Amniotic fluid insulin-like growth factor binding-protein 3 concentration as early indicator of fetal growth restriction

Rapport de synthèse : Introduction : La croissance foetale infra-utérine dépend d'un grand nombre de facteurs maternels, placentaires et foetaux. Une inadéquation d'un ou plusieurs de ces facteurs peut induire un retard de croissance infra-utérin (RCIU) ou au contraire une macrosomie. Les principales causes de RCIU comprennent les infections maternelles, l'éclampsie, les cardiovasculopathies maternelles, la toxicomanie, les malformations foetales et les insuffisances placentaires. Les facteurs endocriniens constituent un petit pourcentage des causes de RCIU, mais méritent que l'on s'y intéresse de plus près. Les facteurs hormonaux les plus importants pour la croissance fatale sont l'insuline et les insuline-like growth factors (IGFs) et non l'hormone de croissance (GH) qui joue un rôle majeur dans la croissance postnatale. Notre attention s'est portée sur IGF-1 qui joue un rôle important dans la croissance intrautérine. Sa biodisponibilité dépend de plusieurs protéines plasmatiques, les IGF-binding proteins (IGFBP 1 à 9). IGFBP-3 est la principale de ces IGFBPs, autant d'un point de vue quantitatif que fonctionnel. Nous avons cherché à déterminer si les concentrations d'IGF-1 et d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre étaient prédictives de la croissance infra-utérine. Les gènes codant pour IGF-1 et IGFBP-3 contenant certaines séquences polymorphiques, nous avons également étudié leur influence sur la croissance foetale. L'analyse du liquide amniotique présente l'avantage de pouvoir être effectuée dès la 14ème semaine d'aménorrhée alors que la biométrie foetale échographique ne permet pas à ce stade de déceler des anomalies de la croissance infra-utérine. Méthode : Nous avons analysé des échantillons de liquide amniotique prélevés entre la 14ème et la 18ème semaine de grossesse chez 196 patientes. Les concentrations d'IGF-1 et d'IGFBP-3 ont été dosées par ELISA, les polymorphismes analysés par PCR. Ces résultats ont été ensuite analysés en fonction du poids de naissance des nouveaux-nés, répartis en trois groupes normal pour l'âge gestationnel (AGA), petit pour l'âge gestationnel (SGA) et grand pour l'âge gestationnel (LGA). Résultats : Les concentrations d'IGFBP3 dans le liquide amniotique sont significativement plus élevées (p = 0.030) dans le groupe SGA par rapport au groupe AGA, d'autant plus quand les taux sont ajustés en fonction de paramètres tels que l'âge gestationnel lors de l'amniocentèse (ANCOVA analysis : p = 0.009). La distribution du polymorphisme VNTR (variable number of tandem repeat) dans la région promotrice d'IGF-1 au sein du groupe SGA est significativement différente de celle du groupe AGA (p = 0.029). En effet, la fréquence de l'association allélique 19CA/20CA est diminuée dans le groupe SGA. Nous n'avons pas identifié de différence de distribution des séquences polymorphiques d'IGFBP-3 entre les différents groupes. Conclusion : Une concentration élevée d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre est associée à un risque plus élevé de retard de croissance alors que l'association allélique 19CA/20CA dans la région polymorphique IGF-1 VNTR est un facteur protecteur.

Transcriptional activation of the macrophage migration-inhibitory factor gene by the corticotropin-releasing factor is mediated by the cyclic adenosine 3',5'- monophosphate responsive element-binding protein CREB in pituitary cells.

Macrophage migration-inhibitory factor (MIF) has recently been identified as a pituitary hormone that functions as a counterregulatory modulator of glucocorticoid action within the immune system. In the anterior pituitary gland, MIF is expressed in TSH- and ACTH-producing cells, and its secretion is induced by CRF. To investigate MIF function and regulation within pituitary cells, we initiated the characterization of the MIF 5'-regulatory region of the gene. The -1033 to +63 bp of the murine MIF promoter was cloned 5' to a luciferase reporter gene and transiently transfected into freshly isolated rat anterior pituitary cells. This construct drove high basal transcriptional activity that was further enhanced after stimulation with CRF or with an activator of adenylate cyclase. These transcriptional effects were associated with a concomitant rise in ACTH secretion in the transfected cells and by an increase in MIF gene expression as assessed by Northern blot analysis. A cAMP-responsive element (CRE) was identified within the MIF promoter region which, once mutated, abolished the cAMP responsiveness of the gene. Using this newly identified CRE, DNA-binding activity was detected by gel retardation assay in nuclear extracts prepared from isolated anterior pituitary cells and AtT-20 corticotrope tumor cells. Supershift experiments using antibodies against the CRE-binding protein CREB, together with competition assays and the use of recombinant CREB, allowed the detection of CREB-binding activity with the identified MIF CRE. These data demonstrate that CREB is the mediator of the CRF-induced MIF gene transcription in pituitary cells through an identified CRE in the proximal region of the MIF promoter.

