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Biomedical subjects

G Raugei

Publications and source records attributed to G Raugei.

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

Differential role of four cysteines on the activity of a low M(r) phosphotyrosine protein phosphatase.

In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.

ATP-Binding Cassette Transporters↗
PubMed1992

Overexpression of a synthetic phosphotyrosine protein phosphatase gene inhibits normal and transformed cell growth.

We studied the level of the cytosolic phosphotyrosine protein phosphatase (PTPase) (originally termed low-M(r) acid phosphatase) in normal NIH/3T3 and in v-erbB-transformed fibroblasts. The level of the enzyme, assayed by ELISA, was inversely related to cell proliferation, normally growing cells had less enzyme than their contact-inhibited counterparts and v-erbB transformants had less enzyme than normal NIH/3T3. In order to overexpress the enzyme and study its effects in normal and transformed cells, we transfected a synthetic gene coding for the PTPase in control NIH/3T3 and v-erbB transformants. The overexpressed enzyme was recognized by antibodies raised against the native enzyme and, in cells overexpressing the PTPase, we observed a marked dephosphorylation of tyrosyl residues of cellular proteins. Cell proliferation, in both normal and v-erbB transformants overexpressing the PTPase, was measured. We observed that PTPase overexpression was accompanied by significantly reduced thymidine incorporation in both cell types, either serum-starved or serum-stimulated. The ability of transformed v-erbB cells to grow in soft agar was also markedly decreased by overexpression of the enzyme. Taken together, our results indicate that overexpression of PTPase might interfere with mitogenic signalling pathways in both normal and transformed cells, and propose a role for PTPase in the control of cell proliferation.

3T3 Cells↗
PubMed1991

Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of lipocortin 1 and 5 in differentiated, but not undifferentiated U-937 cells.

The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.

Annexins↗
PubMed1990

Characterization of the human lipocortin-2-encoding multigene family: its structure suggests the existence of a short amino acid unit undergoing duplication.

Human genomic clones of the gene encoding lipocortin (LIP) 2 (p36) and of three pseudogenes have been isolated and characterized. The LIP2 gene is at least 40 kb long and consists of 13 exons. The three pseudogenes present typical features of retroposons and, together with the gene, probably represent the entire LIP2 multigene family. Chromosomal assignment of the four loci is proposed. The hypothesis that an ancestral unit coding for 15 to 20 amino acids may have been involved in the evolution of the gene is discussed.

Amino Acid Sequence↗
PubMed1990

An inducible enhancer controls the expression of the human interleukin 1 beta gene.

This paper describes regulatory sequences responsible for the control of human interleukin 1 beta gene expression in cells of the monocytic lineage. Hybrid plasmids containing different regions of the interleukin 1 beta gene and the bacterial chloramphenicol acetyltransferase gene were transfected into human monocytic THP-1 or U937 cells. After treatment with 12-O-tetradecanoylphorbol-13-acetate, which triggers cell differentiation, we have identified an enhancer sequence which mediates the induction of gene activity by 12-O-tetradecanoylphorbol-13-acetate. The enhancer, approximately 180 base pairs long, is located between positions -2982 and -2795 upstream from the transcriptional start site. Further analysis has shown that 132 base pairs immediately upstream from the start site of transcription are sufficient to promote constitutive expression of the gene. Additional promoter elements, located within the first intron, maximize the level of gene expression. Although activated monocytes and macrophages are the main producers of interleukin 1 beta, both the enhancer and the constitutive promoter can function in monocytic cells as well as in nonmonocytic HeLa cells.

Base Sequence↗
PubMed1987

Human interleukin-1 beta gene.

We report the nucleotide sequence of the human chromosomal gene which encodes the interleukin-1 beta protein (IL-1 beta). The gene spans a region of 7.5 kb and the coding part is divided into seven exons. Comparison with the homologous mouse gene reveals that the structural organization is conserved through evolution. In addition to this, human and murine IL-1 beta genes show extensive sequence homology within the intervening sequences.

Amino Acid Sequence↗
PubMed1985

Structure and expression of the human haptoglobin locus.

Human genomic clones of the haptoglobin Hp1F and the "haptoglobin related' gene (Hpr) have been isolated. The two genes are adjacent, spanning a region of approximately 21 kb. A comparison of their coding sequences shows that Hpr differs from Hp1F at 28 codons. Northern blot and primer elongation analyses with human liver RNA show that the haptoglobin gene Hp1F appears to be transcribed some 1000-fold less in fetal than in adult liver. In adult liver the amount of Hpr mRNA is at the lower limit of detection, therefore the extent of its expression is at most less than 1000-fold that of the Hp1F gene. No Hpr mRNA can be detected in fetal liver.

