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G Damante

Publications and source records attributed to G Damante.

At least 19 recordsLinked to original sources
PubMed1997

Immunocytochemical expression of tissue specific transcription factor-1 in lung carcinoma.

AIMS: To investigate the immunocytochemical expression of the tissue specific transcription factor-1 (TTF-1) on cytological specimens of small cell lung carcinoma (SCLC) and to establish its value in the cytological diagnosis of lung cancer. METHODS: For each case, the diagnosis was made on cytological specimens and confirmed on subsequent bronchial biopsy specimens. TTF-1 was detected immunocytochemically using the avidinbiotin complex technique with a rabbit antiserum. Expression of TTF-1 was evaluated in 41 cases of SCLC and 17 cases of non-small cell carcinoma (NSCC). The latter were subdivided into eight cases of adenocarcinomas and nine cases of squamous cell carcinomas (SCC). RESULTS: Positive nuclear immunoreactivity to TTF-1 was identified in 38 (92.7%) of the 41 cases of SCLC, in five (62.5%) of eight cases of adenocarcinoma, and one (11%) of nine cases of SCC. A significant difference was observed between the two main groups, SCLC and NSCC. A comparison between SCLC and adenocarcinoma and SCC showed that TTF-1 expression was significantly different. TTF-1 immunoreactivity was not detected in the inflammatory cells of the same cases. CONCLUSIONS: TTF-1 is strictly associated with SCLC; it was weakly expressed in the various subtypes of NSCC. Although TTF-1 is not specific for SCLC, it can be used to highlight neoplastic cells to good effect when a large inflammatory component is present, and to differentiate SCLC from lymphoid infiltrates.

Adenocarcinoma↗
PubMed1996

Analysis of the solution structure of the homeodomain of rat thyroid transcription factor 1 by 1H-NMR spectroscopy and restrained molecular mechanics.

The solution structure of the rat thyroid transcription factor 1 (TTF-1) homeodomain has been elucidated by 1H-NMR and restrained modeling. The TTF-1 homeodomain folds in the same manner as classical homeodomains, with three helices, a loose loop between the first two helices, and a tight turn between helix II and helix III. The typical assembly of the hydrophobic core is maintained and N-capping motifs are identified in helix I and helix III. The N-terminal stretch of helix II exhibits some mobility, similar to the preceding loop region, which may be related to its anomalous capping. The N-terminal decapeptide and the C-terminal octapeptide of the molecule (68 residues long) are disordered. All the previous characteristics are shared by all known isolated homeodomain structures. An important difference among these structures occurs at the C-terminal extension of helix III, which is either disordered or helically folded. In the TTF-1 homeodomain, the C-terminal extension of helix III (residues 51-59) appears structured, albeit not as rigidly as the preceding portion. Analysis of the NOEs and hydrogendeuterium exchange of backbone amides provides evidence for discontinuity between the two moieties of helix III, which is introduced by a tightening or a kink of residues 51-53.

Animals↗
PubMed1996

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5'-TAAT-3'. The homeodomain of thyroid transcription factor 1 (TTF-1HD) binds to sequences containing a 5'-CAAG-3' core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF-1HD by both structural and functional approaches. As already suggested by the three-dimensional structure of TTF-1HD, the DNA binding specificities of the TTF-1, Antennapedia and Engrailed homeodomains, either wild-type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3' end of the core motif 5'-NAAN-3'. The nucleotide at the 5' position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF-1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

Animals↗
PubMed1996

In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases.

Amino Acids↗
PubMed1996

A network of specific minor-groove contacts is a common characteristic of paired-domain-DNA interactions.

Pax proteins are a family of transcription factors conserved during evolution and able to bind specific DNA sequences through a domain called a "paired domain'. The DNA-binding specificity of the Pax-8 paired domain was investigated. Site-selection experiments indicate that Pax-8 binds to a consensus sequence similar to those bound by Pax-2 and Pax-5. When consensus sequences of various paired domains are observed in light of recent structural studies describing paired-domain-DNA interaction [Xu, Rould, Jun, Desplan and Pabo (1995) Cell 80, 639-650], it appears that base-pairs contacted in the minor groove are conserved, while most of the base-pairs contacted in the major groove are not. Therefore a network of specific minor groove contacts is a common characteristic of paired-domain-DNA interactions. The functional importance of such a network was successfully tested by analysing the effect of consensus-based mutations on the Pax-8 binding site of the thyroglobulin promoter.

Animals↗
PubMed1996

TTF-1 gene expression in human lung tumours.

Tissue-specific transcription factors control cell determination and differentiation. TTF-1 is a tissue-specific transcription factor expressed in the thyroid and lung. We investigated the expression of TTF-1 in normal human lung, and in various histopathological types of lung cancers by immunohistochemistry. In normal lung, TTF-1 expression was restricted to bronchial and alveolar epithelial cells. TTF-1 expression was found in 7 of the 29 cases of non-small cell lung carcinomas. In these tumours, the expression of TTF-1 did not correlate with the histological degree of differentiation. Results obtained using RNase protection assay confirmed that TTF-1 was expressed only in a subset of non-small cell carcinomas. TTF-1, as expected, was not expressed in neoplasms having a neuroendocrine cell origin, such as carcinoids. Interestingly, TTF-1 was always expressed in small cell lung carcinomas. These findings indicate that: (i) small cell lung carcinomas could originate from the endothermal cell lineage and (ii) dedifferentiation processes that operate in these neoplasms do not affect molecular mechanisms necessary for TTF-1 gene expression.

