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Biomedical subjects

M Tripodi

Publications and source records attributed to M Tripodi.

At least 19 recordsLinked to original sources
PubMed1995

Temporal and tissue-specific expression of the MET ORF driven by the complete transcriptional unit of human A1AT gene in transgenic mice.

We inserted the sequence coding for the cytoplasmic portion of the human MET receptor into an 18-kb genomic fragment containing the entire human A1AT gene (encoding alpha-1-antitrypsin). Stringent control of gene expression, at the transcriptional, post-transcriptional and translational levels, was ensured by insertion of the MET open reading frame into A1AT, thus maintaining: (i) all the elements that confer tissue-specific transcription initiation, (ii) all the sequences involved in transcript processing and (iii) all the sequences which influence messenger stability and translational efficiency. The expression pattern of this vector in transgenic mice was identical to that of the human A1AT transgene, as well as to that of A1AT in humans with regard to both temporal and tissue-specific regulation.

Animals↗
PubMed1994

Evaluation of a transgenic mouse model for alpha-1-antitrypsin (AAT) related liver disease.

We have attempted to produce a transgenic mouse model of the neonatal liver disease associated with the human PIZ allele. Analysis of a number of transgenic mouse lines carrying either a normal human PIM gene construct or the mutant Z is reported. Using isoelectric focusing analysis of plasma from transgenic mice, we have shown that the human AAT proteins produced in mice are processed in a similar way to their counterparts in humans. By comparing the level of M and Z mRNA in liver with the levels of M and Z proteins in plasma we have inferred that, as in humans, the mutant protein tends to accumulate within the hepatocyte. Accumulation of Z protein has also been demonstrated by immunocytochemistry. Two of the M transgenic lines produce such high levels of the human protein that it, like the Z protein, accumulates as globules. Histological features of livers from 116 mice of different ages and genotypes were examined: 37 non-transgenic, 62 Z transgenic (23 low expressing and 39 high expressing) and 17 M transgenic mice, all high expressing. Cirrhosis or fibrosis was not seen in any animal and we were unable to find any evidence for neonatal liver disease. Some necrosis was seen in all genotypes and this increased significantly with age with one Z line showing significantly more frequent necrosis than any other group. This line, the highest expressing Z line, was back crossed onto 7 different genetic backgrounds but no major differences between the back crosses with respect to liver disease were observed. The mouse model we have developed is compared with other transgenic Z mouse models; none of these is representative of human neonatal liver disease. Our view is that the transgenic animals generated in these experiments may be most useful for investigating the liver manifestations that almost invariably occur in ZZ adults. Alteration of additional factors other than accumulation of Z protein, for example inactivation of the endogenous mouse genes or some environmental challenge, might produce a mouse model with more relevance to neonatal liver disease.

Animals↗
PubMed1994

Combined systemic beta-interferon and thymostimulin administration for metastatic brain tumors. A brief report.

Interferon-beta (IFN-B) plus thymostimulin (Th) were administered for eight weeks, intravenously and by intramuscular route to eight patients with metastatic brain tumors. No patient had a complete response. One had a stable disease and seven a progressive disease during that time. The mechanisms of the action of IFN-B and Th, and the possible therapeutic value are discussed.

Aged↗
PubMed1992

Recognition efficiency of the hepatitis B virus polyadenylation signals is tissue specific in transgenic mice.

The hepatitis B virus genome contains a unique polyadenylation (TATAAA) signal which is differentially utilized in the formation of the various hepatitis B virus transcripts. A head-to-tail multiple-copy insertion of a viral fragment comprising the viral enhancer, the X promoter, the X open reading frame, and the viral poly(A) signal in transgenic mice allowed us to monitor tissue-specific differences in the expression of transcripts initiating from the X promoter. These transcripts are efficiently processed at the first polyadenylation site in the liver, while in the kidney, the brain, and the testis, a portion of the transcripts covers two copies of the transgene, since only the second polyadenylation site is properly recognized. As discussed in this article, this observation suggests a tissue-specific distribution of cellular factors involved in polyadenylation.

