Biomedical subjects

M Piechaczyk

Publications and source records attributed to M Piechaczyk.

At least 55 records · Page 3Linked to original sources

PubMed1994

Differential sensitivity of FOS and JUN family members to calpains.

Degradation of c-fos protein (c-FOS) in the cytoplasm is very rapid in vivo and constitutes a crucial regulation of the nuclear steady-state level through the control of the amount of full-length molecules available for nuclear transport. Using cytoplasmic extracts from various origins, we report herein that c-FOS degradation can be initiated in a calcium-dependent manner which involves cysteine proteases called milli- and micro-calpain. Interestingly, FOS-B, a member of the fos multigene family, as well as all members of the jun family (JUN-B, c-JUN and JUN-D) are also sensitive to calpains albeit to different extents. FRA-2, which is a c-FOS-related protein, is resistant to micro- but not to milli-calpain whereas FRA-1, another member of the fos family, is resistant to both proteases. Given the fact that a work by others (Hiraï et al., 1991b) suggests that calpains can be involved in c-FOS and c-JUN degradation in vivo, our observations raises the possibility of a novel contribution to the regulation of AP-1 transcription complex activity through a differential control of the steady-state level of some of its components that involves calpains.

Animals↗

PubMed1993

Concerted evolution in the GAPDH family of retrotransposed pseudogenes.

In murine rodents the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) multigene family includes more than 300 retroprocessed pseudogenes. Its single functional gene encodes GAPDH, an enzyme of glycolysis. Because of its manageable size, this family is a good model for the study of genome cohesion and evolution. By sequence comparison of several GAPDH pseudogenes in Rattus norvegicus and Mus musculus, we have obtained evidence that (i) the GAPDH family still generates new pseudogenes; we note in each species the beginning of a process of species-specific evolution since the pseudogenes of one genus on average cluster more with one another than they do with those of the other genus, and (ii) the GAPDH family contains diversified subfamilies. These findings suggest a certain level of transcription and transposition of the pseudogenes independent of the functional gene which may result from various mechanisms. The homogenization we observe may be due to the pseudogenes themselves (concerted evolution in a strict sense), which explains the occurrence of long-term homogenization of old sequences and subfamily groupings.

Animals↗

PubMed1992

Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.

We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.

Base Sequence↗

PubMed1992

A major human thyroglobulin epitope defined with monoclonal antibodies is mainly recognized by human autoantibodies.

The antigenic nature of 15 anti-human thyroglobulin (hTg) monoclonal antibody (mAb) epitopes was studied by two different approaches. First, we tested two successive protease-digest products of hTg. Only four mAb from the same cluster of reactivity recognized a low-molecular weight peptide, the other mAb only bound native hTg or high-molecular weight digest fractions. Second, these 15 mAb were used to immunoscreen hTg expression libraries. Only the same four mAb revealed immunoreactive clones corresponding to region 1149-1295 on the hTg primary sequence. After subcloning, this antigenic determinant was reduced to a 102-amino acid peptide (hTg region 1149-1250). The two different methodologies were coherent and complementary, and demonstrated that hTg sequence 1149-1250 is the target for this cluster of four mAb. Moreover, anti-hTg autoantibodies which cross-reacted with these mAb bound the 102-amino acid peptide. This epitope was the one most frequently detected by sera from autoimmune thyroid disease. The data confirm the presence of an immunodominant domain in the central part of the hTg molecule and suggest that this mAb epitope may be a powerful probe for the diagnosis of autoimmune thyroid disorders.

Amino Acid Sequence↗

PubMed1992

Long-term expression of the c-fos protein during the in vitro differentiation of cerebellar granule cells induced by potassium or NMDA.

Levels of the c-fos protein were assayed in mouse cerebellar granule cells during their in vitro development under different culture conditions. When grown in media favoring both their survival and differentiation, i.e. in the presence of 30 mM K+ or 12.5 mM K+ plus 100 microM N-methyl-D-aspartate (NMDA), the c-fos protein becomes detectable in the nucleus of granule cells on and after 6 days and persists to high levels until the culture begins to decline. The protein c-fos appears therefore after the critical period described for the survival effect of K+ depolarization or NMDA receptor stimulation which corresponds to days 2-5 after plating. The c-fos protein remains however scarcely detectable or undetectable throughout the life-span of cells cultured under conditions providing poor survival and differentiation, i.e. in the presence of low K+ (5 or 12.5 mM) alone or when the effect of NMDA is blocked by the NMDA receptor antagonist MK-801. Interestingly, in cortical and striatal neurons, the survival and differentiation of which being not affected by depolarizing media, no c-fos protein is detected whatever the culture conditions tested at least during the first 18 days in vitro. This suggests that long-term expression of the c-fos gene might be related to some aspect of the late in vitro differentiation process of cerebellar granule cells.

