Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class Ia gene H-2K^b under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2K^b heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-K^b construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models. Mol. Reprod. Dev. 50:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Benthic indicators of sediment quality associated with run-of-river reservoirs

Freshwater ecosystems have been fragmented by the construction of large numbers of dams. In addition to disruption of ecological continuity and physical disturbance downstream, accumulation of large amounts of sediment within run-of-river reservoirs constitutes a latent ecotoxic risk to aquatic communities. To date, run-of-river reservoirs and ecotoxic risks associated with contaminated sediment to the biodiversity and functioning of such systems are little studied. Therefore, the main objective of this study was to describe macroinvertebrate assemblages, and the functioning of these systems, and to propose indicators of sediment contamination to integrate in in-situ risk assessment methodology. To identify specific assemblages of run-of-river reservoirs, we first compared macroinvertebrate assemblages and their biotrait profiles (i.e. from a database of biological and ecological traits) in reservoirs (n = 6) and associated river sites (upstream and downstream of dams). Then, we compared responses of assemblages and biotrait profiles to sediment contamination of the banks and channels of reservoirs to select the most useful spatial scale to identify sediment contamination. Nineteen indicator taxa were observed to be specifically associated with channel habitats of reservoirs. Among these, the abundance of three taxa (Tanypodinae (Diptera), Ephemerella (Ephemeroptera) and Atherix (Diptera)) revealed the effect of metal sediment contamination. “Between-reservoirs” differences in their biotrait profile were found along the contamination gradient, with a shift of communities’ composition and functionality, and an increase in functional similarity. Many traits (response traits), for example “maximum size”, “transverse distribution”, “substrate preferences”, “saprobity”, “temperature”, “resistance forms”, and “locomotion”, were specifically linked to contamination of sediments by metals. This study indicates how sediment contamination can change the structural and functional composition of run-of-river reservoir assemblages. Indicator taxa and response traits identified in this study could improve current risk assessment methodology and potentially enable prediction of the risks of contaminated sediments stored in reservoirs in downstream ecosystems.     

The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.     

Role of interferons in the control of Lassa virus replication in human dendritic cells and macrophages

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), which primarily targets human dendritic cells (DC) and macrophages (MP). Massive numbers of viral particles are released with no effect on the viability, activation or maturation of these cells. LV does not inhibit the activation of cells induced by sCD40L or LPS. We report here the consequences of exogenous activation of LV-infected human DC and MP for viral replication. The activation of cells with lipopolysaccharide or exogenous poly(I-C) and the transfection of cells with poly(I-C) strongly inhibited LV replication, at least partly by inducing type I interferon (IFN) synthesis. In contrast, cell stimulation with sCD40L did not induce type I IFN responses or inhibit LV release. Recombinant type I IFNs strongly inhibited LV replication in both cell types, whereas IFNgamma and IFNlambda did not. The modest type I IFN production observed in LV-infected MP, but not in DC, was involved in controlling LV replication in MP. These results provide an explanation for the slower replication of LV in MP than in DC, and suggest that type I IFNs are crucial in the control of LV.     

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26

Summary A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related fig (fms-like gene) fibrovblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.     

Identification and characterization of a novel chemically induced allele at the planar cell polarity gene Vangl2

Planar cell polarity (PCP) signaling controls a number of morphogenetic processes including convergent extension during gastrulation and neural tube formation. Defects in this pathway cause neural tube defects (NTD), the most common malformations of the central nervous system. The Looptail (Lp) mutant mouse was the first mammalian mutant implicating a PCP gene (Vangl2) in the pathogenesis of NTD. We report on a novel chemically induced mutant allele at Vangl2 called Curly Bob that causes a missense mutation p.Ile268Asn (I268N) in the Vangl2 protein. This mutant segregates in a semi-dominant fashion with heterozygote mice displaying a looped tail appearance, bobbing head, and a circling behavior. Homozygote mutant embryos suffer from a severe form of NTD called craniorachischisis, severe PCP defects in the inner hair cells of the cochlea and posterior cristae, and display a distinct defect in retinal axon guidance. This mutant genetically interacts with the Lp allele (Vangl2 ^S464N) in neural tube development and inner ear hair cell polarity. The Vangl2^I268N protein variant is expressed at very low levels in affected neural and retinal tissues of mutant homozygote embryos. Biochemical studies show that Vangl2^I268N exhibits impaired targeting to the plasma membrane and accumulates in the endoplasmic reticulum. The Vangl2^I268N variant no longer physically interacts with its PCP partner DVL3 and has a reduced protein half-life. This mutant provides an important model for dissecting the role of Vangl2 in the development of the neural tube, establishment of polarity of sensory cells of the auditory and vestibular systems, and retinal axon guidance.

     

Allosteric-type control of synaptobrevin cleavage by protect tetanus toxin light chain

Summary The light chain of tetanus neurotoxin (TeNTL chain) has been shown to be endowed with zine endopeptidase activity, selectively directed towards the Gln⁷⁶-Phe⁷⁷ bond of synaptobrevin, a vesicle-associated membrane protein critically involved in neuroexocytosis. In previous reports, truncations at the NH₂- and COOH-terminus of synaptobrevin have shown that the sequence 39–88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well-defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH₂- and COOH-terminal peptides of synaptobrevin, S 27–55 (S₁) and S 82–93 (S₂), to the synaptobrevin fragment S 56–81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S₁ and S₂ with complementary sites present on TeNT as shown by surface plasmon resonance experiments. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT probably involving a conformational change of the toxin. This could accound for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.     

A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four “EF hand” type Ca²⁺ binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca²⁺ ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca_n (n = 0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Complex patterns of hybridization between exotic and native North American poplar species

? Premise of the study: Poplars and their hybrids are seen as important candidates for bioenergy initiatives. However, many concerns have been raised about large-scale plantations of new poplar cultivars. The deployment of such plants with novel traits brings the risk of potential spread of novel genome regions (including exotic genes, transgenes, or other heritable modifications) into natural populations of related species. The possibility of introgression is especially high in poplars because reproductive barriers between species are weak. Knowledge of the frequency of hybridization between cultivated trees and natural populations is one important step in the risk-assessment process. ? Methods: We studied the rate of spontaneous hybridization from two sexually mature poplar plantations into adjacent natural populations of Populus deltoides and P. balsamifera. The two plantations, both in eastern Canada, contain many different complex hybrid clones with components from exotic species, mostly P. nigra, P. trichocarpa, and P. maximowiczii. We analyzed 12 species-specific single nucleotide polymorphisms from six different genes in 5373 offspring sampled from the natural populations. ? Results: Contributions from all three exotics were found in the offspring, confirming low reproductive barriers among poplar species in these sections. The frequency of hybrid offspring varied among pollen donors, recipient populations, and years. ? Conclusions: The remarkably high rate of hybridization that was found in the smallest natural population sampled suggests that small peripheral populations carry a higher risk of introgression. These results could be used as a starting point for developing regulatory guidelines for the introduction of plants with novel traits.