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Biomedical subjects

F E Baralle

Publications and source records attributed to F E Baralle.

At least 19 recordsLinked to original sources
PubMed2000

Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES.

Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).

Animals↗
PubMed1999

Human apolipoprotein A-IV reduces gastric acid secretion and diminishes ulcer formation in transgenic mice.

We have investigated the involvement of human apolipoprotein A-IV (apoA-IV) in gastric acid secretion and ulcer formation in recently generated apoA-IV transgenic mice. Compared to control littermates, transgenic animals showed a gastric acid secretion decreased by 43-77% whereas only slight variations were observed in the different cell population densities within the gastric mucosa. In addition, no variation in gastrin levels was observed. Transgenics were protected against indomethacin-induced ulcer formation, with lesions diminishing by 45 to 64% compared to controls. These results indicate that endogenous apoA-IV expression can regulate gastric acid secretion and ulcer development.

Age Factors↗
PubMed1999

Interaction between the (-170) CRE and the (-150) CCAAT box is necessaryfor efficient activation of the fibronectin gene promoter by cAMP and ATF-2.

The fibronectin promoter contains an ATF/cyclic AMP (cAMP) response element (CRE) site two helical turns upstream of a CCAAT site with which it interacts. We investigated the effects of mutating these (-170) CRE and(-150) CCAAT elements on the promoter activity regulated by three different modulators previously known to act through CRE: ATF-2, cAMP and E1a. While the cooperation seems to play no role in E1a action, integrity of the (-150) CCAAT is necessary for ATF-2 and cAMP efficient activation in a cell-specific manner. These results show that the CRE and CCAAT elements function as a 'composite element' and establish a cell-specific function for CRE-CCAAT synergy.

3T3 Cells↗
PubMed1999

Regulation of the human apolipoprotein AIV gene expression in transgenic mice.

The apolipoprotein (Apo) AI-CIII-AIV gene cluster has a complex pattern of gene expression that is modulated by both gene- and cluster-specific cis-acting elements. In particular the regulation of Apo AIV expression has been previously studied in vivo and in vitro including several transgenic mouse lines but a complete, consistent picture of the tissue-specific controls is still missing. We have analysed the role of the Apo AIV 3' flanking sequences in the regulation of gene expression using both in vitro and in vivo systems including three lines of transgenic mice. The transgene consisted of a human fragment containing 7 kb of the 5' flanking region, the Apo AIV gene itself and 6 kb of the 3' flanking region (-7+6 Apo AIV). Accurate analysis of the Apo AIV mRNA levels using quantitative PCR and Northern blots showed that the 7+6 kb Apo AIV fragment confers liver-specific regulation in that the human Apo AIV transgene is expressed at approximately the same level as the endogenous mouse Apo AIV gene. In contrast, the intestinal regulation of the transgene did not follow, the pattern observed with the endogenous gene although it produced a much higher intestinal expression following the accepted human pattern. Therefore, this animal model provides an excellent substrate to design therapeutic protocols for those metabolic derangements that may benefit from variations in Apo AIV levels and its anti-atherogenic effect.

Animals↗
PubMed1999

Mammalian cell expression of malaria merozoite surface proteins and experimental DNA and RNA immunisation.

The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.

Animals↗
PubMed1999

Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer.

Alternative mRNA splicing of the fibronectin EDI exon is controlled by a purine-rich exonic splicing enhancer (ESE), postulated as a binding site for SR proteins. By using a transient expression alternative splicing assay combined with promoter swapping, we have demonstrated that the promoter can also control EDI splicing, arguing for coupling between the transcription and splicing machineries. We now report that the SR proteins SF2/ASF and 9G8 stimulate EDI splicing in vivo and that their effect requires an intact EDI ESE. Most importantly, we show that sensitivity to these SR proteins critically depends on the promoter structure, suggesting that the transcription machinery modulates their recruitment to the ESE.

Alternative Splicing↗
PubMed1999

Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites.

