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The present manuscript describes the application of two inventory strategies to optimize the supply process within an air conditioning manufacturing facility. These approaches were considered as the air conditioning industry involves variable demand patterns due to its large market (automotive, home, cooling/heating systems, among others). Because the components which are assembled in this factory involve 45 components which have different demand, lead times and inventory management costs, the strategies were adjusted to reduce costs and management complexity. First, inventory management costs were optimized according to the classification of the most important components. Then, periodic and continuous review models were adapted to optimize the management costs of these components. Validation of these strategy through discrete-event computer simulation led to determine their suitability for this case study, significantly reducing inventory management costs while increasing service level. Simulation also led to determine that for specific components, a model can lead to better (and feasible) results than the other and showing no stock-out events. This is an outcome which can be considered by companies to make a better evaluation of the supply strategies during the planning process.

Resumen (Introducción): La evaluación del suministro de servicios ecosistémicos por los diferentes usos del suelo de la agricultura familiar facilita plantear estrategias para el uso sostenible de la tierra. (Objetivo): Se determinaron los efectos de diferentes usos del suelo practicados en la agricultura familiar, sobre la conservación de la diversidad arbórea y el suministro de servicios ecosistémicos críticos: almacenamiento de carbono aéreo y subterráneo y servicios de soporte que provienen del suelo, en la Reserva de Biosfera del Bosque Mbaracayú (Canindeyú, Paraguay). (Metodología): Fue determinada la biodiversidad arbórea, el almacenamiento de carbono aéreo y de suelo, las propiedades químicas y físicas del suelo en 7 fincas con diversos tipos de cobertura: bosque nativo alterado, sistemas agroforestales con yerba mate, cultivo anual de subsistencia y pasturas. En cada uno de los puntos de muestreo, fueron registrados datos de los individuos arbóreos y arbustivos, y fueron tomadas muestras del estrato herbáceo, hojarasca y suelo. (Resultados): Se registró mayor índice de diversidad y almacenamiento de carbono aéreo en bosques nativos alterados y sistemas agroforestales, sin diferencia significativa entre ellos. Los valores de CIC y Ca+2 de suelo fueron más elevados en los bosques nativos alterados comparado con pasturas, y los otros usos presentaron valores intermedios. No hubo diferencias significativas entre los diferentes usos en cuanto al carbono almacenado ni en estabilidad de la estructura del suelo. Sin embargo, las pasturas presentaron índices de calidad de suelo más bajos. (Conclusiones): Se demostró que bosques nativos alterados y sistemas agroforestales aportan más a la conservación de la diversidad arbórea, retención de carbono y calidad del suelo. Abstract (Introduction): The evaluation of ecosystem services in different land use systems in family farming facilitates the development of sustainable land use strategies. (Objective): We studied the effects of different land uses, commonly applied in family farming in the Mbaracayú Forest Biosphere Reserve (Canindeyú, Paraguay), on the conservation of tree diversity and delivery of critical ecosystem services: above and belowground carbon storage and supporting services rendered by soil. (Methods): We determined the diversity of trees, the stocks of carbon above ground and in the soil, as well as chemical and physical soil properties in 7 farms with diverse land use systems: disturbed native forest, agroforestry system with yerba mate, subsistence crops and pastures. In each of the sampling points, data of the tree and shrub individuals were recorded, and samples of the herbaceous stratum, litter and soil were taken. (Results): We found the highest rate of both diversity and above ground carbon storage in the disturbed native forests and agroforestry systems without significant difference between the two. The values of CEC and Ca+2 in the soil were higher in disturbed native forests compared to pastures, while the remaining land uses showed intermediate values. There was no significant difference between the different land use types regarding carbon storage and physical stability of the soils. Nonetheless, the pastures had the lowest soil qualities. (Conclusions): We demonstrated that the altered native forests and agroforest systems with yerba mate promote better the conservation of tree diversity, carbon retention and soil quality.

La proteína fosfatasa 2A (PP2A) es una importante Ser/Thr fosfatasa que participa en la regulación de múltiples procesos celulares. Esto implica que cualquier actividad deficiente de PP2A es responsable de graves patologías graves. Por ejemplo, una de las principales características histopatológicas de la enfermedad de Alzheimer son los ovillos neurofibrilares, que están compuestos principalmente por formas hiperfosforiladas de la proteína tau. Esta tasa alterada de fosforilación de tau se ha correlacionado con la depresión PP2A en pacientes con EA. Con el objetivo de prevenir la inactivación de PP2A en escenarios de neurodegeneración, nos hemos propuesto diseñar, sintetizar y evaluar nuevos ligandos de PP2A capaces de impidir su inhibición. Para lograr este objetivo, los nuevos ligandos PP2A presentan similitudes estructurales con el fragmento central C19-C27 del conocido inhibidor de PP2A ácido okadaico (AO). De hecho, este fragmento central de OA no ejerce acciones inhibidoras. Así, estos compuestos carecen de motivos estructurales inhibidores de PP2A pero, por contra, compiten con los inhibidores de PP2A, recuperando así la actividad fosfatasa. Demostrando esta hipótesis, la mayoría los compuestos mostraron un buen perfil neuroprotector en modelos de neurodegeneración relacionados con el deterioro de PP2A, destacando el derivado 10, denominado ITH12711, como el más prometedor. Este compuesto (1º) restauró la actividad catalítica de PP2A, medida en un sustrato fosfopéptido por Western blot, (2º) demostró una buena penetración cerebral medida por PAMPA, y (3º) evitó el deterioro de la memoria inducido por LPS en ratones sometidos a la prueba de reconocimiento de objetos. Por lo tanto, los prometedores resultados del compuesto 10 validan nuestra aproximación para diseñar nuevos fármacos activadores de PP2A basados en el fragmento central de OA.

