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Biomedical subjects

R Ravazzolo

Publications and source records attributed to R Ravazzolo.

At least 55 records · Page 3Linked to original sources
PubMed1999

OTX2 homeodomain protein binds a DNA element necessary for interphotoreceptor retinoid binding protein gene expression.

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. We have previously demonstrated that a sequence named A element, in the IRBP promoter is essential for IRBP gene transcription in vivo. Here we demonstrate that the human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments.

Animals↗
PubMed1999

Intron 16 insertion of the angiotensin converting enzyme gene and transcriptional regulation.

BACKGROUND: The insertion/deletion (I/D) polymorphism in intron 16 of angiotensin converting enzyme (ACE) is associated with circulating and tissue enzymatic levels, around two-fold higher in homozygous D than homozygous I individuals. The mechanism underlying this quantitative difference is unknown and the hypothesis that the deletion removes a transcriptional silencer located within the intron has been proposed. METHODS: We have set up an assay based on constructs carrying fragments of intron 16, either in the I or in the D form, fused to the Herpes simplex virus thymidine kinase heterologous promoter driving the expression of the CAT reporter gene. These constructs have been used in transfection experiments in ACE expressing cells. RESULTS: Plasmids containing either intronic fragments from I and D subjects or an artificial D fragment derived from an I intron did not show significant difference between I and D in affecting the heterologous promoter activity. The finding of a yet undescribed polymorphism in intron 16 is also reported. CONCLUSION: The above results suggest that the intron 16 deletion by itself has no effect in regulating transcription in our assay system.

Cell Line↗
PubMed1999

Identification of LMX1B gene point mutations in italian patients affected with Nail-Patella syndrome.

Nail-Patella syndrome, or osteo-onychodysplasia, is an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns and nephropathy. Previous studies have demonstrated linkage of the Nail-Patella locus with polymorphic markers on human chromosome 9q34. Recently, point mutations in the LMX1B gene have been identified in Nail-Patella patients and in families with recurrence of Nail-Patella syndrome and open-angle glaucoma. We describe here the identification of additional point mutations in the LMX1B gene in a set of Italian patients affected with Nail-Patella syndrome: two deletions of 1 and 2 bp causing a frameshift in two sporadic patients and nonsense mutations in two familial and one sporadic cases have been identified. All the mutations affect the homeodomain of the LMX1B protein and could cause the Nail-Patella syndrome through a loss of function as well as a dominant negative effect. Haplotype analysis in the two familial cases carrying the same stop codon mutation suggests the presence of a founder effect. Finally, analysis of cDNA clones obtained from human fetal kidney has revealed the existence of two different transcripts of LMX1B gene likely due to an alternative splicing.

Alternative Splicing↗
PubMed1998

An upstream negative regulatory element in human granulocyte-macrophage colony-stimulating factor promoter is recognised by AP1 family members.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine involved in haematopoiesis and host defence. Production of GM-CSF has been detected in tumour cells including the U87MG astrocytoma cell line. Previous studies have been focused on the regulatory role of the proximal region of the GM-CSF promoter. Our studies on the distal region of the promoter in U87MG cells identify a negative cis element (-1377/-1298) which contains a AP1-like site able to bind c-jun and c-fos transcription factors, according to the results of DNA/protein binding assays. Mutagenesis of the AP1-like site eliminates AP1 binding and the negative effect on promoter activity.

Antibodies↗
PubMed1998

Cell-specific regulation of alpha1(III) and alpha2(V) collagen by TGF-beta1 in tubulointerstitial cell models.

