Epitopic specificities of anti-thyroglobulin autoantibodies.
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Antibody Specificity↗
Biomedical subjects
M Piechaczyk
Publications and source records attributed to M Piechaczyk.
Novel anti-digoxin monoclonal antibodies with different binding specificities for digoxin metabolites and other glycosides.
Spleen cells of BALB/c mice immunized with a digoxin-bovine serum albumin conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Seven monoclonal antibodies (MAb) selected by indirect ELISA were produced, purified and characterized. All the MAb were IgG1 isotypes. The apparent equilibrium association constants (Ka) of four of the MAb, determined by Scatchard analysis of the RIA data, ranged from 1 x 10(9) M-1 to 5.9 x 10(9) M-1. The estimated Ka values of the three other MAb were found to be between 4.8 x 10(7) M-1 and 5.9 x 10(8) M-1. Using digoxin and eighteen structurally-related compounds, the seven MAb could be divided into five groups based on their binding specificities assessed by an inhibition immunoenzymatic test. The MAb in Groups I and II, in particular, showed very different specificity profiles: the two MAb in Group I had low cross-reactivity with cardioinactive digoxin metabolites, whereas the high affinity MAb in Group II recognized all the digoxin metabolites tested. The MAb in Group I might be useful in a digoxin immunoassay and the Group II MAb in therapeutic reversal of digoxin intoxication.
Animals↗
AUUUA motifs are dispensable for rapid degradation of the mouse c-myc RNA.
Sequence determinants responsible for c-myc RNA rapid turn-over are localized within the 3' non-coding region which is mainly characterized by the presence of two polyadenylation signals and a high content in A and U. Although the AUUUA/UUAUUUA motif is commonly thought to specify a whole class of unstable RNAs coding for various onco-proteins and cytokines, site-directed mutagenesis showed that both of the two such sequences found in the mouse c-myc RNA are dispensable for rapid RNA degradation. Although less efficient than the whole 3' non-coding region, the last 50 nucleotides of c-myc RNA, mainly made up of U and A and devoid of AUUUA/UUAUUUA motif, are sufficient to confer instability to the coding sequence.
Animals↗
Cerebellar granule cell survival and maturation induced by K+ and NMDA correlate with c-fos proto-oncogene expression.
Persistent depolarization with a high K+ concentration (30 mM) or sustained activation of N-methyl-D-aspartate (NMDA) receptors (12.5 mM K+ plus 100 microM NMDA) enhance both survival and maturation of mouse cerebellar granule neurons in vitro taking as criteria the amount of protein and DNA and the release of endogenous glutamate respectively. K+ and NMDA neurotrophic effects are associated with c-fos protein expression in the nucleus of these cells suggesting that c-fos protein could play a role in the survival and/or maturation of granule neurons.
Animals↗
Significance of the recognition of certain antigenic regions on the human thyroglobulin molecule by natural autoantibodies from healthy subjects.
We have evaluated the epitope specificity of natural antihuman thyroglobulin (hTg) autoantibodies (aAb) in the plasma of healthy individuals. By an indirect ELISA technique, we selected 56 plasma samples with high anti-hTg antibody activity and used the IgG fraction isolated from these plasma to study the antigenic domains on the hTg molecule recognized by the natural anti-hTg aAb. A panel of 15 mAb, coupled to alkaline phosphatase and recognizing six regions (I to VI) on the hTg molecule, served to identify the domains recognized by the natural anti-hTg aAb using a competitive ELISA procedure. A total of 26 of the IgG fractions was found to interact with at least one of the regions defined by our battery of mAb. Region V was recognized by the majority of the IgG fractions. Interestingly, region II was rarely recognized by the same IgG fraction that reacted with region V. Inasmuch as we have previously shown that region II is mainly recognized by aAb in the serum of subjects with various thyroid disorders, we propose that recognition of region V reflects the normal physiologic state of the immune system with respect to the hTg molecule.
Adolescent↗
The muridae glyceraldehyde-3-phosphate dehydrogenase family.
