IB1/JIP-1 controls JNK activation and increased during prostatic LNCaP cells neuroendocrine differentiation.

The scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1) is a modulator of the c-Jun N-terminal kinase (JNK) activity, which has been implicated in pleiotrophic cellular functions including cell differentiation, division, and death. In this study, we described the presence of IB1/JIP-1 in epithelium of the rat prostate as well as in the human prostatic LNCaP cells. We investigated the functional role of IB1/JIP-1 in LNCaP cells exposed to the proapoptotic agent N-(4-hydroxyphenyl)retinamide (4-HPR) which induced a reduction of IB1/JIP-1 content and a concomittant increase in JNK activity. Conversely, IB1/JIP-1 overexpression using a viral gene transfer prevented the JNK activation and the 4-HPR-induced apoptosis was blunted. In prostatic adenocarcinoma cells, the neuroendocrine (NE) phenotype acquisition is associated with tumor progression and androgen independence. During NE transdifferentiation of LNCaP cells, IB1/JIP-1 levels were increased. This regulated expression of IB1/JIP-1 is secondary to a loss of the neuronal transcriptional repressor neuron restrictive silencing factor (NRSF/REST) function which is known to repress IB1/JIP-1. Together, these results indicated that IB1/JIP-1 participates to the neuronal phenotype of the human LNCaP cells and is a regulator of JNK signaling pathway.

Epidermal growth factor receptor: a re-emerging target in glioblastoma.

PURPOSE OF REVIEW: Amplification and overexpression of the epidermal growth factor receptor (EGFR) gene are a hallmark of primary glioblastoma (45%), making it a prime target for therapy. In addition, these amplifications are frequently associated with oncogenic mutations in the extracellular domain. However, efforts at targeting the EGFR tyrosine kinase using small molecule inhibitors or antibodies have shown disappointing efficacy in clinical trials for newly diagnosed or recurrent glioblastoma. Here, we review recent insights into molecular mechanisms relevant for effective targeting of the EGFR pathway. RECENT FINDINGS: Molecular workup of glioblastoma tissue of patients under treatment with small molecule inhibitors has established drug concentrations in the tumor tissue, and has shed light on the effectiveness of target inhibition and respective effects on pathway signaling. Further, functional analyses of interaction of small molecule inhibitors with distinct properties to bind to the active or inactive form of EGFR have provided new insights that will impact the choice of drugs. Finally, vaccination approaches targeting the EGFRvIII mutant featuring a tumor-specific antigen have shown promising results that warrant larger controlled clinical trials. SUMMARY: A combination of preclinical and clinical studies at the molecular level has provided new insights that will allow refining strategies for targeting the EGFR pathway in glioblastoma.

NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP).

NR2E3, also called photoreceptor-specific nuclear receptor (PNR), is a transcription factor of the nuclear hormone receptor superfamily whose expression is uniquely restricted to photoreceptors. There, its physiological activity is essential for proper rod and cone photoreceptor development and maintenance. Thirty-two different mutations in NR2E3 have been identified in either homozygous or compound heterozygous state in the recessively inherited enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), and clumped pigmentary retinal degeneration (CPRD). The clinical phenotype common to all these patients is night blindness, rudimental or absent rod function, and hyperfunction of the "blue" S-cones. A single p.G56R mutation is inherited in a dominant manner and causes retinitis pigmentosa (RP). We have established a new locus-specific database for NR2E3 (www.LOVD.nl/eye), containing all reported mutations, polymorphisms, and unclassified sequence variants, including novel ones. A high proportion of mutations are located in the evolutionarily-conserved DNA-binding domains (DBDs) and ligand-binding domains (LBDs) of NR2E3. Based on homology modeling of these NR2E3 domains, we propose a structural localization of mutated residues. The high variability of clinical phenotypes observed in patients affected by NR2E3-linked retinal degenerations may be caused by different disease mechanisms, including absence of DNA-binding, altered interactions with transcriptional coregulators, and differential activity of modifier genes.

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Decay accelerating factor (DAF) as the host antigen with protective activity to complement killing of schistosomula

The acquisition of host antigens by Schistosoma mansoni was studied by evaluating the resistance of schistosomula to the complement attack mediated by lethal antibody. Schistosomula cultured for 24 hours with intact human erythrocytes (N-HuE) or ghosts of any type of ABO or Rh blood group, showed a marked resistance to complement damage. Sheep red blood cells, pronase-treated N-HuE or erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which are complement-sensitive cells, were unable to protect schistosomula. Schistosomula protected by N-HuE became again susceptible to complement killing after incubation with a monoclonal antibody anti-DAF. These results indicate that, in vitro, host DAF from N-HuE can be acquired by schistosomula surface in a biological active form that protects the parasite from the complement lesion.

Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

Decomposing differences in total factor productivity across firm size

This paper investigates the extent to which the gap in total factor productivity between small and large firms is due to differences in the endowment of factors determining productivity and to the returns associated with these factors. We place particular emphasis on the contribution of differences in the propensity to innovate and in the use of skilled labor across firms of different size. Empirical evidence from a representative sample of Spanish manufacturing firms corroborates that both differences in endowments and returns to innovation and skilled labor significantly contribute to the productivity gap between small and large firms. In addition, it is observed that the contribution of innovation to this gap is caused only by differences in quantity, while differences in returns have no effect; in the case of human capital, however, most of the effect can be attributed to increasing differences in returns between small and large firms.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Wound infection after excision and primary midline closure for pilonidal disease: risk factor analysis to improve patient selection.

BACKGROUND: Excision and primary midline closure for pilonidal disease (PD) is a simple procedure; however, it is frequently complicated by infection and prolonged healing. The aim of this study was to analyze risk factors for surgical site infection (SSI) in this context. METHODS: All consecutive patients undergoing excision and primary closure for PD from January 2002 through October 2008 were retrospectively assessed. The end points were SSI, as defined by the Center for Disease Control, and time to healing. Univariable and multivariable risk factor analyses were performed. RESULTS: One hundred thirty-one patients were included [97 men (74%), median age = 24 (range 15-66) years]. SSI occurred in 41 (31%) patients. Median time to healing was 20 days (range 12-76) in patients without SSI and 62 days (range 20-176) in patients with SSI (P < 0.0001). In univariable and multivariable analyses, smoking [OR = 2.6 (95% CI 1.02, 6.8), P = 0.046] and lack of antibiotic prophylaxis [OR = 5.6 (95% CI 2.5, 14.3), P = 0.001] were significant predictors for SSI. Adjusted for SSI, age over 25 was a significant predictor of prolonged healing. CONCLUSION: This study suggests that the rate of SSI after excision and primary closure of PD is higher in smokers and could be reduced by antibiotic prophylaxis. SSI significantly prolongs healing time, particularly in patients over 25 years.

Role of microRNAs in the pathogenesis of Ewing's sarcoma

SummaryEwing's sarcoma family tumors (ESFT) are the second most frequent cancer of bone in adolescents and young adults. ESFT are characterized by a chromosomal translocation that involves the 5' segment of the EWSR1 gene and the 3' segment of an ets transcription factor family member gene. In 85% of cases the chromosomal translocation generates the fusion protein EWSR1-FLI-1. Recent work from our laboratory identified mesenchymal stem cells (MSC) as the putative cell of origin of ESFT and characterized a CD133+ subpopulation of ESFT cells with tumor initating and self-renewal capacity, known as cancer stem cells (CSC). MicroRNAs (miRNAs) are small non-coding RNA that regulate protein expression at the post-transcriptional level by either repressing translation or destabilizing mRNA. MiRNAs participate in several biological processes including cell proliferation and differentiation. We used miRNA expression profile comparison between MSC and ESFT cell lines and CD133+ ESFT cells and CD133" ESFT cells to investigate the role of miRNAs in ESFT pathogenesis. MiRNA expression profile comparison of MSC and ESFT cell lines identified 35 differentially expressed miRNAs. Among these was down-regulation of let-7a which results, in part, by the direct repression of let-7a-l promoter by EWSR1-FLI-1. Overexpression of let-7a in ESFT cells blocked ESFT tumorigenesis through an High-motility group AT-hook2 (HMGA2)-mediated mechanism.MiRNA profiling of CD133+ ESFT and CD 133" ESFT cells revealed a broad repression of miRNAs in CD133+ ESFT mediated by down-regulation of TARBP2, a central regulator of the miRNA maturation pathway. Down-regulation of TARBP2 in ESFT cell lines results in a miRNA expression profile reminescent of that observed in CD133+ ESFT and associated with increased tumorigenicity. Enhancement of TARBP2 activity using the antibiotic enoxacin or overexpression of miRNA-143 or miRNA-145, two targets of TARBP2, impaired ESFT CSC self-renewal and block ESFT tumorigenicity. Moreover in vivo administration of synthetic let- 7a, miRNA-143 or miRNA-145 blocks ESFT tumor growth.Thus, dysregulation of miRNA expression is a key feature in ESFT pathogenesis and restoration of their expressions might be used as a new therapeutic tool.RésuméLe sarcome d'Ewing est la deuxième tumeur osseuse la plus fréquente chez l'enfant et le jeune adolescent. Le sarcome d'Ewing est caractérisé par une translocation chromosomique qui produit une protéine de fusion EWSR1-FLI-1. Des récents travaux ont identifié les cellules mésenchymateuses souches (MSC) comme étant les cellules à l'origine du sarcome d'Ewing ainsi qu'une sous-population de cellules exprimant le marqueur CD 133, dans le sarcome d'Ewing connu comme les cellules cancéreuses souches (CSC). Ces cellules ont la capacité d'initier la croissance tumorale et possèdent des propriétés d'auto-renouvellement. Les microRNAs (miRNAs) sont de petits ARN qui ne codent pas pour des protéines et qui contrôlent l'expression des protéines en bloquant la traduction ou en dégradant l'ARNm. Les miRNAs participent à différents processus biologiques comme la prolifération et la différenciation cellulaires.Le but de ce travail est d'étudier le rôle des miRNAs dans le sarcome d'Ewing. Un profil d'expression de miRNAs entre les MSC et des lignées cellulaires de sarcome d'Ewing a mis en évidence 35 miRNAs différemment exprimés. Parmi ceux-ci, la répression de let-7a est liée à la répression directe du promoteur de let-7a-l par EWSR-FLI-1. La sur-expression de let-7a dans des lignées cellulaires de sarcome d'Ewing inhibe leur croissance tumorale. Cette inhibition de croissance tumorale est régulée par la protéine high-motility group AT-hook2 (HMGA2).Un profil d'expression de miRNAs entre les cellules du sarcome d'Ewing CD133+ et CD133" montre une sous-expression d'un grand nombre de miRNAs dans les cellules CD133+ par rapport aux cellules CD133". Cette différence d'expression de miRNAs est due à la répression du gène TARBP2 qui participe à la maturation des miRNAs. La suppression de TARBP2 dans des cellules d'Ewing induit un profil d'expression de miRNAs similaire aux cellules CD133+ du sarcome d'Ewing et augmente la tumorigenèse des lignées cellulaires. De plus l'utilisation d'enoxacin, une molécule qui augmente l'activité de TARBP2 ou la sur- expression des miRNA143 ou miRNA-145 dans les CSC du sarcome d'Ewing bloque l'auto- renouvellement des cellules et la croissance tumorale. Finalement, l'administration de let-7a, miRNA-143 ou miRNA-145, dans des souris bloque la croissance du sarcome d'Ewing. Ces résultats indiquent que la dysrégulation des miRNAs participe à la pathogenèse du sarcome d'Ewing et que les miRNAs peuvent être utilisés comme des agents thérapeutiques.

Brain-derived neurotrophic factor enhances the hippocampal expression of key postsynaptic proteins in vivo including the monocarboxylate transporter MCT2.

Brain-derived neurotrophic factor (BDNF) promotes synaptic plasticity via an enhancement in expression of specific synaptic proteins. Recent results suggest that the neuronal monocarboxylate transporter MCT2 is a postsynaptic protein critically involved in synaptic plasticity and long-term memory. To investigate in vivo whether BDNF can modulate the expression of MCT2 as well as other proteins involved in synaptic plasticity, acute injection of BDNF was performed in mouse dorsal hippocampal CA1 area. Using immunohistochemistry, it was found that MCT2 expression was enhanced in part of the CA1 area and in the dentate gyrus 6 h after a single intrahippocampal injection of BDNF. Similarly, expression of the immediate early genes Arc and Zif268 was enhanced in the same hippocampal areas, in accordance with their role in synaptic plasticity. Immunoblot analysis confirmed the significant enhancement in MCT2 protein expression. In contrast, no changes were observed for the glial monocarboxylate transporters MCT1 and MCT4. When other synaptic proteins were investigated, it was found that postsynaptic density 95 (PSD95) and glutamate receptor 2 (GluR2) protein levels were significantly enhanced while no effect could be detected for synaptophysin, synaptosomal-associated protein 25 (SNAP25), αCaMKII and GluR1. These results demonstrate that MCT2 expression can be upregulated together with other key postsynaptic proteins in vivo under conditions related to synaptic plasticity, further suggesting the importance of energetics for memory formation.

Countercyclical Fiscal Policy and Cyclical Factor Utilization

In a neoclassical growth model with monopolistic competition in the product market, the presence of cyclical factor utilization enhances the stabilization role of countercyclical taxes. The costs of varying capital utilization take the form of varying rates of depreciation, which in turn have amplifying effect on investment decisions as well as the volatility of most aggregate variables. This creates an additional channel through which taxes affect the economy, a channel that enhances the stabilization role of countercyclical taxes, with particularly strong effects in the labor market. However, in terms of welfare, countercyclical taxes are welfare inferior due to reduced precautionary saving motives.