Introduction: Systemic vasculitis belongs to a group of rare, serious multisystemic diseases, that without treatment can be life-threatening. Objective: to study and to determine the clinical characteristics and mortality of patients with systemic vasculitis admitted in the period 2000-2020, in two public hospitals in Paraguay. Material and Method: a retrospective, descriptive, observational, cross-sectional study with an analytical component of 41 adult patients with a diagnosis of systemic vasculitis. The diagnosis was made by clinical, laboratory, imaging and histological findings. Risk factors for mortality were established by the chi-square test, considering a p value less than p

Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction. Ministerio de Economía España

Cigarette smoking is associated with increased risk for all histologic types of lung cancer, but why the strength of this association is stronger for squamous cell carcinoma than adenocarcinoma of the lung (SQC-L, ADC-L) is not fully understood. Because nicotine and tobacco-specific nitrosamines contribute to carcinogenesis by activating nicotinic acetylcholine receptors (nAChRs) on lung tumors and epithelial cells, we investigated whether differential expression of nAChR subtypes in these tumors could explain their different association with smoking. Expression of nAChR subunit genes in paired tumor and non-tumor lung specimens from 40 SQC-L and 38 ADC-L patients was analyzed by quantitative PCR. Compared to normal lung, both tumors share: i) transcriptional dysregulation of CHRNA3/CHRNA5/CHRNB4 (α3, α5, β4 subunits) at the chromosomal locus that predisposes to lung cancer; and ii) decreased expression of CHRFAM7A (dupα7 subunit); this last subunit negatively modulates α7-nAChR activity in oocytes. In contrast, CHRNA7 (α7 subunit) expression was increased in SQC-L, particularly in smokers and non-survivors, while CHRNA4 (α4 subunit) expression was decreased in ADC-L. Thus, over-representation of cancer-stimulating α7-nAChR in SQC-L, also potentiated by smoking, and underrepresentation of cancer-inhibiting α4β2-nAChR in ADC-L could explain the different tobacco influences on the tumorigenic process in each cancer type, This study was supported by grants to C. Montiel and F. Arnalich from the Ministry of Economy, Industry and Competitiveness, Government of Spain (SAF2014-56623-R) and Foundation “Mutua Madrileña Investigación Biomédica” (FMM2011), Spain. A.B. is recipient of a fellowship (Beca FPI, Universidad Autónoma Madrid). J.L.C. and C.M.S. are recipients of fellowships (Beca FPU from Ministerio de Educación, Cultura y Deporte and Beca FPI from Ministry of Economy, Industry and Competitiveness, Government of Spain, respectively)

We herein report the design, synthesis, and functional impact of an okadaic acid (OA) small analogue, ITH12680, which restores the activity of phosphoprotein phosphatase 2A (PP2A), whose deficient activity has been implicated in nicotine-mediated tumor progression and chemoresistance in non-small cell lung cancer (NSCLC). For its design, we paid attention to the structure of the PP2A-OA complex, where the C16-C38 OA fragment confers PP2A affinity and selectivity, but it is not involved in the inhibitory effect. Confirming this hypothesis, PP2A activity was not inhibited by ITH12680. By contrast, the compound partially restored OA-exerted PP2A inhibition in vitro. Moreover, flow cytometry and immunoblotting experiments revealed that ITH12680 reversed nicotine-induced cisplatin resistance in NSCLC cells, as it prevented nicotine-induced reduction of Bax expression and inhibited nicotine-mediated activation of cell survival and proliferation kinases, Akt and ERK1/2. Our findings suggest that the rescue of nicotine-inhibited PP2A activity could diminish the resistance to cisplatin treatment observed in NSCLC patients who continue smoking. ispartof: Bioorganic Chemistry vol:100 ispartof: location:United States status: published

Objectives: Tobacco smoking is strongly correlated with the onset and progression of non-small cell lung cancer (NSCLC). By activating α7 nicotinic acetylcholine receptors (α7-nAChRs) in these tumors nicotine and its tobacco-derived nitrosamine, NNK, contribute to these oncogenic processes. Here, we investigated whether the human-specific duplicated form of the α7-nAChR subunit (dupα7) behaves as an endogenous negative regulator of α7-nAChR-mediated tumorigenic activity induced by tobacco in NSCLC cells, similarly to its influence on other α7-nAChR-controlled functions in non-tumor cells. Methods: Two human NSCLC cell lines, lung adenocarcinoma (A549) and squamous cell carcinoma of the lung (SK-MES-1), both wild-type or with stable overexpression of dupα7 (A549dupα7 or SK-MES-1dupα7), were used to investigate in vitro anti-tumor activity of dupα7 on nicotine- or NNK-induced tumor progression. For this purpose, migration, proliferation or epithelial-mesenchymal transition (EMT) were examined. The anti-tumor effect of dupα7 on nicotine-promoted tumor growth, proliferation or angiogenesis was also assessed in vivo in an athymic mouse model implanted with A549dupα7 or A549 xenografts. Results: Overexpression of dupα7 in both cell lines almost completely suppresses the in vitro tumor-promoting effects induced by nicotine (1 μM) or NNK (100 nM) in wild-type cells. Furthermore, in mice receiving nicotine, A549dupα7 xenografts show: (i) a significant reduction of tumor growth, and (ii) decreased expression of cell markers for proliferation (Ki67) or angiogenesis (VEGF) compared to A549 xenografts. Conclusion: Our study demonstrates, for the first time, the in vitro and in vivo anti-tumor capacity of dupα7 to block the α7-nAChR-mediated tumorigenic effects of tobacco in NSCLC, suggesting that up-regulation of dupα7 expression in these tumors could offer a potential new therapeutic target in smoking-related cancers.

