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With its large size, dense atmosphere, methane-based hydrological-like cycle, and diverse surface features, the Saturnian moon Titan is one of the most unique of the outer Solar System satellites. Study of the photochemically produced molecules in Titan's atmosphere is critical in order to understand the mechanics of the atmosphere and, by extension, the interactions between atmosphere, surface, and subsurface water ocean. One example is propyne vapor, a photochemically produced species in Titan's upper atmosphere expected to condense in Titan's stratosphere at lower altitudes. Propyne may also be a trace species in Titan's stratospheric co-condensed ice clouds detected by the Cassini Composite InfraRed Spectrometer. Bulk structural characterization of propyne ice is currently incomplete and is lacking in published laboratory Raman spectra and X-ray diffraction data. Here, we present a laboratory characterization of propyne ice, including the first published X-ray diffraction and Raman spectroscopy results for propyne ice.
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PedvaxHIB® is an effective pediatric vaccine for protecting infants from invasive gram-negative bacterium Haemophilus influenzae type b. It is a highly purified capsular polysaccharide, polyribosylribitol phosphate that is covalently linked to an outer membrane protein complex of Neisseria meningitidis. PRP is first derivatized with an organic linker, followed by the coupling of a butadiamine group, and then at the end terminal, a bromoacetyl group is attached for conjugation with thiolated OMPC. The stability of the bromide group in derivatized PRP is monitored by two different methods, capillary electrophoresis and NMR spectroscopy. The loss of the bromide group is detected by measuring the amount of free bromide ion liberated using capillary electrophoresis and by observing a change in amide proton peaks near the bromide group using NMR. The two methods give similar rate hydrolysis results, therefore both can be employed as quick stability tools for bromoacetylation PRP content during manufacturing.
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Infecciones por Haemophilus , Vacunas contra Haemophilus , Haemophilus influenzae tipo b , Amidas , Anticuerpos Antibacterianos , Proteínas de la Membrana Bacteriana Externa , Vacunas Bacterianas , Bromuros , Niño , Electroforesis Capilar , Infecciones por Haemophilus/prevención & control , Haemophilus influenzae , Humanos , Lactante , Espectroscopía de Resonancia Magnética , Fosfatos , Polisacáridos Bacterianos , ProtonesRESUMEN
Exposure to second-hand smoke (SHS) is an important public health problem. We assessed SHS exposure in restaurants and bars across the European Union (EU) and studied associations with country-level smoke-free legislation. Data of Eurobarometer surveys 2014 and 2017 were used to estimate country-specific prevalence of observing smoking in restaurants and bars, which can be considered a marker of SHS exposure. Additionally, we used multilevel logistic regression models to study associations with comprehensiveness of national smoke-free regulations in restaurants and bars, which were derived from the Tobacco Control Scale. In total, 44,809 people from all 28 EU member states were included in the analysis. The results of the multilevel logistic analysis show that in countries with complete and extensive bans, respondents were less likely to have observed people smoking inside restaurants than in countries with partial bans, which represented the lowest level of smoke-free policy implementation (OR 0.24, 95%CI 0.10-0.57 for complete ban and OR 0.23, 95%CI 0.10-0.54 for incomplete but extensive ban). Also, the prevalence of seeing people smoking in a bar was lower in the countries with an extensive ban (OR 0.23 95%CI 0.11-0.45) and with a complete ban (OR 0.20 95%CI 0.10-0.40). Between 2014 and 2017, SHS exposure in restaurants and bars decreased significantly. Our results confirm that in countries with extensive or complete smoking bans, people were less exposed to SHS in restaurants and bars; and that partial bans are less effective in reducing SHS exposure.
