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Biomedical subjects

M De Felice

Publications and source records attributed to M De Felice.

At least 19 recordsLinked to original sources
PubMed2002

Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland.

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSHTSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pit(dw)pit(dw)) or of a functional TSHR (tshr(hyt)tshr(hyt) and TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.

Animals↗
PubMed2001

Identification of the DNA-binding protein, HrcA, of Streptococcus thermophilus.

HrcA is a negative transcriptional factor controlling the expression of the stress-specific operons dnaK and groESL in several bacteria. Although the HrcA structural gene has been identified in various organisms, studies at the protein level have been so far limited and mostly restricted to Bacillus subtilis. We have identified the HrcA protein of Streptococcus thermophilus and show here that it is a dimer with a native molecular mass of 74.5 kDa and a sequence-specific DNA-binding activity. Partially denatured and inactive S. thermophilus HrcA recovered its binding activity in the presence of the GroEL chaperone.

Bacillus subtilis↗
PubMed2001

The functional ccpA gene is required for carbon catabolite repression in Lactobacillus plantarum.

We report the characterization of the ccpA gene of Lactobacillus plantarum, coding for catabolite control protein A. The gene is linked to the pepQ gene, encoding a proline peptidase, in the order ccpA-pepQ, with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two ccpA transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element (cre) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a ccpA mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, beta-glucosidase and beta-galactosidase, was relieved from carbon catabolite repression in the ccpA mutant strain. In vivo footprinting analysis of the catabolite-controlled bglH gene regulatory region in the ccpA mutant strain showed loss of protection of the cre under repressing conditions.

Bacterial Proteins↗
PubMed2001

Surface display of recombinant proteins on Bacillus subtilis spores.

We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

Amino Acid Sequence↗
PubMed2000

Efficient insertional mutagenesis in Streptococcus thermophilus.

Bacteria have always been considered ideal organisms for genetic analysis. While this is true for some model organisms, like Escherichia coli, Bacillus subtilis and, more recently, Lactococcus lactis, genetic analysis of other organisms is often prevented by lack of valuable tools, like vectors, transposons and methods for transformation, gene inactivation and random insertional mutagenesis. This is the case of the moderately thermophilic bacterium Streptococcus thermophilus, an organism that, in spite of its widespread use for food fermentations, is only poorly characterized. We report here an insertional mutagenesis system that allows efficient random mutagenesis, easy characterization of the interrupted genes and construction of stable null mutations. This may become a powerful S. thermophilus tool for both genetic analysis and construction of 'food-grade' mutants of this biotechnologically relevant microorganism.

Base Sequence↗
PubMed2000

Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus.

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.

Adenosine Triphosphatases↗
PubMed2000

A physical and functional analysis of the newly-identified bglGPT operon of Lactobacillus plantarum.

A newly-identified bglGPT operon of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed three open reading frames encoding (i) a 237-amino acid protein (BglG), (ii) a 577-amino acid protein (BglP) and (iii) a 486-amino acid protein (BglT). BglG, BglP and BglT were shown to be homologous to the BglG family of transcriptional antiterminators, to permeases of the phosphoenolpyruvate-dependent phosphotransferase system and to beta-glucosidases, respectively. Complementation of E. coli mutant strains showed that BglP and BglT are a permease and a beta-glucosidase active on the beta-glucosides, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-glucoside, respectively. BglG was also shown to promote expression of a bglG-lacZ gene fusion in an E. coli bglG(-) background. A ribonucleic antiterminator sequence, the antiterminator-responsive cis-element and a 'catabolite responsive element', were found downstream of the transcriptional start point. Transcription of the operon was repressed 10-fold in L. plantarum cells grown on glucose as compared to ribose.

Bacterial Proteins↗
PubMed2000

Enhanced and feedback-resistant gamma-glutamyl kinase activity of an Escherichia coli transformant carrying a mutated proB gene of Streptococcus thermophilus.

We used a PCR-based method to generate a single base pair mutation in the proB gene of Streptococcus thermophilus, which replaced an aspartic acid with a glycine residue at position 192 of the first proline biosynthetic enzyme gamma-glutamyl kinase. This was the first identified mutation in amino acid biosynthesis in S. thermophilus to our knowledge. The mutation caused an enhanced, feedback-resistant gamma-glutamyl kinase activity and conferred an analogue-resistant phenotype to an Escherichia coli transformant containing the mutated gene.

Amino Acid Sequence↗
PubMed2000

On the fate of ingested Bacillus spores.

Spores of various Bacillus species, including B. subtilis, B. cereus and B. clausii, are used as probiotics, although they are generally absent from the normal microflora of man. We used two nonpathogenic Bacillus species, B. subtilis and B. clausii, to follow the fate of spores inoculated intragastrically in mice. We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species. Both spores and cells were detected in the lymph nodes and spleen of one mouse. Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B. anthracis germination in macrophages. These results indicate that any claimed probiotic effect of B. subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine.

Administration, Oral↗
PubMed1999

Two DNA polymerase sliding clamps from the thermophilic archaeon Sulfolobus solfataricus.

