Functional proteomics mapping of a human signaling pathway

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.     

Plasma ghrelin concentration is a signal of decreased fat free mass in healthy elderly females

This study aimed to evaluate whether circulating ghrelin is associated with changes in different body composition parameters over a 12‐month prospective study period in healthy older females. On 41 postmenopausal women (mean age: 71.0 ± 6.5 years), ghrelin, leptin, insulin resistance (IR), and body composition parameters were assessed before and after the study period. Trunk fat: leg fat ratio (+3.6%), fat free mass (FFM) (?4.1%), glucose (+5.8%), and IR (+7.0%) were significantly changed (P < 0.05), whereas no changes in height, body mass, body mass index, fat mass (FM), %FM, trunk fat, leptin, ghrelin, and insulin were observed as a result of study period. At baseline, ghrelin correlated negatively (r > ?0.306; P < 0.05) with body mass, FM, %FM, trunk fat, FFM, leptin, insulin, and IR. Multivariate linear regression analysis demonstrated that baseline ghrelin concentration was significantly associated only with the mean change in FFM value over the 12‐month study period. In conclusion, basal ghrelin concentration predicted the loss of FFM in healthy elderly females. In addition, these results suggest that circulating ghrelin concentration could be regarded as a signal of decreased FFM in healthy elderly females. Am. J. Hum. Biol., 2009. © 2009 Wiley‐Liss, Inc.     

A rare cause of neonatal seizure: incontinentia pigmenti

Incontinentia pigmenti (IP) is a rare genetic multisystem disorder that may affect many organs including the skin, bone, eyes and the central nervous system. Central nervous system manifestations are seen in 30% of cases with seizures and mental retardation. Seizures occurring as the presenting sign of IP are rarely reported. We report a case of a female newborn with IP who had seizures on day 4 of life, which were followed in her second month by the development of the characteristic cutaneous changes for IP. With this case report, we would like to emphasize the need for inclusion of IP in the differential diagnosis of neonatal seizures.     

Molecular aspects of human FcgammaR interactions with IgG: functional and therapeutic consequences

The binding of IgG antibodies to receptors for the Fc region of IgG (FcgammaR) is a critical step for the initiation and/or the control of effector immune functions once immune complexes have been formed. Site-directed and random mutagenesis as well as domain-swapping, NMR and X-ray cristallography have made it possible to get detailed insights in the molecular mechanisms that govern IgG/FcgammaR interactions and to define some of the structural determinants that impact IgG binding to the various FcgammaR. It has demonstrated the role of particular stretches and individual residues located in the lower hinge region of the CH2 domain and in the CH2 and CH3 domains of the Fc region. The importance of the sugar components linked to asparagine 297 in the binding properties of IgG1, the human IgG isotype the most widely used in antibody-based therapies, has been also highlighted. These data have led to the engineering of a new generation of monoclonal antibodies for therapeutic use with optimized effector functions.     

Natural polymorphisms in the protease gene modulate the replicative capacity of non-B HIV-1 variants in the absence of drug pressure.

BACKGROUND: Genetic variation in the HIV-1 pol gene, which encodes the main targets for anti-HIV drugs, may favors different susceptibility and resistance pathways to antiretroviral agents. Several amino acid substitutions occur frequently in some non-B viruses at positions associated with drug resistance in clade B viruses. The clinical relevance of those polymorphisms is unclear. OBJECTIVE: To evaluate the effect of two natural protease (PR) polymorphisms, K20I and M36I, which are frequently found in non-B subtypes, on the virus replicative capacity in the presence and absence of protease inhibitors (PI). STUDY DESIGN: Infectious HIV-1 clones carrying K20I, M36I or K20I/M36I were designed. Their replication kinetics were analyzed by viral competition in the absence of PI. Susceptibility to six different PI was phenotypically assessed in clones and in recombinant viruses carrying non-B proteases from 16 drug-naive individuals. RESULTS: In the absence of drug, the M36I clone replicated more rapidly than wt (wild type) or the double mutant K20I/M36I. Natural polymorphisms 20I and/or 36I improved the virus replicative capacity under drug pressure, reducing the susceptibility to saquinavir and indinavir, with IC(50) values 2-3.5-fold higher than wt. All but one drug-naive individual carrying non-B viruses were fully susceptibility to all tested PI, suggesting that additional substitutions within the PR might compensate the reduced PI susceptibility caused by K20I and/or M36I. CONCLUSION: Natural PR polymorphisms in non-B HIV-1 variants can influence in vitro the virus replication capacity in the presence and/or absence or certain PI. Hypothetically, the improved viral replication of mutant 36I might favor a more rapid spreading of non-B subtypes of HIV-1.     

Pathological and Genetic Characterization of Bilateral Adrenomedullary Hyperplasia in a Patient with Germline <Emphasis Type="Italic">MAX</Emphasis> Mutation

In recent years, familial pheochromocytoma (PHEO) with germline mutations in the MAX (MYC associated factor X) gene has been reported in a few cases. Here, we investigated a 25–year-old patient with multiple PHEOs associated with a non-sense germline MAX mutation. Preoperative ¹⁸F-FDOPA PET/CT revealed bilateral adrenal involvement with multiple tumors. In addition, both adrenal glands were found to have diffuse or nodular adrenal medullary hyperplasia (AMH), a histopathological feature previously described as a precursor of MEN2- and SDHB-related PHEOs but not MAX. After bilateral adrenalectomy, different paraffin-embedded and frozen samples were analyzed for allelic imbalances of the MAX gene using allelic quantification by pyrosequencing. The expression of the protein MAX was studied by immunohistochemistry. All PHEOs but also nodular AMH exhibited a loss of the normal allele. By contrast, the diffuse AMH did not show loss-of-heterozygosity. Nevertheless, immunohistochemistry demonstrated loss of protein MAX expression in all samples including diffuse hyperplasia, suggesting a causative role of MAX mutation for both PHEOs and AMH. The present case shows that both nodular and diffuse AMH belongs to the spectrum of MAX-related disease. These data support the possible continuum between nodular AMH and PHEO, expanding the qualification of micro-PHEO to nodular AMH.     

Functional assays for evaluation of serogroup B meningococcal structures as mediators of human opsonophagocytosis

Functional flow cytometry and chemiluminescence (CL) assays have been modified to identify serogroup B meningococcal structures that mediate anti-meningococcal opsonophagocytosis. Serogroup B meningococcal outer membrane vesicles (OMV) were adsorbed to fluorescent latex beads (OMV-beads) and opsonized with acute phase and convalescence sera from patients with serogroup B meningococcal disease. Phagocytosis of these beads by human monocytes and polymorphonuclear leukocytes (non-lymphocytes) was dependent on both antigen exposure on the bead surface and on serum opsonization. OMV-beads opsonized with serum from a patient recovering from meningococcal disease, caused 97% of the non-lymphocytes to phagocytose an average of 15.8 beads per cell with a CL response of 46550 mVs, whereas opsonized control beads were phagocytosed by 19% of the non-lymphocytes with 1.1 beads per cell and a CL response of 53 mVs. Increased amounts of functional, anti-OMV opsonins were detected during infection, and opsonized OMV-beads elicited phagocyte responses of similar magnitude to those of opsonized whole meningococci. Phagocyte internalization of OMV-beads was confirmed by confocal laser scanning microscopy. We conclude that epitopes on the meningococcal outer membrane are recognized by anti-meningococcal opsonins in these functional phagocytosis assays, which provide a basis for subsequent evaluation of various purified bacterial components as mediators of human opsonophagocytic responses and hence future vaccine constituents.     

Bulk polymerization of lactides initiated by aluminium isopropoxide, 3. Thermal stability and viscoelastic properties

The thermal stability of poly(L -lactide) and L /D -lactide stereocopolymers polymerized in bulk with aluminium isopropoxide as initiator was investigated by thermogravimetric analysis, and time and temperature dependence of molecular weight and polydispersity. Compared to poly(L -lactide) prepared with stannous octoate as catalyst the thermal stability of polylactides is remarkably enhanced when the ring-opening polymerization is initiated by aluminium alkoxide. Ultimate mechanical properties and viscoelasticity of these melt-stable thermoplastics have also been studied in relation to the polyester enantiomeric composition.     

Predictors for 2-year outcome of major depressive episode

In this 2-year prospective study, we searched for predictive factors influencing the 2-year outcome of major depressive episodes. Demographic characteristics (age, gender, education, employment), illness-related variables (severity, age at onset, number and duration of previous episodes), personality characteristics (DSM-IV personality disorders, trait anxiety, coping style), life context factors (life events before and during the depressive episode, social support, social adjustment), and biological markers (dexamethasone suppression test, thyroid stimulating hormone levels) of 117 inpatients with major depressive episode were assessed. A structural equation model was used to test the proposed correlational structure of the relevant variables. The non-remission of the depressive symptoms by the end of a 6-week acute treatment phase was found to be the most relevant factor predicting sustained non-remission at the end of a 2-year follow-up period. At the end of the sixth week, the severity of depression depended on the level of social support and on the severity of depression at baseline. Among the baseline variables, anxious personality traits and a lower level of education predicted a high level of depressive symptoms at the end of the 2-year follow-up. Life events before and during the depressive episode, and the biological markers at baseline had no direct effect on the outcome. The rapid remission of the depressive symptoms is the most important predictor for the favorable long-term outcome of a depressive episode. Personality characteristics, social support and level of education,--interacting with each other--also play a significant role.     

Priapism in sickle cell anaemia in Senegal: prevalence, attitudes and knowledge

We interviewed 108 sickle cell anaemia patients aged 5 years older on priapism, and 113 healthy subjects in a control group. They were recruited in the out-patient consultation of the two sickle cell anemia care units of the Fann teaching hospital in Dakar. Ten cases of priapism were identified, all in the group of sickle cell patients (the difference is significant, p = 0.004). Prevalence of priapism was 9.3%. Actuarial probability of having priapism was 8.3% by 10 years of age, and 38.9% +/- 5.7 by 20 years of age. Before the interview, only 10.2% of the sickle cell patients and 8% in the control group knew about priapism, and most of them were unaware of its association with sickle cell disease (75% of sickle cell anaemia patients and 80% of control subjects). This lack of information should be improved by an educational program.