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Política para Fumadores , Contaminación por Humo de Tabaco , Unión Europea , Humanos , Prevalencia , RestaurantesRESUMEN
Background: Currently, about 65% of the world's population is covered by at least one MPOWER tobacco control policy measure. The impact of such policies might rely on policy compliance. Objective: This study aims to describe and compare global trends in legislation and compliance of the following three tobacco control policies between 2009 and 2019: direct advertisement, promotion and sponsorship, and smoke-free environments. Method: Data from the six most recent WHO Tobacco Control (2009-2019) reports were used to show the development of and possible associations between legislated policies and policy compliance. Data pertaining to the three indicators direct advertisement, promotion and sponsorship, and smoke-free environments were collected and analysed per country income category, according to the Organization for Economic Co-operation and Development. For each country, we (i) calculated the legislation describing the situation according to the law as a percentage of fulfilled MPOWER measurements and (ii) present the level of compliance (ranging from 0 to 10) for the corresponding policy. Results: Both tobacco control policy legislation and compliance for direct advertising improved worldwide - between 2009 and 2019 the median increased from 37.5% to 87.5% for policy and from 5 to 8 for compliance. In contrast, promotion and sponsorship restrictions hardly developed since 2011 and are especially weak among low- and middle-income countries. With respect to smoke-free environments, global policy legislation increased steadily over time while the relative compliance hardly increased. In 2019 data did not show significant correlations between policy legislation and compliance: direct advertising ρ = -0.003, p = 0.970; promotion and sponsorship ρ = 0.140, p = 0.107; smoke-free environments ρ = 0.158, p = 0.070. Conclusion: There is a clear need to understand the barriers in achieving tobacco control policy compliance and to routinely collect and incorporate data on compliance in research in order to generate a more reliable basis for further improvements in tobacco control.
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Nicotiana , Contaminación por Humo de Tabaco , Adhesión a Directriz , Humanos , Políticas , Uso de TabacoRESUMEN
The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.
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Infecciones Asintomáticas/epidemiología , Interacciones Huésped-Parásitos/genética , Malaria Falciparum/epidemiología , Plasmodium falciparum/patogenicidad , Adolescente , Adulto , Animales , Niño , Preescolar , Enfermedades Endémicas/prevención & control , Eritrocitos/parasitología , Femenino , Genotipo , Humanos , Lactante , Malaria Falciparum/genética , Malaria Falciparum/parasitología , Masculino , Malí/epidemiología , Persona de Mediana Edad , Plasmodium falciparum/genética , Estaciones del Año , Adulto JovenRESUMEN
The study aimed to describe worldwide levels and trends of tobacco control policy by comparing low and middle income countries with other income categories from 2007 to 2014 and to analyze the corresponding relation to recent changes in smoking prevalence. Policy measure data representing years 2007 to 2014 were collected from all available World Health Organization (WHO) reports on the global tobacco epidemic. Corresponding policy percentage scores (PS) were calculated based on MPOWER measures. Age-standardized smoking prevalence data for years 2010 and 2015 were collected from the WHO Global Health Observatory Data Repository. Trends of PS were analysed with respect to WHO region and OECD country income category. Scatter plots and regression analysis were used to depict the relationship between tobacco control policy of 2010 and change in smoking prevalence between 2015 and 2010 by sex and income category. Combined PS for all countries increased significantly from 47% in 2007 to 61% by 2014 (p < 0.001). When grouped by income category and region, policies were strengthened in all categories, albeit with varying progression. By 2014, tobacco control policy legislation had reached 45% in the Least Developed Countries (LDCs), 59% in Low Middle Income Countries (LMICs), 66% in Upper Middle Income Countries (UMICs) and 70% in High Income Countries (HICs). Overall, there was a negative relationship between higher policy scores and change in smoking prevalence. Although policy strengthening had been conducted between 2007 and 2014, room for considerable global improvement remains, particularly in LDCs.
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Países Desarrollados , Países en Desarrollo , Regulación Gubernamental , Política de Salud/legislación & jurisprudencia , Prevención del Hábito de Fumar , Fumar/legislación & jurisprudencia , Humanos , Prevalencia , Fumar/epidemiología , NicotianaRESUMEN
In Germany, vaccination of infants against hepatitis B is recommended since 1995. However, data on long-term immunity is sparse and the necessity of a booster dose remains uncertain. Aims of this study were to assess the long-term persistence of antibodies to the hepatitis B surface antigen (anti-HBs) after immunization during infancy and the effect of a subsequent hepatitis B booster vaccination during adolescence on anti-HBs levels. Patients from a private pediatric practice who had received a full vaccination course of hepatitis B as infants and who were quantitatively tested for anti-HBs during adolescence (pre-booster levels) were included. In those participants who received a hepatitis B booster, post-booster anti-HBs levels were measured. Univariate analyses were conducted to determine factors associated with pre- and post-booster anti-HBs levels, respectively. 106 participants (53% male) were included in the study. At an average of 13.7 y after primary vaccination, 14% of participants had an anti-HBs level of ≥100 IU/l, while 46% were at 10-99 IU/l and 40% had anti-HBs levels of <10 IU/l. In total, 34 received a booster vaccination. Of those, 97% (33/34) had post-booster anti-HBs levels ≥ 100 IU/l, which were independent from pre-booster levels. No other patient characteristics were associated with pre-booster or post-booster anti-HBs≥ 100 IU/l. Although almost half of study participants showed low anti-HBs levels at follow-up, robust responses to booster vaccination suggest that adolescents who received the full vaccination course during infancy are still protected against hepatitis B infection.