The α7 nicotinic receptor subunit and its partially duplicated human-specific dupα7 isoform are coexpressed in neuronal and non-neuronal cells. In these cells, α7 subunits form homopentameric α7 nicotinic acetylcholine receptors (α7-nAChRs) implicated in numerous pathologies. In immune cells, α7-nAChRs are essential for vagal control of inflammatory response in sepsis. Recent studies show that the dupα7 subunit is a dominant-negative regulator of α7-nAChR activity in Xenopus oocytes. However, its biological significance in mammalian cells, particularly immune cells, remains unexplored, as the duplicated form is indistinguishable from the original subunit in standard tests. Here, using immunocytochemistry, confocal microscopy, coimmunoprecipitation, FRET, flow cytometry, and ELISA, we addressed this challenge in GH4C1 rat pituitary cells and RAW264.7 murine macrophages transfected with epitope- and fluorescent protein-tagged α7 or dupα7. We used quantitative RT-PCR of dupα7 gene expression levels in peripheral blood mononuclear cells (PBMCs) from patients with sepsis to analyze its relationship with PBMC α7 mRNA levels and with serum concentrations of inflammatory markers. We found that a physical interaction between dupα7 and α7 subunits in both cell lines generates heteromeric nAChRs that remain mainly trapped in the endoplasmic reticulum. The dupα7 sequestration of α7 subunits reduced membrane expression of functional α7-nAChRs, attenuating their anti-inflammatory capacity in lipopolysaccharide-stimulated macrophages. Moreover, the PBMC's dupα7 levels correlated inversely with their α7 levels and directly with the magnitude of the patients' inflammatory state. These results indicate that dupα7 probably reduces human vagal anti-inflammatory responses and suggest its involvement in other α7-nAChR-mediated pathophysiological processes., This work was supported in part by Ministry of Economy, Industry and Competitiveness, Government of Spain Grants SAF2011-23575, SAF2014-56623-R, and SAF2017-82689-R (to C. M. and F. A.).

The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.

This study explores the presence of mentoring relationships among a sample of 56 mental health professionals in a large county mental health agency. Participants completed the Alleman Mentoring Scales Questionnaire in an online survey. Individuals involved in mentoring relationships experienced more career and psychological enhancing functions in the work place than those who were not in mentoring relationships. Mentoring relationships predicted career and personal development of mental health professionals. Recommendations include incorporating mentoring programs in mental health agencies in order to increase social workers' competence in their jobs.

This article discusses a creative endeavor to establish a university social groupwork club affi liated with the Association for the Advancement of Social Work with Groups, an international professional association. The club's purposes were: 1) to nurture social groupwork; 2) to engage students, practitioners and academics in a collaborative groupwork experience, and; 3) to link members to an international social work organization that supports all aspects of groupwork. The planning, stages, mutual aid and evaluation of the club are described. A longitudinal survey design was used to evaluate members' perceptions of the club. Feedback questionnaires (N = 129) were analyzed for a one-year period. Results indicated that club members benefi ted from mutual aid and experienced professional growth.

Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1beta (IL-1beta) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1beta effect on type A gamma-aminobutyric acid receptors (GABA(A)Rs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABA(A)Rs, respectively. Confocal images in both cell types revealed that IL-1beta increases recruitment of GABA(A)Rs to the cell surface. Moreover, brief applications of IL-1beta to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (I(GABA)); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with I(GABA) recording and confocal images of GABA(A) Rs in oocytes showed that IL-1beta stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABA(A)Rs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABA(A)R, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1beta with increased "GABAergic tone." We propose that through this mechanism IL-1beta might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE.

Understanding precisely the functioning of voltage-gated Ca2+ channels and their modulation by signaling molecules will help clarifying the Ca2+-dependent mechanisms controlling exocytosis in chromaffin cells. In recent years, we have learned more about the various pathways through which Ca2+ channels can be up- or down-modulated by hormones and neurotransmitters and how these changes may condition chromaffin cell activity and catecolamine release. Recently, the attention has been focused on the modulation of L-channels (Cav 1), which represent the major Ca2+ current component in rat and human chromaffin cells. L-channels are effectively inhibited by the released content of secretory granules or by applying mixtures of exogenous ATP, opioids, and adrenaline through the activation of receptor-coupled G proteins. This unusual inhibition persists in a wide range of potentials and results from a direct (membrane-delimited) interaction of G protein subunits with the L-channels co-localized in membrane microareas. Inhibition of L-channels can be reversed when the cAMP/PKA pathway is activated by membrane permeable cAMP analog or when cells are exposed to isoprenaline (remote action), suggesting the existence of parallel and opposite effects on L-channel gating by distinctly activated membrane autoreceptors. Here, the authors review the molecular components underlying these two opposing signaling pathways and present new evidence supporting the presence of two L-channel types in rat chromaffin cells (α1C and α1D), which open new interesting issues concerning Ca2+-channel modulation. In light of recent findings on the regulation of exocytosis by Ca2+-channel modulation, the authors explore the possible role of L-channels in the autocontrol of catecholamine release.

Se presentan dos casos de actinomicosis abdominal relacionada a dispositivo intrauterino de 12 y 13 anos de uso, sin seguimiento ginecologico, que se presentaron como abdomen agudo quirurgico no sospechandose el origen bacteriologico en primera instancia.

