A baromfipestis vírus (NDV) törzsek evolúciós változásának következményei a vakcinázás hatékonyságára = Effect of the evolutionary change of Newcastle disease virus on the protection by vaccination

A baromfipestis (Newcastle-betegség) természetes fertőzését szimuláló, kontakt-ráfertőzési modellt dolgoztak ki, amellyel a vírusfertőzés eddig hozzáférhetetlen jellegzetességeit --fertőzés elleni immunállapotokat és járványvírusok terjedőképességét -- vizsgálták. Kísérletes úton megállapították, hogy korai (háború előtti) járványok törzseinek (II.-IV. genotípusok) terjedőképessége immunizált csirkék között olyan csekély (Imm&#9472;), hogy ez megmagyarázza, miért pusztultak ki ezek a vírusok az 1980-as évekre. Ezzel szemben az 1960-as évek után felbukkant, recens (V.-VIII.) genotípusok vírusai klinikailag védett állatok populációiban is terjednek (Imm+). Az Imm-karakter segítségével és egyéb evolúciós és ökológiai folyamatok elemzésével a virulens NDV genotípusok keletkezésének két mechanizmusát fedték fel. A vakcinázást megelőző időben (<1950-es évek), amikor a gazdapopulációk csak fogékony csirkékből álltak, a régi genetikai csoportok járványvírusai avirulens?virulens átalakulással keletkeztek, és az immunszelekció hiánya miatt, Imm&#9472; karakterűvé váltak. Ezzel szemben, a vakcinázás bevezetését követően felbukkant recens genotípusok az immunkörnyezethez való adaptálódás után gyors diverzifikációval jöttek létre. Ez a jelenség az alapja, hogy a betegség az emergens fertőzések jegyeit viseli magán, nevezetesen, hogy az országok zömében endémiás fertőzést alakítottak ki, sőt egyes vonalaik (VII. csoport) világszerte terjednek. | A Newcastle disease (ND) challenge system simulating natural transmission was established in chickens for the assessment of anti-infection protection afforded by various vaccination methods, as well as for the transmissibility of different epizootic ND virus strains. Based on experimental results it was established that the spreading capacity among immunized chickens, of genotype II.-IV. strains of early (before the war) outbreaks was very low (Imm&#9472;). This serves as an explanation for the extinction of these viruses in the field by the 1980s. By contrast recent virulent viruses (in genotypes V.-VIII.) that emerged in epizootics after the 1960s exhibited a significant degree of transmissibility (Imm+) in clinically protected birds. With the application of character Imm and a number of ecological and evolutionary processes, two mechanisms for the origin of virulent genotypes were revealed. In the period prior to vaccination (<1950s), in the lack of immunological selection, avirulent?virulent transition resulted in Imm&#9472; viruses such as those of the early genotypes. By contrast, emergence of recent genotypes was due to a rapid diversification of a highly adapted virulent ancestor (adaptive radiation). This process may underlie the emergent nature of the disease, namely that some 60% of the countries are endemically infected and certain lineages (genotype VII) spread throughout the world

Enhanced at puberty 1 (EAP1) is a new transcriptional regulator of the female neuroendocrine reproductive axis

The initiation of mammalian puberty and the maintenance of female reproductive cycles are events controlled by hypothalamic neurons that secrete the decapeptide gonadotropin-releasing hormone (GnRH). GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The hierarchical control of the process is unknown, but it requires coordinated regulation of these cell-cell interactions. Here we report the functional characterization of a gene (termed enhanced at puberty 1 [EAP1]) that appears to act as an upstream transcriptional regulator of neuronal networks controlling female reproductive function. EAP1 expression increased selectively at puberty in both the nonhuman primate and rodent hypothalamus. EAP1 encoded a nuclear protein expressed in neurons involved in the inhibitory and facilitatory control of reproduction. EAP1 transactivated genes required for reproductive function, such as GNRH1, and repressed inhibitory genes, such as preproenkephalin. It contained a RING finger domain of the C3HC4 subclass required for this dual transcriptional activity. Inhibition of EAP1 expression, targeted to the rodent hypothalamus via lentivirus-mediated delivery of EAP1 siRNAs, delayed puberty, disrupted estrous cyclicity, and resulted in ovarian abnormalities. These results suggest that EAP1 is a transcriptional regulator that, acting within the neuroendocrine brain, contributes to controlling female reproductive function.This work was supported by grants from the NIH, the National Institute of Child Health and Human Development/NIH (to S.R. Ojeda), the European Society for Paediatric Endocrinology (to H. Jung), the German Research Foundation (to S. Heger), and the European Commission (PIONEER to S. Heger)

