A semistatic microplate-based phytotoxicity test

DOE Office of Scientific and Technical Information (OSTI.GOV)

Radetski, C.M.; Ferard, J.F.; Blaise, C.

1995-02-01

A novel phytotoxicity test is described herein that employs a microplate equipped with membrane-bottomed wells. This MultiScreen[trademark] (Millipore Corp., Bedford, MA) microplate allows performance of a semistatic algal test, in which test medium is renewed periodically. With such a design, the algal test becomes comparable to other short-term tests used to evaluate chronic toxicity of chemicals and effluents. The EC50s obtained for Cu[sup 2+], Cd[sup 2+], Cr[sup 6+], atrazine, and one leachate sample (municipal sludge incinerator residue) with static and semistatic algal microplate tests were compared in this study. The semistatic microplate test revealed greater sensitivity than did the staticmore » microplate test.« less

Scribed transparency microplates mounted on a modified standard microplate.

PubMed

Cheong, Brandon Huey-Ping; Chua, Wei Seong; Liew, Oi Wah; Ng, Tuck Wah

2014-08-01

The immense cost effectiveness of using transparencies as analyte handling implements in microplate instrumentation offers the possibility of application even in resource-limited laboratories. In this work, a standard microplate was adapted to serve as the permanent base for disposable scribed transparencies. The approach is shown to ameliorate evaporation, which can affect assay accuracy when analytes need to be incubated for some time. It also offers assurance against fluorescence measurement errors due to the cross-talk of samples from adjacent wells. Copyright © 2014 Elsevier Inc. All rights reserved.

UAV Monitoring for Enviromental Management in Galapagos Islands

NASA Astrophysics Data System (ADS)

Ballari, D.; Orellana, D.; Acosta, E.; Espinoza, A.; Morocho, V.

2016-06-01

In the Galapagos Islands, where 97% of the territory is protected and ecosystem dynamics are highly vulnerable, timely and accurate information is key for decision making. An appropriate monitoring system must meet two key features: on one hand, being able to capture information in a systematic and regular basis, and on the other hand, to quickly gather information on demand for specific purposes. The lack of such a system for geographic information limits the ability of Galapagos Islands' institutions to evaluate and act upon environmental threats such as invasive species spread and vegetation degradation. In this context, the use of UAVs (unmanned aerial vehicles) for capturing georeferenced images is a promising technology for environmental monitoring and management. This paper explores the potential of UAV images for monitoring degradation of littoral vegetation in Puerto Villamil (Isabela Island, Galapagos, Ecuador). Imagery was captured using two camera types: Red Green Blue (RGB) and Infrarred Red Green (NIR). First, vegetation presence was identified through NDVI. Second, object-based classification was carried out for characterization of vegetation vigor. Results demonstrates the feasibility of UAV technology for base-line studies and monitoring on the amount and vigorousness of littoral vegetation in the Galapagos Islands. It is also showed that UAV images are not only useful for visual interpretation and object delineation, but also to timely produce useful thematic information for environmental management.

Microplates with adaptive surfaces.

PubMed

Akbulut, Meshude; Lakshmi, Dhana; Whitcombe, Michael J; Piletska, Elena V; Chianella, Iva; Güven, Olgun; Piletsky, Sergey A

2011-11-14

Here we present a new and versatile method for the modification of the well surfaces of polystyrene microtiter plates (microplates) with poly(N-phenylethylene diamine methacrylamide), (poly-NPEDMA). The chemical grafting of poly-NPEDMA to the surface of microplates resulted in the formation of thin layers of a polyaniline derivative bearing pendant methacrylamide double bonds. These were used as the attachment point for various functional polymers through photochemical grafting of various, for example, acrylate and methacrylate, polymers with different functionalities. In a model experiment, we have modified poly-NPEDMA-coated microplates with a small library of polymers containing different functional groups using a two-step approach. In the first step, double bonds were activated by UV irradiation in the presence of N,N-diethyldithiocarbamic acid benzyl ester (iniferter). This enabled grafting of the polymer library in the second step by UV irradiation of solutions of the corresponding monomers in the microplate wells. The uniformity of coatings was confirmed spectrophotometrically, by microscopic imaging and by contact angle measurements (CA). The feasibility of the current technology has been shown by the generation of a small library of polymers grafted to the microplate well surfaces and screening of their affinity to small molecules, such as atrazine, a trio of organic dyes, and a model protein, bovine serum albumin (BSA). The stability of the polymers, reproducibility of measurement, ease of preparation, and cost-effectiveness make this approach suitable for applications in high-throughput screening in the area of materials research.

Deciphering cGMP signatures and cGMP-dependent pathways in plant defence

PubMed Central

Meier, Stuart; Madeo, Laura; Ederli, Luisa; Donaldson, Lara; Gehring, Chris

2009-01-01

The second messenger, 3′,5′-cyclic monophosphate (cGMP), is a critical component of many different processes in plants while guanylyl cyclases that catalyse the formation of cGMP from GTP have remained somewhat elusive in higher plants. Consequently, two major aims are the discovery of novel GCs and the identification of cGMP mediated processes. Recently, we have reported temporal signatures of ozone (O3)-induced hydrogen peroxide (H2O2) and nitric oxide (NO) generation, their effect on cGMP generation, and consequent transcriptional changes of genes diagnostic for stress responses in tobacco. We demonstrated that O3 and NO induced early transcriptional activation of the scavenger encoding proteins, alternative oxidase (AOX1a), glutathione peroxidase (GPX) and the induction of ethylene production through aminocyclopropancarboxylic acid synthase (ACS2) are cGMP-independent. By contrast, the early response of the phenylalanine ammonia lyase gene (PALa) and the late response of the gene encoding the pathogenesis-related protein (PR1a) show critical dependence on cGMP. Here we show differential cGMP responses to virulent and avirulent Pseudomonas syringae strains and propose that host-pathogen recognition and/or down-stream processes are transduced by complex cGMP signatures. This is in accordance with the identification of a growing number of multi-domain molecules in Arabidopsis that are reported to contain putative functional GC catalytic centers. PMID:19794847

Radar Image of Galapagos Island

NASA Image and Video Library

1996-10-23

This is an image showing part of Isla Isabella in the western Galapagos Islands. It was taken by the L-band radar in HH polarization from the Spaceborne Imaging Radar C/X-Band Synthetic Aperture Radar on the 40th orbit of NASA’s space shuttle Endeavour.

