When there seem to be no predetermining factors: early child and proximal family risk predicting externalizing behavior in young children incurring no distal family risk.

The main objective of the current study was to examine the impact of two child risk factors, i.e. personality and inhibition, and two proximal family risk factors, i.e. parenting and attachment, and the impact of their cumulative effect on later externalizing behavior among young children incurring no distal family risk. Data were collected in a longitudinal two-wave design from 161 non-referred and referred children aged three to five years at the onset of the study. All of the children were raised in families of middle to high socio-economic status, i.e. their parents were educated to a middle to high level, had access to the job market and lived together as couples. The four risk domains were assessed at the onset of the study, while EB was rated both at the onset of the study and in the 24-month follow-up. Results confirmed that the four risk domains were each both correlates of EB and efficient at discriminating non-referred from referred children; that their combination regardless of their content (cumulative risk) provided a strong prediction of both later EB and non-referred vs referred sample membership. The results are discussed both for research and clinical purposes.

Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles.

Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid substitutions appeared to disrupt necessary interactions between E1 and E2. For some mutants, reductions in HCVpp-associated E1 were associated with the incorporation of a high molecular weight, hyperglycosylated E2 that displayed decreased CD81-binding. Other entry-deficient mutants displayed normal E1E2 incorporation into pseudoparticles and normal CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production. However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry.

Interest of the association clonidine-spironolactone in cirrhotic patients with ascites and activation of sympathetic nervous system.

BACKGROUND: The aim of this study was to examine the effects of spironolactone, clonidine and the association of clonidine-spironolactone on renin-aldosterone and sympathetic systems, renal function, systemic hemodynamics and mobilization of ascites in 32 alcoholic cirrhotic patients with marked increase in sympathetic system. METHODS: Measurements were taken before and after an 8-day treatment with spironolactone (200 mg/day), after an 8-day treatment with clonidine (0.150 mg/day) and 10 days after adjunction of spironolactone (200 mg/day) to clonidine. RESULTS: Three patients abandoned the treatment or were excluded because lack of compliance. Spironolactone alone induced an increase in renin-aldosterone and sympathetic systems without any remarkable increase of natriuresis and body weight loss. Given for 8 days, clonidine alone induced a significant decrease in plasma norepinephrine associated with a significant increase in glomerular filtration rate without effect on natriuresis. In contrast, 10 days after adjunction of spironolactone to clonidine, plasma renin and aldosterone significantly decreased, natriuresis increased (from 7.4 +/- 0.7 to 41.6 +/- 3.2 mEq/24 h) and body weight decreased (from 66.03 +/- 2.3 to 63.5 +/- 2.3 kg) without adverse effects. CONCLUSION: In cirrhotic patients with ascites and marked activation of sympathetic nervous system, spironolactone (200 mg/day) is unable to mobilize ascites. In these patients, after 8 days, clonidine decreases sympathetic activity, increases glomerular filtration rate and after 18 days, decreases plasma renin and aldosterone concentrations allowing a better action of spironolactone. The association clonidine-spironolactone enhances natriuresis and body weight loss.

Induction of hepatitis C virus E1 envelope protein-specific immune response can be enhanced by mutation of N-glycosylation sites.

Deglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.

Efficient delivery of Antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8+ T cells.

The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.

Characterization of major protein phosphatases from selected species of Kluyveromyces. Comparison with protein phosphatases from Yarrowia lipolytica.

Casein phosphatase activities have been identified in five yeast strains grown on Pi-deficient medium. Maximal endocellular activities appeared in the exponential phase. Exocellular phosphatases were significantly produced from Yarrowia lipolytica W-29 and Kluyveromyces marxianus, in the early stationary phase. Major phosphatases from K. marxianus were one heavy acid phosphatase composed of 64-67 kDa subunits, which could be secreted in the medium, and one type 2A protein phosphatase with an apparent molecular mass of 147 kDa and a 52 kDa catalytic subunit dissociated by 80% ethanol treatment. The characteristics of phosphatases purified from K. marxianus were compared with those previously purified from Y. lipolytica.

[Fom orphan receptor to novel neuropeptide: an example of reverse pharmacology]. / De récepteur orphelin à nouveau neuropeptide: un exemple de pharmacologie inverse.

