Search PubMed⌕ Search

Biomedical subjects

R Mantovani

Publications and source records attributed to R Mantovani.

At least 73 records · Page 4Linked to original sources
PubMed1992

Incorporating knowledge to databases--a solution to complex domains.

This paper describes the design of an integrated environment to handle information on congenital heart disease patients. A three-layer model that integrates a database, a knowledge-base and a graphical user-interface is presented. The role of each layer and its connections is discussed. The advantages of using a hypertext front-end system to present and retrieve complex data are also addressed. The current status of the project and its future developments are described in the final section.

Artificial Intelligence↗
PubMed1991

The E alpha promoter: a linker-scanning analysis.

To accurately delineate the DNA sequence elements involved in the transcriptional regulation of the murine MHC class II gene E alpha, we have constructed a set of "linker-scanning" mutants of the E alpha promoter region. These were made by the gapped heteroduplex technique and result in a set of 10-bp replacement mutations, covering the -206 to -6 stretch of the promoter. The effect of these mutations on transcriptional activity was evaluated in several systems, either by transfection into cultured cells or by in vitro transcription. The data points to the now classical X and Y boxes as the most important control elements, either for constitutive expression in B lymphoma cells, or for IFN-gamma-inducible expression in macrophages. Motifs upstream of the X box also play a role, but are somewhat less critical. Overall, we find no marked difference between regulatory strategies in B lymphomas or activated macrophages.

Animals↗
PubMed1989

The same nuclear proteins bind the proximal CACCC box of the human beta-globin promoter and a similar sequence in the enhancer.

Using in vitro assays, we show that nuclear proteins related to the Sp1 and GT-1 factors bind to a CACCC box sequence in the human beta-globin enhancer, adjacent to binding sites for the erythroid-specific factor NFE1 and the ubiquitous factor CP1. The same proteins are known to bind to the proximal, but not to the distal, CACCC, box in the human beta-globin promoter. A C G mutation in the promoter CACCC box, known to cause beta-thalassemia, greatly decreases protein binding to the CACCC box; the same effect is obtained when this mutation is introduced into the enhancer CACCC box.

Base Sequence↗
PubMed1989

The deletion of the distal CCAAT box region of the A gamma-globin gene in black HPFH abolishes the binding of the erythroid specific protein NFE3 and of the CCAAT displacement protein.

Non-deletion Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by great elevation of the synthesis, in adult age, of fetal hemoglobin (HbF), of either the A gamma or G gamma type. Strong genetic evidence indicates point mutations in the G gamma- or A gamma-globin promoter as responsible for overexpression of the mutated gene. Here we report that a 13 nucleotides deletion in the CCAAT box region of the A gamma-globin promoter, associated with greater than 100 fold overexpression of the gene, abolishes the in vitro binding of the ubiquitous factors CP1 and CDP (CCAAT displacement protein) and of the erythroid specific protein NFE3. Loss of NFE3 binding is consistent with a similar effect of the -117 G greater than A HPFH mutation, suggesting a possible role of NFE3 as a negatively acting factor. In addition, loss of CDP binding indicates that this alteration might also contribute to the HPFH phenotype in this particular case, suggesting possible heterogeneity of the mechanisms causing HPFH.

Base Sequence↗
PubMed1989

Increased erythroid-specific expression of a mutated HPFH gamma-globin promoter requires the erythroid factor NFE-1.

The -175 T greater than C mutation in the promoter of the A gamma- or G gamma-globin gene causes a 50-100 fold increase of the expression of the respective gene in adult erythroid cells (Hereditary Persistence of Fetal Hemoglobin). We show here that this mutation increases 3-9 fold the expression of a gamma-CAT reporter plasmid transfected into the erythroid cells K562, but not that of the same plasmid in non erythroid cells. The overexpression of the mutant is abolished by the mutation of the binding site for the erythroid specific factor NFE1; inactivation of the adjacent binding site for the ubiquitous factor OTF1 does not cause overexpression of the normal gamma-globin promoter. Previous results demonstrated that the -175 mutation slightly increases the in vitro binding of NFE1 and almost abolishes that of OTF1; the present functional data indicate that altered binding of NFE1, but not of OTF1, is responsible for the observed overexpression of the mutated promoter.

Adult↗
PubMed1989

Distamycins inhibit the binding of OTF-1 and NFE-1 transfactors to their conserved DNA elements.

We investigated the effects of the antiviral agent distamycin A and of a distamycin derivative (FCE 24517) which possesses antineoplastic activity on the binding of some regulatory proteins to DNA. Both compounds inhibited the binding to DNA of the ubiquitous octamer binding factor OTF 1 and of the erythroid specific GATAAG protein (NFE 1). This was shown using the electrophoretic mobility shift assay on a DNA fragment of human gamma-globin gene promoter (-156 to -201), on the same fragment with a point mutation (T to C mutation) known to have an increased affinity of binding for NFE 1, on a DNA fragment of human histone H2B promoter and on a DNA fragment of mouse alpha 1 globin promoter. The ability of distamycin or of FCE 24517 to inhibit the binding was specific for AT-rich sequences since neither drug inhibited the binding of nuclear protein factors to the sequence CCACACCC of the human beta globin gene. Binding to DNA was investigated by evaluating the drugs' ability to protect selected sequences from DNase I digestion (DNase footprinting). Distamycins binding was highly preferential for DNA sequences containing stretches of AT. These studies indicate that chemicals which have a high degree of DNA sequence-specific binding can selectively inhibit the binding of regulatory proteins to DNA. These effects might be responsible for modification of the transcription of specific genes and might to some extent account for these drugs' antiviral and antineoplastic activities. This approach offers potential for the investigation of new such drugs.

Base Sequence↗
PubMed1989

Patterns and routes of tracheobronchial colonization in mechanically ventilated patients. The role of nutritional status in colonization of the lower airway by Pseudomonas species.

Tracheobronchial colonization by Gram-negative bacteria is common in mechanically ventilated patients. Pseudomonas sp are commonly isolated from the lower airways. We hypothesized that Pseudomonas sp would preferentially colonize the lower airway and would be more common in patients with poor nutritional status. We serially collected 75 pairs of upper and lower respiratory tract cultures from 14 patients treated with mechanical ventilation for at least one week, examined patterns of airway colonization and routes of bacterial entry for Pseudomonas sp and other enteric Gram-negative bacteria (EGNB), and related these findings to host-associated factors, including nutritional status. Pseudomonas sp were the most common species isolates taken from the lower airway, found in nine of 14 patients and in 41.3 percent of all cultures. In contrast to other EGNB, Pseudomonas sp were found significantly (p less than or equal to 0.05) more often in the tracheobronchial tree (31 of 75 cultures) than in the oropharynx (18 of 75 cultures). Primary colonization of the lower airway by Pseudomonas sp was found in four patients, while other EGNB never followed this pattern when subjects were studied with cultures taken every third day. A host-related factor related to lower airway colonization by Pseudomonas species was poor nutritional status, assessed by a multifactorial index (p less than or equal to 0.01). We conclude that in mechanically ventilated patients, Pseudomonas sp colonize the lower airway in a different pattern and by a different route from those of other EGNB. The findings that Pseudomonas sp preferentially colonize the tracheobronchial tree may be important for the design of strategies to prevent airway colonization. The recognition that poor nutritional status, a potentially modifiable host-related factor, favors lower airway growth of Pseudomonas sp suggests one direction for future infection-control efforts.

Adult↗
PubMed1989

DNA sequences regulating human globin gene transcription in nondeletional hereditary persistence of fetal hemoglobin.

Strong genetic evidence supports the idea that point mutations in the promoter of gamma-globin genes overexpressed in adult age [hereditary persistence of fetal hemoglobin (HPFH)] are responsible for the observed phenotype. DNA binding sites for ubiquitous and/or erythroid specific nuclear proteins correlate in location with the positions of point mutations responsible for HPFH. The analysis of the effects of one of these mutations (-175 T greater than C) on in vitro binding of nuclear proteins and on the activity of the mutated promoter in transfection assays indicates that altered binding of the erythroid-specific protein NFE-1 may be responsible for increased activity of the mutated promoter. Other HPFH mutations close to the distal CCAAT box (-117 G greater than A and 13 nucleotide deletions, -114 to -102) have complex effects on in vitro binding of nuclear proteins; their only common effect is the loss of binding of the erythroid-specific factor NFE3. If mechanisms generating the HPFH phenotype are homogeneous, NFE3 might be a negatively acting factor; alternatively, heterogeneous mechanisms might operate and HPFH might additionally be related to loss of binding to the distal CCAAT box region of either NFE1 (-117 HPFH) or of the ubiquitous CCAAT displacement protein-CDP (13 nucleotides deletion). Finally, it is also proposed that increased activity of the HPFH promoters may secondarily cause decreased expression of the delta- and beta-globin genes in cis possibly by competition between gamma- and beta-globin promoters for interaction with common regulatory elements.

