Biomedical subjects
Publications and source records attributed to J Bertin.
Mass spectral fingerprints of detergents in Austrian, Hungarian, Uruguayan, and Chilean gasolines have been obtained using electrospray ionization mass spectrometry. Polymers or copolymers were observed based on ether motifs (inter-peak spacings of 44, 58 and 72 u) in all samples. Austrian gasoline was found to also contain polymers based on isobutene-amine.
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In dairy goats and ewes the use of equine Chorionic Gonadotropin (eCG) as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination (AI). Treatment for induction and synchronization of ovulation consists of a progestagen delivered by vaginal sponge, followed by an eCG injection. In some females, the first injection of eCG induces a humoral response with high concentrations of anti-eCG antibodies in contrast to other females displaying a very low concentration of anti-eCG antibodies. Females eliciting a low response were also poor responders after the following treatments. Conversely, high responders at the first treatment systematically yielded high immune responses upon the following treatment. By a molecular genetic approach using microsatellites we showed that the anti-eCG immune response phenotypes were associated with MHC class II polymorphism. Females with high residual antibody concentrations at the time of eCG injection exhibited a much lower kidding rate than other females did. Lower fertility of these females, inseminated at a fixed time after eCG treatment (43H for goats and 55H for ewes), might be due to the delay in estrus occurrence and the pre ovulatory LH surge. Consequently, under field conditions old females selected for AI are only those with low residual anti-eCG antibody concentrations and old females with high residual antibody concentration are culled from AI breeding because of their low fertility during the previous year. So we have undertaken comparative studies to establish if the anti-eCG immune response is correlated with the global immunity in animals.
In addition to promoting cell survival, neurotrophins also can elicit apoptosis in restricted cell types. Recent results indicate that nerve growth factor (NGF) can induce Schwann cell death via engagement of the p75 neurotrophin receptor. Here we describe a novel interaction between the p75 receptor and receptor-interacting protein 2, RIP2 (RICK/CARDIAK), that accounts for the ability of neurotrophins to choose between a survival-versus-death pathway. RIP2, an adaptor protein with a serine threonine kinase and a caspase recruitment domain (CARD), is highly expressed in dissociated Schwann cells and displays an endogenous association with p75. RIP2 binds to the death domain of p75 via its CARD domain in an NGF-dependent manner. The introduction of RIP2 into Schwann cells deficient in RIP2 conferred NGF-dependent nuclear transcription factor-kappaB (NF-kappaB) activity and decreased the cell death induced by NGF. Conversely, the expression of a dominant-negative version of RIP2 protein resulted in a loss of NGF-induced NF-kappaB induction and increased NGF-mediated cell death. These results indicate that adaptor proteins like RIP2 can provide a bifunctional switch for cell survival or cell death decisions mediated by the p75 neurotrophin receptor.
Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-kappaB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-kappaB induction. Dominant-negative versions of CARD4 block activation of NF-kappaB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.
The CED4/Apaf-1 family of proteins functions as critical regulators of apoptosis and NF-kappaB signaling pathways. A novel human member of this family, called CARD12, was identified that induces apoptosis when expressed in cells. CARD12 is most similar in structure to the CED4/Apaf-1 family member CARD4, and is comprised of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding site (NBS), and a C-terminal domain of leucine-rich repeats (LRR). The CARD domain of CARD12 interacts selectively with the CARD domain of ASC, a recently identified proapoptotic protein. In addition, CARD12 coprecipitates caspase-1, a caspase that participates in both apoptotic signaling and cytokine processing. CARD12 may assemble with proapoptotic CARD proteins to coordinate the activation of downstream apoptotic and inflammatory signaling pathways.
BCL10 belongs to the caspase recruitment domain (CARD) family of proteins that regulate apoptosis and NF-kappaB signaling pathways. Analysis of BCL10-deficient mice has revealed that BCL10 mediates NF-kappaB activation by antigen receptors in B and T cells. We recently identified a subclass of CARD proteins (CARD9, CARD11, and CARD14) that may function to connect BCL10 to multiple upstream signaling pathways. We report here that CARD10 is a novel BCL10 interactor that belongs to the membrane-associated guanylate kinase family, a class of proteins that function to organize signaling complexes at plasma membranes. When expressed in cells, CARD10 binds to BCL10 and signals the activation of NF-kappaB through its N-terminal effector CARD domain. We propose that CARD10 functions as a molecular scaffold for the assembly of a BCL10 signaling complex that activates NF-kappaB.
The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-kappaB signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membrane-associated guanylate kinase (MAGUK) family, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD11 and CARD14 have homologous structures consisting of an N-terminal CARD domain, a central coiled-coil domain, and a C-terminal tripartite domain comprised of a PDZ domain, an Src homology 3 domain, and a GUK domain with homology to guanylate kinase. The CARD domains of both CARD11 and CARD14 associate specifically with the CARD domain of BCL10, a signaling protein that activates NF-kappaB through the IkappaB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-kappaB and induce the phosphorylation of BCL10. These findings suggest that CARD11 and CARD14 are novel MAGUK family members that function as upstream activators of BCL10 and NF-kappaB signaling.
BCL10/CLAP is an activator of apoptosis and NF-kappaB signaling pathways and has been implicated in B cell lymphomas of mucosa-associated lymphoid tissue. Although its role in apoptosis remains to be determined, BCL10 likely activates NF-kappaB through the IKK complex in response to upstream stimuli. The N-terminal caspase recruitment domain (CARD) of BCL10 has been proposed to function as an activation domain that mediates homophilic interactions with an upstream CARD-containing NF-kappaB activator. To identify upstream signaling partners of BCL10, we performed a mammalian two-hybrid analysis and identified CARD9 as a novel CARD-containing protein that interacts selectively with the CARD activation domain of BCL10. When expressed in cells, CARD9 binds to BCL10 and activates NF-kappaB. Furthermore, endogenous CARD9 is found associated with BCL10 suggesting that both proteins form a pre-existing signaling complex within cells. CARD9 also self-associates and contains extensive coiled-coil motifs that may function as oligomerization domains. We propose here that CARD9 is an upstream activator of BCL10 and NF-kappaB signaling.
