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.

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.

9-cis-retinoic acid enhances fatty acid-induced expression of the liver fatty acid-binding protein gene.

The role of retinoic acids (RA) on liver fatty acid-binding protein (L-FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9-cis-Retinoic acid (9-cis-RA) specifically enhanced L-FABP mRNA levels in a time- and dose-dependent manner. The higher induction was found 6 h after addition of 10(-6) M 9-cis-RA in the medium. RA also enhanced further both L-FABP mRNA levels and cytosolic L-FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR), which are known to be activated, respectively, by 9-cis-RA and long chain fatty acid (LCFA), co-operated to bind specifically the peroxisome proliferator-responsive element (PPRE) found upstream of the L-FABP gene. Our result suggest that the PPAR-RXR complex is the molecular target by which 9-cis-RA and LCFA regulate the L-FABP gene.

Analysis of prion strains by PrPSc profiling in sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Prion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrPSc) of a host-encoded normal cellular protein (PrPC). The conversion of PrPC to PrPSc is thought to play a crucial role in the development of prion diseases and leads to PrPSc deposition, mainly in the central nervous system. Sporadic Creutzfeldt-Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability. METHODS AND FINDINGS: To overcome these difficulties, we developed a new quantification protocol for PrPSc by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrPSc profiles. By studying PrPSc profiles and PrPSc type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrPSc distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrPSc type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP). CONCLUSION: PrPSc profiling represents a valuable tool for the molecular classification of human prion diseases and has important implications for their diagnosis by brain biopsy. Our results show that the co-existence of more than one PrPSc type might be influenced by genetic and brain region-specific determinants. These findings provide valuable insights into the generation of distinct PrPSc types.

Genes for normal sleep and sleep disorders.

Sleep and wakefulness are complex behaviors that are influenced by many genetic and environmental factors, which are beginning to be discovered. The contribution of genetic components to sleep disorders is also increasingly recognized as important. Point mutations in the prion protein, period 2, and the prepro-hypocretin/orexin gene have been found as the cause of a few sleep disorders but the possibility that other gene defects may contribute to the pathophysiology of major sleep disorders is worth in-depth investigations. However, single gene disorders are rare and most common disorders are complex in terms of their genetic susceptibility, environmental effects, gene-gene, and gene-environment interactions. We review here the current progress in the genetics of normal and pathological sleep.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Emergence of secretion-defective sublines of Pseudomonas aeruginosa PAO1 resulting from spontaneous mutations in the vfr global regulatory gene.

Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.

Reggies/flotillins regulate E-cadherin-mediated cell contact formation by affecting EGFR trafficking.

The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

Amphibian albumins as members of the albumin, alpha-fetoprotein, vitamin D-binding protein multigene family.

The Xenopus laevis 68-kd and 74-kd albumin amino acid sequences are examined with respect to their relationship to the other known members of the albumin/alpha-fetoprotein/vitamin D-binding protein gene family. Each of the three members of this family presents a unique pattern of conserved regions indicating a differential selective pressure related to specific functional characteristics. Furthermore, an evolutionary tree of these genes was deduced from the divergence times calculated from direct nucleotide sequence comparisons of individual gene pairs. These calculations indicate that the vitamin D-binding protein/albumin separation occurred 560-600 million years (Myr) ago and the albumin/alpha-fetoprotein divergence 280 Myr ago. This observation leads to the hypothesis according to which the albumin/alpha-fetoprotein gene duplication occurred shortly after the amphibian/reptile separation. Consequently, and unlike mammals, amphibians and fishes should lack an alpha-fetoprotein in their serum at larval stages, which is consistent with a recent analysis of serum proteins in Xenopus laevis larvae. This hypothesis now will have to be tested further in additional lower vertebrates.

Eosinophilic esophagitis: Updated consensus recommendations for children and adults.

Eosinophilic esophagitis (EoE) is a clinicopathologic condition of increasing recognition and prevalence. In 2007, a consensus recommendation provided clinical and histopathologic guidance for the diagnosis and treatment of EoE; however, only a minority of physicians use the 2007 guidelines, which require fulfillment of both histologic and clinical features. Since 2007, the number of EoE publications has doubled, providing new disease insight. Accordingly, a panel of 33 physicians with expertise in pediatric and adult allergy/immunology, gastroenterology, and pathology conducted a systematic review of the EoE literature (since September 2006) using electronic databases. Based on the literature review and expertise of the panel, information and recommendations were provided in each of the following areas of EoE: diagnostics, genetics, allergy testing, therapeutics, and disease complications. Because accumulating animal and human data have provided evidence that EoE appears to be an antigen-driven immunologic process that involves multiple pathogenic pathways, a new conceptual definition is proposed highlighting that EoE represents a chronic, immune/antigen-mediated disease characterized clinically by symptoms related to esophageal dysfunction and histologically by eosinophil-predominant inflammation. The diagnostic guidelines continue to define EoE as an isolated chronic disorder of the esophagus diagnosed by the need of both clinical and pathologic features. Patients commonly have high rates of concurrent allergic diatheses, especially food sensitization, compared with the general population. Proved therapeutic options include chronic dietary elimination, topical corticosteroids, and esophageal dilation. Important additions since 2007 include genetic underpinnings that implicate EoE susceptibility caused by polymorphisms in the thymic stromal lymphopoietin protein gene and the description of a new potential disease phenotype, proton pump inhibitor-responsive esophageal eosinophila. Further advances and controversies regarding diagnostic methods, surrogate disease markers, allergy testing, and treatment approaches are discussed.

Fas ligand expression is restricted to nonlymphoid thymic components in situ.

