revealed that the content of type III collagen was greatly reduced in several subjects with joint hypermobility, stretchability and bruisability of skin. When cultured dermal fibroblasts were found to secrete decreased amounts of type III procollagen into medium (about 30-45% the normal amount) and serum type III procollagen aminopropeptide levels were significantly lower than normal values (P less than 0.001).

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.

Type I procollagen in the severe non-lethal form of osteogenesis imperfecta. Defective pro-alpha 1(I) chains in a patient with abnormal proteoglycan metabolism and mineral deposits in the dermis.

We 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.