Several genes coding for different cytokines may affect host susceptibility to tuberculosis. This study investigates the relationship of the single base change polymorphic variants identified in the first intron of interferon-gamma (+874 T/A) and in the promoter region of interleukin-10 gene (-1,082 G/A), with cytokine production by peripheral blood mononuclear cells and tuberculosis susceptibility. We studied a Spanish population of 113 patients with culture-proven pulmonary tuberculosis, 207 healthy close contacts (125 tuberculin reactive and 82 tuberculin negative), and 100 healthy tuberculin-negative control subjects. Multiple logistic regression analysis showed that individuals homozygous for the interferon-gamma (+874) A allele had a 3.75-fold increased risk of developing tuberculosis (95% confidence interval, 2.26-6.23, p = 0.0017). Stimulated production of interferon-gamma by peripheral mononuclear cells from patients with genotype AA was depressed compared with that of non-AA homozygotes at the time of diagnosis and after completion of therapy. Multivariate analysis showed that the presence of an AA genotype and the absolute number of lymphocytes were the only independent predictors of interferon-gamma production. In contrast, the different rates of interleukin-10 production associated with the interleukin-10 polymorphism did not affect susceptibility to tuberculosis. Thus, a genetic defect in the production of interferon-gamma in individuals homozygous for the (+874) A allele could contribute to their increased risk of developing tuberculosis.

The contribution of distinct Ca(2+)-sensitive protein kinases to the regulation of the expression of the synaptosomal-associated protein SNAP-25 was examined in bovine chromaffin cells. Prolonged incubation with high K(+) (38 mM) or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic receptor agonist, significantly increased SNAP-25 protein and mRNA expression, as assessed by immunoblotting and semi-quantitative RT-PCR analysis. Both stimuli preferentially enhanced mRNA coding for the SNAP-25a isoform. Increase of SNAP-25 expression induced by K(+) or DMPP was inhibited over 70% by KN-62 and KN-93, two Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitors, whereas the inactive analogue KN-92 only reduced the expression by 34%. The three compounds also inhibited the high K(+)-elicited [Ca(2+)](i) signal by 40%, suggesting that the effect of KN-62 and KN-93 was a combination of CaMK/ Ca(2+) influx inhibitory actions. Incubation of the cells with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126 reduced protein expression elicited by high K(+) by 50%, but did not modify the response to DMPP. Interestingly, although protein kinase A (PKA) inhibition by H-89 did not affect the high K(+) or DMPP-induced SNAP-25 expression, basal protein levels were significantly modified upon activation or inhibition of this pathway. Basal expression of SNAP-25 was also modified by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, but not by Go6976, a PKC-alpha inhibitor, suggesting that the Ca(2+)-insensitive PKC-epsilon isoform control basal expression of SNAP-25 in these cells. Taken together, these results provide the first evidence that diverse protein kinases might converge in the induction of SNAP-25 expression in chromaffin cells. The preferential contribution of one or another kinase would depend on the physiological or experimental conditions.

Simultaneous recordings of inward whole-cell Ca(2+) channel currents (I(Ca) ) and increments of capacitance as an indication of exocytosis (Delta(Cm)), were performed in voltage-clamped single adrenal chromaffin cells from wild-type and alpha(1A) subunit deficient mice, using the perforated-patch configuration of the patch-clamp technique. Using protocol #1 (one single Ca(2+) channel blocker per cell), to dissect the components of I(Ca), L channels contributed 43%, N channels 35% and P/Q channels 30% to the total I(Ca) of wild-type cells. Using protocol #2 (cumulative sequential addition of 3 microm nifedipine, 1 microm omega-conotoxin GVIA, and 1 microm omega-agatoxin IVA), L, N and P/Q channels contributed 40%, 34% and 14%, respectively, to I(Ca); an R component of around 11% remained. In wild-type mice the changes of Delta(Cm) paralleled those of I(Ca). In alpha(1A) deficient mice the L component of I(Ca) rose to 53% while the P/Q disappeared; the N and R components were similar. In these mice, Delta(Cm) associated to N and R channels did not vary; however, the P/Q component was abolished while the L component increased by 20%. In conclusion, exocytosis was proportional to the relative density of each Ca(2+) channel subtype, L, N, P/Q, R. Ablation of the alpha(1A) gene led to a loss of P/Q channel current and to a compensatory increase of L channel-associated secretion; however, this compensation was not sufficient to maintain the overall exocytotic response, that was diminished by 35% in alpha(1A) -deficient mice. This may be due to altered Ca(2+) homeostasis in these mice, as compared to wild mouse chromaffin cells.

Summary This study aims to determine the influence of the polymorphism within the intron 2 of the interleukin-1 receptor antagonist gene (IL-1RN*) on the outcome of severe sepsis, and to assess its functional significance by correlating this polymorphism with the total production of interleukin-1 receptor antagonist (IL-1Ra) protein determined in stimulated peripheral blood mononuclear cells (PBMC). A group of 78 patients with severe sepsis (51 survivors and 27 nonsurvivors) was compared with a healthy control group of 130 blood donors, and 56 patients with uncomplicated pneumonia. We found a significant association between IL-1RN* polymorphism and survival. Thus, after adjusting for age and APACHE II score, multiple logistic regression analysis showed that patients homozygotes for the allele *2 had a 6·47-fold increased risk of death (95% CI 1·01–41·47, P = 0·04). Besides, compared with patients homozygous or heterozygous for the allele *1, IL-1RN*2 homozygotes produced significantly lower levels of IL-1Ra from their PBMC. Our results suggest that insufficient production of this cytokine might contribute, among other factors, to the higher mortality rate found in severe sepsis patients with the IL-1RN*2 homozygous genotype.

