Search PubMed⌕ Search

Biomedical subjects

A Therwath

Publications and source records attributed to A Therwath.

At least 19 recordsLinked to original sources
PubMed2006

Exhaustive genotyping of the interferon alpha receptor 1 (IFNAR1) gene and association of an IFNAR1 protein variant with AIDS progression or susceptibility to HIV-1 infection in a French AIDS cohort.

We have undertaken a systematic genomic approach in order to explore the role of the interferon alpha (IFN-alpha) pathway in AIDS disease development. As it is very difficult to genotype the IFN-alpha gene itself since it has many pseudo-genes, we have focused our interest on the genetic polymorphisms of the IFN-alpha receptor 1 (IFNAR1). We genotyped the Genetics of Resistance to Immunodeficiency Virus (GRIV) cohort composed of patients with extreme profiles of progression to AIDS, slow progressors (SP) and rapid progressors (RP), as well as seronegative controls (CTR). We identified 19 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) greater than 1% among which two were newly characterized by our study. We found putative associations with AIDS disease development for four SNP alleles and for three haplotypes. The most interesting signals were found for two SNPs in linkage disequilibrium, the SNP IFNAR1_18339 corresponding to a Val168Leu mutation in the extracellular domain of the protein and the intronic SNP, IFNAR1_30127. The intronic SNP IFNAR1_30127 yielded a strong signal both when comparing SP with CTR (P=0.002) and RP with CTR (P=0.005) while IFNAR1_18339 yielded a smaller signal because less patients were analyzed; these SNPs could thus be involved in AIDS progression or in susceptibility to human immunodeficiency virus 1 (HIV-1) infection. Interestingly, two independent studies have previously pointed out the SNP IFNAR1_18339 in susceptibility to multiple sclerosis and to malaria. This is the first work investigating the polymorphisms of the IFNAR1 gene in AIDS. Our results which point out a possible role for the IFN-alpha pathway in susceptibility to HIV-1 infection or progression to AIDS need a necessary confirmation by genomic studies in other AIDS cohorts.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Active immunization against murine TNFalpha peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection.

New lines of treatment targeting cytokines have been successfully developed recently and are now widely used in therapy. They are based on passive administration of cytokine inhibitors either soluble receptors or mAbs and the major example is TNFalpha in rheumatoid arthritis (RA). Since a few years, our group has developed a novel alternative approach targeting cytokines by using active immunization against biologically inactive but immunogenic cytokine derivatives. In the present work, we present a new aspect of this research, based on immunization against specific cytokine peptides chosen by molecular modelling. We could elicit a significant humoral response against four TNFalpha peptides by active immunization, and show that the Abs generated cross-reacted with the native cytokine with good titers as determined by ELISA. Interestingly, during coimmunization experiments with couples of peptides, one showed a clear immunodominant effect over the other. Overall, we could not show the neutralization of TNFalpha biological activity in vitro by the immunized sera, but it seems that it is not a prerequisite to observe clinical efficacy. Indeed, using the LPS/galactosamine-induced shock, we could demonstrate that one of the four peptides tested conferred a clinical protection. These results validate the feasibility and efficacy of active immunization against cytokine peptides, and confirm that active immunization against cytokines could represent in the future an alternative to passive immunization in many diseases.

Amino Acid Sequence↗
PubMed2004

Genomic analysis of Fas and FasL genes and absence of correlation with disease progression in AIDS.

Apoptosis has been suggested as a major mechanism for the CD4(+) T-lymphocyte depletion observed in patients infected with human immunodeficiency virus 1 (HIV-1). To evaluate the impact of genetic variations to apoptosis during progression of acquired immunodeficiency syndrome (AIDS), we have performed an extensive genetic analysis of Fas and Fas ligand ( FasL) genes. The coding regions and promoters of these genes were resequenced in a cohort of 212 HIV-1-seropositive patients presenting extreme disease phenotypes and 155 healthy controls of Caucasian origin. Overall, 33 single nucleotide polymorphisms (SNPs) with an allele frequency >1% were identified and evaluated for their association with disease progression. Among them, 14 polymorphisms were newly characterized. We did not find any statistically significant association of Fas and FasL polymorphisms and haplotypes with AIDS progression.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Apoptosis inhibition mediated by medroxyprogesterone acetate treatment of breast cancer cell lines.