Animals↗
PubMed1991

Disruption of the LF-A1 and LF-B1 binding sites in the human alpha-1-antitrypsin gene has a differential effect during development in transgenic mice.

Previous work in transfected cell lines and in nuclear extracts has led to the identification of two cis-acting elements important for transcription of the human alpha-1-antitrypsin (A1AT) gene, which bind to two liver specific trans-acting factors, LF-A1 and LF-B1. Mutations EM3 and PM1, which abolish the binding of LF-A1 and LF-B1 respectively, drastically reduce transcription activity of the A1AT gene in vitro and in cell culture. The same mutants have now been introduced in a larger DNA context and their effect has been tested in transgenic mice. A stretch of DNA was constructed which carries two transcriptional units: 18 kb of the human retinol binding protein (RBP) gene, driving the expression of the bacterial chloramphenicol acetyl transferase, linked to 17.5 kb containing the entire A1AT coding sequence with additional 5' and 3' flanking sequences. Transcription from the RBP promoter was shown to predominate in liver, and could be used as an internal marker of 'active copy number'. Mutations in the A1AT gene promoter were introduced by homologous recombination in bacterial cells. The results show that base pair substitutions in the binding site for LF-A1 and LF-B1 drastically reduce transcription in non-hepatic adult tissues, yolk sac, and fetal liver, whereas only LF-B1 binding site mutations have a marked, albeit variable, effect in adult liver.

Aging↗
PubMed1990

Generation of small mutation in large genomic fragments by homologous recombination: description of the technique and examples of its use.

We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.

Cloning, Molecular↗
PubMed1988

Assignment of human coagulation factor XII (fXII) to chromosome 5 by cDNA hybridization to DNA from somatic cell hybrids.

Human coagulation factor XII (fXII), a serine protease synthesized in liver and active in plasma, is involved in a wide variety of functions, including blood coagulation, fibrinolysis, bradykinin and complement activation. A complementary DNA (597 bp) encoding amino acid -16 to amino acid 183 of fXII protein was used to determine the chromosomal location of the fXII gene. DNAs from hamster-human somatic cell hybrids were digested with restriction enzymes and hybridized with the fXII cDNA. By the Southern method it was shown that restriction fragments able to hybridize to fXII cDNA are present only in DNA extracted from clones retaining human chromosome 5.

Animals↗
PubMed1988

Expression of human alpha 1-acid glycoprotein genes in cultured cells and in transgenic mice.

The human genome contains three alpha 1-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for slightly different forms of the protein. The major component of human alpha 1-acid glycoprotein found in plasma is coded by AGP-A, which is expressed in liver and in hepatoma cell lines and is induced by inflammatory stimuli. We have studied the regulation of the cloned AGP-A gene by transfection into cell lines of hepatic and nonhepatic origin. Unlike any other liver-specific gene investigated so far, every AGP construct tested was expressed with comparable efficiency in hepatoma and HeLa cells. In contrast, identical constructs in transgenic mice are expressed in a tissue-specific manner and are regulated by acute-phase stimuli. Transgenic mice carrying the cluster of three AGP genes secrete the human protein in the serum, and the corresponding mRNA is mainly derived from the AGP-A gene. The mRNA is liver specific, and its concentration increases several fold following experimentally induced inflammation. Additional transgenic lines carrying only the AGP-A gene showed that sufficient information for tissue-specific and regulated expression is contained within a 6.6-kb segment comprising the whole coding region plus 1.2-kb 5'-flanking and 2-kb 3'-flanking DNA.

Animals↗
PubMed1987

The human alpha-1-antitrypsin gene is efficiently expressed from two tissue-specific promotors in transgenic mice.

Alpha 1-antitrypsin (alpha 1AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and alpha 1AT mutant proteins are associated with hereditary disorders. To investigate the regulation of the normal human alpha 1AT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alpha 1AT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.

Animals↗
PubMed1987

Factor XII gene alteration in Hageman trait detected by TaqI restriction enzyme.

A cDNA for coagulation factor XII has been used to investigate the presence of gene lesions and restriction fragment length polymorphisms in two brothers with Hageman trait and their family. A TaqI polymorphic fragment has been found in the two propositi and in 11 members of the paternal lineage. This polymorphism, absent in the normal population, is correlated with the reduction of factor XII activity and enables the identification of heterozygous factor XII deficiency. Factor XII gene deletion as the cause of Hageman trait in this family has been excluded. A restriction map has been constructed, and the TaqI polymorphic site has been localized within the 5' portion of the gene. The mutation in the polymorphic site is probably the cause of the factor XII deficiency. Data suggest the presence of one factor XII gene per haploid genome.

Collodion↗
PubMed1986

A sequence upstream from the coding region is required for the transcription of the 7SK RNA genes.

We have isolated and characterized two recombinant lambda phages containing sequences homologous to 7SK RNA which code for a RNA 330 nucleotides long in an "in vitro" transcription system. S1 mapping of the transcript shows that this RNA corresponds to the 7SK RNA obtained from human cells, indicating that the two recombinant phages contain genes coding for 7SK RNA. The transcription of these genes is polymerase III dependent. Sequences upstream from the start of transcription are essential for "in vitro" synthesis of 7SK RNA, suggesting that internal promoter elements, if present, are not sufficient to support the synthesis of 7SK RNA. A region of homology with the upstream sequences of the genes for U6 RNA, 7SL RNA and Bombyx mori alanine tRNA is found within 50 bp from the transcription start point. Within the homologous region a motif common to the four genes is a "TATA"-like box, placed at position -30 to -25 of the 7SK RNA gene, which is typical of the polymerase II promoter region.

Base Sequence↗
PubMed1984

Human leukemia K562 cells: relationship between hemin-mediated erythroid induction, cell proliferation and expression of c-abl and c-myc oncogenes.

We have studied the expression of the c-myc and c-abl oncogenes in two human leukemic K562 cell lines which do express hemoglobin genes retaining a differential rate of cell proliferation. Our data indicate that in hemin-induced K562(S) cells the expression of c-abl oncogene decreases and appears to be related to a decrease in the proliferation capacity rather than to the activation of differentiated functions. The K562(hC) cell line, which produces large amounts of Hb Gower 1 retaining an efficient rate of cell proliferation, expresses indeed the c-abl oncogene at high level.

Cell Division↗
PubMed1984

DNA sequences complementary to human 7 SK RNA show structural similarities to the short mobile elements of the mammalian genome.

A complementary DNA clone of 7 SK RNA from HeLa cells was used to study the genomic organization of 7 SK sequences in the human genome. Genomic hybridizations and genomic clones show that 7 SK is homologous to a family of disperse repeated sequences most of which lack the 3' end of the 7 SK RNA sequence. Only few of the genomic K sequences are homologous to both 3' and 5' 7 SK probes and presumably include the gene(s) for 7 SK RNA. The sequence of four genomic 7 SK clones confirms that they are in most cases pseudogenes. Although Alu sequences are frequently found near the 3' and 5' end of K DNA, the sequences immediately flanking the pseudogenes are different in all clones studied. However, direct repeats were found flanking directly the K DNA or the K-Alu unit, suggesting that the K sequences alone or in conjunction with Alu DNA might constitute a mobile element.

Base Sequence↗
PubMed1984

Human leukemia K-562 cells: induction of erythroid differentiation by 5-azacytidine.

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.

Azacitidine↗
PubMed1984

Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x.

Cytoplasmic poly(A)+mRNA from 12-day mouse-yolk-sac erythroid cells has been used to prepare a cDNA library in the plasmid pBR322. One clone containing sequences coding for the alpha-like embryonic globin chain x, pHE52, has been identified by hybrid selection and in vitro translation of the complementary mRNA. The nucleotide sequence of pHE52 confirms that it codes for an embryonic alpha-like globin chain. The insert sequence is 316 nucleotides long, contains the codons corresponding to amino acid residues 43-141, and extends into the 3' untranslated region. An analysis of the nucleotide sequence of pHE52 and the other known alpha globins suggests that the adult-embryonic divergence began approx. 400 million years ago reflecting a difference in the evolutionary history of the alpha- and beta-globin gene complexes.

Amino Acid Sequence↗