The catestatin fragment of chromogranin A is the first known endogenous compound able to inhibit catecholamine release elicited by the activation of neuronal nicotinic acetylcholine receptors (nAChRs) of different animal species and catecholaminergic cell types. However, how catestatin regulates the receptor activity, which subunit combination of the heteropentameric forms of receptor is better blocked by the peptide, or how it affects the different stages of the exocytotic process have not yet been evaluated. To address these questions, we have assayed the effects of catestatin: (first) on the inward currents elicited by ACh (I(ACh)) in voltage-clamped oocytes expressing different combinations of nAChR subunits; and (second) on the cytosolic Ca(2+) concentration, [Ca(2+)](c), and quantal release of catecholamines simultaneously monitored in single adrenal chromaffin cells stimulated with ACh. Catestatin potently blocks all the subtypes of nAChRs studied. Furthermore, it inhibits the α(3)β(4) current in a reversible, noncompetitive, voltage-, and use-dependent manner, a behavior compatible with open-channel blockade. In fura-2-loaded single chromaffin cells, the peptide reduced the [Ca(2+)](c) signal and the total release of catecholamines elicited by ACh; however, catestatin did not modify the kinetics or the last step of the exocytotic process. Our results suggest that catestatin might play an autocrine regulatory role in neuroendocrine secretion through its interaction with different native nAChR subtypes; the extent of receptor blockade by the peptide could be acutely regulated by the intensity and duration of the presynaptic stimulus.

et al., [Background]:Stimulation of the vagus nerve in the so-called cholinergic antiinflammatory pathway (CAP) attenuates systemic inflammation, improving survival in animal sepsis models via α7 nicotinic acetylcholine receptors on immunocompetent cells. Because the relevance of this regulatory pathway is unknown in human sepsis, this pilot study assessed whether the α7 gene expression level in septic patients' peripheral blood mononuclear cells (PBMC) might be used to assess CAP activity and clinical outcome. [Methods]:The PBMCs α7 messenger RNA levels were determined by real-time quantitative reverse-transcription polymerase chain reaction in 33 controls and 33 patients at enrollment and after their hospital discharge. Data were analyzed to find significant associations between α7 level, vagally mediated heart rate variability as an indirect reflection of CAP activity, serum concentrations of different inflammation markers, and clinical course. [Results]: Septic patients' α7 levels were significantly increased and returned to control values after recovery. These α7 levels correlated directly with the vagal heart input and inversely with the magnitude of the patient's inflammatory state, disease severity, and clinical outcome. [Conclusions]:This study reveals that the PBMC α7 gene expression level is a clinically relevant marker for CAP activity in sepsis: the higher the α7 expression, the better the inflammation control and the prognosis., This work was supported by the Spanish Ministerio de Ciencia e Innovación (grant SAF2011-23575 to C. M. and F. A.); the Fundación Mutua Madrileña Investigación Biomédica (grant FMM2011 to C. M. and F. A.); Spanish Ministerio de Educación, Cultura y Deporte (Fellowship of Formación de Personal Universitario to J. L. C.); Spanish Ministerio de Economía y Competitividad (Fellowship of Formación Personal Investigador to C. M. S.), and Chilean Ministerio de Educación (Chle Fellowship to M. C. M.).

Since moderate hyperhomocysteinemia is an independent risk factor for vascular disease by mean of its oxidant effect and glutathione plays a main role as intracellular redox-regulating agent, we have studied for the first time the total intracellular content of homocysteine in aging. Plasma homocysteine concentration, total intracellular and plasma glutathione, and other related thiol compounds such as cysteine and the glutathione catabolite cysteinglycine were also studied. Forty three healthy elderly subjects and twenty seven healthy young ones were studied. The total intracellular peripheral blood mononuclear cell content was higher for homocysteine, cysteine and cysteinglycine, whereas that of the total glutathione was greatly decreased in elderly people with respect to young ones. Elderly subjects showed significantly higher levels than young ones of total plasma homocysteine and cysteinglycine, but not cysteine, whereas total plasma glutathione levels were increased. In addition, elderly subjects showed significantly decreased plasma vitamin E levels and increased concentrations of serum lipid peroxides measured as TBARS (reaction product of malondialdehyde with thiobarbituric acid). The intracellular glutathione content presented significantly negative correlation with serum TBARS, and intracellular and plasma homocysteine levels. These findings show an increase of homocysteine synthesis associated with aging, which in turn can produce an augmented oxidant effect on endothelium, and an impaired intracellular antioxidant capacity leading to an enhanced lipid peroxidation and decreased total intracellular glutathione content.

We studied the effects of the novel Na(+)/Ca(2+) exchange inhibitor KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes. As expected, KB-R7943 blocked the Na(+)-gradient dependent (45)Ca(2+) uptake into chromaffin cells (IC(50) of 5.5 microM); but in addition, the compound also inhibited the (45)Ca(2+) entry and the increase of cytosolic Ca(2+) concentration, [Ca(2+)](c), stimulated by 5 s pulses of ACh (IC(50) of 6.5 and 1.7 microM, respectively). In oocytes expressing alpha(3)beta(4) and alpha(7) nicotinic AChRs, voltage-clamped at -60 mV, inward currents elicited by 1 s pulses of 100 microM ACh (I(ACh)) were blocked by KB-R7943 with an IC(50) of 0.4 microM and a Hill coefficient of 0.9. Blockade of alpha(3)beta(4) currents by KB-R7943 was noncompetitive; moreover, the blocker (0.3 microM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 microM and 1 mM ACh, respectively). Inhibition of alpha(3)beta(4) currents by 0.3 microM KB-R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 microM), the inhibition amounted to 33, 64 and 80% in oocytes voltage-clamped at -40, -60 and -100 mV, respectively. The onset of blockade was faster and the recovery slower at -100 mV; the reverse was true at -40 mV. In conclusion, KB-R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open-channel blocker at the rat brain alpha(3)beta(4) AChR. These results should be considered when KB-R7943 is to be used to study Ca(2+) homeostasis in cells expressing nicotinic AChRs and the Na(+)/Ca(2+) exchanger.

Rat α3β4 or α7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non-toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 μM) applied at regular intervals. The N/P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC inhibited α3β4 currents with an IC50 of 1.3 μM; the blockade was non-competitive and reversible. The α7 currents were unaffected. At 1 μM, ω-conotoxin GVIA (N-type Ca2+ channel blocker) inhibited by 24 and 20% α3β4 and α7 currents, respectively. At 1 μM, ω-agatoxin IVA (a P/Q-type Ca2+ channel blocker) did not affect α7 currents and inhibited α3β4 currents by only 15%. L-type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on α3β4 nicotinic AChRs. The mechanism of α3β4 currents blockade by ω-conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage-dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while ω-toxins did not. These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist. British Journal of Pharmacology (1999) 127, 1375–1387; doi:10.1038/sj.bjp.0702692

We have studied capacitative Ca2+ entry into Xenopus oocytes by depleting intracellular Ca2+ stores with inositol 1,4,5-trisphosphate or thapsigargin. Capacitative Ca2+ entry was evoked by hyperpolarisation and monitored via the Ca(2+)-activated Cl- current. Hyperpolarisation-evoked currents increased with extracellular [Ca2+] in the range 0.9-5 mM, and were reversibly inhibited by extracellular Mg2+ (0.1-10 mM) by up to 60%. Currents were decreased by the voltage-gated Ca2+ channel antagonists omega-conotoxin GVIA, MVIIA and MVIIC (0.3-10 microM) and the inhibition of Ca2+ entry in individual oocytes by omega-conotoxins GVIA and MVIIA was highly heterogeneous, but not additive. Flunarizine (10 microM) and the imidazoles SKF 96365 (10 microM), miconazole (40 microM) and econazole (40 microM) partly blocked Ca2+ entry. Ca2+ entry was unaffected by calciseptine (300 nM) or alpha-bungarotoxin (1 microM). The possibility that these compounds might inhibit the Ca(2+)-activated Cl- current rather than capacitative Ca2+ entry itself was examined by recording the Cl- current activated by the increase in [Ca2+]i activated by the flash photolysis of caged Ca2+. Eicosatetraynoic acid (2-10 microM) markedly inhibited, and La3+ (1 mM but not 100 microM) potentiated the increase in Ca(2+)-activated Cl- current. In contrast, omega-conotoxins and Mg2+ had no effect on the Ca(2+)-activated Cl- current itself. These findings support the hypothesis that capacitative Ca2+ entry into Xenopus oocytes occurs through channels with a pharmacology similar to that of neuronal non-L type voltage-gated Ca2+ channels.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the 'endotoxin tolerant' phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases., This work was supported by grants to C. Montiel and F. Arnalich from the Ministerio de Economía y Competitividad, Government of Spain (SAF2011-23575) and Fundación Mutua Madrileña (FMM2011), Spain.

In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 microM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 microM, respectively. Flunarizine inhibited both types of contractions with IC50 values of 1.7 microM for 5-HT and 2.4 microM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2(+)-activated Cl- channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 microM dotarizine and blocked around 62% by 10 microM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 microM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.

OBJECTIVE To examine the relation of circulating appetite neuropeptides, CCK-8 sulphate (CCK-8s) and beta-endorphin, and the tumour necrosis factor-alpha (TNF-alpha) and soluble TNF receptors (sTNFR) to the anorexia and wasting associated with HIV-infection. DESIGN Cross-sectional analysis. SETTING A university-based HIV/AIDS ambulatory clinic in Madrid, Spain. PARTICIPANTS Thirty-six randomly selected AIDS patients without concomitant diseases or secondary infections were classified into two groups: 19 patients with wasting and 17 with normal body weight, and 18 healthy controls. MEASUREMENTS Nutritional status was evaluated by anthropometry, laboratory parameters and self-report of appetite. Plasma levels of TNF-alpha and sTNFR proteins p55 (sTNFR-p55) and p75 (sTNFR-p75) were determined by enzyme immunoassay, whereas CCK-8s and beta-endorphin levels were measured by radioimmunoassay. RESULTS AIDS patients with wasting had significantly higher plasma concentrations of CCK-8s, but lower levels of beta-endorphin when compared to well-nourished AIDS patients (P < 0.01) or controls (P < 0.001). Mean levels of TNF-alpha, and sTNFR-p55 and sTNFR-p75 were greater in AIDS patients with wasting than in asymptomatic AIDS patients or in controls. No significant association was observed between any of these circulating peptides and the parameters of malnutrition. CONCLUSIONS An activation of the TNF system, together with reciprocal changes in plasma concentrations of two neuropeptides with opposing appetite regulation, that is increased concentrations of CCK-8s but lower levels of beta-endorphin, are associated with the presence of HIV wasting. We hypothesize that these changes may contribute to the development of HIV wasting by producing a pathological inhibition of appetite.