Several reports suggested that steroidogenic hormones could be directly involved in the regulation of apoptosis in vitro, but whether this is due to blocking or promoting mechanism of these hormones remains controversial. However, it was shown that progesterone exhibited a protective effect against the apoptotic process during mouse mammary gland involution in vivo. In this study, we analyzed the effect of medroxyprogesterone acetate (MPA) treatment, an agonist of progesterone, on serum starvation induced apoptosis on breast cancer cell lines. Positive and negative progesterone receptor (PgR+ and PgR-) breast cancer cell lines were treated with MPA (10 nM), either in standard culture conditions or in serum-free medium to induce apoptosis. Cell survival, proliferation and apoptosis were simultaneously analyzed with the expression of apoptosis-related genes measured by a real time quantitative RT-PCR. At non cytotoxic doses, MPA protected PgR+ T47-D, MCF-7 and H466-B cell lines against serum depletion-induced apoptosis, while MPA did not protect PgR-MDA-MB-231 cells against serum depletion induced apoptosis. In PgR+ cell lines and in concordance with the protective effect, the pro-apoptotic HRK and BAK1 mRNAs were up-regulated after apoptosis induction, while they were no more induced in condition of protection against apoptosis after MPA treatment. We also observed, specifically in PgR+ cells, an up-regulation of BCLX-L and BCLX-S and a down-regulation of BCL2 mRNAs, which are specific to the MPA response and unrelated to apoptotic process. Involvement of these genes with regard to the MPA-mediated protection against apoptosis is discussed.

Antineoplastic Agents, Hormonal↗
PubMed1998

Increased prosomal proteins in breast cancer cells and in neighboring normal cells in Parsi and non-Parsi populations.

Monoclonal antibodies were raised against the prosomal proteins p27K, p29K and the prosome-like protein p21K (PLP) from normal breast glandular cells and from benign and malignant tumors. They were used to clarify the involvement of prosomes in tumorigenesis of human breast cells. Immunostaining showed the distribution of prosomes in the cytoplasm and nuclei of cells from European normal women (EN) and Parsi (P) and non-Parsi (NP) benign (B) and malignant (M) tissues. The flow-cytometry studies showed an increased mean percentage of labeled cells, particularly with anti-p27K prosomal protein mAb, in malignant tissue from NP compared to EN. The p21K data indicated an increase in the number of cells labeled by flow-cytometry studies in all groups compared to EN, while p29K-expressing cells were more abundant in NPN, PB, PM and NPM. Intergroup comparison showed that the mean percentage of cells labeled with anti-p27K and anti-p29K was significantly higher in PB than in NPB, as seen by flow cytometry, whereas there was a higher production or accumulation of the p21K (PLP) prosomal protein in NPM than in PM, as seen by immunostaining. By comparison with EN, there were also significantly more normal cells containing the three antigens in the apparently normal tissue in the neighborhood of the tumor in NPM, and more cells containing p21K in PM patients than in EN. As prosomes are involved in the cell differentiation and in the cell cycle control, the changes observed in breast tissues may be related to oncogenic processes. Furthermore, the modified subunit pattern of prosomes in cancer and, possibly, pre-cancer tissue may be of interest for diagnosis purposes.

Breast↗
PubMed1996

Antagonism between estradiol and progestin on Bcl-2 expression in breast-cancer cells.

Bcl-2 is a key protein involved in the control of apoptosis. Our previous studies on breast and endometrium indicated hormonal regulation of bcl-2 in these tissues. In the present work we have analyzed Bcl-2 and Bax protein expressions in MCF-7 and T47-D, 2 hormone-dependent breast-cancer cell lines, by immunoblots. Estradiol markedly increased Bcl-2 protein content, both in short- and in long-term treatments of MCF-7 cells. Two types of anti-estrogens (4-hydroxytamoxifen and RU 58668) were able to reverse this effect. Also, a synthetic progestin (ORG 2058) was able to decrease the Bcl-2 level in T47-D cells. The level of Bax protein, however, was not affected in the same conditions of hormonal treatments. The level of Bcl-2 expression was 4.5-fold higher in MCF-7 than in MDA-MB 231 (an estradiol-independent cell line). From these results, we infer the existence of hormonal regulation of Bcl-2 expression and evoke a novel role for estradiol and progestin in the genesis of breast cancer.