The cell surface receptor Fas (Apo-1/CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in activation-induced death of mature T cells and in killing mediated by cytolytic T cells. The role of the Fas pathway in apoptosis associated with thymic selection events is, however, controversial. Although Fas and FasL are known to be expressed in the thymus, the nature and in vivo localization of FasL-expressing cells have not been determined. Using recently developed anti-FasL Abs in combination with in situ hybridization on tissue sections, we show in this work that FasL-expressing cells are present in the thymus, particularly within the medulla. FasL mRNA was detected readily in thymic stromal cell extracts, but not in isolated thymocytes. Moreover, immunohistochemical analysis of serial tissue sections stained with Abs against FasL in conjunction with epithelial and dendritic cell markers indicated that both thymic epithelial and dendritic cells express FasL in situ. The coexistence of FasL-expressing stromal cells and Fas-expressing thymocytes may have important implications for the role of the Fas pathway in apoptosis associated with thymic selection events.

Mutation screening of patients with Alzheimer disease identifies APP locus duplication in a Swedish patient.

BACKGROUND: Missense mutations in three different genes encoding amyloid-β precursor protein, presenilin 1 and presenilin 2 are recognized to cause familial early-onset Alzheimer disease. Also duplications of the amyloid precursor protein gene have been shown to cause the disease. At the Dept. of Geriatric Medicine, Karolinska University Hospital, Sweden, patients are referred for mutation screening for the identification of nucleotide variations and for determining copy-number of the APP locus. METHODS: We combined the method of microsatellite marker genotyping with a quantitative real-time PCR analysis to detect duplications in patients with Alzheimer disease. RESULTS: In 22 DNA samples from individuals diagnosed with clinical Alzheimer disease, we identified one patient carrying a duplication on chromosome 21 which included the APP locus. Further mapping of the chromosomal region by array-comparative genome hybridization showed that the duplication spanned a maximal region of 1.09 Mb. CONCLUSIONS: This is the first report of an APP duplication in a Swedish Alzheimer patient and describes the use of quantitative real-time PCR as a tool for determining copy-number of the APP locus.

Interstitial cells of Cajal are normally distributed in both ganglionated and aganglionic bowel in Hirschsprung's disease

Résumé Introduction : La chirurgie de la maladie de Hirschsprung est fréquemment compliquée d'une atteinte post-opératoire de la motilité intestinale. Des anomalies du système nerveux entérique (SNE) telles que la dysplasie neuronale intestinale de type B, l'hypoganglionose ou l'aganglionose, présents dans le segment abaissé, peuvent être la cause de certaines de ces complications mais aucune information n'est disponible quant au rôle des cellules interstitielles de Cajal (CIC) sur la motilité intestinale dans la phase post-opératoire. Ces cellules sont considérées avoir un rôle de pacemaker dans le tractus gastro-intestinal. L'objectif de cette étude était de décrire la distribution des CIC dans le segment proximal du côlon réséqué lors de cures chirurgicales de maladie de Hirschsprung et de confronter ces observations à l'évolution clinique post-opératoire. Matériel et Méthodes : L'incidence des complications post-opératoires a été déterminée par une revue rétrospective des dossiers de 48 patients opérés pour maladie de Hirschspung entre 1977 et 1999 et par l'étude histologique et immuno-histochimique des pièces réséquées chez ces patients. Nous avons comparé la distribution des CIC dans le segment proximal du côlon avec celle du côlon sain de 16 enfants contrôles par microscopie optique. L'immunohistochimie au c-Kit a été utilisée pour marquer spécifiquement les CIC sur échantillons paraffinés. Ces résultats ont ensuite été corrélés avec l'étude du SNE de ces mêmes segments, déterminée par immunohistochimie au CD56 et au protein gene product 9.5. Résultats Les complications post-opératoires suivantes furent identifiées : constipation 46%, constipation avec incontinence 15%, entérocolite 8%, décès 4% (probablement sur entérocolite). La distribution des CIC dans les segments proximaux réséqués chez les enfants avec maladie de Hirschsprung était identique à celle observée dans les segments de côlon sain, et ce indépendamment de la distribution normale ou anormale du SNE. Chez les enfants opérés pour maladie de Hirschsprung les segments réséqués présentaient les anomalies d'innervation suivantes : aganglionose 10.4%, hypoganglionose 12.5%, dysplasie neuronale intestinale de type B 6.3%, autres dysganglionoses 14.6%. Aucune relation entre ces anomalies d'innervation et les complications post-opératoires n'a été mise en évidence. Conclusion : La distribution des CIC est normale chez les patient opérés pour maladie de Hirschsprung, et ne contribue donc pas aux atteintes post-opératoires de la motilité intestinale. Cela signifie aussi que le réseau de CIC se développe noinialement dans le côlon humain, même en présence d'une innervation colique anormale ou absente. Abstract: Surgery for Hirschsprung's disease is often complicated by post-operative bowel motility disorders. The impact of intestinal neural histology on the surgical outcome has been previously studied, but no information is available concerning the influence of the distribution of interstitial cells of Cajal (ICC) on these complications. These cells are considered to be pacemakers in the gastrointestinal tract. The aim of this study was to assess the distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease and confront these results with the clinical outcome. Using immunohistochemistry for light microscopy, we compared the pattern of distribution of ICC in the proximal segment of resected bowel in Hirschsprung's disease with that in normal colon. We correlated these results with the corresponding neural intestinal histology determined by CD56 and the protein gene product 9.5 immunohistochemistry. The distribution of ICC in the proximal segment of resected bowel is identical to that of normal colon, regardless of normal or abnormal colon innervation. ICC distribution does not seem to contribute to post-operative bowel motility disorders in patients operated for Hirschsprung's disease.

Negative control of keratinocyte differentiation by Rho/CRIK signaling coupled with up-regulation of KyoT1/2 (FHL1) expression.

Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell-cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal.