Biomedical subjects

M Di Liberto

Publications and source records attributed to M Di Liberto.

13 recordsLinked to original sources

PubMed2004

Brain perfusion abnormalities in patients with euthyroid autoimmune thyroiditis.

PURPOSE: Brain perfusion abnormalities have recently been demonstrated by single-photon emission computed tomography (SPECT) in rare cases of severe Hashimoto's thyroiditis (HT) encephalopathy; moreover, some degree of subtle central nervous system (CNS) involvement has been hypothesised in HT, but no direct evidence has been provided so far. The aim of this study was to assess cortical brain perfusion in patients with euthyroid HT without any clinical evidence of CNS involvement by means of 99mTc-ECD brain SPECT. Sixteen adult patients with HT entered this study following informed consent. METHODS: The diagnosis was based on the coexistence of high titres of anti-thyroid auto-antibodies and diffuse hypoechogenicity of the thyroid on ultrasound in association with normal circulating thyroid hormone and TSH concentrations. Nine consecutive adult patients with non-toxic nodular goitre (NTNG) and ten healthy subjects matched for age and sex were included as control groups. All patients underwent 99mTc-ECD brain SPECT. Image assessment was both qualitative and semiquantitative. Semiquantitative analysis was performed by generation of four regions of interest (ROI) for each cerebral hemisphere--frontal, temporal, parietal and occipital--and one for each cerebellar hemisphere in order to evaluate cortical perfusion asymmetry. The Asymmetry Index (AI) was calculated to provide a measurement of both magnitude and direction of perfusion asymmetry. RESULTS: As assessed by visual examination, 99mTc-ECD cerebral distribution was irregular and patchy in HT patients, hypoperfusion being more frequently found in frontal lobes. AI revealed abnormalities in 12/16 HT patients, in three of the nine NTNG patients and in none of the normal controls. A significant difference in the mean AI was found between patients with HT and both patients with NTNG (p<0.003) and normal controls (p<0.001), when only frontal lobes were considered. CONCLUSION: These results show the high prevalence of brain perfusion abnormalities in euthyroid HT. These abnormalities are similar to those observed in cases of severe Hashimoto's encephalopathy and may suggest a higher than expected involvement of CNS in thyroid autoimmune disease.

Adult↗

PubMed2000

Tumor necrosis factor alpha inhibits type I collagen synthesis through repressive CCAAT/enhancer-binding proteins.

Extracellular matrix (ECM) formation and remodeling are critical processes for proper morphogenesis, organogenesis, and tissue repair. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) inhibits ECM accumulation by stimulating the expression of matrix proteolytic enzymes and by downregulating the deposition of structural macromolecules such as type I collagen. Stimulation of ECM degradation has been linked to prolonged activation of jun gene expression by the cytokine. Here we demonstrate that TNF-alpha inhibits transcription of the gene coding for the alpha2 chain of type I collagen [alpha2(I) collagen] in cultured fibroblasts by stimulating the synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to a previously identified TNF-alpha-responsive element. This conclusion was based on the concomitant identification of C/EBPbeta and C/EBPdelta as TNF-alpha-induced factors by biochemical purification and expression library screening. It was further supported by the ability of the C/EBP-specific dominant-negative (DN) protein to block TNF-alpha inhibition of alpha2(I) collagen but not TNF-alpha stimulation of the MMP-13 protease. The DN protein also blocked TNF-alpha downregulation of the gene coding for the alpha1 chain of type I collagen. The study therefore implicates repressive C/EBPs in the TNF-alpha-induced signaling pathway that controls ECM formation and remodeling.

Amino Acid Sequence↗

PubMed1997

A transcription activator with restricted tissue distribution regulates cell-specific expression of alpha1(XI) collagen.

Different regulatory programs are likely to control expression of the alpha1(XI) collagen (COL11A1) gene in cartilaginous and non-cartilaginous tissues and in coordination with different collagen genes. Here, we report the identification of a cis-acting element that is required for constitutive and tissue-specific activity of the proximal COL11A1 promoter. The element binds an apparently novel activator whose expression is restricted mostly, but not exclusively, to cells of mesenchymal origin. Transient transfection experiments using wild-type and mutant constructs demonstrated the critical contribution of a 45-base pair upstream element (FP9) to promoter activity. The same functional tests and DNA binding assays narrowed down the critical portion of FP9 to a 20-base pair sequence, which consists of an imperfect palindrome with strong homology to the GATA consensus motif. Despite being able to bind GATA proteins in vitro, FP9 is actually recognized by a distinct approximately 100-kDa polypeptide (FP9C) probably belonging to the zinc-finger family of transcription factors. FP9C binding was mostly identified in nuclei from cells of mesenchymal origin, including those actively engaged in COL11A1 transcription. A positive correlation was also established between the level of FP9C binding and the degree of cell differentiation in vitro. Thus, FP9C represents an unusual example of tissue-specific and differentiation-related transcription factor with overlapping expression in hard and soft connective tissues.

3T3 Cells↗

PubMed1997

Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK.