Animals↗

PubMed1992

Tyrosine iodination and iodotyrosyl coupling of the N-terminal thyroid hormone forming site of human thyroglobulin modulate its binding to auto- and monoclonal antibodies.

The present work was aimed at studying the interaction of autoantibodies (aAb) and monoclonal antibodies (mAb) with the N-terminal thyroid hormone forming site of human thyroglobulin (TG). Obtained by CNBr treatment of TG, the peptide (22 kDa) containing the complete major hormonogenic site of human TG was purified in three forms according to the degree of iodination and iodotyrosine coupling: the native, poorly iodinated form (n-22K), the iodinated form containing iodotyrosine but not hormone residues (i-22K) and the form containing thyroid hormone (t-22K). We report that aAb from some patients with autoimmune thyroid diseases showed significant binding to both iodinated 22 kDa forms. Furthermore, a detailed study using mAb evidenced that iodination and coupling induced changes in the antigenicity of the molecule, some occurring without direct implication of iodine or thyroid hormones. The 22 kDa peptide appears as an interesting model to study the antigenic changes induced by the structural modifications in the course of thyroid hormone synthesis. This observation could be relevant to the etiopathogenic process of thyroid autoimmune diseases.

Antibodies, Monoclonal↗

PubMed1992

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker.

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

DNA, Recombinant↗

PubMed1992

Cell targeting by murine recombinant retroviruses.

Cell targeting by murine retroviruses carrying recombinant genes would have numerous applications such as immortalization of under-represented cell types from complex cellular mixtures, increasing therapeutical yields in the case of gene therapy and delivery of specific genes at any location and at any moment of the life-time of living animals. To this aim, we are currently developing techniques that allow binding of viral particles to specific cell membrane markers different from the natural receptors. We have shown that biochemical bi-specific molecular adaptors able to bind both viral particles and cell surface molecules may reveal appropriate for piloting viruses toward specific cell types. More recently, we have undertaken the genetic engineering of the retroviral envelope glycoprotein in order to modify its natural tropism. This approach is discussed below.

Defective Viruses↗

PubMed1991

Autoimmunity to human thyroglobulin. Respective epitopic specificity patterns of anti-human thyroglobulin autoantibodies in patients with Sjögren's syndrome and patients with Hashimoto's thyroiditis.

We evaluated the epitopic specificity pattern of anti-human thyroglobulin (anti-hTg) autoantibodies from patients with primary Sjögren's syndrome (SS). All of the primary SS sera tested contained both IgG and IgM anti-hTg autoantibodies recognizing at least 1 region on hTg; in 65% of the cases, 3 or more regions were recognized. A strong recognition of region II, as is seen in Hashimoto's thyroiditis, was associated with thyroid disorder in primary SS. These results emphasize the importance of region II in autoimmune thyroid disease.

Adult↗

PubMed1991

Evidence for a restricted idiotypic and epitopic specificity of anti-thyroglobulin autoantibodies in patients with autoimmune thyroiditis.

Anti-thyroglobulin (TG) autoantibodies from patients with autoimmune thyroiditis express a cross-reactive alpha idiotype (Id) termed T44 which is not expressed by IgG from normal individuals. The present study demonstrates that the expression of the T44 Id is strongly associated with the recognition by anti-TG autoantibodies of a specific epitopic cluster on human TG. The epitopic reactivity of anti-TG autoantibodies was determined using a competitive inhibition assay with a panel of 15 monoclonal antibodies that define six antigenic clusters on TG. All T44+ autoantibodies from patients recognized cluster II, whereas no anti-TG IgG from healthy individuals reacted with this region. Affinity columns of Sepharose-bound intravenous therapeutic immunoglobulins which contain anti-T44 activity, retained both T44 Id-expressing antibodies and a subset of region II-specific anti-TG autoantibodies from patients with Hashimoto's disease. Restricted idiotypic and epitopic specificity may demarcate disease-associated from natural anti-TG autoantibodies, suggesting that qualitative rather than quantitative criteria should be used to identify pathological autoantibodies.

Antibody Specificity↗

PubMed1991

In vivo interleukin 6 gene expression in the tumoral environment in multiple myeloma.

Whether interleukin 6 (IL 6) is an autocrine or paracrine myeloma cell growth factor in vivo remains unresolved. To identify which cells are producing IL 6 in vivo, we have studied the IL 6 gene expression in bone marrow mononuclear cells (BMMC) of 19 patients with multiple myeloma (MM) and in peripheral blood mononuclear cells (PBMC) of 9 patients with plasma cell leukemia (PCL). We found that the IL 6 gene was transcribed by BMMC of most patients with MM (79%). Further, IL 6 mRNA was not produced by purified myeloma cells from patients with either MM (5 patients) or PCL, but by the bone marrow environment, mainly by monocytes and myeloid cells (CD13+CD15+ cells). For 2 patients with PCL, for whom PBMC and BMMC samples were available, IL 6 mRNA could be detected in BMMC but not in PBMC. Finally, no IL 6 mRNA was detected in five freshly established IL 6-dependent myeloma cell lines. The present data give a clear-cut demonstration of the paracrine origin of IL 6 in vivo in human MM.

