Experimental destruction of olfactory neurons stimulates proliferation and differentiation of local neural precursors and is used as a model to study in vivo mechanisms for degeneration and regeneration of the nervous system. Quiescin-sulfhydryl oxidases (QSOX) have a potential role in the control of the cell cycle or growth regulation and have recently been described in the central nervous system. In mice, we show an expression of QSOX in olfactory mucosa. Northern- and western-blot analysis show that the destruction of olfactory epithelium is associated with a reversible reduction in QSOX expression. Interestingly, QSOX is not localized in olfactory neurons (ON) but in cells of the lamina propria, suggesting that olfactory epithelium destruction may act as a signal of down-regulation of QSOX expression.

In normal endometrial glandular epithelial cells (GEC), 17beta-estradiol (E2) enhances proliferation and c-fos expression only in the presence of growth factors. On the contrary, growth factors are not required for the E2 effects in cancerous cells. Thus, a repression of E2 action could exist in normal cells and be turned off in cancerous cells, allowing a direct estrogen-dependent proliferation. To verify this hypothesis, we established immortalized and transformed cell models, then investigated alterations of E2 effects during oncogenesis. SV40 large T-antigen was used to generate immortalized GEC model (IGEC). After observation of telomerase reactivation, IGEC model was transfected by activated c-Ha-ras to obtain transformed cell lines (TGEC1 and TGEC2). The phenotypic, morphological, and genetic characteristics of these models were determined before studying the E2 effects. In IGEC, the E2 action on proliferation and c-fos expression required the presence of growth factors, as observed in GECs. In TGECs, this action arose in the absence of growth factors. After IGEC transformation, the activation of ras pathway would substitute the priming events required for the release of repression in GEC and IGEC and thus permit direct E2 effects. Our cell models are particularly suitable to investigate alterations of gene regulation by E2 during oncogenesis.

cpQSOx1 is a member of the QSOx family of proteins, expressed in the guinea pig (Cavia porcellus) and ortholog of the rat rQSOx1. In this study, in vitro experiments were conducted and showed that, as other member of this family, cpQSOx1 has a sulfydryl oxidase activity, and is a secreted protein. Then, the expression of this enzyme was researched in the guinea pig brain, as very little information exists yet on the expression of QSOx family members in the central nervous system. By immunohistochemistry, RT-PCR and in situ hybridization, cpQSOx1 is synthesized by neurons throughout the whole guinea pig central nervous system. Reticular structures as the basal forebrain, reticular thalamic nucleus and reticular nuclei of the brainstem contained the densest labeling. These results are discussed in terms of putative roles of this protein in synaptic strengthening and in redox activities.

Objectives : To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification.Design and Methods : TRAP assays were performed by using a TRAPeze(R) telomerase kit with or without [alpha-P-32] -dCTP. Amplification products were electrophoresed in polyacrylamide, gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls.Results : TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n = 24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions.Conclusions : TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification. (C) 2003 The Canadian Society of Clinical Chemists. All rights reserved.

Using differential hybridization of a guinea pig endometrial cell cDNA library, a potentially negatively estrogen-regulated gene, SOX-3, was isolated. According to the nucleotide and protein sequence similarities, SOx-3 belonged to the FAD-linked sulfhydryl oxidase family containing the egg white sulfhydryl oxidase, the rat seminal vesicle sulfhydryl oxidase-2 SOx-2, the quiescence-inducible protein hQ6. The SOX-3 transcript in the guinea pig as well as 5 different mRNAs in human tissues appeared differentially expressed in the tissues studied. In secondary endometrial cell culture, the SOX-3 mRNA level increased during a serum depletion-induced quiescence, decreased when cells enter the G1 phase after serum stimulation, and was restored during the S and G2/M phases. Thus, SOX-3 could be implicated in the negative cell cycle control. The SOx-3 protein appeared to be specific of epithelial cells in the uterus. Its expression level varied during the estrus cycle in the guinea pig, suggesting a regulation by steroid hormones.

We have isolated, in guinea-pig endometrial cells, an estrogen-induced 1.8 kb RNA called gec1. Screening of a guinea-pig genomic library led to identification of gec1 gene consisting of 4 exons and 3 introns. Exon 1 contains the 5′UTR and the ATG initiation codon. A guinea-pig gec1 cDNA was obtained by 5′-RACE. The 351 bp coding sequence shares 76.8% identity with that of the human GABARAP 924 bp cDNA while UTRs of the two cDNAs differ. A gec1 probe from the 3′UTR revealed a 1.9 kb mRNA in human tissues and a human GEC1 cDNA was isolated from placenta. Its coding sequence shares 93 and 79% identity with that of guinea-pig gec1 and human GABARAP, respectively. The human and guinea-pig GEC1 proteins have 100% identity. GEC1 and GABARAP proteins have 87% identity and N terminus featuring a tubulin binding motif. Thus, estrogen-regulated gec1 is a new gene which could encode a microtubule-associated protein.

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC(50) of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID(50) of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.

Conference Abstract| October 01 2000 Characterization of a SV40 T Antigen Immortalized Guinea-pig Endometrial Glandular Epithelial Cell Line (IGEC-1) and c-fos Regulation by 17 β-Estradiol (E2) C. Nemos; C. Nemos 1Laboratoire de Biochimie-Biologie moleculaire Search for other works by this author on: This Site PubMed Google Scholar J. P. Remy-Martin; J. P. Remy-Martin 1Laboratoire de Biochimie-Biologie moleculaire Search for other works by this author on: This Site PubMed Google Scholar B. Cypriani; B. Cypriani 1Laboratoire de Biochimie-Biologie moleculaire Search for other works by this author on: This Site PubMed Google Scholar M. Jouvenot; M. Jouvenot 1Laboratoire de Biochimie-Biologie moleculaire Search for other works by this author on: This Site PubMed Google Scholar P. Adami P. Adami 1Laboratoire de Biochimie-Biologie moleculaire Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2000) 28 (5): A296. https://doi.org/10.1042/bst028a296a Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation C. Nemos, J. P. Remy-Martin, B. Cypriani, M. Jouvenot, P. Adami; Characterization of a SV40 T Antigen Immortalized Guinea-pig Endometrial Glandular Epithelial Cell Line (IGEC-1) and c-fos Regulation by 17 β-Estradiol (E2). Biochem Soc Trans 1 October 2000; 28 (5): A296. doi: https://doi.org/10.1042/bst028a296a Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2000 Biochemical Society2000 Article PDF first page preview Close Modal You do not currently have access to this content.

Pokeweed antiviral protein (PAP) is a soluble leaf protein produced by the plant Phytolacca americana. It is able to specifically remove an adenine residue from the 28 S ribosomal RNA of eukaryotic ribosomes, thus blocking protein synthesis. Although PAP seems to be synthesized constitutively at very high concentrations during plant development, the mechanism of its synthesis remains unclear since the protein is toxic to pokeweed ribosomes. Recently, our group identified an inactive complexed form of PAP (PAPi) which could play a significant role in the plant protection mechanism during PAP synthesis. In order to estimate the concentration of PAP in plant extracts, a double sandwich ELISA was developed. This test enabled the detection and the quantification of PAP in a crude leaf extract at concentrations ranging from 2–1000 ng ml−1. It was specifically developed to discriminate between free PAP and PAPi. This report shows that the use of a monoclonal antibody specific to the C-terminal extremity of mature PAP, combined with an anti-PAP polyclonal antibody did not detect PAPi, unless it was denaturated prior to ELISA. Thus, the specificity of this test was restricted to free PAP and the test proved to be a reliable and unique tool to estimate the integrity of PAPi.

Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80°C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.

Monoclonal antibodies specific to pokeweed antiviral protein (PAP), a ribosome-inactivating protein (RIP), were obtained after six unsuccessful fusions. A special procedure including injections of low doses of purified PAP from spring leaves in a short period was adopted. Some clones highly specific to PAP react with recombinant PAP. One clone cross-reacts with PAP-S isolated from seeds but none cross-reacts with the isoform PAP II isolated from summer leaves. These antibodies represent a useful tool to investigate the mechanisms of PAP biosynthesis and plant protection involving RIPs.

Monoclonal antibodies (mAbs) have been used to study structure-function relationships of (R)-3-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a lipid-requiring mitochondrial membrane enzyme with an absolute and specific requirement for phosphatidylcholine (PC) for enzymic activity. The purified enzyme (apoBDH, devoid of phospholipid and thereby inactive) can be re-activated with preformed phospholipid vesicles containing PC or by short-chain soluble PC. Five of six mAbs cross-react with BDH from bovine heart and rat liver, including two mAbs to conformational epitopes. One mAb was found to be specific for the C-terminal sequence of BDH and served to: (1) map endopeptidase cleavage and epitope sites on BDH; and (2) demonstrate that the C-terminus is essential for the activity of BDH. Carboxypeptidase cleavage of only a few (< or = 14) C-terminal amino acids from apoBDH (as detected by the loss of C-terminal epitope for mAb 3-10A) prevents activation by either bilayer or soluble PC. Further, for BDH in bilayers containing PC, the C-terminus is protected from carboxy-peptidase cleavage, whereas in bilayers devoid of PC the C-terminus is cleaved, and subsequent activation by PC is precluded. We conclude that: (1) the C-terminus of BDH is essential for enzymic activity, consistent with the prediction, from primary sequence analysis, that the PC-binding site is in the C-terminal domain of BDH; and (2) the allosteric activation of BDH by PC in bilayers protects the C-terminus from carboxypeptidase cleavage, indicative of a PC-induced conformational change in the enzyme.

d-3-Hydroxybutyrate dehydrogenase (BDH) is an NAD+-dependent dehydrogenase of the mitochondrial inner membrane involved in the energetic balance between the liver and peripheral organs in mammals. It allows the conversion of ketone bodies (acetoacetate and d-3-hydroxybutyrate) and it is one of the best documented lipid-requiring enzymes with a dependence on lecithins. After release of proteins from the membrane by phospholipase A2 treatment of salt-treated mitochondria, the rat liver enzyme is absorbed on controlled-pore glass beads. After batch washing, the enzyme, devoid of lipids (apoBDH), is specifically eluted at pH 8.05—8.15 with a 0.1 M Tris—1 M LiBr buffer under reducing conditions (5 mM dithiothreitol). It appears that during BDH absorption, the glass beads mimic the phospholipid surface of biomembranes.

Primary biliary cirrhosis is an evolutive and chronic human liver disease characterized by presence of antimitochondrial autoantibodies in the serum. We present the biochemical definition of these autoantigens and show that purified pyruvate dehydrogenase complex contains three of the major mitochondrial antigens of M2 type i.e. the E2 subunit (dihydrolipoamide acetyl transferase), the X subunit and the E1 alpha subunit (pyruvate dehydrogenase), by immunoblotting experiments and inhibition of enzyme activity with several types of serums.

In rats, as in most mammal, ketone bodies are mainly produced in liver while they are metabolized in extrahepatic tissues. The expression of mitochondrial membrane-bound D-beta-hydroxybutyrate dehydrogenase (BDH), a ketone body-converting enzyme, has been estimated by two immunological techniques: immunohistofluorescence and Western blotting. The in situ labeling with anti-BDH antibody shows that the enzyme is expressed differently among the organs. Furthermore, within a given organ there are strong differences according to the cell type. The quantification of the enzyme by immunoblotting reveals that liver mitochondria have the highest content (more than 3% in protein mass). This content is 3,5 and 10 times lower in kidney, heart and brain mitochondria, respectively. Parallel D-beta-hydroxybutyrate dehydrogenase activity measurements on isolated mitochondria show differences in molecular activity of this enzyme according to the tissue origin. Due to the phospholipid requirement of this enzyme these differences in molecular activity are related to specific membrane lipid composition.

The effect of various metabolic conditions inducing an overproduction of ketone bodies in the rat have been studied at the different level of the D-beta-hydroxybutyrate dehydrogenase (BDH) expression: the enzymatic activity and the protein content in isolated mitochondria, and at the translational activity of isolated polysomes. The strongest variations were obtained under a diabete where the BDH expression is largely decreased. Insulin is able to reverse this diabete dramatic effect. The results are in agreement with the absence of direct relationships between the ketone bodies production and the D-beta-hydroxybutyrate dehydrogenase expression but appears to be controlled by insulin at different levels at least at the transcriptional, post translational and catalytic activities.

In order to continue the molecular studies of D-beta-hydroxybutyrate dehydrogenase (BDH) undertaken in our laboratory for several years, we have initiated a genetic approach which consists in the BDH cDNA cloning from a rat liver cDNA library. The immunoscreening method allowed to isolate a clone which exhibits a DNA insert shorter than the expected full length BDH cDNA.

beta-hydroxybutyrate dehydrogenase (BDH), a major protein located in the inner mitochondrial membrane is encoded, as most of mitochondrial proteins, in the nuclear genome. It is synthetized on the free polysomes and post-translationally imported into the mitochondria. The neosynthesized protein is a higher molecular weight precursor. The presequence is cleaved by the matrix protease to give the mature protein. The translocation across the mitochondrial membranes needs energy. The results also indicate that cytosolic factors with low molecular weight are essential in the recognition of precursor by mitochondria and to sort out newly synthetized nuclear encoded mitochondrial proteins from others nuclear encoded proteins.