The crystal structure of the adenine nucleotide exchange factor GrpE in complex with the adenosine triphosphatase (ATPase) domain of Escherichia coli DnaK [heat shock protein 70 (Hsp70)] was determined at 2.8 angstrom resolution. A dimer of GrpE binds asymmetrically to a single molecule of DnaK. The structure of the nucleotide-free ATPase domain in complex with GrpE resembles closely that of the nucleotide-bound mammalian Hsp70 homolog, except for an outward rotation of one of the subdomains of the protein. This conformational change is not consistent with tight nucleotide binding. Two long alpha helices extend away from the GrpE dimer and suggest a role for GrpE in peptide release from DnaK.

Adenosine Diphosphate↗

PubMed1997

Comparative analysis of fibrillar and basement membrane collagen expression in embryos of the sea urchin, Strongylocentrotus purpuratus.

The time of appearance and location of three distinct collagen gene transcripts termed 1 alpha, 2 alpha, and 3 alpha, were monitored in the developing S. purpuratus embryo by in situ hybridization. The 1 alpha and 2 alpha transcripts of fibrillar collagens were detected simultaneously in the primary (PMC) and secondary (SMC) mesenchyme cells of the late gastrula stage and subsequently expressed in the spicules and gut associated cells of the pluteus stage. The 3 alpha transcripts of the basement membrane collagen appeared earlier than 1 alpha and 2 alpha, and were first detected in the presumptive PMC at the vegetal plate of the late blastula stage. The PMC exhibited high expression of 3 alpha at the mesenchyme blastula stage, but during gastrulation the level of expression was reduced differentially among the PMC. In the late gastrula and pluteus stages, both PMC and SMC expressed 3 alpha mRNA, and thus at these stages all three collagen genes displayed an identical expression pattern by coincidence. This study thus provides the first survey of onset and localization of multiple collagen transcripts in a single sea urchin species.

Animals↗

PubMed1995

Overlapping pathways mediate the opposing actions of tumor necrosis factor-alpha and transforming growth factor-beta on alpha 2(I) collagen gene transcription.

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are multifunctional peptides intimately involved in the process of extracellular matrix remodeling. We recently showed that TGF-beta stimulates the human alpha 2(I) collagen gene by increasing the affinity of an Sp1-containing transcriptional complex bound to an upstream sequence termed the TbRE (Inagaki, Y., Truter, S. and Ramirez, F. (1994) J. Biol. Chem. 269, 14828-14834). Here, we report that the TbRE-bound complex also mediates the inhibitory signal of TNF-alpha. Nuclear proteins from cells treated with TNF-alpha bind to the TbRE sequence substantially more strongly than those from untreated cells. Additionally, TNF-alpha increases binding of a second protein complex that recognizes the negatively cis-acting element located immediately next to the TbRE. Thus, we postulate that TNF-alpha counteracts the TGF-beta-elicited stimulation of collagen gene expression through overlapping nuclear signaling pathways. One modifies the TGF-beta-targeted transcriptional complex, probably by reducing its stimulatory effect on collagen transcription. The other acts on the binding of the adjacent factor, presumably by increasing its effectiveness in repressing the activity of the collagen promoter. The convergence of the TGF-beta and TNF-alpha pathways on the same sequence of the alpha 2(I) collagen promoter is yet another example of combinatorial gene regulation achieved through composite response elements.

Base Sequence↗

PubMed1993

Complete primary structure of a sea urchin type IV collagen alpha chain and analysis of the 5' end of its gene.

We isolated several overlapping cDNAs from Strongylocentrotus purpuratus coding for a nonfibrillar collagen chain structurally homologous to the vertebrate type IV collagen chains and arbitrarily termed 3 alpha chain. The deduced amino acid sequence of the sea urchin polypeptide includes a 28-residue signal peptide, a 14-residue amino-terminal non-collagenous segment, a triple-helical domain of 1390 residues containing 23 imperfections, and a 226-residue carboxyl-terminal non-collagenous region. Comparison of the sea urchin amino- and carboxyl-terminal non-collagenous domains with those of the vertebrate type IV collagen chains indicated a high level of sequence identity to the alpha 1 (IV) and alpha 5 (IV) chains. This evolutionary relationship was further strengthened by the analysis of the genomic organization of the 5' portion of the sea urchin gene, which also provided the composition of some of the upstream sequences. In addition, this work demonstrated that our gene product is identical to that encoded by the partial cDNA clone recently isolated by others (Wessel, G. M., Etkin, M., and Benson, S. (1991) Dev. Biol. 148, 261-272) who demonstrated its involvement in the biomineralization process of cultured mesenchyme cells.

Amino Acid Sequence↗

PubMed1993

Pro-alpha 2(V) collagen gene; pairwise analysis of the amino-propeptide coding domain, and cross-species comparison of the promoter sequence.