Three objectives were defined when planning this study: (i) to identify binding sites for [ 125 I]-apamin in intact bovine adrenal medulla chromaffin cells and to estimate their density and selectivity; (ii) to determine whether apamin modified the release of catecholamines evoked by brief pulses of dimethylphenylpiperazinium (DMPP, 1 or 5 μM for 10 sec), histamine (10 μM for 10 sec) or high K + (20, 35 or 70 mM for 10 sec) applied to superfused cells; and (iii) to test whether apamin affected the profiles of the changes in cytosolic Ca 2+ concentrations [Ca 2+ ] i obtained in suspensions of cells loaded with fura-2 and stimulated with DMPP or histamine. At equilibrium, increasing concentrations of [ 125 I]-apamin gave a saturation curve whose Scatchard transformation produced a K d of 132 pM and a B max of 0.72 fmol/10 6 cells. Quinine, tetraethylammonium, charybdotoxin or glibenclamide (blockers of various subtypes of K + channels) did not inhibit [ 125 I]apamin binding. Binding was blocked by apamin and by d -tubocurarine, two blockers of small-conductance Ca 2+ -activated K + channels (SK channels). The number of binding sites for [ 125 I]apamin amounted to approx. 900 per single chromaffin cell, 0.72 sites per μm 2 surface area. Apamin (1 μM) enhanced the secretory response to histamine (10 μM), DMPP (1 or 5 μM) and high K + (20 or 35 mM) by 2–3-fold. The response to 70 mM K + , however, was unaffected. Apamin also enhanced the peak [Ca 2+ ] i increase produced by DMPP or histamine by approx. 30%. Overall, these results strongly support the hypothesis that under physiological conditions, SK channels control some of the electrical activity of chromaffin cells and indirectly, the opening of voltage-dependent Ca 2+ channels, the access of Ca 2+ to the secretory machinery and the rate of catecholamine release to the circulation from the intact adrenal gland.

This study was carried out to define the effects of various Ca2+ channel modulatory drugs on mitochondrial Ca2+ movements. Bovine adrenal medulla mitochondria took up Ca2+ at an initial rate of 6.8 nmol mg protein-1 5 s-1, with a Km of 15 microM and a Bmax of 30 nmol mg protein-1. At 30 microM, neither verapamil, diltiazem, nitrendipine nor Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] affected the initial rate of Ca2+ uptake. Ca(2+)-loaded mitochondria retained their Ca2+ contents in the presence of ruthenium red for at least 30 min. Cinnarizine and flunarizine, but not verapamil, diltiazem, isradipine, Bay K 8644 or nitrendipine, caused a fast and dramatic Na(+)-independent Ca2+ loss. Other Ca2+ channel antagonists assayed such as penfluridol, R56865 [N-[1-(4-(4-fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2- benzothiazolamine], lidoflazine, R87926 [(+)-(S)-4-(2-benzothiazolyl-methylamino)-alpha-[(3,4-difluorophenoxy ) methyl] 1 piperidine] and sabeluzole, also had a mitochondrial Ca2+ depleting effect which seemed to be directly related to their octanol/water partition coefficient. The Na(+)-dependent Ca2+ efflux from mitochondria was completely inhibited by diltiazem and greatly blocked by nitrendipine. Isradipine caused a moderate blockade and Bay K 8644 and verapamil had no effect. All these data open the possibility of developing novel Ca2+ channel antagonists having selective actions on plasmalemmal Ca2+ channels, and others with additional and different effects on mitochondrial Ca2+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)

El pdf del artículo es el manuscrito de autor.-- et al., [Introduction]: Plasma vascular endothelial growth factor (VEGF) was shown to increase during acute hypoglycemia and could mediate rapid adaptation of the brain. In this study we examined the neuroendocrine response in patients with type 2 diabetes mellitus (T2DM) in hypoglycemic coma or with acute neuroglycopenic symptoms. [Methods]: We prospectively studied 135 consecutive T2DM patients admitted for severe hypoglycemia during a 2-year period. We collected clinical variables and measured plasma concentrations of VEGF, epinephrine, norepinephrine, cortisol and growth hormone at admission and 30. min afterwards. [Results]: Thirty two patients developed hypoglycemic coma and 103 did not lose consciousness. Median plasma VEGF level of coma patients was 3.1-fold lower at baseline than that of non-coma patients, and even 5.3-fold lower 30. min afterwards. Plasma epinephrine concentration was significantly lower just at baseline in coma patients. On the contrary, there were no differences in concentrations of the other hormones. Multivariate logistic regression analysis showed that VEGF concentration (OR 0.68; CI 0.51-0.95) was a protective factor against the development of coma. [Conclusions]: VEGF and epinephrine responses to acute hypoglycemia are reduced in T2DM patients who develop hypoglycemic coma. An increased plasma VEGF concentration appeared to be a protective factor against the development of hypoglycemic coma. © 2011 Elsevier Ltd., This work was supported by a Grant from Plan Nacional I+D+I (SAF 2008-05347), and from Fundación Mutua Madrileña de Investigación Médica to Francisco Arnalich (PI 8945/2011) and to Carmen Montiel (research project 2008).

We describe here the effects of PCA50941 (a novel 1,4-dihydropyridine derivative) comparatively with Bay K 8644 on various parameters in bovine adrenal chromaffin cells. The binding of [ 3 H](+)-isradipine to bovine adrenal medulla plasma membranes was inhibited similarly by PCA50941 and Bay K 8644 at various [Ca 2+ ] o suggesting a common binding site for both compounds on the dihydropyridine receptor. In voltage-clamped chromaffin cells PCA50941 (1 μM) and Bay K 8644 (1 μM) shifted the I-V relationship of whole-cell Ca 2+ currents by about 5–10 mV towards more hyperpolarizing potentials. At −20 mV, PCA50941 enhanced I Ca by 195 ± 16% and Bay K 8644 by 288 ± 51%. Stimulation of fura 2-loaded chromaffin cell suspensions with 17.7 K + /0.5 Ca 2+ increased 3-fold the basal [Ca 2+ ] i . PCA50941 increased further the K + -evoked peak to 655 nM, and Bay K 8644 to 1129 nM. In the presence of 5 mM Ca 2+ , PCA50941 or Bay K 8644 increased the [Ca 2+ ] peaks to 427 and 350 nM, respectively. PCA50941 potentiated the release of catecholamines from perfused bovine adrenal glands evoked by 30 s pulses of 17.7 mM K + in a manner dependent on the [Ca 2+ ] o . Thus at 1, 2.5, 5 and 10 mM Ca 2+ , secretion was 2.3-, 3.8-, 5- and 4-fold greater than in control glands. Bay K 8644 enhanced the K + -induced response 3- and 9-fold at [Ca 2+ ] o of 0.25 or 0.5 mM, respectively; at higher [Ca 2+ ] o the potentiation was similar to that of PCA50941. The results support the idea that Bay K 8644 and the novel dihydropyridine derivative PCA50941 potentiates the K + -evoked release of catecholamines from bovine chromaffin cells by enhancing Ca 2+ entry through L-type Ca 2+ channels.