The 40-50 kDa merozoite surface antigen (MSA2) is a candidate molecule for use in a malaria vaccine. The gene for MSA2 from the 3D7 isolate of Plasmodium falciparum was amplified by polymerase chain reaction and cloned into the bacterial expression vector pGEX-3X to obtain a fusion protein of MSA2 with Schistosoma japonicum glutathione S-transferase. The recombinant fusion protein was used to immunize rabbits. After four injections, the sera had Western blotting and immunofluorescence titres of 10(-6). Immune sera, and immunoglobulin (Ig)G, F(ab)'2, F(ab) prepared from the immune sera, were assessed for their effects on the growth of 3D7 parasites in vitro by microscopy and a [3H]-hypoxanthine incorporation assay. The antibodies did not significantly inhibit red blood cell invasion and parasite growth when added to cultures as 10% v/v serum or as immunoglobulin preparations at concentrations up to 200 microg ml(-1). However, in the presence of IgG or F(ab)'2, but not F(ab), antibodies to MSA2, the proportions of red blood cells invaded by more than one merozoite increased significantly. Multiple invasion is attributed to merozoites cross-linked by bivalent antibodies, attaching to and subsequently invading the same red cell. These observations have a bearing on the evasion of host immune responses by the parasite and the use of full-length recombinant MSA2 protein in a malaria vaccine.

Animals↗
PubMed1999

Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9.

The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3' splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG)(n)repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3' splice site sequences, the extent of exon 9 skipping in CFTR mRNA.

Alternative Splicing↗
PubMed1999

Regulation of fibronectin EDA exon alternative splicing: possible role of RNA secondary structure for enhancer display.

The fibronectin primary transcript undergoes alternative splicing in three noncoordinated sites: the cassette-type EDA and EDB exons and the more complex IIICS region. We have shown previously that an 81-nucleotide region within the EDA exon is necessary for exon recognition and that this region contains at least two splicing-regulatory elements: a polypurinic enhancer (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). Here, we have analyzed the function of both elements in different cell types. We have mapped the ESS to the nucleotide level, showing that a single base change is sufficient to abolish its function. Testing of the ESE and ESS elements in heterologous exons, individually or as part of the complete EDA regulatory region, showed that only the ESE element is active in different contexts. Functional studies coupled to secondary-structure enzymatic analysis of the EDA exon sequence variants suggest that the role of the ESS element may be exclusively to ensure the proper RNA conformation and raise the possibility that the display of the ESE element in a loop position may represent a significant feature of the exon splicing-regulatory region.

Alternative Splicing↗
PubMed1998

Regulation of the fibronectin EDA exon alternative splicing. Cooperative role of the exonic enhancer element and the 5' splicing site.

Alternatively spliced exons generally contain weak splicing sites, and exonic and/or intronic regulatory elements recognised by trans-acting auxiliary splicing factors. The EDA exon of the fibronectin gene is a typical example of an exon bearing a purine-rich exon splicing enhancer (ESE) element recognised by members of the SR phosphoprotein family. The regulatory region that governs splicing in the human EDA exon also contains an exon splicing silencer (ESS) element. We have cloned the mouse EDA genomic region, and we show that the ESE and the ESS elements, although they have base differences, can be replaced by the human elements without significant change in the exon inclusion/exclusion ratio. This fact suggests a common splicing regulatory mechanism across species. We demonstrate in vivo the functional activity of the mouse ESE element in splicing. We also show that the trans-acting factors recognising this element cooperate with the 5' splicing site of the EDA exon to facilitate proper exon recognition. Indeed, a strong 5' splicing site overrides the ESE function in exon recognition. However, the presence of a strong 3' splicing site is not sufficient to compensate for the absence of the splicing enhancer. Our data provide in vivo evidence of the interplay between the exonic splicing regulatory elements and the splicing sites, leading finally to subtle regulation of alternative splicing.

3T3 Cells↗
PubMed1998

Expression of RGD minus fibronectin that does not form extracellular matrix fibrils is sufficient to decrease tumor metastasis.

Fibronectin (FN) is a plasma and extracellular matrix (ECM) glycoprotein, the expression of which may modulate the invasive and metastatic abilities of cancer cells. LMM3 is a cell line derived from the highly metastatic mouse mammary adenocarcinoma MM3 and is unable to express FN both at protein and mRNA levels. To study the role of FN in the metastatic process, LMM3 cells were stably transfected with 2 variants (wt and RGD-minus) of a full length human FN cDNA. All analyzed clones secreted recombinant FN and although none assembled FN in the ECM they showed an in vitro reduced migratory ability and an increased adhesive capacity. FN-producing cells were assayed for experimental and spontaneous metastasis. All clones tested showed a significant reduction in the number of experimental lung metastasis when compared with a control clone. Similar trends were observed for spontaneous metastatic ability. Our results indicate that the expression of FN that lacks the well-recognized RGD cell-binding site and that does not form ECM fibrils, is sufficient to decrease the metastatic potential of cancer cells. Our results also suggest that an RGD-independent mechanism may be acting in the prevention of metastasis.