Carcinoid Tumor↗
PubMed1996

Expression of thyroid transcription factor 1 gene can be regulated at the transcriptional and posttranscriptional levels.

The complete structure of the gene for thyroid transcription factor 1 (TTF-1), both in rats and humans, has been determined. The rat TTF-1 gene shows three transcriptional start sites and contains two introns, one of which is alternatively spliced. Nuclear run-on and transient transfection experiments indicate that TTF-1 gene expression can be controlled at different levels. Using thyroid and nonthyroid cell lines, it can be shown that transcriptional mechanisms are involved in controlling thyroid-specific expression of the TTF-1 gene. In contrast, in thyroid cells expressing an activated Ki-ras oncogene, the steady-state level of TTF-1 mRNA is greatly reduced, while transcription of the TTF-1 gene is only moderately affected, suggesting that the accumulation of TTF-1 mRNA can be regulated by a posttranscriptional, Ras-sensitive mechanism.

Animals↗
PubMed1995

Redundant domains contribute to the transcriptional activity of the thyroid transcription factor 1.

The thyroid transcription factor 1 (TTF-1) is a homeodomain-containing protein implicated in the activation of thyroid-specific gene expression. Here we report that TTF-1 is capable of activating transcription from thyroglobulin and, to a lesser extent, thyroperoxidase gene promoters in nonthyroid cells. Full transcriptional activation of the thyroglobulin promoter by TTF-1 requires the presence of at least two TTF-1 binding sites. TTF-1 activates transcription via two functionally redundant transcriptional activation domains that as suggested by competition experiments, could use a common intermediary factor.

Animals↗
PubMed1995

Definition of the DNA-binding specificity of TTF-1 homeodomain by chromatographic selection of binding sequences.

The homeodomain of the thyroid transcription factor-1 (TTF-1HD) shows a peculiar DNA-binding specificity, preferentially recognizing sequences having the 5'-CAAG-3' core motif. In order to detail the DNA-binding specificity of this protein, a TTF-1HD-Sepharose column chromatography was used. A sequential selection and amplification of sequences was performed. TTF-1HD binding activity for selected and unselected sequences was measured. The presence of the 5'-CAAG-3' core motif was necessary, but not sufficient, to obtain the maximal binding activity for TTF-1HD. However, several of the selected sequences do not contain the 5'-CAAG-3' core motif and are bound by TTF-1HD only 2-fold less with respect to sequences bound with the highest affinity. Therefore, these data indicate that TTF-1HD specifically recognizes a spectrum of sequences wider than previously determined.

Base Sequence↗
PubMed1994

Analysis of the conformation and stability of rat TTF-1 homeodomain by circular dichroism.

The conformational stability of TTF-1HD has been determined by CD-monitored thermal denaturation and isothermal urea unfolding studies. The Gibbs free energy of stabilization found are 1.44 and 1.26 kcal.mol-1, respectively. TTF-1HD exhibits a Tm of 42 degrees C and a delta Cp of 80 cal.mol-1.K-1 indicating that TTF-1HD, when free in solution, is a mobile flexible segment folded into loose helices. Such a flexibility would be relevant for the DNA-binding function of this homeodomain. In fact, a small reduction of the alpha-helical content of TTF-1HD significantly modifies its DNA-binding activity.

Animals↗
PubMed1994

Expression of thyroid-specific transcription factors TTF-1 and PAX-8 in human thyroid neoplasms.

TTF-1 and PAX-8 are tissue-specific transcription factors expressed in the thyroid follicular cells, contributing to the maintenance of the differentiated phenotype. In fact, it has been demonstrated that TTF-1 and PAX-8 are able to activate transcription from thyroglobulin and thyroperoxidase (TPO) promoters, the transcriptional activity of which is in vivo restricted only to the thyroid follicular cell. In order to gain insight into how these transcription factors control in vivo the differentiation of the thyroid cell and to have a better molecular characterization of human thyroid tumors, TTF-1, PAX-8, thyroglobulin, and TPO mRNA levels were measured in nonmalignant and malignant human thyroid tissues. Results indicate that the expression of TTF-1 and PAX-8 is not sufficient per se for the expression of the thyroid-differentiated phenotype. Furthermore, in follicular adenomas, PAX-8 mRNA levels are strictly related to TPO mRNA levels, suggesting that the amount of PAX-8 could play a role in the modulation of TPO gene expression. TTF-1 mRNA is always well detectable in papillary carcinomas and, in contrast, always absent in anaplastic carcinomas. Identical results were obtained when the expression of TTF-1 protein was investigated using immunohistochemistry. Thus, TTF-1 gene expression could be a molecular marker in order to distinguish these two types of thyroid neoplasms.