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Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/inmunología , Hepatitis B/prevención & control , Adolescente , Niño , Preescolar , Estudios de Cohortes , Femenino , Alemania , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Humanos , Lactante , Recién Nacido , Masculino , Estudios Retrospectivos , Factores de TiempoRESUMEN
Polysaccharide based-vaccines have been successful in providing protection in adults from bacterial infections, however they are not as effective in infants or young children. To enhance the immune response in these high risk groups, the polysaccharide is conjugated with a carrier protein such as cross-reacting material 197 (CRM197). The CRM197 protein has been well-characterized biochemically and biophysically using various analytical techniques however, none of these have been CE-based methods. Of the various CE techniques, imaged capillary isoelectric focusing (icIEF) is a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. Applications of icIEF technique using Convergent Bioscience icIEF instrumentation with whole-field imaging technology are presented and discussed in this paper. These applications include rapid method development to establish a CRM197 identity test for product release, a concentration assay for upstream and downstream in-process product development, and CRM197 stability with respect to its charge heterogeneity under accelerated temperature stress. The data presented demonstrates the utility of the icIEF method as a multifunctional assay because it can screen for better product candidates during early stage clonal selection as well as support in-process and final product characterization throughout CRM197 development.
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Proteínas Bacterianas/química , Toxina Diftérica/química , Portadores de Fármacos/química , Electroforesis Capilar/métodos , Focalización Isoeléctrica/métodos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Toxina Diftérica/análisis , Portadores de Fármacos/análisis , Mutación , Vacunas/químicaRESUMEN
Integrin-linked kinase (ILK) is a major structural adaptor protein governing signaling complex formation and cytoskeletal dynamics. Here, through the use of conditional knock-out mice, we demonstrate a requirement for ILK in oligodendrocyte differentiation and axonal myelination in vivo. In conjunction, ILK-deficient primary oligodendrocytes are defined by a failure in process extension and an inability to form myelin membrane upon axonal contact. Surprisingly, phosphorylation of the canonical downstream targets Akt and GSK3ß is unaffected following ILK loss. Rather, the defects are due in part to actin cytoskeleton dysregulation with a correspondent increase in active RhoA levels. Morphological rescue is possible following Rho kinase inhibition in an oligodendrocyte subset. Furthermore, phenotypic severity correlates with environmental complexity; oligodendrocytes are severely malformed in vitro (a relatively simple environment), but undergo phenotypic recovery in the context of the whole animal. Together, our work demonstrates ILK as necessary for normal oligodendrocyte development, reinforces its role as a bridge between the actin cytoskeleton and cell membrane, and highlights the overarching compensatory capacity of oligodendrocytes in response to cellular milieu.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Oligodendroglía/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Simulación de Dinámica MolecularRESUMEN
Capillary electrophoresis (CE) is a versatile analytical method used to characterize glycoproteins. We have used several modes of CE separation such as CE-SDS gel, imaged capillary isoelectric focusing (icIEF), and capillary zone electrophoresis (CZE) to study therapeutic glycoprotein products. CE-SDS gel is applied to characterize the glycan occupancy and number of glycosylation sites, and icIEF is used to study the charge heterogeneities due to sialic acids in glycoproteins. To further characterize the glycoprotein, removal of N-linked glycans is necessary and a CZE technique is employed to analyze each glycan moiety. Examples from a monoclonal antibody, erythropoietin, and granulocyte colony-stimulating factor are presented here to demonstrate the utility of these CE modes. The details of sample preparation and separation conditions for each CE mode are described in this chapter.