Adenocarcinoma↗
PubMed1998

Functional analysis of the interaction between HCV 5'UTR and putative subunits of eukaryotic translation initiation factor eIF3.

Translation initiation in Hepatitis C Virus is controlled by the presence of an internal ribosome entry site element (IRES) principally located in its 5' untranslated region (UTR). Mutation/deletion analyses have shown that the integrity of this structure is essential for initiation of cap-independent protein synthesis. We have developed a strategy to swap the position of the two major domains (II and III) on the 5'UTR sequence. The aim was to further characterize this mechanism by preserving domain-specific interactions but possibly losing contacts that require any interdomain geometry. The expression of dicistronic mRNAs containing these different UTRs showed that the positioning of the different domains on the 5'UTR is essential for efficient IRES functioning. We then used these mutants to identify cellular factors implicated in IRES activity. Using UV crosslinking assays we found that domain III makes direct contact with two proteins (p170/p120) which can be associated with efficient IRES activity. In particular, we have mapped the binding sites of these proteins and shown that p120 binds to the apical loop segment of domain III, whilst p170 binds in the stem portion, independently of domain III position or context. Finally, we provide evidence showing that p170 and p120 represent two subunits of eukaryotic initiation factor eIF3: p170 and p116/p110.

Animals↗
PubMed1998

The A/G polymorphism in the -78 position of the apolipoprotein A-I promoter does not have a direct effect on transcriptional efficiency.

A promoter polymorphism A/G at position 78 bp upstream of the transcription initiation site characterizes the human apolipoprotein A-I gene. Some studies correlated the higher Apo A-I levels or increased Apo A-I transcription efficiency with the A allele, while other studies did not confirm these results. We have investigated the in vitro effects of this transition on the transcriptional efficiency of ApoAI gene by creating two sets of identical constructs with the whole Apo A-I promoter, carrying the A or the G, linked to the complete ApoAI gene. The relative activity of the two promoter alleles was determined through a quantitative RT-PCR system after transient tranfections of human HepG2 cell line in basal state and after stimulation with retinoic acid or 17beta-estradiol. Our results exclude differences in promoter activity linked to the A or G promoter alleles either in basal or in stimulated conditions. The data suggest that the A/G polymorphism does not directly affect the transcriptional efficiency of ApoAI gene, although it may be in linkage disequilibrium with other regulatory sequences and the combination of these elements may explain the contradictory results of the ApoAI gene expression.

Adenine↗
PubMed1998

The neutralizing antibody response against a conserved region of human immunodeficiency virus type 1 gp41 (amino acid residues 731-752) is uniquely directed against a conformational epitope.

Amino acids 731-752 (731PRGPDRPEGIEEEGGERDRDRS752) of the transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1) are conserved in most virus isolates and are controversially reported to be implicated in virus neutralization. The humoral response in infected patients against this region is poor and humans immunized with gp160 show high levels of antibodies against the peptide but poor neutralization titres. Nonetheless, several groups have succeeded in obtaining neutralizing antibodies against this sequence using different antigen-presenting systems. In order to identify the sequence(s) against which the neutralizing response was generated, we used the flock house virus (FHV) antigen-presenting system to analyse neutralizing antisera from mice immunized with a cowpea mosaic virus (CPMV) chimera expressing the 731-752 sequence. Data show that the neutralizing response is uniquely directed against a conformational epitope mapping to the ERDRD portion of this sequence, although the major antibody response, which is non-linear, and is not neutralizing, is against an IEEE epitope. These results provide an explanation for the controversy regarding the immunogenicity of this region of gp41 and suggest that this conformational epitope, in the absence of the non-neutralizing epitope, should be considered for a subunit vaccine. In addition, this study highlights the usefulness of antigen-presenting systems that preserve epitope conformation in the investigation of immune responses.

Animals↗
PubMed1998

Localization of the different hepatitis C virus core gene products expressed in COS-1 cells.

