Functional connectivity is preserved but reorganized across several anesthetic regimes.

Under anesthesia, systemic variables and CBF are modified. How does this alter the connectivity measures obtained with rs-fMRI? To tackle this question, we explored the effect of four different anesthetics on Long Evans and Wistar rats with multimodal recordings of rs-fMRI, systemic variables and CBF. After multimodal signal processing, we show that the blood-oxygen-level-dependent (BOLD) variations and functional connectivity (FC) evaluated at low frequencies (0.031-0.25 â€‹Hz) do not depend on systemic variables and are preserved across a large interval of baseline CBF values. Based on these findings, we found that most brain areas remain functionally active under any anesthetics, i.e. connected to at least one other brain area, as shown by the connectivity graphs. In addition, we quantified the influence of nodes by a measure of functional connectivity strength to show the specific areas targeted by anesthetics and compare correlation values of edges at different levels. These measures enable us to highlight the specific network alterations induced by anesthetics. Altogether, this suggests that changes in connectivity could be evaluated under anesthesia, routinely used in the control of neurological injury.

Transmission and immunity: the importance of heterogeneity in the fight against malaria.

The complex relationship between transmission and parasite prevalence in humans is an important issue. Using a large dataset matching estimates of malaria transmission and Plasmodium falciparum prevalence in African children, a stimulating study published in Nature provides evidence that heterogeneity in susceptibility crucially determines the prevalence of infection. Moreover, it suggests that children who clear infections are not immune to new infections, irrespective of the amount of transmission. It is important to question the relevance of such results based on mathematical models when discussing host-parasite interactions, especially their implications for public health interventions.

[Epigenetics and cancer]. / Epigénétique et cancer.

The term epigenetics encompasses all the modifications that are stable across cell generations, but which do not imply any change in DNA sequence. Post-translational modifications of the histones and DNA methylation are the most studied types of epigenetic information due to their major impact on transcription. The link between epigenetics and cancer arises from the fact that epigenetic deregulations frequently participate in tumorigenesis by inactivation of tumour-suppressor genes. Since these deregulations are reversible, hopes of treatment rely on a better understanding of the maintenance mechanisms of the epigenetic information. Among the different pathways of transcription inhibition, DNA methylation is the simplest and one of the best characterized at the present time. Inhibitors of DNA methyltransferases are currently under clinical trials and already show promising results.

A family of human zinc finger proteins that bind methylated DNA and repress transcription.

In vertebrates, densely methylated DNA is associated with inactive transcription. Actors in this process include proteins of the MBD family that can recognize methylated CpGs and repress transcription. Kaiso, a structurally unrelated protein, has also been shown to bind methylated CGCGs through its three Krüppel-like C2H2 zinc fingers. The human genome contains two uncharacterized proteins, ZBTB4 and ZBTB38, that contain Kaiso-like zinc fingers. We report that ZBTB4 and ZBTB38 bind methylated DNA in vitro and in vivo. Unlike Kaiso, they can bind single methylated CpGs. When transfected in mouse cells, the proteins colocalize with foci of heavily methylated satellite DNA and become delocalized upon loss of DNA methylation. Chromatin immunoprecipitation suggests that both of these proteins specifically bind to the methylated allele of the H19/Igf2 differentially methylated region. ZBTB4 and ZBTB38 repress the transcription of methylated templates in transfection assays. The two genes have distinct tissue-specific expression patterns, but both are highly expressed in the brain. Our results reveal the existence of a family of Kaiso-like proteins that bind methylated CpGs. Like proteins of the MBD family, they are able to repress transcription in a methyl-dependent manner, yet their tissue-specific expression pattern suggests nonoverlapping functions.

The human enhancer blocker CTC-binding factor interacts with the transcription factor Kaiso.

