Recombinant Collagen Engineered to Bind to Discoidin Domain Receptors Functions as a Receptor Inhibitor

A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities

A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets

Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcgammaRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca(2+) concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcgammaRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the G(i)-coupled ADP receptor P2Y12, but was not affected by blockade of the G(q)-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca(2+) mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca(2+) signaling. In platelets stimulated by cross-linking of FcgammaRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the G(i)-coupled alpha(2A)-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a G(q)-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the G(q)-coupled thromboxane A(2) receptor by was completely inhibited by ADP scavengers under conditions in which intracellular Ca(2+) mobilization was unaffected. Inhibition of -induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a G(i)-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B

Prenatal Exposure to Ethanol Causes Differential Effects in Nerve Growth Factor and its Receptor in the Basal Forebrain of Preweaning and Adult Rats

In this study we investigated nerve growth factor (NGF) levels in the cortex and hippocampus of the offspring of pregnant female Sprague-Dawley rats receiving a single intragastric administration of acute ethanol on the 15th day of gestation and compared them with a control group of rats that received an injection of sucrose. We also examined the distribution of the low-affinity NGF receptor, p75NGFR, on NGF-responsive neurons that are localized in the septum and the nucleus of Meynert, which receive the respective trophic support from the hippocampus and the cortex. In the ethanol-treated group, the results show that at post-natal age 15 days, the NGF septohippocampal pathways were markedly affected. At day 15, the NGF level was significantly higher in the offspring of ethanol-treated rats. By day 40, NGF values in both groups decreased to similar levels. At day 60, however, the NGF level in the ethanol-treated animals decreased to a significantly lower value than that of the control group, which remained essentially unchanged. In parallel, at day 60 the numbers of septal cholinergic neurons expressing p75NGFR were also significantly lower in ethanol-treated rats than in control animals. Because ethanol is known to induce neurological disorders, as well as deficits in cell proliferation and differentiation, the results suggest that one cause of the deleterious effects induced by ethanol is the low availability of NGF during certain stages of postnatal brain development

Control de inventarios en una Pyme de indumentaria femenina

1. INTRODUCCIÓN - 1.1 PROBLEMA - 1.2 MARCO TEÓRICO - 1.3 METODOLOGÍA - 1.4 OBJETIVOS DEL TRABAJO - 1.4 LÍMITES O ALCANCE DEL TRABAJO - 2. REVISIÓN BIBLIOGRÁFICA - 2.1 INVENTARIOS - 2.2 ITEMS INDIVIDUALES O STOCK KEEPING UNITS - 2.3 COSTOS DEL INVENTARIO - 2.4 OTROS FACTORES DE IMPORTANCIA - 2.4.1 Demanda - 2.4.2 El tiempo de reposición (Lead Time) (L) - 2.5 SISTEMAS DE INVENTARIOS - 2.5.1 Modelo de inventario para un solo período - 2.5.2 Sistemas de inventarios para varios períodos - 2.5.3 Modelo EOQ - 2.5.4 Modelo de la cantidad fija de la orden con existencias de reserva - 2.6 MODELO PARA PERÍODOS FIJOS DE TIEMPO - 2.6.1 Modelos para periodos fijos con existencias de reserva - 2.6 PRONÓSTICO DE DEMANDA - 2.6.1 Características de los pronósticos - 2.6.2 Métodos de pronóstico - 2.6.3 Método utilizado para pronosticar la demanda por series de tiempo - 2.7 MÉTODOS - 2.7.1 Sistemas de pronósticos para demanda estacional; Modelo Holt Winter - 2.7.2 Medidas de errores de pronósticos - 2.7.3 Estimación de los errores de pronóstico extendidos al lead time - 2.8 DISTRIBUCIONES DE TALLES - 3. METODOLOGÍA EMPLEADA - 3.1 DISTRIBUCIÓN DE TALLES - 3.2 CLASIFICACIÓN A, B, C - 3.3 CÁLCULO DE EOQ Y PUNTO DE REORDEN - 3.4 PRONÓSTICOS DE DEMANDA - 3.5 IMPACTO FINANCIERO - 4. DESARROLLO - 4.1 DISTRIBUCIÓN DE TALLES - 4.2 ANÁLISIS ABC - 4.2 CÁLCULO DE EOQ Y PUNTO DE REORDEN - 4.3 PRONÓSTICOS DE DEMANDA - 4.4 CÁLCULO DEL EOQ Y PUNTO DE REORDEN - 4.5 IMPACTO FINANCIERO - 5. LÍMITES DURANTE EL TRABAJO - 6. CONCLUSIONES - 7. BIBLIOGRAFÍA - ANEXO I -El siguiente proyecto se sitúa en un comercio, ubicado en la provincia de La Rioja. El mismo se dedica a la compra y venta de indumentaria femenina. Sostiene el nombre de fantasía “Le chic”. Le chic tiene una trayectoria de 15 años en el rubro, es una empresa familiar compuesta por su dueño, fundador y responsable del área de finanzas y gerencia general de la misma, Cr. Balduini Gerardo, junto a la Cra. Salman María que se encarga del área de ventas, compra y reposición de mercadería. Le chic cuenta con tres empleadas dedicadas a las ventas, con 4 horas de trabajo establecidas por cada una, repartiéndose los turnos en cuanto a conveniencia y necesidades de la empresa. Todas reciben un monto fijo de pago mensual, y en las épocas del año de fiestas o acontecimientos importantes (esencialmente noviembre, diciembre y enero,) se contrata a corto plazo una empleada extra para cubrir la creciente clientela estacional. Las compras de mercadería se realizan en base a la experiencia de los dueños, sin un sistema fijo de control de inventarios, y sin conocer de manera cuantitativa la demanda que enfrentan. Las compras se realizan desde tres puntos distintos del país, Córdoba, Buenos Aires y Santa Fe, haciendo encargos de las mismas o realizando viajes (ya que al tratarse de ropa, el tacto y la personalización es importante) comprando y trayendo la misma en el mismo viaje. Tomando en cuenta el tiempo de viaje o encargo, la mercadería está disponible en el comercio 48hs después de realizado el pedido, y en exhibición otras 24hs después de articularla y fijarle un precio. El siguiente trabajo se realiza tras analizar el comportamiento del comercio después de tantos años de funcionamiento y con una creciente cantidad de mercadería (2000 Prendas en stock aproximadamente). Sin embargo, el mismo no cuenta aún con un óptimo sistema de inventarios, ni con un sistema de compras adecuado, contando con sobrecompras de un 15% - 20% del total de las compras al inicio de cada temporada. Los montos de las compras se toman basando las mismas en la experiencia de sus dueños, no controlando las cantidades necesarias a reponer, y utilizando las ganancias únicamente para reponer mercadería que en algunos casos no es central en la venta, ya que al controlar únicamente los montos finales de venta, no se conocen las prendas con mayor rango de ventas, asimismo no se conoce con exactitud los ciclos estacionales marcados y qué relación existe entre ellos y los montos a reponer dependiendo de la estación anual en que se encuentre. Se producen entonces dos problemas sobre compras de mercadería o faltantes de la misma. El primero de ellos provoca liquidaciones de final de temporada de excesivas cantidades de mercadería reponiendo el costo nominal de las mismas provocando una pérdida real monetaria del 15% - 20%. El segundo provoca que dado que se trata de un rubro de alta competencia en el mercado, una falta de satisfacción de la demanda es pagada con la perdida de la demanda en ese instante del tiempo, por lo que es necesario contar con un mínimo inventario de seguridad necesario para cada estación anual. Por lo tanto se procederá a proponer un sistema de control de inventarios y de compras para reposición del mismo, permitiendo un ahorro promedio de 15% de las compras. Por otro lado, se propondrá un sistema de pronóstico de demanda para cuantificar los ciclos estacionales y que las compras de mercadería se realicen en proporción y necesidad de los mismos, aunque no puede ser totalmente exacto el pronóstico, el mismo permitirá minimizar errores y de esa manera reducir costos.Fil: Balduini Salman, Maximiliano. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina

Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation

AbstractRecent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the β2 subunit of the Na+/K+-ATPase, as verified according to the recently proposed “β-profiling test” [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+-ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+-activated RBCs, suggesting a possible role for this GTPase in membrane shedding

Tunneling nanotubes and mesenchymal stem cells: new insights into the role of melatonin in neuronal recovery

none5sìEfficient cell-to-cell communication is essential for tissue development, homeostasis, and the maintenance of cellular functions after injury. Tunneling nanotubes (TNTs) have emerged as a new important method of cell-to-cell communication. TNTs are primarily established between stressed and unstressed cells and can transport a variety of cellular components. Mitochondria are important trafficked entities through TNTs. Transcellular mitochondria transfer permits the incorporation of healthy mitochondria into the endogenous network of recipient cells, changing the bioenergetic profile and other functional properties of the recipient and may allow the recipient cells to recuperate from apoptotic processes and return to a normal operating state. Mesenchymal cells (MSCs) can form TNTs and transfer mitochondria and other constituents to target cells. This occurs under both physiological and pathological conditions, leading to changes in cellular energy metabolism and functions. This review summarizes the newly-described capacity of melatonin to improve mitochondrial fusion/fission dynamics and promote TNT formation. This new evidence suggests that melatonin’s protective effects could be attributed to its ability to prevent mitochondrial damage in injured cells, reduce senescence, and promote anastasis, a natural cell recovery phenomenon that rescues cells from the brink of death. The modulation of these new routes of intercellular communication by melatonin could play a key role in increasing the therapeutic potential of MSCs.openLuchetti, Francesca; Carloni, Silvia; Nasoni, Maria G.; Reiter, Russel J.; Balduini, WalterLuchetti, Francesca; Carloni, Silvia; Nasoni, Maria G.; Reiter, Russel J.; Balduini, Walte

Bone Marrow Osteoblastic Niche: A New Model to Study Physiological Regulation of Megakaryopoiesis

BACKGROUND: The mechanism by which megakaryocytes (Mks) proliferate, differentiate, and release platelets into circulation are not well understood. Growing evidence indicates that a complex regulatory mechanism, involving cellular interactions, composition of the extracellular matrix and physical parameters such as oxygen tension, may contribute to the quiescent or permissive microenvironment related to Mk differentiation and maturation within the bone marrow. METHODOLOGY/PRINCIPAL FINDINGS: Differentiating human mesenchymal stem cells (hMSCs) into osteoblasts (hOSTs), we established an in vitro model for the osteoblastic niche. We demonstrated for the first time that the combination of HSCs, Mks and hypoxia sustain and promote bone formation by increasing type I collagen release from hOSTs and enhancing its fibrillar organization, as revealed by second harmonic generation microscopy. Through co-culture, we demonstrated that direct cell-cell contact modulates Mk maturation and differentiation. In particular we showed that low oxygen tension and direct interaction of hematopoietic stem cells (HSCs) with hOSTs inhibits Mk maturation and proplatelet formation (PPF). This regulatory mechanism was dependent on the fibrillar structure of type I collagen released by hOSTs and on the resulting engagement of the alpha2beta1 integrin. In contrast, normoxic conditions and the direct interaction of HSCs with undifferentiated hMSCs promoted Mk maturation and PPF, through a mechanism involving the VCAM-1 pathway. CONCLUSIONS/SIGNIFICANCE: By combining cellular, physical and biochemical parameters, we mimicked an in vitro model of the osteoblastic niche that provides a physiological quiescent microenvironment where Mk differentiation and PPF are prevented. These findings serve as an important step in developing suitable in vitro systems to use for the study and manipulation of Mk differentiation and maturation in both normal and diseased states

Outside-In Signalling Generated by a Constitutively Activated Integrin αIIbβ3 Impairs Proplatelet Formation in Human Megakaryocytes

BACKGROUND: The interaction of megakaryocytes with matrix proteins of the osteoblastic and vascular niche is essential for megakaryocyte maturation and proplatelet formation. Fibrinogen is present in the vascular niche and the fibrinogen receptor α(IIb)β(3) is abundantly expressed on megakaryocytes, however the role of the interaction between fibrinogen and α(IIb)β(3) in proplatelet formation in humans is not yet fully understood. We have recently reported a novel congenital macrothrombocytopenia associated with a heterozygous mutation of the β(3) subunit of α(IIb)β(3). The origin of thrombocytopenia in this condition remains unclear and this may represent an interesting natural model to get further insight into the role of the megakaryocyte fibrinogen receptor in megakaryopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Patients' peripheral blood CD45+ cells in culture were differentiated into primary megakaryocytes and their maturation, spreading on different extracellular matrix proteins, and proplatelet formation were analyzed. Megakaryocyte maturation was normal but proplatelet formation was severely impaired, with tips decreased in number and larger in size than those of controls. Moreover, megakaryocyte spreading on fibrinogen was abnormal, with 50% of spread cells showing disordered actin distribution and more evident focal adhesion points than stress fibres. Integrin α(IIb)β(3) expression was reduced but the receptor was constitutively activated and a sustained, and substrate-independent, activation of proteins of the outside-in signalling was observed. In addition, platelet maturation from preplatelets was impaired. CONCLUSIONS/SIGNIFICANCE: Our data show that constitutive activation of α(IIb)β(3)-mediated outside-in signalling in human megakaryocytes negatively influences proplatelet formation, leading to macrothombocytopenia