Introduction Concentrations of plasma cell-free DNA are increased in various diseases and have shown some prognostic value in many patient groups, including critically ill patients. Pathophysiological processes behind the need for mechanical ventilation and the treatment itself could raise plasma levels of cell-free DNA. We evaluated levels of plasma cell-free DNA and their prognostic value in patients needing mechanical ventilation. Methods We studied prospectively 580 mechanically ventilated critically ill patients. Blood samples were taken at study admission (Day 0) and on Day 2. Plasma cell-free DNA concentrations were measured by real-time quantitative PCR assay for the β-globin gene and are expressed as genome equivalents (GE)/ml. Results Median (interquartile range, IQR) plasma cell-free DNA concentration was 11,853 GE/ml (5,304 to 24,620 GE/mL) at study admission, and 11,610 GE/mL (6,411 to 21,558 GE/mL) on Day 2. Concentrations at admission were significantly higher in 90-day non-survivors than survivors, 16,936 GE/mL (7,262 to 46,866 GE/mL) versus 10,026 GE/mL (4,870 to 19,820 GE/mL), P < 0.001. In a multivariate logistic regression analysis plasma cell-free DNA concentration over 16,000 GE/ml remained an independent predictor of 90-day mortality (adjusted odds ratio 2.16, 95% confidence interval CI 1.37 to 3.40). Positive likelihood ratio of plasma cell-free DNA at admission for the prediction of 90-day mortality was 1.72 (95% CI 1.40 to 2.11). Conclusions Plasma levels of cell-free DNA were significantly higher in non-survivors than survivors. Plasma DNA level at baseline was an independent predictor of 90-day mortality. However, its clinical benefit as a prognostic marker seems to be limited.

Prolonged stimulation with nicotine (50 μM) enhanced the secretion of catecholamines from perfused cat adrenal glands. The profile of secretion consisted of a quick activation phase to peak of 7.68 μg/min followed by a second inactivation phase which exhibited a t 1 2 of 3.75 min. Sustained stimulation with a solution enriched in K+ (59 mM) also evoked a transient secretory response, with a peak release of 8.62 μg/2 min and a t 1 2 for inactivation of 4.8 min. Co2+ (10 mM) blocked the nicotinic response by 58% and the K+-evoked secretory response by over 96%. In the presence of Co2+; (5 mM), continuous perfusion with nicotine produced a transient but large initial secretory response; the gradual decrease of the extracellular Co2+ concentration, [Co2+]0, as a continuous ramp allowed the development of a second component of secretion which inactivated later on. When the glands were continuously stimulated with 59 mM K+ in the presence of Co2+, the first component of secretion was missing: the second component appeared as [Co2+]0 decreases as a ramp. In similar experiments performed in low-Na+ solution (10 mM Na+), only the first secretion component evoked by nicotine was observed. This finding suggests that the second component of secretion depends on Na+ entry through the nicotinic receptor, on the ensuing cell depolarization and on Ca2+ entry through voltage-dependent Ca2+ channels. These data are compatible with the idea that the nicotinic receptor-mediated catecholamine secretory response in the cat adrenal chromaffin cell has two components: the first seem to be triggered by Ca2+ entry through the nicotinic receptor channels, and the second by Ca2+ entry through voltage-dependent Ca2+ channels. The data also suggest that Co2+ ions protect against the Ca2+-dependent desensitization of nicotinic receptors.

The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.

Diagnosing patients with acute mesenteric ischemia (AMI) in the emergency ward is challenging. This study assesses the usefulness of plasma DNA in patients with clinically suspected AMI.130 consecutive patients who underwent laparotomy were studied. Cell-free plasma DNA was measured by real-time quantitative PCR assay for the beta-globin gene. The primary endpoint was the accuracy of plasma DNA for predicting 30-day mortality.Surgery revealed AMI in 99 patients and alternative diagnoses in 31 patients. Forty-six patients with AMI died (46.6%) as compared to 6 (19.4%) in the non-AMI group (p0.05). The DNA concentration at admission was significantly higher in patients with AMI (median 7340 GE/ml, versus, 2735 GE/ml, p0.01) and in AMI patients who died (8830 GE/ml, versus 4970 GE/ml, p0.05). The area under the ROC curves for plasma DNA as a marker for mesenteric ischemia and independent predictor for 30-day mortality were 0.708 (95% CI 0.701-0.890) and 0.815 (95% CI 0.735-0.894). Multiple logistic regression analysis showed that the risk of hospital mortality increased 1.52-fold for every 1000 GE/ml increase in plasma DNA.Plasma DNA levels may be a useful biomarker in predicting the outcome of patients with AMI.

A pilot study examined the relationship between job satisfaction and perceived mentoring among 56 mental health supervisors and practitioners in a county mental health agency. Participants completed the Alleman Mentoring Activities Questionnaires and the Job Descriptive Index and Job in General Scale. Practitioners who perceived they were involved in mentoring relationships with supervisors were more satisfied with their jobs than those who perceived that they were not involved in mentoring relationships. The mentoring functions of sponsoring, assigning challenging tasks, and demonstrating trust predicted job satisfaction. Recommendations include incorporating mentoring functions in supervisory training to increase mental health professionals’ job satisfaction.