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. The immunolabeling with antibodies against sm-specific a-actin (which was taken as a marker of myofibroblasts) showed that, already in low-passage cultures, the percentage of myofibroblasts was higher in the nodule-derived cell populations, and progressively increased with increasing passages. This suggests that myofibroblasts have higher proliferation potential than control fibroblasts. Myofibroblasts were also found to undergo polyploidization and hypertrophy, especially in high-passage cultures. Based on these results, it may be hypothesized that in fibroblastic rheumatism the development of the typical nodules could depend on the intrinsic capability of myofibroblats of proliferating faster than normal fibroblasts and of becoming polyploid and hypertrophic. Nodule-derived cells in culture synthesized slightly less collagen and non-collagen proteins than did the control fibroblasts; this suggests that the increased fibrosis observed in nodules in situ could be likely dependent on a reduced degradation of the extracellular matrix components.

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 < 0.05), and particularly in collagen (-40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.

Transient beneficial effect of GH replacement therapy and topical GH application on skin ulcers in a boy with prolidase deficiency.

A diagnostic examination for short stature in a boy with chronic ulcers of the feet due to prolidase deficiency, a rare disorder associated with intractable ulcers of the skin, led to the diagnosis of growth hormone (GH) deficiency. Replacement treatment with r-hGH associated with the topical application of a GH-containing ointment when the boy was 13 years old resulted in complete but transitory healing of the ulcers, which can probably be attributed to the growth-promoting effects of GH on dermal connective tissue.

5-Thienyltryptamine derivatives as serotonin 5-HT1B/1D receptor agonists: potential treatments for migraine.

A series of 5-(2- or 3-thienyl)tryptamine derivatives (9) has been synthesized and shown to be potent and selective 5-HT1D versus 5-HT1B receptor agonists and, therefore, potential treatments for migraine.

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." The characteristic arrays of orthogonal interfibrillar bridges were missing and the extracellular matrix was totally disorganized in matrices produced by fibroblasts taken postmortem from skin of an electively aborted fetus which did not express decoron in culture, thus supporting the shape module hypothesis. Biglycon, dermatan sulfate, heparan sulfate, collagen, and hyaluronan were produced by these cells but did not contribute to a normal extracellular matrix. A similar electron histochemical and biochemical survey of extracellular matrices produced by seven normal and eight osteogenesis imperfecta cell lines from donors of different ages and both sexes showed no comparable disruptions of their matrices. This investigation appears to be the first to demonstrate systematically proteoglycan:collagen interactions in matrices produced by cultured human cells.

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.

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. 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.

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. The usefulness of sodium dodecyl sulphate as surfactant is reported: refined electropherograms were achieved using 35 mM sodium borate, pH 8.3 containing 65 mM sodium dodecyl sulphate. Almost all peaks were baseline separated, collected and sequenced. This allowed us to define the exact imidodipeptide composition of patients' urine. The possibility of identifying and thus quantifying each single peak means that comparison of urinary imidodipeptide excretion patterns from different patients can be made and the hypothesis that peptide patterns can be correlated with differing clinical severity can be investigated.

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. Electropherograms of urine samples from control subjects and four patients with prolidase deficiency were compared. The presence of imidodipeptides normally absent in urine was evident in patients' urine. Further analysis of urine samples enabled identification of excreted imidodipeptides and the pattern of excretion appeared to be heterogeneous for different patients. This method appears to be useful for identification of imidodipeptides in biological samples, as an efficient aid in diagnosis of PD, and as a method for providing more information about this disease.

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.

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). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat-denatured samples.

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. There was considerable heterogeneity in age at onset of symptoms (varying from 3-17 years), mental retardation and clinical manifestations (asymptomless to very severe). Prolidase activity was determined in hemolysates of patient erythrocytes and cultured dermal fibroblasts. RESULTS: Prolidase activity was found to be deficient, especially against gly-pro. Erythrocyte and fibroblast enzyme was also separated into two forms, a major isoform (I) and a minor one (II) by fast protein liquid chromatography, and activity against different iminodipeptide substrates was tested. Isoform I activity was markedly reduced in all patients as compared to normal controls, while isoform II activity appeared to be unaltered. CONCLUSIONS: We were unable to find any correlation between degree of enzyme activity loss and severity of symptoms.

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. 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.

[Ehlers-Danlos syndrome. Description of 2 clinical cases]. / La sindrome di Ehlers-Danlos. Descrizione di due casi clinici.

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. Biochemical tests carried out on cutaneous fibroblast cultures excluded the presence type I collagen alterations and an altered secretion of type I or III procollagen secretion. The Authors discuss the attribution of cases presented within the context of the various forms of Ehlers-Danlos syndrome in relation to clinical findings and the results of the biochemical tests.

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 3.4% of normal activity after 8 and 45 days, respectively. Urinary iminodipeptide levels following transfusion remained unaltered.

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. Collagen fibrils did not show ultrastructural alterations, but they were distributed into a higher number of small bundles, and their diameters shifted towards lower values compared to age-matched controls. The elastin volume density was slightly reduced in the patients, especially in the femoral areas. In both forearm and femoral dermis, elastin fibers were significantly more numerous and smaller than in controls. Furthermore, elastin fibers were apparently normal in the forearm dermis, whereas appeared polymorphic and cribriform in the femoral skin. The results focused on the importance of efficient proline re-utilization for normal collagen and elastin synthesis and deposition. The differences between femoral and forearm skin regions from both clinical and ultrastructural points of view, may depend on mechanisms that regulate circulation and, possibly, on other factors which modulate the phenotypic expression of mesenchymal cells.

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. The interaction between seaprose and alpha 1-proteinase inhibitor was also studied with SDS-PAGE. Finally, the elastolytic activity of seaprose was checked by means of bovine elastin solubilization. We found that seaprose cleaves preferentially the substrate containing a Phe residue in the P1 position. The inhibitory profile showed that seaprose is a serine-proteinase that cannot be inhibited by alpha 1-proteinase inhibitor. The SDS-PAGE revealed that alpha 1-proteinase inhibitor, after incubation with seaprose, underwent a limited proteolysis. Finally, seaprose 10(-2) M and 10(-3) M was able to solubilize bovine elastin. We conclude that seaprose is a serine-proteinase able to inactivate human alpha 1-proteinase inhibitor with limited proteolysis at (or near) the active site and that it has mild elastinolytic capacity.

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. Collagen heterotrimers containing abnormal chains were overmodified along the entire triple helical domain and showed an unusually low denaturation temperature, so far found only in lethal cases. The mild phenotype in the family is probably due to the fact that abnormal type I collagen molecules are more likely to be degraded than utilized in the extracellular matrix.