Nitric oxide synthase-independent release of nitric oxide induced by KCl in the perfused mesenteric bed of the rat

The aim of the present study was to test whether the contractile responses elicited by KCl in the rat mesenteric bed are coupled to the release of nitric oxide (NO). Contractions induced by 70 mM KCl were coincident with the release of NO to the perfusate. The in vitro exposure to the nitric oxide synthase (NOS) inhibitor L-N(ω)-nitro-L-arginine methyl ester, L-NAME (1-100 μM) potentiated the vascular responses to 70 mM KCl and, unexpectedly, increased the KCl-stimulated release of NO. Moreover, even after the chronic treatment with L-NAME (70 mg/kg/day during 4 weeks), the KCl-induced release of NO was not reduced, whereas the potentiation of contractile responses was indeed achieved. The possibility that NOS had not been completely inhibited under our experimental conditions can be precluded because NOS activity was significantly inhibited after both L-NAME treatments. After the in vitro treatment with 1 to 100 μM L-NAME, the inhibition of NOS was concentration-dependent (from 50% to 90%). With regard to the basal release of NO, the inhibition caused by L-NAME was not concentration-dependent and reached a maximum of 40%, suggesting that basal NO outflow is only partially dependent on NOS activity. An eventual enhancement of NOS activity caused by KCl was disregarded because the activity of this enzyme measured in homogenates from mesenteric beds perfused with 70 mM KCl was significantly reduced. On the other hand, endothelium removal, employed as a negative control, almost abolished NOS activity, whereas the incubation with the Ca2+ ionophore A23187, employed as a positive control, induced an increase in NOS activity. It is concluded that in the mesenteric arterial bed of the rat, the contractile responses elicited by depolarization through KCl are coincident with a NOS-independent release of NO. This observation, which differs from the results obtained with noradrenaline, do not support the use of KCl as an alternative contractile agent whenever the participation of NO is under study. (C) 2000 Elsevier Science B.V.Fil: Mendizabal, Victoria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Poblete, I.. Pontificia Universidad Católica de Chile; ChileFil: Lomniczi, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Besuhli, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Huidobro Toro, J. P.. Pontificia Universidad Católica de Chile; ChileFil: Adler, Edda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentin

Control of Salivary Secretion by Nitric Oxide and Its Role in Neuroimmunomodulation

Abstract: In many in vivo systems exposure to endotoxins (LPS) leads to the co‐induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), which is important to the regulation of the function of different systems during infection. In submandibular glands (SMG) neural (n)NOS is localized in neural terminals and in striated, granular convoluted and excretory ducts, endothelial (e) NOS in vascular endothelium and ducts, and iNOS in macrophages and in tubules and ducts. In normal adult male rats, injection of an inhibitor of NOS decreased the stimulated salivary secretion and a donor of NO potentiated it, indicating that NO exerts a stimulatory role. A single high dose of LPS (5 mg/kg, i.p.) induced an increase in NOS activity measured by the 14C‐citrulline method, increased PGE content almost 100% as measured by RIA, and blocked stimulated salivary secretion. The administration of a specific iNOS inhibitor, aminoguanidine (AG), with LPS not only decreased NOS activity but significantly decreased PGE content, indicating that NO triggered the activation of COX‐2. LPS increased conversion of labeled arachidonate to prostaglandins (PGs) showing that COX was induced. Since a PGE1 analogue blocked stimulated salivation, the LPS‐induced inhibition of salivation is probably due to release of PGs. Therefore, the use of inhibitors of iNOS and COX‐2 could be very useful to increase salivation during infection since saliva has antimicrobial actions.Fil: Besuhli, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Lomniczi, A.. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Elverdín, Juan Carlos. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Suburo, Angela Maria. Universidad Austral; ArgentinaFil: Faletti, Alicia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: McCann, Samuel M.. State University of Louisiana; Estados Unido