Herein, we report the identification and characterization of two DNA polymerase processivity factors from the thermoacidophilic archaeon Sulfolobus solfataricus. They, referred to as 039p (244 amino acid residues, 27 kDa) and 048p (249 amino acid residues, 27 kDa), present significant primary structure similarity to eukaryotic proliferating cell nuclear antigen (PCNA). We demonstrate that both 039p and 048p form oligomers in solution and are able to substantially activate the synthetic activity of the single-subunit family B DNA polymerase from S. solfataricus (Sso DNA pol B1) on poly(dA)-oligo(dT) as a primer-template. This stimulatory effect is the result of enhanced DNA polymerase processivity, as indicated by the analysis of the elongation products on polyacrylamide gels. Activation of Sso DNA pol B1 synthetic activity was also observed on linear primed single-stranded M13 mp18 DNA as a template. By immunoblot analysis using specific rabbit antisera, 039p and 048p were both detected in the logarithmic and stationary phases of S. solfataricus growth curve. This is the first report of the identification and biochemical characterization of two distinct DNA polymerase processivity factors from the same organism. The significance of these findings for the understanding of the DNA replication process in Archaea is discussed.

Amino Acid Sequence↗
PubMed1999

Molecular genetics of congenital hypothyroidism.

Congenital thyroid gland defects - resulting in reduced production of the hormones triiodothyronine (T3) and thyroxine (T4) - can be a consequence of either reduced or absent thyroid tissue (thyroid dysgenesis) or, less frequently, of impairment in the biochemical mechanisms responsible for hormone biosynthesis (thyroid dyshormonogenesis). Recent studies have revealed how mutations in the genes encoding either transcription factors or the thyroid stimulating hormone receptor cause, in humans or in mouse models, thyroid dysgenesis. This demonstrates, for the first time, the heritability of this condition. New genes responsible for thyroid dyshormonogenesis have also been discovered.

Animals↗
PubMed1999

Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice.

The Pic-1, Oct-1,2, Unc-86 (POU) transcription factor Oct-4 is specifically expressed in the germ cell line, and a previous study has indicated that the expression of the lacZ gene inserted into an 18 kb genomic fragment encompassing the Oct-4 gene can come close to mimicking the endogenous embryonic expression pattern of Oct-4 in transgenic mice. In the present study transgenic mice expressing green fluorescent protein (GFP) in the germ cell line were generated using the same Oct-4 genomic fragments and the expression pattern was analyzed in detail through all stages of germ cell development. The GFP expressing primordial germ cells were first detected as early as 8.0 days post-coitum (d.p.c.; early head fold stage) at the base of the allantois in living embryos. The GFP expression was thereafter found in both male and female germ cells at all developmental stages except in male germ cells after differentiating into type A spermatogonia in the postnatal testis. There was also a lower level of expression in female germ cells in the prophase of the first meiotic division. These transgenic mice therefore proved to be powerful tools for isolating living germ cells at various developmental stages to study their nature and to isolate new genes.

Animals↗
PubMed1998

Amino acid residues involved in determining the processivity of the 3'-5' exonuclease activity in a family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus.

Herein, we report on the mutational analysis of a 70-amino acid segment (region 1, residues 438-508) of family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol). Region 1, which lies between the Exo III sequence and the similarity motif D- -SLYP, connects the exonuclease and polymerase domains of Sso DNA pol. Two C-terminally deleted forms of the enzyme, proteins N438 (residues 1-438) and N508 (residues 1-508), were overproduced in the recombinant form and biochemically characterized. They contain the three evolutionarily conserved Exo motifs, but differ in the extent of the C-terminal deletion, since only N508 includes region 1. Both have been found to retain a Mn2+-dependent 3'-5' exonuclease activity, whose thermal stability appears to be increased in comparison to that of the full-sized enzyme. Assays for processive 3'-5' exonuclease activity, carried out with the heparin trap method on a 24-base oligonucleotide, have revealed that protein N508, as well as the full-length Sso DNA pol, retains a level of processivity of the degradative function substantially higher than that for protein N438. In addition, six site-specific mutations have been introduced at the highly conserved Y-GG/A motif, which has been found within Sso DNA pol region 1. All mutant proteins (Lys491Ile, Tyr495Ser, Lys496Ile, Gly497Ala, and Ala498Val) display increased processivity of their 3'-5' exonuclease activity, with the exception of protein Tyr495Phe. By a steady-state kinetic analysis of the exonucleolytic reaction on a 24-base oligonucleotide, the above site-specific mutations have been found to affect Km values consistently with the observed differences in the processivity values, whereas the effect on the kcat values seems to be less important. The results from this mutational analysis indicate that region 1 is involved in determining the processivity of the proofreading function, directly interacting with the nucleic acid substrate.

Amino Acids↗
PubMed1998

Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains.

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

Amino Acid Sequence↗
PubMed1998

Domain organization and biochemical features of Sulfolobus solfataricus DNA polymerase.

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3'-5' exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction.

Catalytic Domain↗
PubMed1998

A mouse model for hereditary thyroid dysgenesis and cleft palate.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2-/- results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

Animals↗
PubMed1998

Expression of the bglH gene of Lactobacillus plantarum is controlled by carbon catabolite repression.

A newly identified bglH gene coding for a phospho-beta-glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the beta-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from -3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression of bglH expression.

Amino Acid Sequence↗