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Eritropoyetina/química , Factor Estimulante de Colonias de Granulocitos/química , Oligosacáridos/aislamiento & purificación , Animales , Anticuerpos Monoclonales/química , Secuencia de Carbohidratos , Electroforesis Capilar/métodos , Electroforesis Capilar/normas , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/química , Glicosilación , Humanos , Focalización Isoeléctrica/métodos , Focalización Isoeléctrica/normas , Datos de Secuencia Molecular , Neuraminidasa/química , Oligosacáridos/química , Procesamiento Proteico-Postraduccional , Estándares de ReferenciaRESUMEN
CE-based methods have increasingly been applied to the analysis of a variety of different type proteins. One of those techniques is imaged capillary isoelectric focusing (icIEF), a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. It offers many advantages over the traditional labor-intensive IEF slab gel method and even standard cIEF with on-line detection technologies with regard to method development, reproducibility, robustness, and speed. Here, specific examples are provided for biopharmaceutical glycoprotein products such as mAbs, erythropoietin (EPO), and recombinant Fc-fusion proteins, though the technique can be adapted for many other therapeutic proteins. Applications of iCIEF using a Convergent Bioscience instrument (Toronto, Canada) with whole-field imaging technology are presented and discussed. These include a quick method to establish an identity test for many protein-based products, product release, and stability evaluation of glycoproteins with respect to charge heterogeneity under accelerated temperature stress, different pH conditions, and in different formulations. Finally, characterization of glycoproteins using this iCIEF technology is discussed with respect to biosimilar development, clone selection, and antigen binding. The data presented provide a "taste'' of what icIEF method can do to support the development of biopharmaceutical glycoprotein products from early clone screening for better product candidates to characterization of the final commercial products.
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Productos Biológicos/química , Biofarmacia/métodos , Electroforesis Capilar/métodos , Glicoproteínas/química , Focalización Isoeléctrica/métodos , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Productos Biológicos/análisis , Células CHO , Cricetinae , Cricetulus , Estabilidad de Medicamentos , Eritropoyetina/análisis , Eritropoyetina/química , Glicoproteínas/análisis , Humanos , Ratones , Pichia , Unión Proteica , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/químicaRESUMEN
BACKGROUND: Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. It is caused by mutations/deletions of the survival motor neuron 1 (SMN1) gene and is typified by the loss of spinal cord motor neurons, muscular atrophy, and in severe cases, death. The SMN protein is ubiquitously expressed and various cellular- and tissue-specific functions have been investigated to explain the specific motor neuron loss in SMA. We have previously shown that the RhoA/Rho kinase (ROCK) pathway is misregulated in cellular and animal SMA models, and that inhibition of ROCK with the chemical Y-27632 significantly increased the lifespan of a mouse model of SMA. In the present study, we evaluated the therapeutic potential of the clinically approved ROCK inhibitor fasudil. METHODS: Fasudil was administered by oral gavage from post-natal day 3 to 21 at a concentration of 30 mg/kg twice daily. The effects of fasudil on lifespan and SMA pathological hallmarks of the SMA mice were assessed and compared to vehicle-treated mice. For the Kaplan-Meier survival analysis, the log-rank test was used and survival curves were considered significantly different at P < 0.05. For the remaining analyses, the Student's two-tail t test for paired variables and one-way analysis of variance (ANOVA) were used to test for differences between samples and data were considered significantly different at P < 0.05. RESULTS: Fasudil significantly improves survival of SMA mice. This dramatic phenotypic improvement is not mediated by an up-regulation of Smn protein or via preservation of motor neurons. However, fasudil administration results in a significant increase in muscle fiber and postsynaptic endplate size, and restores normal expression of markers of skeletal muscle development, suggesting that the beneficial effects of fasudil could be muscle-specific. CONCLUSIONS: Our work underscores the importance of muscle as a therapeutic target in SMA and highlights the beneficial potential of ROCK inhibitors as a therapeutic strategy for SMA and for other degenerative diseases characterized by muscular atrophy and postsynaptic immaturity.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/crecimiento & desarrollo , Atrofia Muscular Espinal/tratamiento farmacológico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Células del Asta Anterior/efectos de los fármacos , Células del Asta Anterior/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Marcha/efectos de los fármacos , Longevidad/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Placa Motora/efectos de los fármacos , Placa Motora/patología , Placa Motora/fisiopatología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular Espinal/patología , Atrofia Muscular Espinal/fisiopatología , Miogenina/metabolismo , Fenotipo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Análisis de Supervivencia , Proteína 2 para la Supervivencia de la Neurona Motora/deficiencia , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.
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Movimiento Celular/fisiología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Apoptosis , Células HeLa , Humanos , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Pez CebraRESUMEN
The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 â 2)-ß-Man-(1 â 2)-ß-Man-(1 â 2)-α-Man-(1 â 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple ß-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.
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Manosidasas/metabolismo , Proteínas de la Membrana/metabolismo , Oligosacáridos/biosíntesis , Oligosacáridos/química , Pichia/metabolismo , Humanos , Manosidasas/genética , Proteínas de la Membrana/genética , Pichia/genética , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin--PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26(LacZ) and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreER(t) line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology.