BACKGROUND: TGF-beta1 modulates the cellular expression of extracellular matrix (ECM) in several renal cell systems in vitro and is considered a determinant of ECM accumulation in tubulointerstitial fibrosis. METHODS: We evaluated the effects of TGF-beta1 on collagen transcription, expression, and removal of the relevant collagens by rat tubuloepithelial cells (NRK 52E) and both rat and monkey interstitial fibroblasts (NRK 49F, CV1) in vitro. RESULTS: TGF-beta1 upregulated the expression of alpha1(III) collagen by fibroblasts (+300%) without affecting its removal. In parallel, a threefold increment of COL3A1 mRNA was found. Experiments of cell transfection employing CV1 fibroblasts as the unique suitable model, and chimaeric constructs of COL3A1 and COL5A2 promoters fused to the luciferase reporter gene, demonstrated a twofold stimulation of a large 1436 COL3A1 promoter construct and negligible effects on shorter fragments, suggesting the presence of a positive responsive element in a region of COL3A1 promoter between -1375 and -579. TGF-beta1 did not influence COL5A2 mRNA and the relative promoter activity in renal fibroblasts. With NRK 52E cell line, TGF-beta1 induced comparable increment of both alpha1(III) collagen expression (+300%) and COL3A1 mRNA (+300%) without affecting the COL3A1 promoter activity of any constructs. TGF-beta1 also upregulated the expression of alpha2(V) collagen chain (+500%) and COL5A2 mRNA (+500%) with a stimulatory effect (+100%) on a 1177 bp fragment of COL5A2 promoter. In this case a relevant inhibitory effect of TGF-beta1, on removal of alpha2(V) by supernatants of NRK 52E was also observed, indicating a double regulatory role of the cytokine on both transcription and removal of this component of ECM. CONCLUSION: Taken together these data indicate that TGF-beta1 is a potent stimulator of alpha1(III) collagen expression by renal fibroblast cell lines in vitro, the basic mechanism being stimulation of COL3A1 transcription. With renal epithelial cell lines, TGF-beta1 mainly upregulated the expression of type V collagen with the most relevant effect on stimulation of collagen transcription and inhibition of its removal. Tubular epithelial cells and renal fibroblasts should play distinct roles in renal fibrosis induced by TGF-beta1 in vivo.

Animals↗
PubMed1997

Sequence and characterisation of the RET proto-oncogene 5' flanking region: analysis of retinoic acid responsiveness at the transcriptional level.

The RET proto-oncogene encodes a receptor tyrosine kinase expressed during neural crest development. RET expression is enhanced in vitro by several differentiating agents, including retinoic acid (RA), which up-regulates RET expression in neuroblastoma cell lines. In the present work we sequenced and analysed a 5 kbp genomic fragment 5' to RET. Three deletion fragments of this region were tested for their RA inducibility in transient transfection assays and failed to support the hypothesis of a direct transcriptional activation. Finally, our functional analysis of a candidate RA response element present in the RET promoter provides new hints for the understanding of the interaction between nuclear receptors and their specific recognition sites.

Base Sequence↗
PubMed1997

c-Rel and p65 subunits bind to an upstream NF-kappaB site in human granulocyte macrophage-colony stimulating factor promoter involved in phorbol ester response in 5637 cells.

To further clarify the complex transcriptional regulation of the human GM-CSF gene, which was extensively investigated in activated T cells, we have studied the role of an upstream NF-kappaB like site in the 5637 non-lymphoid cell line, which derives from a bladder carcinoma and constitutively produces GM-CSF. This sequence, named the A element, has an active role on GM-CSF transcription and is responsive to the tumor promoter PMA in transient transfection experiments. We describe here a heterodimeric binding complex of NF-kappaB subunits (c-Rel and p65) which is identical to the one obtained using the HIV-LTR-kappaB site as recognition sequence and different from the one (c-Rel and p50) observed with nuclear extracts from Mo T-lymphoid HTLV-II infected cells.

Cell Nucleus↗
PubMed1996

An upstream positive regulatory element in human GM-CSF promoter is recognized by NF-kappa B/Rel family members.

To further extend the previous analysis of cis-acting elements and cognate trans-acting factors that contribute to GM-CSF transcriptional regulations, we have examined a promoter region between -1742 and -2010. DNase I footprinting assays showed four protected sequences named A, B, C and D. DNA transfections in the T-lymphoid Mo cell line, which constitutively expresses GM-CSF, indicated that the A element, located between -2002 and -1984, has a positive role on transcription. Further characterization by electrophoretic mobility shift assays in the presence of different competitor oligonucleotides showed that this element binds a factor of the NF-kappa B/Rel family.

Base Sequence↗
PubMed1996

The deletion polymorphism of the angiotensin I-converting enzyme gene is associated with target organ damage in essential hypertension.

