An mRNA region of the canine distemper virus fusion protein gene lacking AUG codons can promote protein expression.

Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.

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

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth. STUDY DESIGN: Amniotic fluid samples (14-18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR. RESULTS: Amniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group (P=0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P=0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group (P=0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR=0.289, 95%CI=0.1-0.9, P=0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups. CONCLUSIONS: High IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.

MyHits: a new interactive resource for protein annotation and domain identification.

The MyHits web server (http://myhits.isb-sib.ch) is a new integrated service dedicated to the annotation of protein sequences and to the analysis of their domains and signatures. Guest users can use the system anonymously, with full access to (i) standard bioinformatics programs (e.g. PSI-BLAST, ClustalW, T-Coffee, Jalview); (ii) a large number of protein sequence databases, including standard (Swiss-Prot, TrEMBL) and locally developed databases (splice variants); (iii) databases of protein motifs (Prosite, Interpro); (iv) a precomputed list of matches ('hits') between the sequence and motif databases. All databases are updated on a weekly basis and the hit list is kept up to date incrementally. The MyHits server also includes a new collection of tools to generate graphical representations of pairwise and multiple sequence alignments including their annotated features. Free registration enables users to upload their own sequences and motifs to private databases. These are then made available through the same web interface and the same set of analytical tools. Registered users can manage their own sequences and annotations using only web tools and freeze their data in their private database for publication purposes.

Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1). In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.

Modulation of the gap junction protein Connexin36 in neurons in a mouse model of transient focalcerebral ischemia

Intercellular communication is achieved at specialized regions of the plasma membrane by¦gap junctions. Gap junctions are transmembrane channels allowing direct contacts between¦the cytoplasms of neighboring cells. Each cell participates with one hemichannel, or¦connexon, made of six protein subunits named connexins. Thanks to these junctions, cells¦potentially share a pool of small molecules and metabolites, such as nucleotides, amino acids¦and second messengers.¦In an ischemic (i.e. non-perfused) territory of the brain, irreversible damage progresses over¦time from the centre of the most severe flow reduction to the periphery with less disturbed¦perfusion. Functionally impaired tissue can survive and recover if sufficient reperfusion is reestablished¦within a limited time period, which depends on various factors and mechanisms¦modulating the signaling pathways leading to cell death.¦Observations were made indicating the presence of electrical coupling between neurons which¦resist better to an ischemic insult. This electrical coupling is likely to be mediated by¦Connexin36 (Cx36), a neuron specific connexin isoform. It was demonstrated in the past that¦global ischemia induces a selective upregulation of Cx36 expression in regions with neurons¦that survive the insult whereas others undergo apoptosis and die. These observations raise the¦possibility that the neuronal gap junction Cx36 might play a role in the destiny of neurons¦after cerebral ischemia.¦The aim of this work was to characterize the regulation of Connexin36 in a mouse model of¦transient focal cerebral ischemia by immunofluorescence and Western blot analysis. Our¦immunofluorescence results suggest a specific increase in Cx36 in the penumbral region of¦the ischemic hemisphere.

Promoter and transcription analysis of penicillin-binding protein genes in Streptococcus gordonii.

An optimally cross-linked peptidoglycan requires both transglycosylation and transpeptidation, provided by class A and class B penicillin-binding proteins (PBPs). Streptococcus gordonii possesses three class A PBPs (PBPs 1A, 1B, and 2A) and two class B PBPs (PBPs 2B and 2X) that are important for penicillin resistance. High-level resistance (MIC, > or =2 microg/ml) requires mutations in class B PBPs. However, although unmutated, class A PBPs are critical to facilitate resistance development (M. Haenni and P. Moreillon, Antimicrob. Agents Chemother. 50:4053-4061, 2006). Thus, their overexpression might be important to sustain the drug. Here, we determined the promoter regions of the S. gordonii PBPs and compared them to those of other streptococci. The extended -10 box was highly conserved and complied with a sigma(A)-type promoter consensus sequence. In contrast, the -35 box was poorly conserved, leaving the possibility of differential PBP regulation. Gene expression in a penicillin-susceptible parent (MIC, 0.008 microg/ml) and a high-level-resistant mutant (MIC, 2 microg/ml) was monitored using luciferase fusions. In the absence of penicillin, all PBPs were constitutively expressed, but their expression was globally increased (1.5 to 2 times) in the resistant mutant. In the presence of penicillin, class A PBPs were specifically overexpressed both in the parent (PBP 2A) and in the resistant mutant (PBPs 1A and 2A). By increasing transglycosylation, class A PBPs could promote peptidoglycan stability when transpeptidase is inhibited by penicillin. Since penicillin-related induction of class A PBPs occurred in both susceptible and resistant cells, such a mutation-independent facilitating mechanism could be operative at each step of resistance development.

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity.

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.

Different epitopes of the LACK protein are recognized by V beta 4 V alpha 8 CD4+ T cells in H-2b and H-2d mice susceptible to Leishmania major.

After inoculation of Leishmania major, a rapid production of IL-4 by LACK-specific CD4+ T cells has been shown to drive Th2 cell development in susceptible mice i.e. BALB/c and C57BL/6 mice rendered susceptible by neutralization of IFN-gamma at the onset of infection. Here, we showed that peptide AA 156-173 induced an early IL-4 mRNA expression not only in BALB/c mice but also in resistant B10.D2 mice when IFN-gamma is neutralized. Epitope mapping of LACK protein demonstrated that peptide containing AA 293-305 induced early IL-4 mRNA transcripts in susceptible H-2b mice i.e. BALB/b and resistant C57BL/6 mice when IFN-gamma is neutralized. Stringently, the early IL-4 response to the H-2d (AA 156-173) or the H-2b (AA 293-305) epitopes occurred in V beta 4 V alpha 8 CD4+ T cells from either H-2d or H-2b susceptible mice, respectively.

Pathological Reorganization of NMDA Receptors Subunits and Postsynaptic Protein PSD-95 Distribution in Alzheimer's Disease.

In Alzheimer's disease (AD), synaptic alterations play a major role and are often correlated with cognitive changes. In order to better understand synaptic modifications, we compared alterations in NMDA receptors and postsynaptic protein PSD-95 expression in the entorhinal cortex (EC) and frontal cortex (FC; area 9) of AD and control brains. We combined immunohistochemical and image analysis methods to quantify on consecutive sections the distribution of PSD-95 and NMDA receptors GluN1, GluN2A and GluN2B in EC and FC from 25 AD and control cases. The density of stained receptors was analyzed using multivariate statistical methods to assess the effect of neurodegeneration. In both regions, the number of neuronal profiles immunostained for GluN1 receptors subunit and PSD-95 protein was significantly increased in AD compared to controls (3-6 fold), while the number of neuronal profiles stained for GluN2A and GluN2B receptors subunits was on the contrary decreased (3-4 fold). The increase in marked neuronal profiles was more prominent in a cortical band corresponding to layers 3 to 5 with large pyramidal cells. Neurons positive for GluN1 or PSD-95 staining were often found in the same localization on consecutive sections and they were also reactive for the anti-tau antibody AD2, indicating a neurodegenerative process. Differences in the density of immunoreactive puncta representing neuropile were not statistically significant. Altogether these data indicate that GluN1 and PSD-95 accumulate in the neuronal perikarya, but this is not the case for GluN2A and GluN2B, while the neuropile compartment is less subject to modifications. Thus, important variations in the pattern of distribution of the NMDA receptors subunits and PSD-95 represent a marker in AD and by impairing the neuronal network, contribute to functional deterioration.

The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP-N-acetylglucosamine 4-epimerisation.

Mutations in the Bacillus subtilis gene that affect the activity of the uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 4-epimerase (EC 5.1.3.7) were shown to map to galE, the structural gene of the UDP-glucose (Glc) 4-epimerase (EC 5.1.3.2). This genetic evidence that the same enzyme can catalyse the epimerisation of hexoses as well as of their N-acetylated forms is confirmed by in vitro assays with purified enzyme. It appears that in B. subtilis, Gne (GneA, GalE) is involved in two distinct and essential functions, i.e., cell detoxification under certain growth conditions and the biosynthesis of anionic cell wall polymers. We discuss the evidence that such enzymes capable of utilizing both UDP-hexoses and UDP-N-acetylhexosamines are present in other organisms.

Augmented epithelial multidrug resistance-associated protein 4 expression in peritoneal endometriosis: regulation by lipoxin A(4).

OBJECTIVE: To compare the expression of the prostaglandin (PG) E(2) transporter multidrug resistance-associated protein 4 (MRP4) in eutopic and ectopic endometrial tissue from endometriosis patients with that of control subjects and to examine whether MRP4 is regulated by the antiinflammatory lipid lipoxin A(4) (LXA(4)) in endometriotic epithelial cells. DESIGN: Molecular analysis in human samples and a cell line. SETTING: Two university hospitals and a private clinic. PATIENT(S): A total of 59 endometriosis patients and 32 age- and body mass index-matched control subjects undergoing laparoscopy or hysterectomy. INTERVENTION(S): Normal, eutopic, and ectopic endometrial biopsies as well as peritoneal fluid were obtained during surgery performed during the proliferative phase of the menstrual cycle. 12Z endometriotic epithelial cells were used for in vitro mechanistic studies. MAIN OUTCOME MEASURE(S): Tissue MRP4 mRNA levels were quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and localization was analyzed with the use of immunohistochemistry. Cellular MRP4 mRNA and protein were quantified by qRT-PCR and Western blot, respectively. PGE(2) was measured in peritoneal fluid and cell supernatants using an enzyme immunoassay (EIA). RESULT(S): MRP4 was expressed in eutopic and ectopic endometrium, where it was overexpressed in peritoneal lesions and localized in the cytoplasm of glandular epithelial cells. LXA(4) attenuated MRP4 mRNA and protein levels in endometriotic epithelial cells in a dose-dependent manner, while not affecting the expression of enzymes involved in PGE(2) metabolism. Investigations employing receptor antagonists and small interfering RNA revealed that this occurred through estrogen receptor α. Accordingly, LXA(4) treatment inhibited extracellular PGE(2) release. CONCLUSION(S): We report for the first time that MRP4 is expressed in human endometrium, elevated in peritoneal endometriosis, and modulated by LXA(4) in endometriotic epithelial cells.

Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein.

Cell separation, or abscission, is a highly specialized process in plants that facilitates remodeling of their architecture and reproductive success. Because few genes are known to be essential for organ abscission, we conducted a screen for mutations that alter floral organ shedding in Arabidopsis. Nine recessive mutations that block shedding were found to disrupt the function of an ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) we have named NEVERSHED (NEV). As predicted by its homology to the yeast Age2 ARF-GAP and transcriptional profile, NEV influences other aspects of plant development, including fruit growth. Co-localization experiments carried out with NEV-specific antiserum and a set of plant endomembrane markers revealed that NEV localizes to the trans-Golgi network and endosomes in Arabidopsis root epidermal cells. Interestingly, transmission electron micrographs of abscission zone regions from wild-type and nev flowers reveal defects in the structure of the Golgi apparatus and extensive accumulation of vesicles adjacent to the cell walls. Our results suggest that NEV ARF-GAP activity at the trans-Golgi network and distinct endosomal compartments is required for the proper trafficking of cargo molecules required for cell separation.

Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.