The influence of Bone Morphogenic Protein-2 on the consolidation phase in a distraction osteogenesis model.

We asked whether locally applied recombinant-Bone Morphogenic Protein-2 (rh-BMP-2) with an absorbable Type I collagen sponge (ACS) carrier could enhance the consolidation phase in a callotasis model. We performed unilateral transverse osteotomy of the tibia in 21 immature male rabbits. After a latency period of 7 days, a 3-weeks distraction was begun at a rate of 0.5mm/12h. At the end of the distraction period (Day 28) animals were randomly divided into three groups and underwent a second surgical procedure: 6 rabbits in Group I (Control group; the callus was exposed and nothing was added), 6 rabbits in Group II (ACS group; receiving the absorbable collagen sponge soaked with saline) and 9 rabbits in Group III (rh-BMP-2/ACS group; receiving the ACS soaked with 100μg/kg of rh-BMP-2, Inductos(®), Medtronic). Starting at Day 28 we assessed quantitative and qualitative radiographic parameters as well as densitometric parameters every two weeks (Days 28, 42, 56, 70 and 84). Animals were sacrificed after 8 weeks of consolidation (Day 84). Qualitative radiographic evaluation revealed hypertrophic calluses in the Group III animals. The rh-BMP-2/ACS also influenced the development of the cortex of the calluses as shown by the modified radiographic patterns in Group III when compared to Groups I and II. Densitometric analysis revealed the bone mineral content (BMC) was significantly higher in the rh-BMP-2/ACS treated animals (Group III).

Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel diseases (IBDs), Crohn's disease, and ulcerative colitis (UC), are multifactorial disorders, characterized by chronic inflammation of the intestine. A number of genetic components have been proposed to contribute to IBD pathogenesis. In this case-control study, we investigated the association between two common vitamin D-binding protein (DBP) genetic variants and IBD susceptibility. These two single nucleotide polymorphisms (SNPs) in exon 11 of the DBP gene, at codons 416 (GAT>GAG; Asp>Glu) and 420 (ACG>AAG; Thr>Lys), have been previously suggested to play roles in the etiology of other autoimmune diseases. METHODS: Using TaqMan SNP technology, we have genotyped 884 individuals (636 IBD cases and 248 non-IBD controls) for the two DBP variants. RESULTS: On statistical analysis, we observed that the DBP 420 variant Lys is less frequent in IBD cases than in non-IBD controls (allele frequencies, P=0.034; homozygous carrier genotype frequencies, P=0.006). This inverse association between the DBP 420 Lys and the disease remained significant, when non-IBD participants were compared with UC (homozygous carrier genotype frequencies, P=0.022) or Crohn's disease (homozygous carrier genotype frequencies, P=0.016) patients separately. Although the DBP position 416 alone was not found to be significantly associated with IBD, the haplotype DBP_2, consisting of 416 Asp and 420 Lys, was more frequent in the non-IBD population, particularly notably when compared with the UC group (Odds ratio, 4.390). CONCLUSION: Our study adds DBP to the list of potential genes that contribute to the complex genetic etiology of IBD, and further emphasizes the association between vitamin D homeostasis and intestinal inflammation.

Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing.

Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP.

Primate adult brain cell autotransplantation produces behavioral and biological recovery in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonian St. Kitts monkeys.

The potential for "replacement cells" to restore function in Parkinson's disease has been widely reported over the past 3 decades, rejuvenating the central nervous system rather than just relieving symptoms. Most such experiments have used fetal or embryonic sources that may induce immunological rejection and generate ethical concerns. Autologous sources, in which the cells to be implanted are derived from recipients' own cells after reprogramming to stem cells, direct genetic modifications, or epigenetic modifications in culture, could eliminate many of these problems. In a previous study on autologous brain cell transplantation, we demonstrated that adult monkey brain cells, obtained from cortical biopsies and kept in culture for 7 weeks, exhibited potential as a method of brain repair after low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused dopaminergic cell death. The present study exposed monkeys to higher MPTP doses to produce significant parkinsonism and behavioral impairments. Cerebral cortical cells were biopsied from the animals, held in culture for 7 weeks to create an autologous neural cell "ecosystem" and reimplanted bilaterally into the striatum of the same six donor monkeys. These cells expressed neuroectodermal and progenitor markers such as nestin, doublecortin, GFAP, neurofilament, and vimentin. Five to six months after reimplantation, histological analysis with the dye PKH67 and unbiased stereology showed that reimplanted cells survived, migrated bilaterally throughout the striatum, and seemed to exert a neurorestorative effect. More tyrosine hydroxylase-immunoreactive neurons and significant behavioral improvement followed reimplantation of cultured autologous neural cells as a result of unknown trophic factors released by the grafts. J. Comp. Neurol. 522:2729-2740, 2014. © 2014 Wiley Periodicals, Inc.