Kinematics of the Danakil microplate

NASA Astrophysics Data System (ADS)

Eagles, Graeme; Gloaguen, Richard; Ebinger, Cynthia

2002-10-01

A refinement and extrapolation of recent motion estimates for the Danakil microplate, based on ancient kinematic indicators in the Afar region, describes the evolution of a microplate in the continental realm. The Danakil horst is an elevated part of this microplate, exposing a Precambrian basement within the Afar depression, the site of the Nubia-Somalia-Arabia triple junction. We compare evidence for strike- or oblique-slip faults in data from the Afar depression and southern Red Sea to small circles about published poles of rotation for the Danakil microplate with respect to Nubia. A reconstruction about the preferred pole reunites lengths of a Precambrian shear zone on the Nubia and Danakil sides and preserves a uniform basement fabric strike through Nubia, Danakil and Yemen. Since at least magnetic chron C5 (˜11 Ma) Danakil rotated about a different pole with respect to Nubia than either Somalia or Arabia, but between chrons C5 and C2A Nubia-Danakil motion was a close approximation to Nubia-Somalia motion. Since C2A relative motions of the Danakil microplate have been independent of movements on any of the neighbouring plate boundaries. We relate this to the onset of oceanic-type accretion within Afar. The resulting eastwards acceleration of Danakil was accommodated by westwards propagation of the Gulf of Aden rift that became the new, discrete, plate boundary between the Danakil microplate and the Somalia plate. Present-day activity suggests that the Red Sea and Aden rifts will link through Afar, thereby isolating the Danakil horst as a microcontinent on the Arabian margin.

Exploring conservation discourses in the Galapagos Islands: A case study of the Galapagos giant tortoises.

PubMed

Benitez-Capistros, Francisco; Hugé, Jean; Dahdouh-Guebas, Farid; Koedam, Nico

2016-10-01

Conservation discourses change rapidly both at global and local scales. To be able to capture these shifts and the relationships between humans and nature, we focused on a local and iconic conservation case: the Galapagos giant tortoises (Chelonoidis spp.). We used the Q methodology to contextualize conservation for science and decision making and to explore the multidimensionality of the conservation concept in Galapagos. The results indicate four prevailing discourses: (1) Multi-actor governance; (2) giant tortoise and ecosystems conservation; (3) community governance; and (4) market and tourism centred. These findings allow us to identify foreseeable points of disagreement, as well as areas of consensus, and to discuss the implication of the findings to address socio-ecological conservation and sustainability challenges. This can help the different involved stakeholders (managers, scientists and local communities) to the design and apply contextualized conservation actions and policies to contribute to a better sustainable management of the archipelago.

Environmental Education in the Galapagos: 2007 Report to the Charles Darwin Foundation

ERIC Educational Resources Information Center

Stepath, Carl M.

2007-01-01

Background: "Environmental education in the Galapagos: 2007 report to the Charles Darwin Foundation" is a report to the Charles Darwin Foundation (CDF) about the researchers observations about the status of environmental education in the Galapagos in 2006 and 2007. Purpose: This paper reports on environmental education in the Galapagos…

Long-term isolation of a highly mobile seabird on the Galapagos

USGS Publications Warehouse

Hailer, Frank; Schreiber, E.A.; Miller, Joshua M.; Levin, Iris I.; Parker, Patricia G.; Chesser, R. Terry; Fleischer, Robert C.

2011-01-01

The Galapagos Islands are renowned for their high degree of endemism. Marine taxa inhabiting the archipelago might be expected to be an exception, because of their utilization of pelagic habitats--the dispersal barrier for terrestrial taxa--as foraging grounds. Magnificent frigatebirds (Fregata magnificens) have a highly vagile lifestyle and wide geographical distribution around the South and Central American coasts. Given the potentially high levels of gene flow among populations, the species provides a good test of the effectiveness of the Galapagos ecosystem in isolating populations of highly dispersive marine species. We studied patterns of genetic (mitochondrial DNA, microsatellites and nuclear introns) and morphological variation across the distribution of magnificent frigatebirds. Concordant with predictions from life-history traits, we found signatures of extensive gene flow over most of the range, even across the Isthmus of Panama, which is a major barrier to gene flow in other tropical seabirds. In contrast, individuals from the Galapagos were strongly differentiated from all conspecifics, and have probably been isolated for several hundred thousand years. Our finding is a powerful testimony to the evolutionary uniqueness of the taxa inhabiting the Galapagos archipelago and its associated marine ecosystems.

Long-term isolation of a highly mobile seabird on the Galapagos

PubMed Central

Hailer, Frank; Schreiber, E. A.; Miller, Joshua M.; Levin, Iris I.; Parker, Patricia G.; Chesser, R. Terry; Fleischer, Robert C.

2011-01-01

The Galapagos Islands are renowned for their high degree of endemism. Marine taxa inhabiting the archipelago might be expected to be an exception, because of their utilization of pelagic habitats—the dispersal barrier for terrestrial taxa—as foraging grounds. Magnificent frigatebirds (Fregata magnificens) have a highly vagile lifestyle and wide geographical distribution around the South and Central American coasts. Given the potentially high levels of gene flow among populations, the species provides a good test of the effectiveness of the Galapagos ecosystem in isolating populations of highly dispersive marine species. We studied patterns of genetic (mitochondrial DNA, microsatellites and nuclear introns) and morphological variation across the distribution of magnificent frigatebirds. Concordant with predictions from life-history traits, we found signatures of extensive gene flow over most of the range, even across the Isthmus of Panama, which is a major barrier to gene flow in other tropical seabirds. In contrast, individuals from the Galapagos were strongly differentiated from all conspecifics, and have probably been isolated for several hundred thousand years. Our finding is a powerful testimony to the evolutionary uniqueness of the taxa inhabiting the Galapagos archipelago and its associated marine ecosystems. PMID:20861041

Life Cycle of Mantle Plumes: A perspective from the Galapagos Plume (Invited)

NASA Astrophysics Data System (ADS)

Gazel, E.; Herzberg, C. T.