Nociceptin, a neuropeptide 17 amino acid residues in length, is the first novel bioactive substance to have been discovered by functional genomics. Nociceptin was isolated from a rat brain extract as the endogenous ligand to an orphan receptor, ORL1 (Opioid Receptor-Like 1), structurally akin to opioid receptors, whose cDNA had been cloned from a human brain stem library. The peptide is processed from a larger precursor polypeptide, prepro-nociceptin, which is widely distributed in the nervous system. Nociceptin is primarily an inhibitory neuropeptide that acts on neurons to depress synaptic transmission. Nociceptin's wide spectrum of pharmacological actions hints at several potential therapeutic applications for ORL1 receptor agonists and/or antagonists, particularly for treating pain states, stress and anxiety, cognitive defects, and drug addiction. A major challenge is now the discovery and pharmacological in vivo profiling of ORL1 receptor ligands that are active by medically practical routes of administration.

[Biological factors influencing response to diuretics in patients with cirrhosis and ascites]. / Facteurs biologiques influençant la réponse aux diurétiques des malades atteints de cirrhose en décompensation ascitique.

PURPOSES: To examine the biological factors influencing response to diuretics in patients with cirrhosis and ascites. METHODS: Sixty-nine patients were evaluated. Patients were classified into 3 groups: group 1: "good responders" (responding to spironolactone 200 mg/day), group 2: "bad responders" (responding to spironolactone doses above 200 mg/day or requiring addition of furosemide), and group 3: "non-responders" (not responding to spironolactone 400 mg/day and furosemide 160 mg/day). RESULTS: There were 30 patients in group 1, 24 in group 2 and 15 in group 3. The degree of activation of the renin-aldosterone and sympathetic system in group 2 was significantly higher than in group 1 and lower than in group 3. Natriuresis in group 2 (11 +/- 0.7 mEq/24h) was significantly below group 1 (20 +/- 2 mEq/24h) and above group 3 (5 +/- 0.6 mEq/24h). CONCLUSIONS: In patients with cirrhosis and ascites, the degree of activation of the renin-aldosterone and sympathetic nervous system influences diuretic response of ascites and is estimated by measured baseline natriuresis.

Regulation of nociceptin mRNA expression in the septum by dopamine and adenosine systems.

Most effects of nociceptin are related to blockade of stress and anxiolytic-like effects. This neuropeptide is highly expressed in septal nuclei, which are involved in response to stressful situations. Dopamine and adenosine may have modulatory effects on stress behaviour by acting on septal neurons. We therefore analysed the regulation of septal nociceptin expression using quantitative in situ hybridization following manipulations of adenosine and dopamine neurotransmission. No difference was observed between wild-type and A2A receptor-deficient mice. In both genotypes, chronic treatments with caffeine, an equipotent A1 and A2A adenosine receptor antagonist, did not significantly modify nociceptin expression. 6-Hydroxydopamine-induced dopamine depletion was also without effect. These results demonstrate that dopamine and adenosine are not involved in the regulation of septal nociceptin expression in spite of the involvement of these three neurotransmitters in stress and anxiety behaviours.

On the spatial disposition of the fifth transmembrane helix and the structural integrity of the transmembrane binding site in the opioid and ORL1 G protein-coupled receptor family.

Evidence from statistical cluster analyses of a multiple sequence alignment of G protein-coupled receptor seven-helix folds supports the existence of structurally conserved transmembrane (TM) ligand binding sites in the opioid/opioid receptor-like (ORL1) and amine receptor families. Based on the expectation that functionally conserved regions in homologous proteins will display locally higher levels of sequence identity compared with global sequence similarities that pertain to the overall fold, this approach may have wider applications in functional genomics to annotate sequence data. Binding sites in models of the kappa-opioid receptor seven-helix bundle built from the rhodopsin templates of Baldwin et al. (1997) [J. Mol. Biol., 272, 144-164] and Herzyk and Hubbard (1998) [J. Mol. Biol., 281, 742-751] are compared. The Herzyk and Hubbard template is found to be in better accord with experimental studies of amine, opioid and rhodopsin receptors owing to the reduced physical separation of the extracellular parts of TM helices V and VI and differences in the rotational orientation of the N-terminal of helix V that reveal side chain accessibilities in the Baldwin et al. structure to be out of phase with relative alkylation rates of engineered cysteine residues in the TM binding site of the alpha(2A)-adrenergic receptor. TM helix V in the Baldwin et al. template has been remodelled with a different proline kink to satisfy experimental constraints. A recent proposal that rotation of helix V is associated with receptor activation is critically discussed.

Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin.

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe(10),Tyr(14)]nociceptin ([Bpa(10),Tyr(14)]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa(10),Tyr(14)]noc binds the receptor with high affinity (K(i) approximately 0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC(50) approximately 0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa(10),Tyr(14)]noc results in the irreversible labeling of a glycoprotein of approximately 65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (approximately 6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of approximately 3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu(295) --> Asp mutant receptor also yields a single labeled fragment of approximately 6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296-302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa(10)]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile(300) within the experimentally identified photoreactive region.

Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein.

The addition of N-linked oligosaccharides to Asn-X-(Ser/Thr) sites is catalyzed by the oligosaccharyltransferase, an enzyme closely associated with the translocon and generally thought to have access only to nascent chains as they emerge from the ribosome. However, the presence of the sequon does not automatically ensure core glycosylation because many proteins contain sequons that remain either nonglycosylated or glycosylated to a variable extent. In this study, hepatitis C virus (HCV) envelope protein E1 was used as a model to study the efficiency of N-glycosylation. HCV envelope proteins, E1 and E2, were released from a polyprotein precursor after cleavage by host signal peptidase(s). When expressed alone, E1 was not efficiently glycosylated. However, E1 glycosylation was improved when expressed as a polyprotein including full-length or truncated forms of E2. These data indicate that glycosylation of E1 is dependent on the presence of polypeptide sequences located downstream of E1 on HCV polyprotein.

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.

Time-co-ordinated control of glycogen synthase, protein phosphatase 2A and protein kinase CK2 during culture growth in Yarrowia lipolytica in relation to glycogen metabolism.

In the growth course of the lipolytic yeast Yarrowia lipolytica, the activities of protein phosphatase 2A (PP2A) and glycogen synthase (GS) rise during the exponential phase and concomitantly glycogen storage occurs in the cells. There is also an increase in the independence ratio (RI) indicating a shift from an inactive phosphorylated GS form to an active dephosphorylated GS form. During the early stationary phase, an increase in protein kinase CK2 (CK2) activity, a reversion of RI variation and a glycogen content decrease are observed. GS activity proved to be a good indicator of early culture growth phase. Experiments carried out with enzymes purified from Y. lipolytica show strong RI variations upon the action of CK2 and PP2Ac, and 32P incorporation into GS protein through phosphorylation by CK2. GS activity would be controlled by the sequential action of PP2A and CK2.

Functional inactivation of the nociceptin receptor by alanine substitution of glutamine 286 at the C terminus of transmembrane segment VI: evidence from a site-directed mutagenesis study of the ORL1 receptor transmembrane-binding domain.

A site-directed mutagenesis approach has been used to gain insight into the molecular events whereby the heptadecapeptide nociceptin binds and activates the opioid receptor-like 1 (ORL1) receptor, a G protein-coupled receptor. Alanine mutation, in the human ORL1 receptor, of transmembrane amino acid residues that are conserved in opioid receptors, Asp(130) and Tyr(131) in transmembrane segment (TM) III, Phe(220) and Phe(224) in TM V, and Trp(276) in TM VI, yields mutant receptors with reduced affinity, and proportionally decreased reactivity, toward nociceptin. Least to most deleterious in this respect are Ala substitutions of Phe(220) approximately W276A < Tyr(131) << Phe(224) 10,000 nM compared with 0.8 nM at the wild-type receptor). In all respects, this mutant receptor appears to be functionally inactive, indicating that residue Gln(286) may play a pivotal role in ORL1 receptor-mediated transduction of the nociceptin signal.

Overphosphorylation of milk caseins by a recombinant protein kinase CK2 catalytic subunit.

Milk caseins have been phosphorylated by a recombinant protein kinase CK2 catalytic subunit from Schizosaccharomyces pombe (rspCK2alpha). Phosphate incorporation stoechiometries into purified caseins and into native phosphocaseinate, a substrate exhibiting a micellar-like structure, were determined. We incorporated 2.01 mol of P/mol of alpha-casein, 6.46 mol of P/mol of beta-casein, up to 0. 29 mol of P/mol of kappa-casein in 4 h, and more than 1.36 mol of P/mol of casein into phosphocaseinate under optimized conditions. Phosphocaseinate was sequentially phosphorylated; beta-caseins being labeled at first; alpha-caseins being labeled later; and to a lower extend, kappa-caseins were the last to be phosphorylated. The solubility of phosphocaseinate micelles increased by 1.34 from 65 to 87%, and its rennetting time was increased 2.88 times. These results are discussed in terms of plausible structural organization of caseins micelles and the effect of phosphorylation on their structure.

