Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a K_d of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.     

Increase in epidermal growth factor receptor and its messenger ribonucleic acid levels with differentiation of human trophoblast cells in culture

Epidermal growth factor receptor (EGFR) expression was studied during the differentiation of human trophoblast cells in culture. In vitro, intravillous mononuclear cytotrophoblasts aggregate and fuse within 24 h to form a syncytium. This morphological differentiation was associated with a significant twofold increase in specific ¹²⁵I-EGF binding capacity (P < 0.01). Scatchard analyses showed an apparent rise in the number of high-affinity binding sites (0.33 ± 0.04 and 0.63 ± 0.07 pmol/mg protein at 24 and 48 h, respectively), with no change in their affinity (1.34 and 1.42 × 10^?10 mol/L). Affinity labeling of ¹²⁵I-EGF in cultured trophoblast cells followed by SDS-PAGE and autoradiography revealed a band of 175 KDa corresponding to EGFR, the intensity of which increased with the time in culture. EGF-dependent phosphorylation of membrane proteins from cultured trophoblast cells revealed major phosphorylated proteins of 170 KDa (EGFR) and 35 KDa, which were both increased at 48 h, indicating a rise in EGFR-kinase activity during syncytium formation. Northern blot analysis of EGFR-mRNA, followed by hybridization with a ³²P-cDNA probe for EGFR, revealed an increase in EGFR gene expression in syncytiotrophoblasts, as compared to cytotrophoblasts. Thus, the increase in bioactive EGFR observed during the differentiation of trophoblast cells was due to an increase in their synthesis. Cultured trophoblast cells are therefore a good model of spontaneous up-regulation of EGFR expression with cell differentiation. © 1993 Wiley-Liss, Inc.     

Cystic fibrosis mutations in North American populations of French ancestry: Analysis of Quebec French-Canadian and Louisiana Acadian families

A 3-bp deletion (ΔF508) in the cystic fibrosis (CF) gene is the mutation on the majority of CF chromosomes. We studied 112 CF families from North American populations of French ancestry: French-Canadian families referred from hospitals in three cities in Quebec and from the Saguenay-Lac St. Jean region of northeastern Quebec and Acadian families living in Louisiana. ΔF508 was present on 71%, 55%, and 70% of the CF chromosomes from the major-urban Quebec, Saguenay-Lac St. Jean, and Louisiana Acadian families, respectively. A weighted estimate of the proportion of ΔF508 in the French-Canadian patient population of Quebec was 70%. We found that 95% of the CF chromosomes with ΔF508 had D7S23 haplotype B, the most frequent haplotype on CF chromosomes. In the Saguenay-Lac St. Jean families, 86% of the CF chromosomes without ΔF508 had the B haplotype, compared with 31% for the major-urban Quebec and Louisiana Acadian families. The incidence of CF in the Saguenay-Lac St. Jean population was 1/895 live-born infants.     

DNA relatedness among serovars ofListeria monocytogenes sensu lato

Sixty-six strains ofListeria monocytogenes (as defined in the eighth edition ofBergey's Manual of Determinative Bacteriology) were characterized by DNA relatedness. These strains formed five distinct DNA relatedness groups: (i)L. monocytogenes sensu stricto (30 strains) including the type spain ATCC 15313; (ii) serovar 5 strains (9 strains) corresponding to L. bulgarica; (iii) L. inocua (11 strains of serovars 6a, 6b, 4ab, and undesignated ones) including the reference strains; (iv) six strains of serovars 6a and 6b; (v) ten strains of various serovars. These five groups were clearly distinct fromL. grayi (4 strains) andL. murrayi (3 strains).     

The First Steps of Adaptation of Escherichia coli to the Gut Are Dominated by Soft Sweeps

The accumulation of adaptive mutations is essential for survival in novel environments. However, in clonal populations with a high mutational supply, the power of natural selection is expected to be limited. This is due to clonal interference - the competition of clones carrying different beneficial mutations - which leads to the loss of many small effect mutations and fixation of large effect ones. If interference is abundant, then mechanisms for horizontal transfer of genes, which allow the immediate combination of beneficial alleles in a single background, are expected to evolve. However, the relevance of interference in natural complex environments, such as the gut, is poorly known. To address this issue, we have developed an experimental system which allows to uncover the nature of the adaptive process as Escherichia coli adapts to the mouse gut. This system shows the invasion of beneficial mutations in the bacterial populations and demonstrates the pervasiveness of clonal interference. The observed dynamics of change in frequency of beneficial mutations are consistent with soft sweeps, where different adaptive mutations with similar phenotypes, arise repeatedly on different haplotypes without reaching fixation. Despite the complexity of this ecosystem, the genetic basis of the adaptive mutations revealed a striking parallelism in independently evolving populations. This was mainly characterized by the insertion of transposable elements in both coding and regulatory regions of a few genes. Interestingly, in most populations we observed a complete phenotypic sweep without loss of genetic variation. The intense clonal interference during adaptation to the gut environment, here demonstrated, may be important for our understanding of the levels of strain diversity of E. coli inhabiting the human gut microbiota and of its recombination rate.     

Comprehensive profiling of 8p11-12 amplification in breast cancer

In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.     

Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance.     

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.     

Combinations of cytokines promote survival of mice and limit acute radiation damage in concert with amelioration of vascular damage

Recovery from hematopoietic aplasia is a predominant factor in the survival of total-body-irradiated mice within 30 days after exposure. However, other radiation-induced pathophysiological events have been shown to play a role, among which an inflammatory reaction must be considered. In the present study, we evaluated the therapeutic potential of a hematopoietic growth factor (thrombopoietin, Tpo) and pleiotropic cytokines (Il4 or Il11), used alone or in combination, on the survival of mice, hematopoietic reconstitution, inflammatory reaction and vascular changes. All treatments including Tpo induced a higher level of survival than did treatment with a placebo, with combinations being the most efficient. The increased survival could not be explained solely by an improved hematopoietic recovery. Treatments with Tpo also reduced the level of the chemokine KC in plasma and the level of expression of mRNA for inflammatory and coagulation proteins in the lungs of irradiated mice. In addition, radiation- induced vascular hyperpermeability was reduced with the use of Tpo. In summary, our results show that Tpo may improve survival by limiting vascular leakage, which in turn could limit inflammatory reactions and the ensuing tissue damage.     

Molecular and genealogical characterization of the R1443X BRCA1 mutation in high-risk French-Canadian breast/ovarian cancer families

The Quebec population contains about six-million French Canadians, descended from the French settlers who colonized Nouvelle-France between 1608 and 1765. Although the relative genetic contribution of each of these founders is highly variable, altogether they account for the major part of the contemporary French-Canadian gene pool. This study was designed to analyze the role of this founder effect in the introduction and diffusion of the BRCA1 recurrent R1443X mutant allele. A highly conserved haplotype, observed in 18 French-Canadian families and generated using 17 microsatellite markers surrounding the BRCA1 locus, supports the fact that the R1443X mutation is a founder mutation in the Quebec population. We also performed haplotyping analysis of R1443X carriers on 19 other families from seven different nationalities; although the same alleles are shared for three markers surrounding the BRCA1 gene, distinct haplotypes were obtained in four families, suggesting multiple origins for the R1443X mutation. Ascending genealogies of the 18 French Canadian families and of controls were reconstructed on an average depth of 10 generations. We identified the founder couple with the highest probability of having introduced the mutation in the population. Based on the descending genealogy of this couple, we detected the presence of geographical concentration in the diffusion pattern of the mutation. This study demonstrates how molecular genetics and demogenetic analyses can complement each other to provide findings that could have an impact on public health. Moreover, this approach is certainly not unique to breast cancer genetics and could be used to understand other complex traits.Other members of the BCLC Haplotype Group involved in this study are listed in Appendix 1Other members of INHERIT BRCAs involved in this study are listed in Appendix 2H. Vézina and F. Durocher contributed equally to this work and should be regarded as joint first author