The activity of the renin-angiotensin-aldosterone system is thought to play a significant role in the development of target organ damage in essential hypertension. An insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene has recently been associated with increased risk for left ventricular hypertrophy and coronary heart disease in the general population. The D allele is associated with higher levels of circulating ACE and therefore may predispose to cardiovascular damage. The study presented here was performed to investigate the association between the ACE genotype, microalbuminuria, retinopathy, and left ventricular hypertrophy in 106 patients with essential hypertension. ACE gene polymorphism was determined by polymerase chain reaction technique. Microalbuminuria was evaluated as albumin-to-creatinine ratio (A/C) in three nonconsecutive first morning urine samples (negative urine culture) after a 4-wk washout period. Microalbuminuria was defined as A/C between 2.38 to 19 (men) and 2.96 to 20 (women). Hypertensive retinopathy was evaluated by direct funduscopic examination (keith-Wagener-Barker classification) and left ventricular hypertrophy by M-B mode echocardiography. The distribution of the DD, ID, and II genotypes was 27, 50, and 23%, respectively. The prevalence of microalbuminuria, retinopathy, and left ventricular hypertrophy was 19, 74, and 72% respectively. There were no differences among the three genotypes for age, known duration of disease, body mass index, blood pressure, serum glucose, uric acid, and lipid profile. DD and ID genotypes were significantly associated with the presence of microalbuminuria (odds ratio, 8.51; 95% confidence interval, 1.07 to 67.85; P = 0.019), retinopathy (odds ratio, 5.19; 95% confidence interval, 1.71 to 15.75; P = 0.005) and left ventricular hypertrophy (odds ratio, 5.22; 95% confidence interval, 1.52 to 17.94; P = 0.016). Furthermore, patients with DD and ID genotypes showed higher levels of A/C (3.6 +/- 0.9, DD; 2.6 +/- 0.7, ID; 0.9 +/- 0.2 mg/mmol, II; P = 0.0015 by analysis of variance) and increased left ventricular mass index (152 +/- 4.7, DD + ID versus 133 +/- 5.7 g/m2, II; P = 0.01) compared with II patients. The D allele was significantly more frequent in patients with microalbuminuria (odds ratio, 2.59; 95% confidence interval, 1.24 to 5.41; P = 0.013) and in those with retinopathy (odds ratio, 2.44; 95% confidence interval, 1.21 to 4.90; P = 0.015). Multiple regression analyses performed among the entire cohort of patients demonstrated that ACE genotype significantly and independently influences the presence of retinopathy, left ventricular hypertrophy, and microalbuminuria. In conclusion, the D allele of the ACE gene is associated with microalbuminuria as well as with retinopathy and left ventricular hypertrophy, and seems to be an independent risk factor for target organ damage in essential hypertension.

Adult↗
PubMed1995

A single cis-acting element in a short promoter segment of the gene encoding the interphotoreceptor retinoid-binding protein confers tissue-specific expression.

Interphotoreceptor retinoid-binding protein (IRBP) is the major protein component of the interphotoreceptor matrix. IRBP has a highly restricted tissue-specific expression in retinal photoreceptor cells and in a subgroup of pinealocytes. With the purpose of understanding how transcriptional regulation contributes to the expression of human IRBP, we have studied a short promoter fragment (from -123 to +18, relative to the transcription start site). We demonstrate, by analysis of the expression of the lacZ reporter gene fused to this short promoter fragment in transgenic mice, that it is sufficient to confer tissue-specific expression in retinal photoreceptors and in pinealocytes. DNA/protein binding assays, performed to identify binding sites for tissue-specific trans-acting factors, have shown that an element between -45 and -58 binds a factor present only in nuclear extracts of retinoblastoma-derived cell lines, which express IRBP. An element further upstream, between -86 and -106, binds apparently ubiquitous factors. Site-directed mutagenesis was performed to disrupt a GATTAA motif included in the -45 to -58 binding site and a second inverted GATTAA motif present shortly upstream. In transgenic mice bearing the mutated version of the promoter fragment, the expression of the reporter gene was completely abolished, thus suggesting that this element is essential for tissue-specific expression. A GATTAA motif appears in the 5'-flanking regions of several photoreceptor-specific genes, suggesting that this could be the recognition site for a photoreceptor-specific factor.