2009-12-01

Hotspots are localized sources of heat and magmatism considered as modern-day evidence of mantle plumes. Some hotspots are related to massive magmatic production that generated Large Igneous Provinces (LIPS), an initial-peak phase of plume activity with a mantle source hotter and more magmatically productive than present-day hotspots. Geological mapping and geochronological studies have shown much lower eruption rates for OIB compared to lavas from Large Igneous Provinces LIPS such as oceanic plateaus and continental flood provinces. Our study is the first quantitative petrological comparison of mantle source temperatures and extent of melting for OIB and LIP sources. The wide range of primary magma compositions and inferred mantle potential temperatures for each LIP and OIB occurrence suggest that this rocks originated form a hotspot, a spatially localized source of heat and magmatism restricted in time. Extensive outcrops of basalt, picrite, and sometimes komatiite with circa 65-95 Ma ages occupy portions of the pacific shore of Central and South America included in the Caribbean Large Igneous Province (CLIP). There is general consensus of a Pacific-origin of CLIP and most studies suggest that it was produced by melting in the Galapagos mantle plume. The Galapagos connection is consistent with isotopic and geochemical similarities with lavas from the present-day Galapagos hotspot. A Galapagos link for rocks in South American oceanic complexes (eg. the island of Gorgona) is more controversial and requires future work. The MgO and FeO contents of lavas from the Galapagos related lavas and their primary magmas have decreased since the Cretaceous. From petrological modeling we infer that these changes reflect a cooling of the Galapagos mantle plume from a potential temperature of 1560-1620 C in the Cretaceous to 1500 C at the present time. These temperatures are higher than 1350 C for ambient mantle associated with oceanic ridges, and provide support for the mantle

75 FR 235 - Endangered and Threatened Wildlife and Plants; Final Rule To List the Galapagos Petrel and...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-05

... petrel (native to New Zealand), Galapagos petrel (native to the Galapagos Islands, Ecuador), and the... phaeopygia), is a large, long-winged gadfly petrel that is endemic to the Galapagos Islands, Ecuador (BLI..., or projected sites of present occurrence of a species, excluding cases of vagrancy.'' Because this...

Chitosan-coated polystyrene microplate for covalent immobilization of enzyme.

PubMed

Zhang, Yaodong; Li, Li; Yu, Caihong; Hei, Tingting

2011-10-01

Microplates made of polystyrene have been widely used for immunoassays. Protein molecules that have been immobilized on a hydrophobic polystyrene microplate by passive adsorption lose their activity and suffer considerable denaturation. A new chitosan-coated microplate suitable for the covalent immobilization of enzymes has been developed. The primary amino groups of chitosan were exploited for this covalent coupling of proteins. The optical transmittance of the chitosan-coated microplate, at wavelengths of 400-800 nm, was estimated to be suitable for its application in chromogenic reaction-based bioassays. The immobilization efficiency of the chitosan-coated microplate was demonstrated to be far superior to that of a conventional microplate when tested using acetylcholinesterase (AChE) and β-glucosidase as model biomolecules, and the chitosan-coated microplate may thus have potential applications in biosensing and bioreactor systems. © Springer-Verlag 2011

Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense.

PubMed

O'Neal, Lindsey; Ryu, Min-Hyung; Gomelsky, Mark; Alexandre, Gladys

2017-09-15

Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense , c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state. IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and

Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense

PubMed Central

O'Neal, Lindsey; Ryu, Min-Hyung; Gomelsky, Mark

2017-01-01

ABSTRACT Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state. IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and

Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5.

PubMed

Granovsky, A E; Artemyev, N O

2001-06-15

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.

An Extensive Alien Plant Inventory from the Inhabited Areas of Galapagos

PubMed Central

Guézou, Anne; Trueman, Mandy; Buddenhagen, Christopher Evan; Chamorro, Susana; Guerrero, Ana Mireya; Pozo, Paola; Atkinson, Rachel

2010-01-01

Background Plant invasions are causing habitat degradation in Galapagos. Problems are concentrated on the four inhabited islands. Plants introduced to rural areas in the humid highlands and urban areas on the arid coast act as foci for invasion of the surrounding Galapagos National Park. Methodology/Principal Findings Here we present results of the most comprehensive inventory to date of alien vascular plants in the inhabited areas of Galapagos. The survey was conducted between 2002 and 2007, in 6031 properties (97% of the total) on Floreana, Isabela, San Cristobal and Santa Cruz Islands. In total 754 alien vascular plant taxa were recorded, representing 468 genera in 123 families. Dicotyledons represented 554 taxa, monocotyledons 183, there were 7 gymnosperms and 10 pteridophytes. Almost half (363) of the taxa were herbaceous. The most represented families were Fabaceae (sensu lato), Asteraceae and Poaceae. The three most recorded species in the humid rural areas were Psidium guajava, Passiflora edulis and Bryophyllum pinnatum, and in the dry urban areas, Aloe vera, Portulaca oleracea and Carica papaya. In total, 264 (35%) taxa were recorded as naturalized. The most common use for taxa was ornamental (52%). Conclusions/Significance This extensive survey has increased the known alien vascular flora of Galapagos by 257 species, giving a ratio of alien to native taxa of 1.57∶1. It provides a crucial baseline for plant invasion management in the archipelago and contributes data for meta analyses of invasion processes worldwide. A repeat of the survey in the future would act as an effective early detection tool to help avoid further invasion of the Galapagos National Park. PMID:20421999

Earth observations of the Galapagos Islands taken from OV-105 during STS-99.