Blotting, Western↗
PubMed1992

Bacteriophage lambda as a cloning vector.

Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations. Unwanted restriction sites have been altered and arranged together into suitable polylinkers. The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts. Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts. Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40. The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL. Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors. In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis. In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype. Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish. Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.

Bacteriophage lambda↗
PubMed1986

Structure and expression of the chicken epidermal growth factor receptor gene locus.

Similarity between the carboxyl-terminal portion of the human epidermal growth factor (EGF) receptor and the deduced protein sequence of the chicken-derived oncogene v-erbB, of avian erythroblastosis virus strain H, has suggested that the chicken cellular erbB locus, c-erbB, might be part of a longer EGF-receptor gene in the chicken, whose entire coding capacity remained to be defined. The c-erbB locus spans more than 20 X 10(3) base pairs (20 kbp) of DNA and contains at least 1.8 kbp homologous to the v-erbB oncogene. We show here that human EGF receptor cDNA and chicken genomic DNA share homology not only within the c-erbB locus but also within a 25.1-kbp DNA region situated 5' to this locus. The 3' region of the EGF receptor overlaps, in sequence homology, the c-erbB locus. The EGF receptor/c-erbB locus in chicken generates six related but distinctly different mRNAs of sizes 12, 9, 5, 3.6, 3.2 and 2.6 kb. The transcripts of 12, 9, and 3.6 kb contain sequences coding for both the extracellular EGF-binding domain of the receptor and the intracellular tyrosine kinase domain. The 12-kb and 9-kb transcripts, which have already been shown to contain the sequences coding for the v-erbB, were found to possess, in addition, sequences that encode the entire chicken EGF receptor. The 3.2-kb and 2.6-kb mRNAs are homologous only to the 5' portion of the EGF receptor gene. These results therefore indicate that the c-erbB locus, initially defined by homology to the viral transforming gene, corresponds to the 3' region of the EGF receptor gene in the chicken genome. The multiple, related, chicken EGF receptor RNA transcripts reported here are reminiscent of the various human EGF receptor RNA transcripts observed in normal and transformed cells.

Alpharetrovirus↗
PubMed1984

Altered globin gene transcription pattern and the presence of a 7-8 kb alpha A globin gene transcript in avian erythroblastosis virus-transformed cells.

Immature chick erythroid cells transformed by avian erythroblastosis virus (AEV) display an altered pattern of globin gene transcription leading to the abortive phenotypic expression of such transcripts. Detectable adult globin gene-specific RNA sequences, confined exclusively to the nucleus, are uniquely of the alpha A type. The alpha A globin-specific sequences occur in transcripts 7-8 kb long from which the 5' contiguous alpha D gene product is absent, and also in fragments smaller than 9S globin mRNA. The implication of this observation for schemes of post-transcriptional regulation of gene expression and viral transformation are discussed.

Alpharetrovirus↗
PubMed1982

In vitro transformation by avian erythroblastosis virus recruits cells of erythroid lineage with varying degree of differentiation.

We have previously shown that AEV infection of leukosis-free chickens provokes an erythroleukemia in the infected birds, and that the target cell for virus transformation is most likely an early pro-erythroblast comparable to the BFU-E. The virus infection correlates with a block in differentiation which was studied at a specific cellular gene level, namely that of the globin genes. In live birds, the viral transformation has been found to affect the transcriptional activity to two out of three adult globin genes and the phenotypic expression of the third. Indeed, the alpha A gene that is transcribed is silent as well, since its transcription product is fully eliminated in the nucleus following an "abortive processing". Chick bone marrow cells can also be transformed in vitro by AEV in which case the virus recruits, as targets, cells of the erythroid lineage belonging to a wider spectrum and hence in varying degrees of differentiation. In contrast to the results obtained with in vivo transformed cells, molecular hybridization with globin cDNA probes showed that in the erythroblasts transformed in vitro under tissue culture conditions all the three adult globin genes are transcriptionally active. In fact, low but detectable amounts of globin mRNA sequences are present in both the nuclear and cytoplasmic compartment of the cell prior to exposure to any chemical inducers of red cell differentiation. Judging from our present results, we may propose that AEV transformation in vitro does not shut off out only modulates the already functioning erythroid cell differentiation program.

