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S Garofalo

Publications and source records attributed to S Garofalo.

At least 37 records · Page 2Linked to original sources
PubMed1995

Transgenic mice with deficiencies in cartilage collagens: possible models for gene therapy.

We address three issues that are important when considering somatic gene therapy approaches to osteoarthritis (OA) and related syndromes. First, only those diseases for which a precise molecular etiology has been established should be contemplated for somatic gene therapy. Second, DNA sequences should be identified that restrict expression of correcting genes to chondrocytes; we discuss the use of transgenic mice to identify such sequences. Third, we emphasize the usefulness of establishing animal models that mimic human OA syndromes by genetic manipulations. These transgenic models should be essential for testing gene therapy approaches in vivo.

Animals↗
PubMed1994

Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice.

Studies on the function of extracellular matrix components of cartilages and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.

Animals↗
PubMed1994

Normal long bone growth and development in type X collagen-null mice.

To investigate the role of type X collagen in skeletal development, we have generated type X collagen-null mice. Surprisingly, mice without type X collagen were viable and fertile and had no gross abnormalities in long bone growth or development. No differences were detected between the type X collagen-null mice and controls when growth plates of both newborn and 3-week old mice were examined by histology and by immunostaining for extracellular matrix components of bone including osteopontin, osteocalcin and type II collagen. Our results suggest that type X collagen is not required for long bone development. However, mice and humans with dominant acting type X collagen mutations have bone abnormalities, suggesting that only the presence of abnormal type X collagen can modify bone growth and development.

Animals↗
PubMed1993

Assembly of cartilage collagen fibrils is disrupted by overexpression of normal type II collagen in transgenic mice.

Cartilage collagen fibrils, which are characterized by their thin, uniform diameters, are formed of a multicomponent system of three collagen types (II, IX, and XI) and interacting proteoglycans. We have used a genetic approach to test whether the proper assembly of this multiprotein structure was altered by overexpression of one of its normal components. Here we show that in transgenic mice in which the normal mouse alpha 1(II) collagen is overexpressed, thick abnormal collagen fibrils are generated. Mice that showed the highest expression of the transgene also displayed a larger proportion of abnormal fibrils and died at birth. We propose that an imbalance among the constituents of the cartilage collagen fibrils disrupts the mechanism that controls their assembly. The results show the applicability of the transgenic mice system to studies of complex multicomponent protein assemblies in intact animals.

Animals↗
PubMed1993

The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern.

Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3'-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3'-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice.

Amino Acid Sequence↗
PubMed1992

Specific hybridization probes for mouse alpha 2(IX) and alpha 1(X) collagen mRNAs.

We have used polymerase chain reaction (PCR) technology and available cross-species sequence information to construct cDNA probes for mouse alpha 2(IX) and alpha 1(X) collagen transcripts. Sequencing confirmed the identification of the clones. Northern analysis proved sufficient divergence of the cloned sequences from other collagen transcripts: specific detection of the mouse 2.9 kb alpha 2(IX) and 3.3 kb alpha 1(X) collagen mRNAs was seen under normal hybridization and washing conditions.

Amino Acid Sequence↗
PubMed1992

Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain.

We have generated transgenic mice by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7. This mutation was expected to disturb the assembly and processing of the homotrimeric type II collagen molecule in cartilage. Expression of transgene mRNA at levels equivalent or higher than the endogenous mRNA in the offspring of two founder animals resulted in a severe chondrodysplastic phenotype with short limbs, hypoplastic thorax, abnormal craniofacial development, and other skeletal deformities. The affected pups died at birth due to respiratory distress. Light microscopy of epiphyseal growth plates of transgenic pups demonstrated a marked reduction in cartilaginous extracellular matrix and disruption of the normal organization of the growth plate. The zone of proliferating chondrocytes was greatly reduced whereas the zone of hypertrophic chondrocytes was markedly increased extending deep into the diaphysis suggestive of a defect in endochondral ossification. Electron microscopic examination revealed chondrocytes with extended RER, a very severe reduction in the amount of cartilage collagen fibrils, and abnormalities in their structure. We postulate that the deletion in the alpha 1(II) collagen acts as a dominant negative mutation disrupting the assembly and secretion of type II collagen molecules. The consequences of the mutation include interference with normal endochondral ossification. These mice constitute a valuable model to study the mechanisms underlying human chondrodysplasias and normal bone formation.