NASA Image and Video Library

2000-03-28

STS099-753-032 (11-22 February 2000) ---This 70mm photograph, photographed from the Space Shuttle Endeavour, centers on the two westernmost Galapagos Islands--seahorse-shaped Isla Isabela and the smaller round Isla Fernandina to its west. All of the 19 islands in the chain are volcanic in origin, and the craters of several of the shield volcanoes are visible as circular features on each of the islands. The two islands shown in this picture contain the most active volcanoes of the Galapagos. Fernandina last erupted in January-February 1995, with red-hot lava pouring into the sea. After 20 years of inactivity, Cerro Azul on Isla Isabela, last erupted in September-October 1998. Cerro Azul is the southwesternmost volcano on Isla Isabela. At 82 miles long, Isla Isabela is the largest of the islands, and comprises half of the land area of the archipelago. The islands are famous for their unique flora and fauna. Charles Darwin's observations of these species in 1835 contributed to the formation of his ideas on natural selection. Some of the most unique species include flightless cormorants, Galapagos penguins, giant land tortoises, and Galapagos finches. The range of Galapagos penguins is restricted to these western islands where upwelling enriches the ocean productivity, and the adaptation of a typically Antarctic bird family to the equator is an ecological marvel. Giant land tortoises are thought to have the oldest lifespans of any animal on Earth, but, scientists say, they have been driven near to extinction. During the most recent eruption of Cerro Azul, one tortoise was killed and many had to be relocated. The 13 species of Galapagos finches on the islands, although varied in form and lifestyle, are the descendants of an ancestor that happened to colonize this isolated archipelago. The human population of the entire archipelago is about 10,000.

Periodic GMP Matrices

NASA Astrophysics Data System (ADS)

Eichinger, Benjamin

2016-07-01

We recall criteria on the spectrum of Jacobi matrices such that the corresponding isospectral torus consists of periodic operators. Motivated by those known results for Jacobi matrices, we define a new class of operators called GMP matrices. They form a certain Generalization of matrices related to the strong Moment Problem. This class allows us to give a parametrization of almost periodic finite gap Jacobi matrices by periodic GMP matrices. Moreover, due to their structural similarity we can carry over numerous results from the direct and inverse spectral theory of periodic Jacobi matrices to the class of periodic GMP matrices. In particular, we prove an analogue of the remarkable ''magic formula'' for this new class.

Crust-Lithosphere-Asthenosphere Dynamics in Mantle Plume Provinces with Emphasis on the Galapagos =

NASA Astrophysics Data System (ADS)

Orellana, Felipe

Hotspot tracks, which most geoscientists attribute to the effects of mantle plumes on the overlying lithospheric plates, are characterized by distinct bathymetry, gravity signatures, structural geology, volcanology, petrology, and geochemistry; motivating us to try to understand the dynamics behind the space-time-histories of these systems. Making use of classical geodynamic paradigms, such as highly-viscous fluids (Stokes flow, for the mantle and/or lithosphere), elastic plate behavior, and heat flow, we develop conceptual frameworks to explain a number of distinct hotspot track features, and present quantitative models aimed at elucidating their origins. There is much diversity among the population of mantle plume hotspot tracks on the Earth's oceanic crust. For example, there are marked differences in the style of their bathymetry, as well as in their gravimetric signals, and also in the isotopic signature of extruded lavas. At the same time, important underlying differences are given by the age of the lithospheric plates under which the mantle plumes are impinging, lithospheric elastic thickness, the heat (or buoyancy) flux of individual mantle plumes, their melt production, crustal thickening, the proximity of spreading centers, etc. In the first chapter of this dissertation, making use of scaling theory, we show that for most oceanic hotspot tracks, the character of bathymetric expression (primarily rough vs. smooth topography) can be explained by three independent primary underlying factors - plate thickness, or equivalently plate age; plate speed; and plume buoyancy flux - combined into a single parameter, R, the ratio of plume heat flux to the effective thermal capacity of the moving plate overlying the plume. The Galapagos archipelago (off the west coast of equatorial South America), part of a >20 Ma old hotspot track formed by the underlying Galapagos mantle plume, currently exhibits a broad geographic distribution of volcanic centers of surprisingly

Plasma-Treated Microplates with Enhanced Protein Recoveries and Minimized Extractables

PubMed Central

Weikart, Christopher M.; Klibanov, Alexander M.; Breeland, Adam P.; Taha, Ahmad H.; Maurer, Brian R.; Martin, Steven P.

2016-01-01

SiO2 Medical Products, Inc. (SiO) has developed a proprietary technology that greatly enhances protein recoveries and reduces extractables from commercial microplates used for bioanalytical assays and storage of biologics. SiO technology is based on plasma treatment that chemically modifies the surface of polypropylene with predominantly hydrogen-bond-acceptor uncharged polar groups. The resultant surface resists nonspecific protein adsorption over a wide range of protein concentrations, thereby eliminating the need to passivate (and hence potentially contaminate) the microplates with blocking proteins. High shelf-life stability and cleanliness of the plasma-treated microplates have been demonstrated using five different proteins for two common microplate formats. The protein recovery performance of plasma-treated microplates is found to be higher compared with commercial low-protein-binding microplates. PMID:27651466

India-Eurasia collision triggers formation of an oceanic microplate

NASA Astrophysics Data System (ADS)

Matthews, Kara; Müller, Dietmar; Sandwell, David

2016-04-01

Detailed mapping of seafloor tectonic fabric in the Indian Ocean, using high-resolution satellite-derived vertical gravity gradient data, reveals an extinct Pacific-style oceanic microplate - the Mammerickx Microplate - west of the Ninetyeast Ridge. It is one of the first Pacific-style microplates to be mapped outside the Pacific basin, suggesting that geophysical conditions during formation probably resembled those that have dominated at eastern Pacific ridges. The microplate formed at the Indian-Antarctic ridge and is bordered by an extinct ridge in the north and pseudofault in the south, whose conjugate is located north of the Kerguelen Plateau. Independent microplate rotation is indicated by asymmetric pseudofaults and rotated abyssal hill fabric, also identified in multibeam data. Magnetic anomaly picks and age estimates calculated from published spreading rates suggest formation during chron 21o (~47.3 Ma). Plate reorganizations can trigger ridge propagation and microplate development, and we propose that formation of the Mammerickx Microplate is linked with the initial 'soft' stage of the India-Eurasia collision. The collision altered the stress regime at the Indian-Antarctic ridge, leading to a change in segmentation and ridge propagation from an establishing transform fault. Fast Indian-Antarctic spreading that preceded microplate formation, and Kerguelen Plume activity may have facilitated ridge propagation via the production of thin and weak lithosphere. However, both factors had been present for tens of millions of years and are therefore unlikely to have triggered the event. Prior to the collision, this combination of fast spreading and plume activity was responsible for the production of a wide region of undulate seafloor to the north of the extinct ridge and 'W' shaped lineations that record back and forth ridge propagation. Microplate formation provides a means of dating the onset of the India-Eurasia collision, and is completely independent of and

Laryngotracheal reconstruction with resorbable microplate buttressing.

PubMed

Javia, Luv R; Zur, Karen B

2012-04-01

In patients undergoing laryngotracheal reconstruction (LTR), malacic segments of trachea can pose challenges to successful reconstruction. Malacic segments may inadequately support cartilage grafts used in augmentation surgery, sometimes requiring cricotracheal or tracheal resections. We describe a novel technique of LTR with resorbable microplate buttressing of malacic lateral tracheal segments. Retrospective case series. Review of technique, treatment outcomes, and complications of seven children with subglottic stenosis and tracheomalacia requiring a microplate-augmented LTR technique. Seven infants ranging from 26 months to 9 years of age successfully underwent LTR for subglottic stenosis. Six children had a grade III subglottic stenosis. The seventh child had grade II subglottic stenosis, bilateral vocal fold paralysis, an elliptical cricoid, and an obstructing giant suprastomal fibroma. Five children underwent a double-stage LTR with resorbable microplates sutured bilaterally to support severely malacic lateral tracheal segments. A cricotracheal resection would not have been feasible in one child due to the resection length and inadequate tracheal mobilization. Two children underwent a single-stage LTR with unilateral application of a microplate. Six children were decannulated within 3 months and continue without airway symptoms or complications. One child, who is just over 2 months from reconstructive surgery, is being setup for decannulation. No complications were encountered. LTR with resorbable microplate buttressing of malacic lateral tracheal segments is technically feasible, safe, and can avoid more extensive surgery requiring tracheal resection. Further experience may support the use of this technique in challenging airway reconstructions. Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.

Assesing Geographic Isolation of the Galapagos Islands

NASA Astrophysics Data System (ADS)

Orellana, D.; Smith, F.

2016-06-01

The Galapagos Archipelago is one of the most important ecological spots in the planet due its unique biodiversity, active geology, and relatively well-preserved ecosystems. These characteristics are strongly based on the geographical isolation of the islands. On the one hand this isolation allowed the evolution processes that gave the islands their international fame and on the other hand it kept them from major human impacts that affected the vast majority of the Earth's surface. Galapagos' geographical isolation is therefore of mayor value, but it is rapidly diminishing due to the increase of marine and air transportation among islands and with the rest of the world. This increased accessibility implies enhanced risks for the ecological dynamics on the archipelago (e.g. increased risk of biological invasions, uncontrolled tourism growth, more water and energy consumption). Here, we introduce a general accessibility model to assess geographical isolation of the Galapagos Islands. The model aims to characterize accessibility in terms of human mobility by evaluating travel time to each point of the archipelago using all available transportation modalities. Using a multi criteria cost surface for marine and land areas, we estimated travel time for each surface unit using the fastest route and mode of transportation available while considering several friction factors such as surface type, slope, infrastructure, transfer points, legal restrictions, and physical barriers. We created maps to evaluate the isolation of different islands and places, highlighting the potential risks for several habitats and ecosystems. The model can be used for research and decision-making regarding island conservation, such as estimating spreading paths for invasive species, informing decisions on tourism management, and monitoring isolation changes of sensitive ecosystems.

Isabela, Galapagos Islands as seen from STS-59

NASA Technical Reports Server (NTRS)

1994-01-01

Space Radar Laboratory (SRL) scientists will use these photographs of Isabela in the Galapagos island chain, with the radar iamagery, to discriminate among different ages of basalt flows, and different ecosystems of shrub communities, on these equatorial volcanic islands.

ASEAN GMP and pharmaceutical industries in Indonesia.

PubMed

Soesilo, S; Sitorus, U

1995-01-01

Indonesia was appointed by the ASEAN Technical Cooperation in Pharmaceutical as a focal point and to coordinate the development of practical guidelines for the implementation of GMP. The ASEAN GMP Guidelines were endorsed by the ASEAN Technical Cooperation in Pharmaceutical in 1988, which among others required separation of Beta-Lactam dedicated facilities and three degrees of cleanliness for production areas. As it was realised that drug manufacturers in developing countries need more detailed guidelines to be able to implement the GMP, an Operational Manual for GMP was also prepared for providing examples of SOPs lay-outs, documentation etc. It was agreed by the technical cooperation group to leave the implementation of GMP to each member country. However, the ASEAN Manual for Inspection of GMP was drafted and endorsed by the group and training of ASEAN Drug Inspectors was organized to support the implementation. The ASEAN GMP is being implemented in Indonesia through a five-year, stepwise implementation plan, starting in 1989.

Galapagos Islands taken by the STS-109 crew

NASA Image and Video Library

2002-03-10

STS109-718-102 (1-12 March 2002) --- The astronauts on board the Space Shuttle Columbia took this 70mm picture featuring the Galapagos Islands. For orientation purposes, north is towards the bottom of the view. Most of the largest island in the Galapagos group, Isla Isabela, stretches across the middle of the frame. The circular feature on this island at bottom is Volcano Wolf (1707 meters in altitude). Volcano Darwin (1280 meters in sea level) is the next volcano above and to the left, partly ringed with cloud. The single island top right is Isla Fernandina, the top of another volcano (1547 meters). Recent lava flows appear as darker surfaces and the older surfaces appear green, as a result of unusual rains and vegetational greening in this normally arid part of the world. The Equator passes exactly through Volcano Wolf, roughly left to right.

Guidelines for Microplate Selection in High Content Imaging.

PubMed

Trask, Oscar J

2018-01-01

Since the inception of commercialized automated high content screening (HCS) imaging devices in the mid to late 1990s, the adoption of media vessels typically used to house and contain biological specimens for interrogation has transitioned from microscope slides and petri dishes into multi-well microtiter plates called microplates. The early 96- and 384-well microplates commonly used in other high-throughput screening (HTS) technology applications were often not designed for optical imaging. Since then, modifications and the use of next-generation materials with improved optical clarity have enhanced the quality of captured images, reduced autofocusing failures, and empowered the use of higher power magnification objectives to resolve fine detailed measurements at the subcellular pixel level. The plethora of microplates and their applications requires practitioners of high content imaging (HCI) to be especially diligent in the selection and adoption of the best plates for running longitudinal studies or larger screening campaigns. While the highest priority in experimental design is the selection of the biological model, the choice of microplate can alter the biological response and ultimately may change the experimental outcome. This chapter will provide readers with background, troubleshooting guidelines, and considerations for choosing an appropriate microplate.

Microplates in liquid chromatography--new solution in clinical research? A review.

PubMed

Krcmova, Lenka; Solichova, Dagmar; Solich, Petr

2013-10-15

Microplates are routinely used in Radio- or Immuno-assays. Recently, microplates have found use not only in analytical but also in the pre-analytical phase in bioanalyses (sample storage, sample preparation). New connection of this technology to liquid chromatography could be economical, fast and simple solution for many routine laboratories handling large sequences of biological samples. This review summarises the application of microplates in bioanalytical laboratories. Different types of sorbents, materials and shapes of microplates are discussed, and the main advantages and disadvantages of microplates used in clinical research are presented. Copyright © 2013 Elsevier B.V. All rights reserved.

Oceanic microplate formation records the onset of India-Eurasia collision

NASA Astrophysics Data System (ADS)

Matthews, Kara J.; Dietmar Müller, R.; Sandwell, David T.

2016-01-01

Mapping of seafloor tectonic fabric in the Indian Ocean, using high-resolution satellite-derived vertical gravity gradient data, reveals an extinct Pacific-style oceanic microplate ('Mammerickx Microplate') west of the Ninetyeast Ridge. It is one of the first Pacific-style microplates to be mapped outside the Pacific basin, suggesting that geophysical conditions during formation probably resembled those that have dominated at eastern Pacific ridges. The microplate formed at the Indian-Antarctic ridge and is bordered by an extinct ridge in the north and pseudofault in the south, whose conjugate is located north of the Kerguelen Plateau. Independent microplate rotation is indicated by asymmetric pseudofaults and rotated abyssal hill fabric, also seen in multibeam data. Magnetic anomaly picks and age estimates calculated from published spreading rates suggest formation during chron 21o (∼47.3 Ma). Plate reorganizations can trigger ridge propagation and microplate development, and we propose that Mammerickx Microplate formation is linked with the India-Eurasia collision (initial 'soft' collision). The collision altered the stress regime at the Indian-Antarctic ridge, leading to a change in segmentation and ridge propagation from an establishing transform. Fast Indian-Antarctic spreading that preceded microplate formation, and Kerguelen Plume activity, may have facilitated ridge propagation via the production of thin and weak lithosphere; however both factors had been present for tens of millions of years and are therefore unlikely to have triggered the event. Prior to the collision, the combination of fast spreading and plume activity was responsible for the production of a wide region of undulate seafloor to the north of the extinct ridge and 'W' shaped lineations that record back and forth ridge propagation. Microplate formation provides a precise means of dating the onset of the India-Eurasia collision, and is completely independent of and complementary to timing

GALAPAGOS-C: analysis of galaxy morphologies using high-performance computing methods

NASA Astrophysics Data System (ADS)

Hiemer, Andreas; Barden, Marco; Kelvin, Lee S.; Häußler, Boris; Schindler, Sabine

2014-11-01

We present GALAPAGOS-C, a code designed to process a complete set of survey images through automation of source detection (via SEXTRACTOR), postage stamp cutting, object mask preparation, sky background estimation and complex two-dimensional light profile Sérsic modelling (via GALFIT). GALAPAGOS-C is designed around the concept of MPI-parallelization, allowing the processing of large data sets in a quick and efficient manner. Further, GALAPAGOS-C is capable of fitting multiple-Sérsic profiles to each galaxy, each representing distinct galaxy components (e.g. bulge, disc, bar), in addition to the option to fit asymmetric Fourier mode distortions. The modelling reliability of our core single-Sérsic fitting capability is tested thoroughly using image simulations. We apply GALAPAGOS-C to the Space Telescope A901/902 Galaxy Evolution Survey to investigate the evolution of galaxy structure with cosmic time and the dependence on environment. We measure the distribution of Sérsic indices as a function of local object density in the A901/902 cluster sample to provide one of the first measures of the Sérsic index-density relation. We find that the fraction of galaxies with a high Sérsic index (2.5 < n < 7.0) is higher in denser environments (˜35 per cent), halving towards sparsely populated regions (˜15 per cent). The population of low Sérsic index galaxies (0.4 < n< 1.6) is lower in denser environments (˜35 per cent), increasing towards sparsely populated regions (˜60 per cent). The population of intermediate Sérsic index galaxies (1.6 < n < 2.5) approximately follows the trend of the high Sérsic index types.

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

PubMed Central

2015-01-01

ABSTRACT Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114

Radar Image of Galapagos Island

NASA Technical Reports Server (NTRS)

1994-01-01

This is an image showing part of Isla Isabella in the western Galapagos Islands. It was taken by the L-band radar in HH polarization from the Spaceborne Imaging Radar C/X-Band Synthetic Aperture Radar on the 40th orbit of the space shuttle Endeavour. The image is centered at about 0.5 degree south latitude and 91 degrees west longitude and covers an area of 75 by 60 kilometers (47 by 37 miles). The radar incidence angle at the center of the image is about 20 degrees.

The western Galapagos Islands, which lie about 1,200 kilometers (750 miles) west of Ecuador in the eastern Pacific, have six active volcanoes similar to the volcanoes found in Hawaii. Since the time of Charles Darwin's visit to the area in 1835, there have been over 60 recorded eruptions of these volcanoes. This SIR-C/X-SAR image of Alcedo and Sierra Negra volcanoes shows the rougher lava flows as bright features, while ash deposits and smooth pahoehoe lava flows appear dark. A small portion of Isla Fernandina is visible in the extreme upper left corner of the image.

The Galapagos Islands are one of the SIR-C/X-SAR supersites and data of this area will be taken several times during the flight to allow scientists to conduct topographic change studies and to search for different lava flow types, ash deposits and fault lines.

Spaceborne Imaging Radar-C and X-Synthetic Aperture Radar (SIR-C/X-SAR) is part of NASA's Mission to Planet Earth. The radars illuminate Earth with microwaves allowing detailed observations at any time, regardless of weather or sunlight conditions. SIR-C/X-SAR uses three microwave wavelengths: L-band (24 cm), C-band (6 cm) and X-band (3 cm). The multi-frequency data will be used by the international scientific community to better understand the global environment and how it is changing. The SIR-C/X-SAR data, complemented by aircraft and ground studies, will give scientists clearer insights into those environmental changes which are caused by nature and those changes

A continuous perfusion microplate for cell culture.

PubMed

Goral, Vasiliy N; Zhou, Chunfeng; Lai, Fang; Yuen, Po Ki

2013-03-21

We describe a 96-well microplate with fluidically connected wells that enables the continuous fluid perfusion between wells without the need for external pumping. A single unit in such a perfusion microplate consists of three wells: a source well, a sample (cell culture) well in the middle and a waste well. Fluid perfusion is achieved using a combination of the hydrostatic pressure generated by different liquid levels in the wells and the fluid wicking through narrow strips of a cellulose membrane connecting the wells. There is an excellent correspondence between the observed perfusion flow dynamics and the flow simulations based on Darcy's Law. Hepatocytes (C3A cells) cultured for 4 days in the perfusion microplate with no media exchange in the cell culture well had the same viability as hepatocytes exposed to a daily exchange of media. EOC 20 cells that require media conditioned by LADMAC cells were shown to be equally viable in the adjacent cell culture well of the perfusion microplate with LADMAC cells cultured in the source well. Tegafur, a prodrug, when added to primary human hepatocytes in the source well, was metabolized into a cytotoxic metabolite that kills colon cancer cells (HCT 116) cultured in the adjacent cell culture well; no toxicity was observed when only medium was in the source well. These results suggest that the perfusion microplate is a useful tool for a variety of cell culture applications with benefits ranging from labor savings to enabling in vivo-like toxicity studies.

The Origin of Widespread Long-lived Volcanism Across the Galapagos Volcanic Province

NASA Astrophysics Data System (ADS)

O'Connor, J. M.; Stoffers, P.; Wijbrans, J. R.; Worthington, T. J.

2005-12-01

40Ar/39Ar ages for rocks dredged (SO144 PAGANINI expedition) and drilled (DSDP) from the Galapagos Volcanic Province (Cocos, Carnegie, Coiba and Malpelo aseismic ridges and associated seamounts) show evidence of 1) increasing age with distance from the Galapagos Archipelago, 2) long-lived episodic volcanism at many locations, and 3) broad overlapping regions of coeval volcanism. The widespread nature of synchronous volcanism across the Galapagos Volcanic Province (GVP) suggests a correspondingly large Galapagos hotspot melting anomaly (O'Connor et al., 2004). Development of the GVP via Cocos and Nazca plate migration and divergence over this broad melting anomaly would explain continued multiple phases of volcanism over millions of years following the initial onset of hotspot volcanism. The question arising from these observations is whether long-lived GVP episodic volcanism is equivalent to `rejuvenescent' or a `post-erosional' phase of volcanism that occurs hundreds of thousands or million years after the main shield-building phase documented on many mid-plate seamount chains, most notably along the Hawaiian-Emperor Seamount Chain? Thus, investigating the process responsible for long-lived episodic GVP volcanism provides the opportunity to evaluate this little understood process of rejuvenation in a physical setting very different to the Hawaiian-Emperor Chain (i.e. on/near spreading axis versus mid-plate). We consider here timing and geochemical information to test the various geodynamic models proposed to explain the origin of GVP hotspot volcanism, especially the possibility of rejuvenated phases that erupt long after initial shield-building.

The Thermal Evolution of the Galapagos Mantle Plume: Insights from Al-in-Olivine Thermometry

NASA Astrophysics Data System (ADS)

Trela, J.; Gazel, E.; Sobolev, A. V.; Class, C.; Bizimis, M.; Jicha, B. R.; Batanova, V. G.; Denyer, P.