Alpharetrovirus↗
PubMed1981

Modification of the methylation pattern in the vicinity of the chicken globin genes in avian erythroblastosis virus transformed cells.

Having previously found a reduced transcription of globin genes and an abortive processing of the already transcribed globin pre-mRNA in Avian Erythroblastosis Virus (AEV) transformed cells (1), we compared the genomic DNA of these cells with that of normal chicken erythroblasts, using 32-P-labelled cDNA probes specific for the beta, alpha A and alpha D globin sequences. Restriction endonuclease digestion, electrophoresis of digests in agarose gels, Southern blotting and hybridization were carried out. Our results show that the overall genome organization is not disturbed in the immediate neighbourhood of the adult globin genes; the observed restriction fragments are identical for both DNAs after EcoRI, HindIII, BamHI and XbaI digestion, using the beta, alpha A and alpha D globin cDNA probes. However, we observe specific modifications at some methylation sites in the beta, beta-like and alpha D regions: after HpaII or MspI digestion in the alpha D region and after HhaI digestion in the beta and beta-like region, heavier bands appear in the transformed cell DNA in addition to the ones observed in normal DNA. This implies that, at some specific sites, the transformed cell DNA is more methylated than the normal erythroblast DNA. The possible significance of this observation is discussed.

Alpharetrovirus↗
PubMed1979

On pre-messenger RNA and transcriptions. A review.

From the present review integrating old and new data emerge a few principles of gene expression in eukaryotes, and an infinite variety of possible mechanistic details generating the overal pattern. The few principles, most of which are not fundamentally new, may thus be summarized. 1) The eukaryotic genome is subdivided into transcriptional units: into transcriptons which are subject to individual activation controlled at DNA level. 2) Viral genomes may contain one or a few transcriptons, while cells of multicellular organisms contain from 3 x 10(3) in diptera up to an estimated 2 x 10(5) in birds and mammals. 3) Transcriptons may include one or several coding sequences. 4) Transcriptons vary considerably in size: in mammals and birds their size spectrum falls into the 2,000 to 20,000 bp range. 5) Units of coding information constituting one message (genes) and, possibly, units of regulative information are frequently broken up and stored within the transcripton in sub-genic blocks (of so far unknown significance) in general located at a certain distance from the 5' and 3' transcript terminals which are determined by the promotor and terminator signals. 6) The gene, in its specific definition as the functional unit underlying the phenotype, is in general constituted posttranscriptionally by the processing mechanisms from the mosaic of its genomic subunits in the transcripton; segments of coding, service and regulative sequences are recombined within themselves and with each other, polygenic transcripts separate into their unit messages. 7) Activated transcriptons produce pre-mRNA; these primary transcripts are colinear with the DNA of the transcriptional unit. 8) Primary pre-mRNA is processed into secondary pre-mRNA's by extragenic cleavage and intragenic ("splicing") processing, giving rise stepwise to functional mRNA. During this process chemical modifications as methylation, 5'-terminal capping and 3'-terminal polyadenylation take place. 9) Translation yields either potentially functional polypeptides or polycistronic polyproteins subject to further processing. 10) Processing is a regulated process; it involves many of the possible phases and mechanisms of post-transcriptional regulation (cf. 39, 40).

Animals↗
PubMed1978

Post-transcriptional suppression of globin gene expression in cells transformed by avian erythroblastosis virus.

Cells transformed by avian erythroblastosis virus were grown in vitro for up to 5 months. After a few days in culture, synthesis of hemoglobin was undetectable and could not be induced by dimethyl sulfoxide. As shown by globin cDNA hybridization to nuclear and cytoplasmic RNA carried to Crot values of 10(5) moles of nucleotide per liter X sec, globin genes in these cells are transcribed into pre-mRNA, but no trace of globin mRNA appears in the cytoplasm. The implications of this observation for schemes of post-transcriptional regulations and viral transformation are discussed.

Alpharetrovirus↗