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Biomedical subjects

M E Bianchi

Publications and source records attributed to M E Bianchi.

At least 19 recordsLinked to original sources
PubMed2004

Molecular mechanisms in male determination and germ cell differentiation.

Sex determination and gametogenesis are key processes in human reproduction, and any defect can lead to infertility. We describe here the molecular mechanisms of male sex determination and testis formation; defects in sex determination lead to a female phenotype despite the presence of a Y chromosome, more rarely to a male phenotype with XX chromosomes, or to intersex phenotypes. Interestingly, these phenotypes are often associated with other developmental malformations. In testis, spermatozoa are produced from renewable stem cells in a complex differentiation process called spermatogenesis. Gene expression during spermatogenesis differs to a surprising degree from gene expression in somatic cells, and we discuss here mechanistic differences and their effect on the differentiation process and male fertility.

Animals↗
PubMed2004

Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function.

High mobility group box protein 1 (HMGB1) has been considered as a ubiquitous nuclear protein with an architectural function, but even early reports have described its presence outside of the nucleus. Today, we have only started to understand the extranuclear and extracellular functions of HMGB1: we know that it participates in developmental and differentiation processes, triggers and modulates many of the inflammatory cascades in the body, and may even be involved in the metastatic invasion programme of cancer cells. Given such diverse roles, it is important to know which cells express HMGB1, where, and how much. The present review deals with the expression pattern of HMGB1 and provides evidence that, far from being housekeeping, the HMGB1 gene is tightly regulated. This can have implications for therapeutic intervention on inflammatory diseases as well as cancer.

Apoptosis↗
PubMed2001

Spatially precise DNA bending is an essential activity of the sox2 transcription factor.

Sox proteins, a subclass of high mobility group box proteins, govern cell fate decisions by acting both as classical transcription factors and architectural components of chromatin. We aimed to demonstrate that the DNA bending activity of Sox proteins is essential to regulate gene expression. We focused on mouse Sox2, which participates in the transactivation of the Fgf4 (fibroblast growth factor 4) gene in the inner cell mass of the blastocyst. We generated six substitutions in the high mobility group box of Sox2. One mutant showed a reduced DNA bending activity on the Fgf4 enhancer (46 degrees instead of 80 degrees), which resulted in more powerful transactivation compared with the wild type protein. We then selected two single-base mutations in the Fgf4 enhancer that make the DNA less bendable by the Sox2 protein. Again, a different DNA bend (0 degrees and 42 degrees instead of 80 degrees) resulted in a different activation of transcription, but in this case reduced bending corresponded to decreased transcription. We found that the opposite effect on transcription of similar DNA bending angles is due to a 20 degrees difference in the relative orientation of the DNA bends, proving that a correct three-dimensional geometry of enhanceosome complexes is necessary to promote transcription.

Animals↗
PubMed2001

Nestin is a neuroepithelial target gene of thyroid transcription factor-1, a homeoprotein required for forebrain organogenesis.

Thyroid transcription factor-1 (TTF-1, also known as NKX2.1 and T/EBP), a transcription factor belonging to the NKX-2 family of homeodomain-containing genes, plays an essential role in the organogenesis of the thyroid gland, lung, and ventral forebrain. Nestin is an intermediate filament protein strongly expressed in multipotential neuroepithelial stem cells and rapidly down-regulated during postnatal life. Here we show that stable fibroblastic clones expressing TTF-1 acquire a phenotype reminiscent of neuroepithelial cells in culture and up-regulate the endogenous nestin gene. TTF-1 transactivates in HeLa and NIH3T3 cells a reporter gene driven by a central nervous system-specific enhancer element from the second intron of the rat nestin gene, where it recognizes a DNA-binding site (NestBS) whose sequence resembles a nuclear hormone/cAMP-responsive element very different from canonical TTF-1 binding sites. Nuclear extracts from the head of mouse embryos form a retarded complex with NestBS of the same mobility of the extracts obtained from TTF1-expressing clones, which is either abolished or supershifted in the presence of two different antibodies recognizing the TTF-1 protein. Thus, the neuroepithelial marker nestin is a direct central nervous system-specific target gene of TTF-1, leading to the hypothesis that it might be the effector through which TTF-1 plays its role in the organogenesis of the forebrain.

3T3 Cells↗
PubMed2001

Thermodynamics of HMGB1 interaction with duplex DNA.

The high mobility group protein HMGB1 is a small, highly abundant protein that binds to DNA in a non-sequence-specific manner. HMGB1 consists of 2 DNA binding domains, the HMG boxes A and B, followed by a short basic region and a continuous stretch of 30 glutamate or aspartate residues. Isothermal titration calorimetry was used to characterize the binding of HMGB1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To elucidate the contribution of the different structural motifs to DNA binding, calorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), and the full-length HMGB1 protein. Thermodynamically, binding of HMGB1 and all truncated constructs to duplex DNA was characterized by a positive enthalpy change at 15 degrees C. From the slopes of the temperature dependence of the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.105 +/- 0.05 kcal mol(-1) K(-1) were determined for box A and full-length HMGB1, respectively. Significant differences in the binding characteristics were observed using full-length HMGB1, suggesting an important role for the acid tail in modulating DNA binding. Moreover, full-length HMGB1 binds differently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperative, had a larger apparent binding site size, and proceeded with a much larger unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).

Aspartic Acid↗
PubMed2001

General transcription factors bind promoters repressed by Polycomb group proteins.

To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, we used formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Here we show that binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and that depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. We further show that PcG proteins interact in vitro with GTFs. We suggest that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription.

Amino Acid Sequence↗
PubMed2001

Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f).

The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.

Amino Acid Sequence↗
PubMed2001

The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.

Animals↗
PubMed2001

Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and maintain Fab-7 PRE silencing.

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. We have investigated whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome segregation associated with ph mutations. We propose that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression.

Animals↗
PubMed2001

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2.

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.

Amino Acid Sequence↗
PubMed2001

Palivizumab in infants with gestational age < or = 28 weeks and bronchopulmonary dysplasia.

We report data from 11 patients < or = 28 weeks' gestation with severe bronchopulmonary dysplasia (BPD) prophylactically treated with palivizumab for prevention of respiratory syncytial virus (RSV) infection. All babies were receiving pharmacologic respiratory therapy at the moment of discharge from neonatal intensive care unit. Babies received 15 mg/kg i.m. palivizumab every 4 weeks to a max of 5 doses during the period November-March. We compared them with 8 similar infants that did not require therapy at discharge, nor were given any placebo. The treated infants did not present significant side effects. No baby in both groups was infected with RSV during the period of observation. We conclude from these preliminary data that palivizumab did not present contraindications in infants < or = 28 weeks' gestation with severe BPD requiring pharmacologic therapy at discharge. More data are required to evaluate efficacy to prevent RSV infection in these infants.

Analysis of Variance↗
PubMed2000

Cloning and expression pattern of a zebrafish homolog of forkhead activin signal transducer (FAST), a transcription factor mediating Nodal-related signals.

Forkhead activin signal transducer (FAST) is a member of the winged-helix family of DNA-binding proteins that has been implicated in mesoderm induction and left-right axis specification during embryonic development in Xenopus and mouse. We have cloned and characterized a zebrafish FAST homolog. Zebrafish fast is expressed maternally and zygotically. Transcripts start regionalizing and decline in level during gastrulation. During somitogenesis, fast is expressed bilaterally in the lateral plate mesoderm, like its mouse homolog. In addition, zebrafish fast is also expressed bilaterally in the dorsal diencephalon, where the nodal-related cyclops gene is only expressed on the left side. It remains to be demonstrated whether FAST expression in the brain can mediate Nodal-induced asymmetric development.

Amino Acid Sequence↗
PubMed2000

[Clinical features in subjects with congenital anomalies of the corpus callosum].

The corpus callosum is a connecting structure between the two cerebral hemispheres. Its development occurs between 8th and 20th week of gestation. Anomalies of the corpus callosum are divided into malformative or acquired anomalies and may be isolated or associated with other cerebral malformations. The diagnosis of an alteration of the corpus callosum is fundamentally based on neuroradiological examinations. In our study we evaluate the major clinical findings, the psychomotor development, the electroencephalographic and neuroradiological features in a group of 14 children with congenital callosal anomalies, trying to single out how the associated cerebral malformations interfered with the clinical manifestations and especially the prognosis. It was not possible to highlight a distinctive symptom indicative of the presence of a corpus callosum anomaly; nevertheless in 11/14 children there was a psychomotor delay of varying degree. 42% of our subjects presented epilepsy, with a typical West syndrome in 5/6. Children affected by isolated callosum anomalies did not present any significant clinical manifestations. Finally, we conclude that the determinating factors in the severity of the clinical-electroencephalographic picture as a whole, and especially in the prognosis of our patients are the cerebral malformations associated to the callosum anomalies.

Agenesis of Corpus Callosum↗
PubMed1999

The human gene coding for HCN2, a pacemaker channel of the heart.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, underlying 'pacemaker' currents (I(f)/Ih), are involved in pacemaker activity of cardiac sinoatrial node myocytes and central neurons. Several cDNAs deriving from four different genes were recently identified which code for channels characterized by six transmembrane domains and a cyclic nucleotide binding domain. We report here the identification of the human HCN2 gene and show that its functional expression in a human kidney cell line generates a current with properties similar to the native pacemaker f-channel of the heart. The hHCN2 gene maps to the telomeric region of chromosome 19, band p13.3. This is the first identification of a genetic locus coding for an HCN channel.

Amino Acid Sequence↗
PubMed1999

High mobility group protein 1 interacts specifically with the core domain of human TATA box-binding protein and interferes with transcription factor IIB within the pre-initiation complex.

The high mobility group (HMG) box domain has defined a family of proteins, mostly transcription factors, that specifically interacts with DNA on the minor groove and sharply bends it. The founding member of the family, HMG1, does not specifically recognize regular B-DNA but is recruited to DNA by interaction with other transcription factors and TATA box-binding protein (TBP). However, conflicting effects of HMG1 on transcription have been reported. We show that the interaction between HMG1 and TBP is species-specific. This interaction in turn affects the interaction of TBP with transcription factor (TF) IIB and is competed by TFIIA. A primary binding site was mapped to the H2' alpha-helix in the highly conserved core domain of human TBP. On HMG1, the primary binding site was only in the HMG box A, and HMG box A was also sufficient to interact with native TFIID. Both HMG boxes efficiently repressed transcription in vitro as fusions to the Gal4-DNA binding domain. Additionally, HMG box B showed a weak level of activation at very low amounts. These results suggest a general involvement of HMG1 at the early stages of polymerase II transcription that may result in subtle activation or repression of individual genes.

Carrier Proteins↗
PubMed1999

Expression patterns of zebrafish sox11A, sox11B and sox21.

We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21, and two related to mammalian and chicken Sox-11. Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation and become restricted later to the developing central nervous system (CNS); expression in adults is undetectable. sox21 is expressed in the forebrain, midbrain and hindbrain, but maximally at the midbrain-hindbrain junction; sox11A,B have a widespread and dynamic expression in the CNS, but in contrast to sox21 are absent at the midbrain-hindbrain boundary.

Amino Acid Sequence↗