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Baculovirus P35 is a universal substrate-inhibitor of the death caspases. Stoichiometric inhibition by P35 is correlated with cleavage of its reactive site loop (RSL) and formation of a stable P35.caspase complex through a novel but undefined mechanism. The P35 crystal structure predicts that the RSL associates with the beta-sheet core of P35 positioning the caspase cleavage site at the loop's apex. Here we demonstrate that proper interaction between the RSL and the beta-sheet core is critical for caspase inhibition, but not cleavage. Disruption of RSL interaction with the beta-sheet by substituting hydrophobic residues of the RSL's transverse helix alpha1 with destabilizing charged residues caused loss of caspase inhibition, without affecting P35 cleavage. Restabilization of the helix/sheet interaction by charge compensation from within the beta-sheet partially restored anti-caspase potency. Mutational effects on P35 helix/sheet interactions were confirmed by measuring intermolecular helix/sheet association with the yeast two-hybrid system. Moreover, the identification of P35 oligomers in baculovirus-infected cells suggested that similar P35 interactions occur in vivo. These findings indicate that P35's anti-caspase potency depends on a distinct conformation of the RSL which is required for events that promote stable, post-cleavage interactions and inhibition of the target caspase.
The nematode CED-4 protein and its human homolog Apaf-1 play a central role in apoptosis by functioning as direct activators of death-inducing caspases. A novel human CED-4/Apaf-1 family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that mediate disease resistance in plants. CARD4 interacted with the serine-threonine kinase RICK and potently induced NF-kappaB activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-kappaB and apoptotic signaling pathways and may be a component of the host innate immune response.
We report the discovery of a unique renal-cell carcinoma operated 9 years earlier on a thyroidectomy operative specimen. Intra-thyroid metastasis from kidney cancer is uncommon. The average delay to discovery as a painless thyroid nodule is estimated around 6 years following nephrectomy. Pre-operative fine needle aspiration and cytology provide the diagnosis. Prognosis of unique thyroid metastasis of kidney cancer is good despite the distant spread. Surgery is the most common therapeutic attitude.
The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.
To identify novel antiapoptotic proteins encoded by DNA viruses, we searched viral genomes for proteins that might interfere with Fas and TNFR1 apoptotic signaling pathways. We report here that equine herpesvirus type 2 E8 protein and molluscum contagiosum virus MC159 protein both show sequence similarity to the death effector domains (DEDs) of the Fas/TNFR1 signaling components FADD and caspase-8. Yeast two-hybrid analysis revealed that E8 protein interacted with the caspase-8 prodomain whereas MC159 protein interacted with FADD. Furthermore, expression of either E8 protein or MC159 protein protected cells from Fas- and TNFR1-induced apoptosis indicating that certain herpesviruses and poxviruses use DED-mediated interactions to interfere with apoptotic signaling pathways. These findings identify a novel control point exploited by viruses to regulate Fas- and TNFR1-mediated apoptosis.
Fas- and tumor necrosis factor receptor 1 (TNFR1)-induced apoptosis is mediated by the interaction of FADD with caspase-8. Here, we report that the bovine herpesvirus 4 (BHV4) BORFE2 gene encodes a protein that inhibits Fas- and TNFR1-induced apoptosis and contains death effector domains (DEDs). Using the yeast two-hybrid system, we found that the BORFE2 protein interacts with the prodomain of caspase-8. Furthermore, we show that BHV4 BORFE2 is a member of a family of DED-containing proteins that includes other gamma-2 herpesviruses, such as Kaposi's sarcoma-associated herpesvirus and herpesvirus saimiri.
Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.
During transcription of the late strand of polyomavirus DNA, RNA polymerase II stalls and accumulates nearby the binding sites on viral DNA recognized by polyomavirus large T antigen. Stalling by RNA polymerases is eliminated when thermolabile large T antigen is inactivated by using a temperature-sensitive virus mutant (J. Bertin, N.-A. Sunstrom, P. Jain, and N. H. Acheson, Virology 189:715-724, 1992). To determine whether stalling by RNA polymerases is mediated through the interaction of large T antigen with one or more of its binding sites, viable polyomavirus mutants that contain altered large-T-antigen-binding sites were constructed. Point mutations were introduced by site-directed mutagenesis into the multiple, clustered G(A/G)GGC pentanucleotides known to be the target sequence for large T-antigen binding. Mutation of the G(A/G)GGC pentanucleotides in the first two binding sites encountered by RNA polymerases in the intergenic region (sites C and B) had no detectable effect on stalling as measured by transcriptional run-on analysis. However, mutation of the two GAGGC pentanucleotides in binding site A, which lies adjacent to the origin of viral DNA replication, eliminated stalling by RNA polymerases. We conclude that binding of large T antigen to site A blocks elongation by RNA polymerase II. Further characterization of virus containing mutated site A did not reveal any effects on early transcription levels or on virus DNA replication. However, the mutant virus gave rise to small plaques, suggesting impairment in some stage of virus growth. Stalling of RNA polymerases by large T antigen bound to the intergenic region of viral DNA may function to prevent transcription from displacing proteins whose binding is required for the normal growth of polyomavirus.