Animals↗
PubMed1994

A negative cis-acting G-fer element participates in the regulation of expression of the human H-ferritin-encoding gene (FERH).

Ferritin (Fer) is the major iron storage protein in man. Its synthesis is regulated both at the translational and transcriptional levels. In previous studies on transcriptional regulation of the human H-ferritin-encoding gene (FERH), a 160-bp promoter segment was analyzed [Bevilacqua et al., Gene 111 (1992) 255-260]. In order to obtain a more complete view of the elements involved in the transcriptional regulation of FERH, we have studied, in a further upstream region of the human FERH promoter (pFERH), a sequence between -272 and -291, named G-fer, because it contains a stretch of ten G, which binds a nuclear factor present in different cell types. DNA-binding assays and competition experiments suggest that the factor binding to G-fer has binding properties very similar to inhibitory factor-1 (IF-1), an ubiquitous factor that interacts with G-rich elements in the promoters of the mouse type-I collagen genes. DNA transfection experiments in HeLa cells, using either a wild-type or mutated pFERH fused to a reporter gene, showed that a 3-bp substitution mutation, that abolished the binding of the specific factor to G-fer, increased the promoter activity, thus suggesting an inhibitory role for the G-fer element and its cognate trans-acting factor.

Binding Sites↗
PubMed1992

Lactoferrin binding sites and nuclear localization in K562(S) cells.

Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated. In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA-linked protein in nuclear extracts.

Binding Sites↗
PubMed1991

Purification and functional characterization of a DNA-binding protein that interacts with a negative element in the mouse alpha 1(I) collagen promoter.

In an effort to understand the regulation of expression of the mouse type I collagen genes, we have purified to homogeneity a transcription factor, called inhibitory factor 2 (IF-2), that binds to the mouse alpha 1(I) collagen promoter upstream of the proximal CCAAT motif. IF-2 was purified to homogeneity from mouse lymphocyte nuclear extracts by ion-exchange chromatography and two different steps of DNA affinity chromatography, one using the wild-type IF-2-binding site as affinity ligand and one using a mutated IF-2-binding site that has a higher affinity for the factor. Renaturation of active DNA-binding proteins purified through several chromatography steps from sodium dodecyl sulfate-polyacrylamide gels identified two polypeptides of 120 and 100 kDa, respectively, capable of binding specifically to an IF-2-binding site. DNA transfection experiments of NIH 3T3 fibroblasts using an alpha 1(I) promoter-CAT chimeric gene in which mutations were introduced that either improved or decreased the binding affinity of IF-2 for its recognition site, strongly suggest that IF-2 acts as a transcriptional inhibitor of the mouse alpha 1(I) collagen gene. DNA-binding studies with similar concentrations of purified IF-2 and CBF (CCAAT-binding factor) indicate that CBF inhibits binding of IF-2, whereas IF-2 inhibits CBF binding more weakly.

Animals↗
PubMed1991

A fibroblast-specific factor binds to an upstream negative control element in the promoter of the mouse alpha 1(I) collagen gene.

We have identified, in nuclear extracts of NIH-3T3 fibroblasts, a factor which binds a sequence between -339 and -361 in the mouse alpha 1(I) collagen promoter. A 3-base pair substitution mutation introduced in this promoter element, which abolishes the binding of the factor, increases the activity of the promoter 4-fold in DNA transfection experiments. This suggests that this element is a negative regulator of transcription. The factor which binds to the -339 to -361 sequence is present in type I collagen-producing cell lines but not in a variety of other cell types, suggesting that it is involved in the tissue-specific regulation of the alpha 1(I) gene.

Animals↗
PubMed1990

Synthesis of a 60 kD nuclear DNA binding protein induced by cytosine arabinoside in the HL 60 leukemic cell line.

Cytosine arabinoside (ara-C) is being employed at low dosage as differentiative rather than a cytotoxic agent in the therapy of leukemias. We have analyzed nuclear proteins from HL 60 leukemic cells treated with ara-C and have observed increased expression of a 60 kD protein in a dose-dependent fashion. This protein is actively synthesized, as assessed by labeled methionine incorporation. Using DNA cellulose affinity chromatography we could also demonstrate DNA binding properties of the 60 kD protein.

Cell Line↗