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Linaje de la Célula/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Proteína Proteolipídica de la Mielina/genética , Oligodendroglía/citología , Oligodendroglía/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Animales Recién Nacidos , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Integrasas/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Proteína Proteolipídica de la Mielina/metabolismo , Vaina de Mielina/metabolismo , Especificidad de Órganos/genética , Transgenes/genética , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Increased smooth muscle tone is a significant component of benign prostatic hyperplasia, the onset of which correlates with age and declining serum testosterone levels. This study investigates the effects of androgens on key regulators of smooth muscle tone: intracellular calcium ([Ca(2+)](i)) and cyclic adenosine monophosphate (cAMP) in human cultured prostatic stromal cells (HCPSC). METHODS: HCPSC were cultured in the absence or presence of dihydrotestosterone (DHT; 3, 30, and 300 nM) or testosterone (0.3-300 nM) alone or in the presence of flutamide (10 microM). Changes in [Ca(2+)](i) were determined in FURA-2AM (10 microM) loaded cells. Changes in cAMP were determined by Alpha Screen(R) assay. RESULTS: Up to 32% of cultured cells exhibited spontaneous elevations of [Ca(2+)](i). The frequency of these elevations was reduced by nifedipine (10 microM), ryanodine (1 microM), and the adenylate cyclase inhibitor MDL 12,330A (20 microM). Compared to steroid-free cells, a 3-day incubation of cells with testosterone (only 3 nM) elevated basal, but not peak [Ca(2+)](i). In the presence of flutamide, all concentrations of testosterone tested elevated basal, but not peak [Ca(2+)](i). DHT (30 and 300, but not 3 nM) lowered peak and basal [Ca(2+)](i). Increased testosterone concentration dependently decreased resting cell cAMP (pIC(50): 7.64 +/- 0.29 nM). CONCLUSIONS: These findings demonstrate that some HCPSC have the ability to spontaneously and transiently elevate [Ca(2+)](i). The magnitude of these [Ca(2+)](i) peaks, along with resting levels of calcium and cAMP, appear to be regulated by androgens.
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Adenilil Ciclasas/metabolismo , Andrógenos/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Hiperplasia Prostática/metabolismo , Inhibidores de Adenilato Ciclasa , Anciano , Antagonistas de Andrógenos/farmacología , Inhibidores Enzimáticos/farmacología , Flutamida/farmacología , Humanos , Iminas/farmacología , Inmunohistoquímica , Masculino , Microscopía Fluorescente , Nifedipino/farmacología , Hiperplasia Prostática/enzimología , Hiperplasia Prostática/patología , Rianodina/farmacología , Células del Estroma/enzimología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Spinal muscular atrophy (SMA) is an inherited disease resulting in the highest mortality of children under the age of two. SMA is caused by mutations or deletions in the survival motor neuron 1 (SMN1) gene, leading to aberrant neuromuscular junction (NMJ) development and the loss of spinal cord alpha-motor neurons. Here, we show that Smn depletion leads to increased activation of RhoA, a major regulator of actin dynamics, in the spinal cord of an intermediate SMA mouse model. Treating these mice with Y-27632, which inhibits ROCK, a direct downstream effector of RhoA, dramatically improves their survival. This lifespan rescue is independent of Smn expression and is accompanied by an improvement in the maturation of the NMJs and an increase in muscle fiber size in the SMA mice. Our study presents evidence linking disruption of actin cytoskeletal dynamics to SMA pathogenesis and, for the first time, identifies RhoA effectors as viable targets for therapeutic intervention in the disease.
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Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/mortalidad , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína de Unión al GTP rhoA/metabolismo , Amidas/administración & dosificación , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Atrofia Muscular Espinal/genética , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Médula Espinal/enzimología , Médula Espinal/metabolismo , Sobrevida , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
We have discovered a novel bacterial polysaccharide structural element, 3-O-acetylglycerol, in the Streptococcus pneumoniae ST11A polysaccharide: This moiety was elucidated through a combination of homonuclear and heteronuclear 1D and 2D NMR experiments using (1)H, (13)C, and (31)P in various combinations. The 3-O-acetylglycerol moiety is substoichiometrically O-acetylated in ST11A; yet, key connectivities that unequivocally show O-acetylation at the glycerol are provided by the long-range correlations from the acetate methyl groups to the glycerol in the (1)H-(13)C HMBC spectrum. Additionally, we clarify the (1)H-(31)P assignments previously presented.