CTC-binding factor (CTCF) is a DNA-binding protein of vertebrates that plays essential roles in regulating genome activity through its capacity to act as an enhancer blocker. We performed a yeast two-hybrid screen to identify protein partners of CTCF that could regulate its activity. Using full-length CTCF as bait we recovered Kaiso, a POZ-zinc finger transcription factor, as a specific binding partner. The interaction occurs through a C-terminal region of CTCF and the POZ domain of Kaiso. CTCF and Kaiso are co-expressed in many tissues, and CTCF was specifically co-immunoprecipitated by several Kaiso monoclonal antibodies from nuclear lysates. Kaiso is a bimodal transcription factor that recognizes methylated CpG dinucleotides or a conserved unmethylated sequence (TNGCAGGA, the Kaiso binding site). We identified one consensus unmethylated Kaiso binding site in close proximity to the CTCF binding site in the human 5' beta-globin insulator. We found, in an insulation assay, that the presence of this Kaiso binding site reduced the enhancer-blocking activity of CTCF. These data suggest that the Kaiso-CTCF interaction negatively regulates CTCF insulator activity.

Recombinant bovine lactoperoxidase as a tool to study the heme environment in mammalian peroxidases.

The cDNA encoding bovine lactoperoxidase (LPO) has been expressed in CHO cells. The recombinant LPO was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. rLPO exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Engineering of rLPO into a myeloperoxidase (MPO)-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in MPO. However, the resulting bovine LPO mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of MPO, underlining the complex nature of interactions in the heme vicinity.

[Mutagenesis of the human histamine H1 receptor and design of new antihistamine agents]. / Mutagénèse du récepteur histaminique H1 humain et design de nouveaux agents antihistaminiques.

The binding cavity of histamine and histamine antagonists is explored using site directed mutagenesis of the human histamine H1 receptor and the amino acids involved in ligand binding are identified. Whereas Asp107 and Phe199 are important for both agonists and antagonists, two additional amino acids (Asn198 and Trp103) are required for efficient histamine binding. The binding site of antagonists is best defined as resulting from a strong ionic bond to Asp107, an orthogonal interaction between one of the aromatic rings with Phe199, and probably a hydrophobic interaction between the second aromatic ring and the lipophilic amino acids of the upper part of TMIV and TMV. This is consistent with structure-activity data of most described antagonists.

Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

Correct in vivo processing of a chimeric ubiquitin-proapolipoprotein A-I fusion protein in baculovirus-infected insect cells.

The cDNA coding for human proapolipoprotein A-I was expressed as a ubiquitin fusion under the control of the polyhedrin promoter in baculovirus-infected Sf9 Spodoptera frugiperda insect cells. The fusion protein was expressed at high level and was quantitatively cleaved in vivo. The cleaved product was purified and its N-terminal amino acid sequence was established. The data showed that authentic proapolipoprotein A-I has been produced, and thus demonstrated the existence in Spodoptera frugiperda insect cells of a specific ubiquitin hydrolase activity.

Production of authentic human proapolipoprotein A-I in Escherichia coli: strategies for the removal of the amino-terminal methionine.

Several methods were compared with respect to the production of authentic, N-terminal methionine-free proapolipoprotein A-I in engineered Escherichia coli bacteria. A first approach consisted of treating the purified methionylated recombinant protein with an amino-peptidase, purified from Aeromonas proteolytica. A second series of strategies was based on the construction of proapo A-I encoding cassettes carrying built-in recognition sites suitable for specific in vitro cleavage of the products with kallikrein and enterokinase, respectively. Along the same line, a fusion between ubiquitin and proapo A-I was produced in E. coli with the prospect to achieve post-purification cleavage with yeast ubiquitin hydrolase. Finally, proapo A-I was fused to the signal peptide of the bacterial outer membrane protein, OmpA, aiming at an in situ conversion to authentic proapo A-I during secretion to the bacterial periplasm. The data showed that, out of these five systems, the OmpA signal peptide system and, to a lesser extent, the one involving the fusion to ubiquitin were the most efficient in yielding authentic proapo A-I from engineered Escherichia coli.

Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization.

A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable lambda PL promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product.

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli.

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.

Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clones.

Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal any significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.