[Quantitative angiography: update for peripheral vascular applications]. / Angiographie quantitative: mise au point pour des applications vasculaires périphériques.

Visual estimation of vessel diameter is unreliable and observer-dependent. With digital angiography, software for accurate quantification of arterial diameter are available. They allow: (1) selection of equipment of appropriate size (balloons, stents, etc) during revascularization procedures, (2) objective evaluation of the results of such procedures, (3) comparison between follow-up measurements and initial measurements and (4) provide reproducible data in the case of multicenter studies. The widespread availability of digital systems has been accompanied by a dramatic increase in the number of software packages for quantification. The present article reviews some of the currently available methods for quantification, presents the advantages and pitfalls of each system, and outlines their limits.

Nociceptin receptors in the rat spinal cord during morphine tolerance.

The anatomical localization of nociceptin receptors was examined by in vitro quantitative autoradiography techniques in rat spinal cord sections by using [(125)I-Tyr(14)]nociceptin. [(125)I-Tyr(14)]nociceptin appeared to interact with a single class of binding sites (K(D)=0.1 nM) present in the grey matter in all laminae of the spinal cord from cervical to sacral levels. Pre-incubation of sections in the presence of 150 mM NaCl, did not modify the radioligand affinity but significantly augmented the number of accessible binding sites and increased specific binding of [(125)I-Tyr(14)]nociceptin differentially on each laminae. In particular, the superficial layers of the dorsal horn exhibited the highest density of sites after pre-wash. Continuous intrathecal infusion of morphine produced a tolerance accompanied by a significant increase in nociceptin site density in the superficial layers. Thus, nociceptin binding sites may have different properties dependent upon the layer and may be up-regulated during the process of opioid-induced tolerance.

Distribution of the nociceptin and nocistatin precursor transcript in the mouse central nervous system.

The distribution of prepronociceptin messenger RNA, the recently identified endogenous ligand of the ORL1 receptor (opioid receptor-like-1), has been studied in the adult mouse central nervous system using in situ hybridization. Prepronociceptin is a new peptide precursor that generates, upon maturation, at least three bioactive peptides: nociceptin, noc2 and the recently described nocistatin. Considering both the density of labeled neurons per region and their intensity of labeling, the distribution of prepronociceptin messenger RNA-containing neurons can be summarized as follows: the highest level of prepronociceptin messenger RNA expression was detected in the septohippocampal nucleus, bed nucleus of the stria terminalis, central amygdaloid nucleus, and in selective thalamic nuclei such as the parafascicular, reticular, ventral lateral geniculate and zona incerta. High to moderate levels of prepronociceptin messenger RNA expression were detected in the lateral, ventral and medial septum, and were evident in brainstem structures implicated in descending antinociceptive pathways (e.g., the gigantocellular nucleus, raphe magnus nucleus, periaqueductal gray matter), and also observed in association with auditory relay nuclei such as the inferior colliculi, lateral lemniscus nucleus, medioventral preolivary nucleus and lateral superior nucleus. A moderate level of prepronociceptin messenger RNA expression was observed in the medial preoptic nucleus, ventromedial preoptic nucleus, periventricular nucleus, pedonculopontine tegmental nucleus, solitary tract nucleus and spinal trigeminal nucleus. A weak level of prepronociceptin messenger RNA expression was present in some areas, such as the cerebral cortex, endopiriform cortex, hippocampal formation, medial amygdaloid nucleus, anterior hypothalamic area, medial mammillary hypothalamic nuclei, retrorubral field and substantia nigra pars compacta. No labeled cells could be found in the caudate-putamen, nucleus accumbens and ventral tegmental area. The present data confirm that nociceptin is expressed in a broad array of regions of the central nervous system. In good correlation with the presently known physiological actions of nociceptin, they include, amongst others, brain areas conveying/integrating pain and auditory sensory afferences.