CCAAT/enhancer-binding protein family members recruit the coactivator CREB-binding protein and trigger its phosphorylation.

CCAAT/enhancer-binding protein (C/EBP) family members are transcription factors involved in important physiological processes, such as cellular proliferation and differentiation, regulation of energy homeostasis, inflammation, and hematopoiesis. Transcriptional activation by C/EBPalpha and C/EBPbeta involves the coactivators CREB-binding protein (CBP) and p300, which promote transcription by acetylating histones and recruiting basal transcription factors. In this study, we show that C/EBPdelta is also using CBP as a coactivator. Based on sequence homology with C/EBPalpha and -beta, we identify in C/EBPdelta two conserved amino acid segments that are necessary for the physical interaction with CBP. Using reporter gene assays, we demonstrate that mutation of these residues prevents CBP recruitment and diminishes the transactivating potential of C/EBPdelta. In addition, our results indicate that C/EBP family members not only recruit CBP but specifically induce its phosphorylation. We provide evidence that CBP phosphorylation depends on its interaction with C/EBPdelta and define point mutations within one of the two conserved amino acid segments of C/EBPdelta that abolish CBP phosphorylation as well as transcriptional activation, suggesting that this new mechanism could be important for C/EBP-mediated transcription.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin ß (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

The metal ion binding protein Cnnm4 is mutated in rod-cone dystrophy/amelogenesis imperfecta syndrome

Purpose:To identify the gene causing rod-cone dystrophy/amelogenesis imperfecta Methods:Homozygosity mapping was performed using the Affymetrix 50K XbaI array in one family and candidate genes in the linked interval were sequenced with ABI Dye Terminator, vers. 1 in the index patient of 3 families. The identified mutations were screened in normal control individuals. Expression analyses were performed on RNA extracted from the brain, various parts of the eye and teeth; immunostaining was done on mouse eyes and jaw and knock-down experiments were carried out in zebrafish embroys. Results:Sequencing the coding regions of ancient conserved domain protein 4 (CNNM4), a metal ions transporter, revealed a 1-base pair duplication (p.L438fs) in family A, a p.R236Q mutation in family B and a p.L324P in family C. All these mutations were homozygous and involved very conserved amino acids in paralogs and orthologs. Immunostaining and RT-PCR confirmed that CNNM4 was strongly expressed in various parts of the eye and in the teeth. Morpholino experiments in zebrafish showed a loss of ganglion cells at 5 days post fertilization. Conclusions:The rod-cone dystrophy/amelogenesis imperfecta syndrome is caused by mutation in CNNM4 and is due to aberrant metal ion homeostasis.

Penicillin-binding protein gene alterations in Streptococcus uberis isolates presenting decreased susceptibility to penicillin.

Streptococcus uberis is an environmental pathogen commonly causing bovine mastitis, an infection that is generally treated with penicillin G. No field case of true penicillin-resistant S. uberis (MIC > 16 mg/liter) has been described yet, but isolates presenting decreased susceptibility (MIC of 0.25 to 0.5 mg/liter) to this drug are regularly reported to our laboratory. In this study, we demonstrated that S. uberis can readily develop penicillin resistance in laboratory-evolved mutants. The molecular mechanism of resistance (acquisition of mutations in penicillin-binding protein 1A [PBP1A], PBP2B, and PBP2X) was generally similar to that of all other penicillin-resistant streptococci described so far. In addition, it was also specific to S. uberis in that independent resistant mutants carried a unique set of seven consensus mutations, of which only one (Q(554)E in PBP2X) was commonly found in other streptococci. In parallel, independent isolates from bovine mastitis with different geographical origins (France, Holland, and Switzerland) and presenting a decreased susceptibility to penicillin were characterized. No mosaic PBPs were detected, but they all presented mutations identical to the one found in the laboratory-evolved mutants. This indicates that penicillin resistance development in S. uberis might follow a stringent pathway that would explain, in addition to the ecological niche of this pathogen, why naturally occurring resistances are still rare. In addition, this study shows that there is a reservoir of mutated PBPs in animals, which might be exchanged with other streptococci, such as Streptococcus agalactiae, that could potentially be transmitted to humans.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

The fibrinogen- and fibronectin-binding domains of Staphylococcus aureus fibronectin-binding protein A synergistically promote endothelial invasion and experimental endocarditis.

Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties--as well as elastin binding--and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by > or =10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells.

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

Polymorphisms in fibronectin binding protein A of Staphylococcus aureus are associated with infection of cardiovascular devices.

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.