Animals↗
PubMed1991

Reduced amounts of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycine-to-cysteine mutation in the mouse type II procollagen alpha 1-chain gene.

We have generated transgenic mice harboring a glycine-to-cysteine mutation in residue 85 of the triple helical domain of mouse type II collagen. The offspring of different founders displayed a phenotype of severe chondrodysplasia characterized by short limbs and trunk, cranio-facial deformities, and cleft palate. The affected pups died of acute respiratory distress caused by an inability to inflate lungs at birth. Staining of the skeleton showed a severe retardation of growth for practically all bones. Light microscopic examination indicated a decrease in cartilage matrix density, a severe disorganization of growth plate architecture, and the presence of streaks of fibrillar material in the cartilage matrix. Electron microscopic analysis showed a pronounced decrease in the number of typical thin cartilage collagen fibrils, distension of the rough endoplasmic reticulum of chondrocytes, and the presence of abnormally large banded collagen fibril bundles. The level of expression of the mutant type II procollagen alpha 1 chain transgene in cartilage tissues was approximately equal to that of the endogenous gene in two of the strains. We propose that the principal consequence of the mutation is a considerable reduction in density of the typical thin cartilage collagen fibrils and that this phenomenon causes the severe disorganization of the growth plate. We also postulate that the abnormal thick collagen fibrils are probably related to a defect in crosslinking between the collagen molecules. The cartilage anomalies displayed by these transgenic mice are remarkably similar to those of certain human chondrodysplasias.

Animals↗
PubMed1990

Extinction and activation of the thyroglobulin promoter in hybrids of differentiated and transformed thyroid cells.

Thyroglobulin gene expression was repressed in a rat thyroid cell line transformed with Kirsten murine sarcoma virus. Expression of a dominant selectable marker driven by the thyroglobulin promoter was also inhibited. Somatic cell hybridization of transformed and differentiated thyroid cells resulted in extinction of thyroglobulin gene expression. When transformed cells carrying a dominant selectable marker driven by the thyroglobulin promoter were fused to differentiated cells and expression of this marker was selected, we obtained stable hybrid cell lines expressing both the endogenous and the exogenous thyroglobulin promoters. Although the expression of v-ras remained unchanged compared with expression in the parental transformed cells, transformation was suppressed in the hybrid cell lines. The other thyroid differentiation markers, iodide uptake and thyroid-stimulating hormone-dependent growth, were inhibited in all the hybrids tested. We show that activity of the thyroglobulin promoter correlates with the presence of a thyroid nuclear factor that binds the promoter at position -60 from the transcription start site. Loss of this factor accompanies the extinction of thyroglobulin gene expression in hybrids selected for expression of a non-thyroid-specific promoter.

Animals↗
PubMed1983

[Prevalence of hyperkinetic arrhythmias in a healthy infantile and adolescent population: correlations between standard ECG, stress test and dynamic ECG].

We evaluated 250 males and 250 females 4 to 17.99 years old, randomly recruited among school-children. Cardiac and other diseases were excluded. All the subjects were studied with a standard ECG iun the standing and supine position for a minute. Three hundred children underwent a treadmill exercise test according to Bruce's protocol and 50 of them had a 24-hour ambulatory ECG monitoring. Sinus arrhythmia was present in 94% of the children in supine position and in 87% of them while they were standing. Only one child had atrial and ventricular premature beats. In a boy a slow ventricular tachycardia was recorded. One child had a WPW and a girl a short PR interval; both were asymptomatic. The exercise test did not induce any arrhythmias and the slow ventricular tachycardia, in the previously mentioned boy was suppressed during exercise and reappeared during the recovery period. The premature beats were greatly reduced in the child who had them during the standard ECG recording. The 24-hour ambulatory ECG monitoring showed only minor degrees of premature atrial and ventricular beats in 24% of the children studied by this method. The maximum number of premature beats was 39 in 24 hours and never exceeded the first grade of Lown's classification. We conclude that a good correlation exists among the three employed techniques in the study of the prevalence of tachyarrhythmias in healthy children. This is the first study that compares these three techniques in a pediatric population.

Adolescent↗