Bone Marrow↗

PubMed1991

Highly specific radioimmunoassay for digoxin using a monoclonal antibody selected for lack of interference by digoxin-like immunoreactive substances in cord blood sera.

Four digoxin radioimmunoassays (RIA) were evaluated using four antidigoxin monoclonal antibodies (MAb) with different binding specificities for digoxin metabolites and other glycosides. These RIA have been used to measure the apparent digoxin concentrations in the sera of patients treated with digoxin, in cord blood sera and in rat intestine aqueous extracts. Two MAb strongly recognized digoxin-like immunoreactive substances (DLIS) in cord blood sera and in rat intestine aqueous extract. In contrast, one MAb (2C2), which recognizes active digoxin metabolites and does not cross-react with inactive metabolites nor with digotoxin, showed no reactivity with DLIS in the cord blood sera tested. This MAb was used in a new digoxin RIA that was rapid, sensitive, reproducible, and specific for digoxin and its active metabolites.

Antibodies, Monoclonal↗

PubMed1991

Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene.

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

Animals↗

PubMed1991

Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

Animals↗

PubMed1990

Nuclear localization of c-Fos, but not v-Fos proteins, is controlled by extracellular signals.

We report here that transport of the protein product of the c-fos proto-oncogene from the cytoplasm, where it is synthesized, into the nucleus, where it operates as part of the AP-1 transcription complex, is not spontaneous but depends on the continuous stimulation of cells by serum factors. A labile protein inhibitor of transport, the effect of which is reversed by cAMP, is responsible for retention of c-Fos protein within the cytoplasm of serum-starved fibroblasts. In contrast, v-Fos proteins transduced by the murine retroviruses FBJ and FBR, which remain nuclear in the absence of serum, evade the translocation control, which therefore appears to contribute to their tumorigenic potential.

Animals↗

PubMed1990

Stable expression and function of EBV/C3d receptor following genomic transfection into murine fibroblast L cells.

The Epstein-Barr virus (EBV) and the C3d component of complement bind to the same cell surface receptor (EBVR/CR2) which is part of the B lymphocyte differentiation antigen recognized by the monoclonal antibodies (mAb) of the cluster of differentiation 21 (CD21). To analyze EBV and C3d binding to this receptor, mouse fibroblasts were transfected with human genomic DNA and rare CD21-positive cells were selected and cloned by cell sorting. The presence of the human gene in host cell DNA as well as its transcription product were assayed with a cloned EBVR/CR2 cDNA by Southern and Northern blotting analysis, respectively. A glycoprotein of apparent molecular mass of 140 kDa, similar to that found in human B lymphocytes, was immunoprecipitated with anti-CD21 mAb and proved to be functional since both C3d and EBV bound efficiently and specifically to mouse cells expressing EBVR/CR2. However, no expression of EBV nuclear antigens, early antigens and viral capsid antigens was detected in cells exposed to EBV. This indicates that the presence of EBVR/CR2 is not sufficient to allow full infection of mouse fibroblasts.

Animals↗

PubMed1990

Antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I.

An antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I (VHSV I) was developed. The assay employs two monoclonal antibodies (mAb) directed against distinct epitopes of the viral envelope glycoprotein (Gp). The antigen bound by the capture mAb (A17) was detected by addition of a second mAb (L7) conjugated to horseradish peroxidase, followed by addition of the enzyme substrate. The technique is highly sensitive, enabling detection of the virus at a protein concentration as low as 1 ng/ml of total proteins (1.5 fmol of envelope Gp per ml) in purified preparations. VHSV I was also detected in culture supernatants (5 x 10(5) PFU/ml) and in extracts of kidney and spleen of rainbow trout infected experimentally (5 x 10(5) PFU/ml). The assay was highly specific: infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus, spring viraemia of carp virus, pike fry rhabdovirus, eel rhabdovirus and perch rhabdovirus could not be detected by the antigen-capture ELISA for VHSV I.

Antibodies, Monoclonal↗

PubMed1990

MoMuLV-derived self-inactivating retroviral vectors possessing multiple cloning sites and expressing the resistance to either G418 or hygromycin B.

To facilitate cloning procedures in recombinant murine leukemia virus-derived retroviruses, we have constructed vectors that both carry a polylinker with multiple restriction sites and express resistance to either G418 or hygromycin B. Our vectors are self-inactivating retroviruses that suppress interferences between LTR enhancers and internal promoters and avoid transcriptional stimulation of host cell genes. They can also be used as expression vectors in direct transfection assays, since no translation initiation codon lies between the 5' LTR and the cloning polylinker.

Base Sequence↗