Base Sequence↗
PubMed1988

The effects of HPFH mutations in the human gamma-globin promoter on binding of ubiquitous and erythroid specific nuclear factors.

Genetic evidence indicates that single point mutations in the gamma-globin promoter may be the cause of high expression of the mutated gene in the adult period (Hereditary Persistence of Fetal Hemoglobin, HPFH). Here we show that one of these mutations characterized by a T----C substitution at position -175 in a conserved octamer (ATGCAAAT) sequence, abolishes the ability of a ubiquitous octamer binding nuclear protein to bind a gamma-globin promoter fragment containing the mutated sequence; however, the ability of two erythroid specific proteins to bind the same fragment is increased three to five fold. DMS interference and binding experiments with mutated fragments indicate that the ubiquitous protein recognizes the octamer sequence, while the erythroid specific proteins B2, B3 recognize flanking nucleotides. Competition experiments indicate that protein B2 corresponds to an erythroid-specific protein known to bind to a consensus GATAG sequence present at several locations in alpha, beta and gamma-globin genes. Although the distal CCAAT box region of the gamma-globin gene shows a related sequence, an oligonucleotide including this sequence does not show any ability to bind the above mentioned erythroid protein; instead, it binds a different erythroid specific protein, in addition to a ubiquitous protein. The -117 G----A mutation also known to cause HPFH, and mapping two nucleotides upstream from the CCAAT box, greatly decreases the binding of the erythroid-specific, but not that of the ubiquitous protein, to the CCAAT box region fragment.

Base Sequence↗
PubMed1988

An erythroid specific nuclear factor binding to the proximal CACCC box of the beta-globin gene promoter.

We have used the gel retardation and DNAase I assays to investigate the binding of nuclear proteins to the human beta-globin promoter. Upon incubation with beta-globin promoter fragments containing the duplicated CACCC boxes, nuclear proteins from human erythroid cells generate complexes yielding four retarded bands in acrylamide gels; the three slowest bands are common to both erythroid and non erythroid cells. The fast band is present only in K562 erythroleukemic cells induced to differentiation and hemoglobin accumulation and in fetal and adult erythroblasts, but absent in uninduced K562 cells. Binding occurs on a short DNA region including the proximal CACCC box, and is not significantly competed by excess gamma-globin fragments containing the CACCC box; the CACCC box appears to be essential for this binding, as shown by the failure of a fragment containing a natural beta-thalassemic mutation (-87, C----G) to bind significantly to nuclear factors. These data suggest that the erythroid specific CACCC binding factor might play a role in the developmental activation of beta-globin transcription.

Base Sequence↗
PubMed1988

Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter.

A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3' end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

Base Sequence↗
PubMed1987

A protein factor binding to an octamer motif in the gamma-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.

Base Sequence↗
PubMed1987

The 3' ends of the deletions of Spanish delta beta zero-thalassemia and black HPFH 1 and 2 lie within 17 kilobases.

Spanish delta beta zero-thalassemia, a mild thalassemic condition characterized by increased level of hemoglobin (Hb) F production during adult life, is known to be due to a large deletion starting within the beta globin gene cluster and extending beyond the 3' breakpoint of any other similar deletional defects so far identified. By molecular cloning and by genomic mapping we now demonstrate that the deletion of Spanish delta beta zero-thalassemia ends at approximately 11 and 17 kilobases (kb) downstream to the 3' endpoints of black hereditary persistence of fetal hemoglobin (HPFH) type 1 and 2, respectively. As suggested by the complete characterization of this and other deletional defects involving the beta globin gene cluster, the 5' and 3' breakpoints of several deletions cluster in rather restricted DNA areas, further strengthening the idea that common molecular mechanisms may operate in causing these deletions.

Base Sequence↗