In this study we have investigated the subcellular localization in transfected COS-1 cells of the two major forms of the hepatitis C virus core protein: the immature protein of 191 residues (p21) and its proteolytically cleaved product of 173 residues (p19). In this study, and unlike previous investigations, we have been able to distinguish separately the localization of p21 from p19. This was achieved by the addition of a C-terminal HSV Tag to the p21 full coding sequence, and exploiting the fact that it is subsequently lost in the p19 product. In order to obtain an accurate localization of both p21 and p19 we used a mouse anti-HSV Tag MAb together with a human anti-core MAb (B12.F8) to perform double immunofluorescence studies. The results have shown that p21 is always localized around the nuclear envelope. On the other hand, p19 can be found diffused in the cytoplasm to different degrees. These in vivo results reinforce the proposed links between the regulated processing of the hepatitis C virus core protein and the possibility that this may contribute towards the regulation of its diverse biological functions.

Animals↗
PubMed1998

New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign cholesteryl ester storage disease (CESD). To identify the molecular basis of the different phenotypes we have characterised the LAL gene mutations in three new patients with LAL deficiency. A patient with WD was homozygote for a null allele Y303X. The other two patients, with CESD, presented either homozygosity for T267I or compound heterozygosity consisting of Q64R and an exon 8 donor splice site substitution (G-->A in position -1). The mutants T267I and Q64R and the previously reported L273S, G66V, and H274Y CESD substitutions, overexpressed in stable clones, were found to be fully glycosylated and show an enzymatic activity of 3-8% of that of normal LAL. On the other hand, the delta254-277 mutant protein derived from exon 8 skipping and the Y303X protein were totally inactive. By transient transfection of hybrid minigene constructs, the CESD G-->A (-1) substitution resulted in partial exon inclusion, thus allowing the production of a small amount of normal LAL mRNA and hence of a functional enzyme. In contrast, a G-->A substitution observed in WD at position + 1 of the same exon 8 donor site resulted in complete exon skipping and the sole production of an inactive delta254-277 protein. In conclusion, LAL genotypes determine the level of residual enzymatic activity, thus explaining the severity of the phenotype.

Amino Acid Substitution↗
PubMed1997

Biochemical and molecular characterization of hereditary myeloperoxidase deficiency.

Hereditary myeloperoxidase (MPO) deficiency is a neutrophil disorder characterized by the lack of peroxidase activity. Cytochemical, biochemical, spectroscopic, immunocytochemical, and genetic studies were carried out on a 5-year-old MPO-deficient subject and on her parents. The father was also MPO-deficient, whereas the mother had 24% of normal MPO activity. Although the typical absorption spectrum of MPO was absent in both the father and daughter, the father's neutrophils, and not those of the daughter, contained material antigenically related to MPO. In the MPO gene of the father, two mutations were found, each located in a different allele: a T --> C transition, causing the nonconservative replacement M251T and a 14-base deletion within exon 9. The M251T substitution occurred in the carboxy-terminal region of the light chain that is included in the heme pocket. The daughter inherited the 14-base deletion from her father. The study of the MPO mRNAs present in liquid cultures of granulocyte precursors surprisingly showed that the same genetic defect, ie, the 14-base deletion, seemed to exhibit different mRNA phenotypes in the father and the daughter. In fact, mRNA derived from the 14-base-deleted allele was not found in the father and an aberrantly spliced MPO mRNA with a 77-base deletion of exon 9, which includes the 14-base deletion and leads to the generation of a premature stop codon, was found in the daughter. The possibility that Delta77 mRNA could derive from other mutations linked to the Delta14 allele was dismissed because no sequence differences were found in the region (exons and exon-intron junctions). Our data indicate that the alteration of the mRNA context caused by the 14-base deletion provide a basis for the 77-base deletion in the mRNA processing. Since the granulocyte precursors from the liquid cultures of the father were more differentiated than those from the daughter, the observed different behavior of the 14-base-deleted allele in the father and daughter may be the result of a differentiation-stage dependent control of altered spliced mRNA, which may be tolerated during the early stages of differentiation but degraded at later stages. In the liquid cultures of the daughter's cells, in addition to the mRNA with the 77-base deletion, a mRNA with the wild type sequence was also found. This mRNA was inherited from the mother, since no mutations were found in her MPO cDNA and MPO gene. The MPO defect might be caused by a regulatory mutation that induces the MPO gene switch off at an early stage of granulocyte differentiation.

Adult↗