Base Sequence↗
PubMed1985

Molecular evidence of triplication in the haptoglobin Johnson variant gene.

The protein and gene structure of the Hp Johnson variant (Hp3) were analyzed in two related heterozygous individuals. The molecular weight (23 kd) and amino acid composition of Hp3 alpha chain were in agreement with the triplicated structure first suggested by Smithies in 1964. Direct gene analysis by Southern blotting showed a three-fold tandem repeat of the same 1.7 kb DNA segment implicated in the Hp2 gene duplication. On the basis of these data a nine exon model for the Hp3 gene is proposed.

Amino Acid Sequence↗
PubMed1985

A new restriction fragment length polymorphism in the haptoglobin gene region.

Direct gene analysis of the haptoglobin gene region was carried out by Southern blotting using an Hp cDNA as probe. Two types of polymorphism were observed: one due to intragenic duplication, is characterized by a constant fragment length difference of 1700bp observed with several enzymes and by complete correspondence with the protein molecular weight polymorphism; the second type, due to point mutation, was represented by two additional restriction sites for Eco RI and Pst I, with a frequency comparable to that of other genes. These two mutations segregated together in families, suggesting that the recently described Hp related gene is closely linked to the Hp gene. Moreover, they were completely associated with each other. The evolutionary significance of this finding is discussed.

Alleles↗
PubMed1984

Cloning and sequencing of a full length cDNA coding for a human apoferritin H chain: evidence for a multigene family.

We have cloned a segment of cDNA from human liver coding for an apoferritin subunit, probably an H chain. Sequence comparison with the available protein sequence shows that our clone corresponds to a ferritin subunit present as a minor species in human spleen and placenta, but as major species in HeLa cells. Northern blot analysis shows the existence of only one band of similar size in human liver, HeLa cells, Daudi lymphoma and Hep3B hepatoma cell lines. In contrast, Southern blot analysis provides evidence for a multigene family.

Amino Acid Sequence↗
PubMed1983

Cloning and sequencing of a full length cDNA coding for human retinol-binding protein.

We have isolated and sequenced a cDNA clone coding for human Retinol Binding Protein. The sequence indicates that Retinol Binding Protein is synthesized as a single polypeptide chain precursor which is then matured to the secreted protein by removal of a leader peptide. Southern and Northern blot analysis suggest that the gene is present in one or few copies per haploid genome and is transcribed in a single mRNA species.

Amino Acid Sequence↗
PubMed1983

Sequence of human haptoglobin cDNA: evidence that the alpha and beta subunits are coded by the same mRNA.

We have isolated and sequenced a cDNA clone coding for human haptoglobin. Our sequence shows that haptoglobin is very likely synthesized as a single polypeptide chain which is then cleaved at an Arg residue to generate its two characteristic alpha and beta subunit. Southern blot analysis suggests that there are at least two copies of the haptoglobin gene per haploid genome.

Amino Acid Sequence↗
PubMed1983

Cloning of several cDNA segments coding for human liver proteins.

A human cDNA library was constructed using M13 derivative vectors. The simple and rapid procedures for sequencing single-stranded DNA by the dideoxy chain termination method allowed a screening of individual clones directly by DNA sequence analysis. Some of these clones were identified as coding for: serum albumin, alpha1-antitrypsin, retinol-binding protein, prothrombin, haptoglobin, and metallothionein. Furthermore, a clone coding for aldolase B was tentatively identified on the basis of high sequence homology with rabbit muscle aldolase.

Amino Acid Sequence↗
PubMed1983

Common and interchangeable elements in the promoters of genes transcribed by RNA polymerase iii.

We have shown that the 34 bp internal control region of the somatic 5S RNA gene from Xenopus borealis can be split into two separable components. A maxigene carrying an insertion between nucleotide 71 and nucleotide 74 of the coding region is actively transcribed in the nucleus of X. laevis oocytes, giving rise to a maxitranscript with initiation and termination points identical with those of the wild-type transcript. The first 11 bases of the 5S RNA gene promoter are shown to be structurally and functionally homologous with the first component (box A) of the promoter for tRNA genes. This was shown by constructing hybrid 5S RNA-tRNAPro and tRNAPro-5S RNA genes that were efficiently transcribed in the X. laevis oocytes. Initiation of transcription appears to be a complex phenomenon in which both components of the internal promoter play a role.

Animals↗