Adenocarcinoma, Follicular↗
PubMed1994

Sequence-specific DNA recognition by the thyroid transcription factor-1 homeodomain.

The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1HD within the protein-DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gln at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.

Antennapedia Homeodomain Protein↗
PubMed1994

Different molecular mechanisms are involved in the multihormonal control of glucose transport in FRTL5 rat thyroid cells.

We investigated the molecular mechanisms by which TSH, insulin and IGF-1 modulate the glucose transport system in FRTL5 cells. We found that TSH, insulin and IGF-1 increased the glucose transporter Glut-1 specific mRNA levels 6, 8 and 5 fold over control, respectively. The effect on Glut-1 mRNA was evident after 2 hours, followed by an increased Glut-1 protein expression in whole cells, as judged by western blot analysis, after 5 hours of stimulation with all the hormones studied. In contrast, plasma membrane Glut-1 increased (300-400% over control) after 2 hours of stimulation with TSH (10 mU/ml), dibutyryl-cAMP (1mM), IGF-1 (10 ng/ml) and insulin (10 nM). These data indicate that the glucose transport system is under multihormonal control in FRTL5 cells. Two different mechanisms are involved in TSH, IGF-1 and insulin stimulation of the glucose transport: a) neosynthesis of Glut-1 by activation of gene expression; b) recruitment of carriers from the intracellular pool to the plasma membrane.

Animals↗
PubMed1993

Structural study of rat thyroid transcription factor 1 homeodomain (TTF-1 HD) by nuclear magnetic resonance.

The 500 MHz 1H NMR spectrum of a 68-residue peptide, encompassing the rat thyroid transcription factor 1 homeodomain (TTF-1 HD), was fully assigned using standard 2D NMR methodology. The secondary structure elements and their spatial organization were determined and led to a structure very similar to that previously described for other homeodomains and expected also for TTF-1 HD from homology modeling predictions. The three-dimensional arrangement of the three helix fragments of TTF-1 HD preserves the helix-turn-helix motif commonly occurring in many classes of DNA-binding proteins.

Amino Acid Sequence↗
PubMed1993

Effect of salt concentration on TTF-1 HD binding to specific and non-specific DNA sequences.

The Thyroid Transcription factor 1 (TTF-1) recognizes specific DNA sequences by a Homeodomain (TTF-1 HD). The TTF-1 HD DNA-binding properties with both specific and non-specific DNA sequences were investigated. TTF-1 HD exists as a monomer in solution and as a monomer binds DNA. At 75 mM KCl, its relative binding affinity with a specific DNA sequence is about 50 fold higher than with a non-specific DNA sequence. Increase of KCl concentration reduces the apparent binding affinity both to specific and non-specific DNA sequences. However, non-specific binding is more sensitive than specific binding to the increase of salt concentration. When DNA-binding reactions are performed at temperature and salt concentration close to the intracellular environment, TTF-1 HD binds the specific sequence with an affinity at least 1000 fold higher respect to the non-specific sequence.

Animals↗
PubMed1993

Cross-linking of HLA class II antigens modulates the release of tumor necrosis factor-alpha by the EBV-B lymphoblastoid cell line JY.

In addition to their functional role as peptide-binding proteins HLA class II Ag can also act as signal-transducing molecules. The present study showed that cross-linking of HLA class II Ag by the anti-HLA-DR mAb L243 or by the anti-HLA-DR,-DP mAb IVA12 significantly (p < 0.05) increased the release of TNF-alpha by the EBV-B lymphoblastoid cell line JY. In contrast, the anti-HLA-DR mAb 2.06 or the superantigens staphylococcal exotoxin toxic shock syndrome toxin-1 and staphylococcal enterotoxin B that bind to HLA-DR,-DQ Ag did not affect the release of TNF-alpha by JY cells. The accumulation of TNF-alpha in the culture medium of JY cells peaked at 24 h, decreased thereafter, and was found to be dependent on the dose of mAb L243 or mAb IVA12 used to cross-link HLA class II Ag. mAb L243 or staphylococcal exotoxin toxic shock syndrome toxin-1 enhanced the spontaneous homotypic aggregation of JY cells and mediated a dose-dependent inhibition of JY cell proliferation. These phenomena were not mediated by TNF-alpha released in response to cross-linking of HLA class II Ag; polyclonal anti-TNF-alpha neutralizing antibody did not affect JY cell aggregation and the inhibition of JY cell proliferation mediated by mAb L243. In contrast, TNF-alpha secreted by JY cells enhanced a nuclear factor-kB-like activity through the binding to the 75-kDa TNF-alpha receptor. These results demonstrate an additional role of HLA class II Ag as signal-transducing molecules regulating the production of bioactive TNF-alpha by EBV-B cells. The release of TNF-alpha after the triggering of HLA class II molecules could be relevant to different aspects of B cell biology and might play a role in the pathogenesis of human diseases in which antibodies cross-reactive to HLA class II Ag have been identified.

Antibodies, Monoclonal↗