Type V collagen is a minor represented and poorly characterized fibrillar collagen type. Previous cDNA cloning experiments showed that the amino-propeptide of the pro-alpha 2(V) chain shares structural features in common with pro-alpha 1(I), pro alpha 1(II) and pro-alpha 1(III) collagens. In the present paper, this analysis was extended to the gene level. Accordingly the exon/intron arrangement of the amino-propeptide coding domain was compared among pro-alpha 1(I) (COL1A1), pro alpha 1(II) (COL2A1), pro-alpha 1(III) (CO13A1) and pro-alpha 2(V) (COL5A2) collagen genes. This revealed that COL3A1 is the most closely related gene to COL5A2. Based on the assumption that critical regulatory elements might be phylogenetically conserved, we also compared the promoter sequences of the mouse and human COL5A2 genes. This revealed the highest level of sequence homology (97%) in a 52-bp segment which was previously shown to be essential in conferring cell type specificity to the human promoter.

Amino Acid Sequence↗

PubMed1992

Identification of an upstream regulatory region essential for cell type-specific transcription of the pro-alpha 2(V) collagen gene (COL5A2).

The transcriptional features of the human alpha 2(V) collagen gene (COL5A2) were examined by transfection experiments coupled to various DNA binding assays. This approach identified an upstream region essential for the cell type-specific expression of the COL5A2 promoter. Within this region are two nuclear factor-binding sites, FP-A and FP-B, responsible for the formation of distinct DNA-protein complexes. Mutations introduced across each of the two binding sites eliminated the formation of the cognate complex and decreased promoter activity by about 3-fold (FP-A) and 40-fold (FP-B) in transfection experiments. Competition experiments using recognition sequences for known transcription factors exhibiting some similarity to the FP-A- and FP-B-binding sites failed to inhibit COL5A2/protein interactions. Thus, COL5A2 expression appears to be under the positive control of a short regulatory sequence likely to harbor two novel nuclear factor-binding sites.

Base Sequence↗

PubMed1990

Complex and diversified regulatory programs control the expression of vertebrate collagen genes.

The collagens represent a family of structurally related but genetically distinct proteins whose function is essential to maintaining the integrity of vertebrate organs. In addition to their supportive roles, collagens influence a variety of developmental programs and physiological processes. Transcription of collagen genes is controlled by a series of complex interactions between cis-acting regulatory elements and trans-acting nuclear factors that have positive or negative effects on gene expression. Collagen synthesis relies on the timely utilization of diversified regulatory programs that employ tissue and cell-type specific promoters and enhancers. Some of these programs lead to the production of structurally variant chains in different tissues, while others shut down synthesis of a specific collagen type during cell differentiation. Still others control collagen expression in distinct cell lineages. The number, complexity, and variety of the mechanisms leading to the diversified expression of the collagen genes illustrate the unique contribution of this family of proteins to multicellular organogenesis.

Animals↗

PubMed1985

Analysis of the promoter region and the N-propeptide domain of the human pro alpha 2(I) collagen gene.

We have located the exon coding for the start site of transcription of the human pro alpha 2(I) collagen gene. Comparison with the homologous region of other fibrillar collagen genes has confirmed the existence of a consensus sequence (CATGTCTA-n-TAGACATG) capable of forming a hairpin secondary structure possibly involved in the regulation of collagen biosynthesis. Sequence comparison of the chromosomal regions at the 5' end of the pro alpha 1(I) and pro alpha 2(I) collagen genes failed to identify unique DNA elements potentially mediating common regulatory signals. Sequencing of four exons coding for the N-terminal propeptide has determined most of its structure and it has implied the existence of smaller coding units similar to the 11 and 18 bp exons originally described in the avian gene.

Animals↗

PubMed1983

Analysis of the 3' end of the human pro-alpha 2(I) collagen gene. Utilization of multiple polyadenylation sites in cultured fibroblasts.

Three overlapping genomic clones covering 28 kilobases of the human pro-alpha 2(I) collagen gene have been isolated from a lambda phage library. The analysis of 12 introns and 12 exons in the 3' end region has shown that the human gene has a structure remarkably similar to that reported for the homologous chicken gene. One large intron, in the alpha-chain domain, contains an AluI sequence flanked by short direct repeats; a second AluI sequence is present 4 kilobases downstream from the termination codon. The analysis of the exon coding for the 3'-untranslated region has revealed that the pro-alpha 2(I) collagen gene transcribes at least four different mRNAs in cultured fibroblasts. The colinearity and exact location of the termini of these transcripts was determined by Northern blots, R-looping analysis, S1 protection, and DNA sequencing. The ends of two transcripts are closely preceded by the canonical polyadenylation signal (AAUAAA), whereas two of its variations (AUUAAA and AUUAA) precede the ends of the other two transcripts.

Base Sequence↗

PubMed1977

Studies on the structure and possible function of the RNA "cap" in developing sea urchins.

Paracentrotus lividus embryos at the hatching blastula stage very quickly incorporate radioactivity from labeled nucleosides (except uridine) or 32P- or methyl-labeled methionine into a portion of the RNA that has been identified as a "cap". The most probable sequence of this cap is m7G (5') ppp (5')mAmpCp. A very active "capping" and methylation of the "cap" of preexisting RNA molecules was shown to occur at the blastula stage.

Animals↗