Although only one gene is known to be functional, numerous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences are scattered throughout Mus musculus and Rattus rattus genomes. In this report we show that: (1) GAPDH pseudogenes are repeated to comparable extents, at least 400 copies, in 12 other Muridae species; (2) the complete, or nearly so, sequence of GAPDH messenger RNA is amplified, and a high proportion, if not all of these copies, are intronless; (3) GAPDH pseudogenes are preferentially located in heavily methylated and DNAse I-insensitive regions of chromatin; and (4) the presence of atypical GAPDH-related mRNAs in different cellular contexts raises the possibility that more than one GAPDH gene is transcribed.
Animals↗
A versatile and potentially general approach to the targeting of specific cell types by retroviruses: application to the infection of human cells by means of major histocompatibility complex class I and class II antigens by mouse ecotropic murine leukemia virus-derived viruses.
A technique for delivering genes carried by recombinant retroviruses into specific cell types could have numerous applications in oncology, developmental biology, and gene therapy. As a first step toward this remote goal we designed a procedure allowing in vitro cell targeting by retroviruses. Biotinylated antibodies against the viral envelope protein on one side, and against specific cell membrane markers on the other side, were bridged by streptavidin and used to link the virus to the host. The method was successfully used to infect human cells with ecotropic murine retroviruses by means of major histocompatibility complex class I and class II antigens and appears easily adaptable to other cell membrane markers. Moreover, the sequential protocol we designed, although allowing infection of human cells, requires less stringent safety constraints than would handling of amphotropic virus stocks.
Animals↗
Novel immunoradiometric assay of thyroglobulin in serum with use of monoclonal antibodies selected for lack of cross-reactivity with autoantibodies.
A multisite immunoradiometric assay for measurement of serum thyroglobulin (Tg), designated Magnogel-IRMA-Tg, has been developed, involving magnetic microbeads (Magnogel). This assay is based on the use of five anti-Tg monoclonal antibodies (MAbs) directed against three antigenic regions on the Tg molecule that are not recognized by anti-Tg autoantibodies (aAbs). Four of these MAbs, directed against two antigenic domains, were coupled to the magnetic beads and were used to trap the serum antigen. Another MAb, directed against the third region, was iodinated and served as the labeled second antibody. The Magnogel-IRMA-Tg technique is reproducible, rapid, and sensitive (lower detection limit, 3 micrograms/L). The assay reliably measures serum Tg in the presence of anti-Tg aAbs.
Antibodies, Monoclonal↗
In situ and in vitro evidence for intragenic binding of nuclear factors at the murine c-myc locus.
A block to transcriptional elongation within the c-myc proto-oncogene has been previously observed in a large number of different mouse and human cell types and its release is a potentially important element in the pathogenesis of some malignancies. We show here that the chromatin around the mouse c-myc exon 1-intron 1 boundary is differentially accessible to restriction enzymes in purified nuclei. Using a combination of in situ exonuclease III protection assay with in vitro footprints and gel band shifts, we have shown the existence of a stable nucleoprotein complex in this same region in mouse erythroleukemia cell nuclei. This situation is not peculiar to these cells and we have shown that the accessibility of the two BglII sites present at the beginning of intron 1 seems to depend not only upon the transcriptional state, but also upon the structural integrity of the gene.
Animals↗
Altered myc gene transcription and intron-induced stabilization of myc RNAs in two mouse plasmacytomas.
Accumulation of unusually high amounts of larger-than-normal c-myc mRNAs occurs in two mouse plasmacytomas, TEPC 1165 and TEPC 2027. Southern blot and DNA sequence analyses showed that both tumors have undergone translocations of immunoglobulin heavy chain loci to positions 5' of the c-myc gene promotors resulting in removal of DNA sequences encoding a negative transcriptional regulatory element. In contrast to other mouse plasmacytomas, TEPC 1165 and TEPC 2027 rearranged myc genes show increased transcription, partially explaining their abundance of myc RNA. Similar to other mouse plasmacytomas, the abundance of myc RNA in TEPC 1165 and TEPC 2027 is also influenced by increased stability of structurally atypical myc RNAs. Two myc mRNAs are found in TEPC 2027, a 2.4 kb species including all 3 myc exons and a 4.0 kb species with the 3 exons plus the first intron. The two major myc mRNAs in TEPC 1165, 3.0 and 3.9 kb species, also include all three myc exons plus portions of the first intron. S1 nuclease protection analyses show that the 5' initiation and 3' untranslated (UT) regions of the unusual TEPC 1165 RNAs are normal showing that the size differences arise solely from inclusion of first intron sequences in the large myc RNAs. DNA sequence analysis showed that the presence of first intron sequences in the large myc RNAs is due to mutations affecting the splice donor region at the 3' end of exon 1 in both tumors. SDS-PAGE analysis of immunoprecipitated TEPC 1165 and TEPC 2027 myc proteins showed them to be of normal electrophoretic mobility but no more abundant than in a pre-B cell line 18-81 that contains at least 10 fold less myc RNA. The 4.0 kb myc mRNA of TEPC 2027 is atypically stable while the 2.4 kb myc mRNA undergoes normal rapid turnover within the same cell, demonstrating that the presence of first intron sequences in the large myc RNA stabilizes it despite the presence of 3' UT and putative exon 1 destabilizing sequences. These results show that myc intron 1 sequences can counteract the effect of 3' UT region destabilizing sequences in myc RNA and suggest that the increased myc RNA stability noted in TEPC 1165 and TEPC 2027 is largely due to the presence of the intron 1 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6.
To explore the mechanisms involved in the pathogenesis of human multiple myeloma (MM), we investigated the potential role of interleukin-6 (IL-6), a B-cell differentiation factor in humans, and a growth factor for rat/mouse heterohybridomas and murine plasmacytomas. Using a heterohybridoma assay, we found that two well-documented human myeloma cell lines, RPMI 8226 and U266, did not secrete IL-6 and did not express RNA messengers for IL-6. Neutralizing antibodies to IL-6 did not inhibit their proliferation, and recombinant IL-6 did not stimulate it. Taken together, these data show that IL-6 is not the autocrine growth factor of these human myeloma cell lines. A high production of IL-6 was found in the bone marrows of patients with fulminating MM, compared with patients with inactive or slightly active MM, or to healthy donors. This IL-6 production was assigned to adherent cells of the bone-marrow environment but not to myeloma cells. A spontaneous proliferation of myeloma cells freshly isolated from patients was observed in short-term cultures. Recombinant IL-6 was able to amplify it two- to threefold. The spontaneous proliferation of the myeloma cells was inhibited by anti-IL-6 antibodies and reinduced by recombinant IL-6. After 2 to 3 weeks of culture, the myeloma-cell proliferation progressively declined and no IL-6-dependent myeloma cell lines could be obtained despite repeated additions of fresh IL-6 and costimulation with other cytokines such as tumor necrosis factor (TNF)beta, or IL-1 beta. These data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6.
Aged↗
Role of RNA structures in c-myc and c-fos gene regulations.
Proto-oncogenes c-myc and c-fos are subjected to a complex set of controls operating both at the transcriptional and post-transcriptional levels. We report here that: (i) antisense transcription occurs at the murine c-myc locus. However, its biological significance remains to be established; (ii) transcription of both genes is regulated in various situations by a block to elongation of nascent RNA chains. In the case of c-myc, the blockade involves a RNA structure whose nature remains unknown; (iii) elements responsible for the high degree of instability of c-myc and c-fos mRNAs reside in their 3' non-coding regions. A U-rich region, reminiscent of that present in the granulocyte-monocyte colony-stimulating factor mRNA destabilizer, is likely to be involved in the rapid degradation of c-fos mRNA; (iv) exon 1 substitution by intron 1-derived sequences lessens or negates the effect of the 3' destabilizer in abnormal c-myc RNAs from Burkitt's lymphomas and mouse plasmacytomas.
Animals↗
Interferons and oncogenes in the control of cell growth and differentiation: working hypothesis and experimental facts.
This short review summarizes available evidence for (i) growth regulatory properties of exogenous as well as recently described autocrine IFNs, (ii) down-regulation of cellular oncogene expression with emphasis on c-myc and (iii) the possible involvement of the IFN-regulated 2-5A pathway at these levels. Initially described as a part of the IFN-induced antiviral mechanism, this double-stranded RNA-activated pathway leads to the preferential degradation of viral mRNAs in IFN-treated virus-infected cells probably through localized activation at the site of virus replication. Such mechanisms could be involved in the regulation of the stability of rapidly turning over mRNAs as for instance c-myc mRNA in IFN-treated cells. Whatever the elegance of the concept, however, experimental evidence is essentially circumstantial; tools developed in our group to strengthen the demonstration are briefly described.