Supplements to article: A novel transcription complex that selectively modulates apoptosis of breast cancer cells through regulation of FASTKD2

The materials provided here are supplemental tables and figures to an article to be published in 'Molecular and Cellular Biology.'(This refers to the article.) We previously reported that expression of NRIF3 (Nuclear Receptor Interacting Factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (a.k.a IRF-2BP2), the cellular target of NRIF3, was identified as a transcriptional repressor and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF2BP1 (Interferon Regulatory Factor-2 Binding Protein 1) and EAP1 (Enhanced At Puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF2BP1, and EAP1 occurs through the conserved C4 zinc-fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the Death Domain region of NRIF3 (DD1). The DIF-1 complex was found to repress FASTKD2, a putative pro-apoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown while expression of FASTKD2 leads to apoptosis of both breast and non-breast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer

EAP1 regulation of GnRH promoter activity is important for human pubertal timing

The initiation of puberty is orchestrated by an augmentation of gonadotropin-releasing hormone (GnRH) secretion from a few thousand hypothalamic neurons. Recent findings have indicated that the neuroendocrine control of puberty may be regulated by a hierarchically organized network of transcriptional factors acting upstream of GnRH. These include enhanced at puberty 1 (EAP1), which contributes to the initiation of female puberty through transactivation of the GnRH promoter. However, no EAP1 mutations have been found in humans with disorders of pubertal timing. We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited delayed puberty (DP). Variants were analyzed for rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. We identified one in-frame deletion (Ala221del) and one rare missense variant (Asn770His) in EAP1 in two unrelated families; these variants were highly conserved and potentially pathogenic. Expression studies revealed Eap1 mRNA abundance in peri-pubertal mouse hypothalamus. EAP1 binding to the GnRH1 promoter increased in monkey hypothalamus at the onset of puberty as determined by chromatin immunoprecipitation. Using a luciferase reporter assay, EAP1 mutants showed a reduced ability to trans-activate the GnRH promoter compared to wild-type EAP1, due to reduced protein levels caused by the Ala221del mutation and subcellular mislocation caused by the Asn770His mutation, as revealed by western blot and immunofluorescence, respectively. In conclusion, we have identified the first EAP1 mutations leading to reduced GnRH transcriptional activity resulting in a phenotype of self-limited DP.Peer reviewe

Elucidating the genetic architecture of reproductive ageing in the Japanese population.

Population studies elucidating the genetic architecture of reproductive ageing have been largely limited to European ancestries, restricting the generalizability of the findings and overlooking possible key genes poorly captured by common European genetic variation. Here, we report 26 loci (all P < 5 × 10-8) for reproductive ageing, i.e. puberty timing or age at menopause, in a non-European population (up to 67,029 women of Japanese ancestry). Highlighted genes for menopause include GNRH1, which supports a primary, rather than passive, role for hypothalamic-pituitary GnRH signalling in the timing of menopause. For puberty timing, we demonstrate an aetiological role for receptor-like protein tyrosine phosphatases by combining evidence across population genetics and pre- and peri-pubertal changes in hypothalamic gene expression in rodent and primate models. Furthermore, our findings demonstrate widespread differences in allele frequencies and effect estimates between Japanese and European associated variants, highlighting the benefits and challenges of large-scale trans-ethnic approaches

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as 112RRQKRF117 at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif 112RRQKRF117 in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs (112KRQKRF117, 112RRQRRF117 and 112RRRKRF117) but not an avirulence motif (112GRQGRL117) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds