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G Cetta

Publications and source records attributed to G Cetta.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Adult↗
PubMed1998

Micellar electrokinetic chromatography: a convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity.

High performance capillary electrophoresis (HPCE) has been exploited as an analytical method alternative to current procedures for the determination of proteolytic activity of elastases from different sources. Due to some drawbacks with capillary zone electrophoresis (CZE), the mode of operation employed for the assay of elastolytic activity was micellar electrokinetic chromatography (MEKC). Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM SDS and 15% v/v methanol, separation of intact peptide substrate from products of proteolytic reaction was easily achieved in a fused-silica capillary of 50 cm effective length x 75 microm ID. This allowed us to determine the rate of hydrolysis of substrates and to calculate the kinetic parameters Km and k(cat) of the proteases investigated. A comparison of these data with those obtained from high performance liquid chromatography (HPLC)-based experiments showed that MEKC is a convenient technique for studying protease kinetics.

Aspergillus fumigatus↗
PubMed1998

Proteoglycan sulfation in cartilage and cell cultures from patients with sulfate transporter chondrodysplasias: relationship to clinical severity and indications on the role of intracellular sulfate production.

Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been associated with a family of chondrodysplasias that includes diastrophic dysplasia (DTD), atelosteogenesis type 2 (AO2) and the lethal condition achondrogenesis type 1B (ACG1B). There is a correlation between the nature of the mutations and the clinical phenotype, but our understanding of the pathophysiology of the disorder, which involves defective sulfation of cartilage proteoglycans, is far from complete. To evaluate the degree of proteoglycan undersulfation in vivo, we have extracted chondroitin sulfate proteoglycans from cartilage of twelve patients with sulfate transporter chondrodysplasias and analyzed their disaccharide composition by HPLC after digestion with chondroitinase ABC. The amount of non-sulfated disaccharide was elevated in patients' samples (controls, 5.5%+/-2.8 (n=10); patients, 11% to 77%), the highest amount being present in ACG1B patients, indicating that undersulfation of chondroitin sulfate proteoglycans occurs in cartilage in vivo and is correlated with the clinical severity. To investigate further the biochemical mechanisms responsible for the translation of genotype to phenotype, we have studied fibroblast cultures of patients with DTD, AO2 and ACG1B, and controls, by double-labelling with [35S]sulfate and [3H]glucosamine. The incorporation of extracellular sulfate, estimated by the 35S/3H ratio in proteoglycans, was reduced in all patients' cells, with ACG1B cells showing the lowest values. However, disaccharide analysis of chondroitin sulfate proteoglycans showed that these were normally sul fated or only moderately undersulfated; marked undersulfation was observed only after addition of the artificial glycosaminoglycan-chain initiator, beta-D-xyloside, to the culture medium. These results suggest that, while utilization of extracellular sulfate is impaired, fibroblasts can replenish their intracellular sulfate pool by oxidizing sulfur-containing compounds (such as cysteine) and thus partially rescue PG sulfation under basal conditions. This rescue pathway becomes insufficient when GAG synthesis rate is stimulated by beta-D-xyloside. These findings may explain why phenotypic consequences of DTDST mutations are restricted to cartilage, a tissue with high GAG synthesis rate and poor vascular supply, and imply that pharmacological therapy aimed at restoring the intracellular sulfate pool might improve PG sulfation in DTD and related disorders.

Anion Transport Proteins↗
PubMed1997

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.

Dipeptidases↗
PubMed1997

Undersulfation of cartilage proteoglycans ex vivo and increased contribution of amino acid sulfur to sulfation in vitro in McAlister dysplasia/atelosteogenesis type 2.

Mutations in the diastrophic dysplasia sulfate transporter gene cause a family of chondrodysplasias including, in order of increasing severity, diastrophic dysplasia, atelosteogenesis type 2 and achondrogenesis type 1B. McAlister dysplasia is a lethal chondrodysplasia considered on the basis of minor radiographic features to be a disorder different from atelosteogenesis type 2. Here, we demonstrate that McAlister dysplasia arises from mutations in the diastrophic dysplasia sulfate transporter gene and that this disorder essentially coincides on molecular and biochemical grounds with atelosteogenesis type 2. The fetus affected by McAlister dysplasia we have studied is a compound heterozygote for mutations leading to R279W and N425D substitutions in the diastrophic dysplasia sulfate transporter. Proteoglycan sulfation was studied in epiphyseal cartilage and in chondrocyte cultures of the patient by high performance liquid chromatography of chondrotinase digested proteoglycans; a high amount of non-sulfated disaccharide was observed as a consequence of the alteration of the transporter function caused by the mutations. However, sulfated disaccharides were detectable even if in low amounts, both in cultured cells and tissue. Functional impairment of the sulfate transporter was demonstrated in vitro by reduced incorporation of [35S]sulfate relative to [3H]glucosamine in proteoglycans synthesized by chondrocytes and by sulfate-uptake assays in fibroblasts. Parallel in vitro studies in a patient with achondrogenesis 1B indicated that the severity of the clinical phenotype seems to be correlated to the residual activity of the sulfate transporter. The capacity of fibroblasts to use cysteine as an alternative source of sulfate was evaluated by double-labeling experiments. Relative incorporation of [35S]cysteine-derived sulfate in the glycosaminoglycan chains was increased in the patient's cells, indicating that, in vitro, the catabolism of sulfur-containing amino acids can partially compensate for intracellular sulfate deficiency. Residual sulfation observed in proteoglycans extracted from cartilage suggests that this mechanism may be operating also in vivo.

Anion Transport Proteins↗
PubMed1997

Mutation producing alternative splicing of exon 26 in the COL1A2 gene causes type IV osteogenesis imperfecta with intrafamilial clinical variability.

We have characterized a familial form of osteogenesis imperfecta (OI). Following the identification by ultrasound of short limbs and multiple fractures in a fetus at 25 weeks of gestation, the family was referred with a provisional diagnosis of severe OI. We detected subtle clinical and radiological signs of OI in the father and in the paternal grandmother of the proposita, who had never received a diagnosis of OI. Linkage analysis indicated COL1A2 as the disease locus. Heteroduplex analysis of reverse transcription-polymerase chain reaction (RT-PCR) amplification products of pro alpha2(I) mRNA from an affected member and subsequent sequencing of the candidate region demonstrated the presence of normal transcripts and a minority of transcripts lacking exon 26 (54 bp) of COL1A2. Sequencing of PCR-amplified genomic DNA identified an A --> G transition in the moderately conserved +3 position of the IVS 26 donor splice site. The mutant pre-mRNA molecules were alternatively spliced, yielding both full-length and deleted transcripts that represented less than 30% of the total pro alpha2(I) mRNA. The biochemical data on type I collagen synthesized by dermal fibroblasts showed intracellular retention of the mutant protein; failure to detect the shortened alpha2(I) chains either in the medium or in the cell layer may be the consequence of their instability at physiological temperature. These observations justified the mild resulting phenotype.

Adult↗
PubMed1997

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.

Adolescent↗
PubMed1997

Stability of type I collagen CNBr peptide trimers.

As indices of triple helix stability of type I collagen CNBr peptide homotrimers, deltaG degrees for monomer-trimer transitions and melting temperatures were obtained from circular dichroism measurements at increasing temperatures. The data were compared with the stability of the parent native molecule. Peptides were found to have a lower stability than the whole collagen molecule. The general implication is that the coordinated water molecules play a key role in determining collagen triple helical stability and high cooperativity at melting. Other factors (monomer stability, ionic and hydrophobic factors, variations of composition, specific sequences) could also contribute towards peptide stability; these factors could explain the data obtained in the case of peptide alpha1(I) CB3.

Collagen↗
PubMed1997

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.

Adolescent↗
PubMed1997

Phenotypic comparison of an osteogenesis imperfecta type IV proband with a de novo alpha2(I) Gly922 --> Ser substitution in type I collagen and an unrelated patient with an identical mutation.

We examined the type I collagen synthesized by cultured dermal fibroblasts from a patient affected with osteogenesis imperfecta (OI) type IV. Both normal and abnormal trimers were produced. The mutant collagen molecules were excessively modified intracellularly, had a melting temperature 4 degrees C lower than the control, were secreted at a reduced rate, and underwent delayed processing to mature alpha chains.Molecular investigations identified a G --> A transition in one COL1A2 allele, resulting in a Gly922 --> Ser substitution in the alpha2(I) chain. The proband's mutation was demonstrated to arise "de novo" by the absence of the mutant allele restriction enzyme pattern from parental genomic DNA.We analyzed the insoluble extracellular matrix deposited by long-term cultured fibroblasts from our patient and from a previously described unrelated individual who carries an identical substitution. In both cases, the mutant chain constituted 10-15% of the total alpha chains deposited.We also present here the first detailed comparison of phenotype between unrelated OI patients with an identical collagen mutation. These two patients are both Caucasian females, ages 8 and 9 years, each diagnosed as type IV OI by the Sillence classification. They have a similar phenotype including moderate skeletal fragility with several femur fractures, dentinogenesis imperfecta, wormian bone, and reduced height and weight. We conclude that this phenotype is related both to the location of this mutation and to the similar extent of matrix incorporation by the mutant chains. Molecular and biochemical studies of unrelated individuals with identical amino acid substitutions in type I collagen resulting in either similar or dissimilar clinical outcomes will make a significant contribution to identifying the factors involved in the modulation of the OI phenotype.

Cells, Cultured↗
PubMed1996

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.

Dipeptidases↗
PubMed1996

Undersulfation of proteoglycans synthesized by chondrocytes from a patient with achondrogenesis type 1B homozygous for an L483P substitution in the diastrophic dysplasia sulfate transporter.

Achondrogenesis type 1B is an autosomal recessive, lethal chondrodysplasia caused by mutations in the gene encoding a sulfate/chloride antiporter of the cell membrane (Superti-Furga, A., Hästbacka, J., Wilcox, W. R., Cohn, D. H., van der Harten, J. J., Rossi, A., Blau, N., Rimoin, D. L., Steinmann, B., Lander, E. S., and Gitzelmann, R.(1996) Nat. Genet. 12, 100-102). To ascertain the consequences of the sulfate transport defect on proteoglycan synthesis, we studied the structure and sulfation of proteoglycans in cartilage tissue and in fibroblast and chondrocyte cultures from a fetus with achondrogenesis 1B. Proteoglycans extracted from epiphyseal cartilage and separated on agarose gels migrated more slowly than controls and stained poorly with alcian blue. The patient's cultured cells showed reduced incorporation of [35S]sulfate relative to [3H]glucosamine, impaired uptake of sulfate, and higher resistance to chromate toxicity compared to control cells. Epiphyseal chondrocytes cultured in alginate beads synthesized proteoglycans of normal molecular size as judged by gel filtration chromatography, but undersulfated as judged by ion exchange chromatography and by the amount of nonsulfated disaccharide. High performance liquid chromatography analysis of chondroitinase-digested proteoglycans showed that sulfated disaccharides were present, although in reduced amounts, indicating that at least in vitro, other sources of sulfate can partially compensate for sulfate deficiency. A t1475c transition causing a L483P substitution in the eleventh transmembrane domain of the sulfate/chloride antiporter was present on both alleles in the patient who was the product of a consanguineous marriage. The results indicate that the defect of sulfate transport is expressed in both chondrocytes and fibroblasts and results in the synthesis of proteoglycans bearing glycosaminoglycan chains which are poorly sulfated but of normal length.

Achondroplasia↗
PubMed1996

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.

Animals↗
PubMed1996

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.

Blotting, Northern↗
PubMed1996

Serum type I and type III procollagen peptide levels in sarcoidosis.

Type I and type III are the most abundant collagens in the lung. The aim of our study was to compare type I and III procollagen peptides in sera of sarcoid patients. Sixty eight patients with sarcoidosis were studied (19 with newly recognized disease, 7 with relapsing disease, 15 with chronic disease, and 27 in stable remission). Thirty healthy volunteers served as controls. The levels of procollagen I and III peptides were determined by radioimmunoassay. Angiotensin-converting enzyme (ACE) level was evaluated by means of a colorimetric assay. In patients with newly recognized sarcoidosis, both serum procollagen I and III peptide levels were increased with respect to controls (p=0.0014 and p<0.00001, respectively). There was a poor correlation between levels of procollagen I and III (r=0.26), whereas there was a closer correlation between procollagen III and ACE (r=0.69). Procollagen I peptide level did not identify patients in roentgenological stage III. In conclusion, in patients with newly recognized sarcoidosis there is a significant increase in the serum level of procollagen I peptide. However, procollagen I peptide is not a marker of sarcoid patients with fibrosis, ie. stage III disease. Its clinical usefulness seems to be weaker than that of procollagen III peptide.

Adult↗
PubMed1994

A base substitution at IVS-19 3'-end splice junction causes exon 20 skipping in pro alpha 2(I) collagen mRNA and produces mild osteogenesis imperfecta.

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the proband's fibroblasts showed the presence of two populations of alpha 2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patient's pro alpha 2(I) mRNA: normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3' consensus splicing site. As a consequence pro alpha 2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported.

Base Sequence↗