Animals↗
The regulatory strategies of c-myc and c-fos proto-oncogenes share some common mechanisms.
There is evidence for both transcriptional and post-transcriptional levels of regulation of c-fos and c-myc proto-oncogenes. Transcription of both genes can be regulated at the level of initiation. However, it was recently shown in various situations for c-myc, and in one case for c-fos, that these genes can also be down-regulated by a block to elongation of nascent RNA chains. Both c-myc and c-fos mRNAs are known to be extremely unstable (half-lives around 10-15 min) and c-myc RNA turnover has been shown to be modulated under various physiological situations. Atypical c-myc RNAs found in certain mouse plasma cell tumors (MPCs) and Burkitt, lymphomas (BLs) are significantly and sometimes dramatically more stable than their normal counterparts. In this review we report that: i) transcriptional control elements reside in murine c-myc and c-fos first exons. Daudi cells provide an example of c-myc activation via removal of this block to elongation; ii) elements necessary for the rapid degradation of c-fos and c-myc RNAs reside in their 3' non-coding regions; iii) these destabilizing elements can be counteracted by atypical 5' sequences found in abnormal c-myc transcripts from BLs and mouse plasmocytomas.
Animals↗
Sequence determinants of c-myc mRNA turn-over: influence of 3' and 5' non-coding regions.
Normal c-myc RNAs are very unstable with a half-life of less than 30 min whereas those rearranged in 5', as found in Burkitt's lymphomas and mouse plasmacytomas, are significantly more stable. To learn about the sequence determinants controlling their turnover, we have studied naturally occurring and artificially constructed c-myc RNAs rearranged in 5' or 3'. The first conclusion is that sequences necessary for rapid c-myc RNAs turnover are localized in their 3' untranslated region. The second conclusion is that stabilization of truncated c-myc RNAs in tumors does not result from deletion of the non-coding first exon but rather from its replacement by intronic and/or exogenous sequences. This latter conclusion rests on two lines of evidence: (i) deleting the 5' rearranged sequences from the relatively stable MOPC 315 RNA restores its complete instability (pSV c-myc 1); (ii) reciprocally, appending intron 1 sequences 5' to otherwise unstable germline c-myc exons 2 and 3 have a dramatic stabilizing effect (pIM 0).
Animals↗
Regulation of c-fos gene expression in hamster fibroblasts: initiation and elongation of transcription and mRNA degradation.
Rapid and transient activation of both c-fos transcription and mRNA accumulation occurs when resting CCL39 hamster fibroblasts are serum-stimulated to grow. By using several combinations of serum and cycloheximide, a protein synthesis inhibitor, we showed that: i) addition of cycloheximide to resting cell elicits an increase in c-fos gene transcription located within the first 540 bases of the unit, suggesting that an "attenuation-like" mechanism, similar to that observed for c-myc, might be essential for c-fos transcriptional regulation; ii) it also prevents both transcriptional shutoff and mRNA degradation in serum-stimulated cells; iii) upon removal of cycloheximide, mRNA degradation resumes rapidly; deletion of a 130 bases long segment in the 3 non-coding region leads to a stabilization of c-fos mRNA lending experimental support to a putative destabilizer element within this sequence.
Animals↗
Antigenic domains on the human thyroglobulin molecule recognized by autoantibodies in patients' sera and by natural autoantibodies isolated from the sera of healthy subjects.
We determined the regions on the human thyroglobulin (hTg) molecule recognized by anti-hTg autoantibodies (aAbs) in the sera of patients with Hashimoto's thyroiditis, Graves' disease, and thyroid carcinoma and by anti-hTg natural aAbs isolated from the sera of healthy subjects. Fifteen anti-hTg monoclonal antibodies (MAbs) directed against six distinct antigenic regions were used for this study. The anti-hTg aAbs in the patients' sera recognized mainly region II and occasionally region IV. The natural aAbs were present in the serum at low concentrations; consequently, we isolated and concentrated them for this investigation. The isolated natural aAbs inhibited the interaction of the anti-hTg MAbs with the majority of the antigenic regions identified. Region II was not well recognized, however, by these natural aAbs. This difference in specificity between the anti-hTg aAbs and the anti-hTg natural aAbs may have diagnostic significance.
Autoantibodies↗
Production and characterization of highly specific anti-methotrexate monoclonal antibodies.
Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.
Animals↗