2016-12-01

The mantle plume hypothesis is widely accepted for the formation of large igneous provinces (LIP) and many ocean island basalts (OIB). Petrologic models support a mantle plume origin by indicating high mantle temperatures (>1500 °C) for some plume-melts relative to melts generated at ambient mid ocean ridge conditions (1350 °C). Mantle plumes forming LIPs and OIBs provide our primary source of information on the geochemical and lithological heterogeneity of the lower mantle. The Galapagos hotspot represents one of the most thermally and geochemically heterogeneous plumes on the planet, sustaining long-lived isotopic and lithological heterogeneity over its 90 Ma evolution. Previous petrologic studies showed that the Galapagos plume secularly cooled over time and that the decrease in the plume's temperature correlates with an increase in a recycled (pyroxenite) component. We used Al-in-olivine thermometry to show that maximum olivine crystallization temperatures confirm secular cooling of the Galapagos plume. Olivines from the early melting stages of the plume at 90 Ma (Caribbean LIP) record the highest crystallization temperatures (1600 °C). Olivines from the current archipelago record the lowest temperatures of only 1300 °C. The largest decrease in temperature occurred between 90 and 70 Ma ( 200 °C decrease) and coincides with the plume head-tail transition. Olivines from the 60-90 Ma-old accreted Galapagos-tracks in Costa Rica and Panama record higher Ni, Fe/Mn, and lower Ca contents than those from the present-day archipelago, indicating a higher abundance of pyroxenite (recycled oceanic crust) entrained in parts of the plume head that melted to form the Caribbean LIP. However, the Galapagos plume was pyroxenite-rich for 40 Ma thus pyroxenite-entrainment goes beyond the plume-tail transition. Our results suggest that hotter regions of the Galapagos plume entrained larger amounts of dense, recycled components due to their greater buoyancy; however, this

Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5: Insights into Nucleotide Specificity, Dimerization, and cGMP-Dependent Conformational Change

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heikaus, Clemens C.; Stout, Joseph R.; Sekharan, Monica R.

2008-08-15

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular NOEs.

Philippine microplate tectonics and hydrocarbon exploration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallagher, J.J. Jr.

1986-07-01

Hydrocarbon traps in the Philippine Islands developed during a long, complex history of microplate tectonics. Carbonate and clastic stratigraphic traps formed during Mesozoic and early Cenozoic rifting and drifting. Hydrocarbons, generated in deep rift basins, migrated to the traps during drifting. Later Cenozoic compressional tectonic activity and concomitant faulting enhanced some traps and destroyed others. Seismic data offshore from Palawan Island reveal the early trap histories. Later trap histories can be interpreted from seismic, outcrop, and remote-sensing data. Understanding the microplate tectonic history of the Philippines is the key to interpreting trap histories.

Multiple lineages of Avian malaria parasites (Plasmodium) in the Galapagos Islands and evidence for arrival via migratory birds.

PubMed

Levin, I I; Zwiers, P; Deem, S L; Geest, E A; Higashiguchi, J M; Iezhova, T A; Jiménez-Uzcátegui, G; Kim, D H; Morton, J P; Perlut, N G; Renfrew, R B; Sari, E H R; Valkiunas, G; Parker, P G

2013-12-01

Haemosporidian parasites in the genus Plasmodium were recently detected through molecular screening in the Galapagos Penguin (Spheniscus mendiculus). We summarized results of an archipelago-wide screen of 3726 endemic birds representing 22 species for Plasmodium spp. through a combination of molecular and microscopy techniques. Three additional Plasmodium lineages were present in Galapagos. Lineage A-infected penguins, Yellow Warblers (Setophaga petechia aureola), and one Medium Ground Finch (Geospiza fortis) and was detected at multiple sites in multiple years [corrected]. The other 3 lineages were each detected at one site and at one time; apparently, they were transient infections of parasites not established on the archipelago. No gametocytes were found in blood smears of infected individuals; thus, endemic Galapagos birds may be dead-end hosts for these Plasmodium lineages. Determining when and how parasites and pathogens arrive in Galapagos is key to developing conservation strategies to prevent and mitigate the effects of introduced diseases. To assess the potential for Plasmodium parasites to arrive via migratory birds, we analyzed blood samples from 438 North American breeding Bobolinks (Dolichonyx oryzivorus), the only songbird that regularly migrates through Galapagos. Two of the ephemeral Plasmodium lineages (B and C) found in Galapagos birds matched parasite sequences from Bobolinks. Although this is not confirmation that Bobolinks are responsible for introducing these lineages, evidence points to higher potential arrival rates of avian pathogens than previously thought. Linajes Múltiples de Parásitos de Malaria Aviar (Plasmodium) en las Islas Galápagos y Evidencia de su Arribo por Medio de Aves Migratorias. © 2013 Society for Conservation Biology.

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation.

PubMed

Kim, Jeong Joo; Lorenz, Robin; Arold, Stefan T; Reger, Albert S; Sankaran, Banumathi; Casteel, Darren E; Herberg, Friedrich W; Kim, Choel

2016-05-03

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Copyright © 2016 Elsevier Ltd. All rights reserved.

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

DOE PAGES

Kim, Jeong Joo; Lorenz, Robin; Arold, Stefan T.; ...

2016-04-07

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Here, although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-raymore » scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG.« less

Photosensitizer adhered to cell culture microplates induces phototoxicity in carcinoma cells.

PubMed

Ziegler, Verena; Kiesslich, Tobias; Krammer, Barbara; Plaetzer, Kristjan

2013-01-01

In vitro experiments in plastic receptacles are the basis of characterization of new photosensitizers (PSs) for the photodynamic therapy. We recently reported that lipophilic PSs adhere to cell culture microplates in a kinetic-like manner (Engelhardt et al., 2011). In the current study, we examined the interaction and phototoxic effects of the microplate-adhered PS in cancer cells. Therefore, we preloaded microplates with hypericin, Foscan, PVP-hypericin, or aluminum (III) phthalocyanine tetrasulfonate chloride (AlPCS4) for 24 hours and measured the PS distribution after addition of A431 human carcinoma cells: following another 24 hours up to 68% of hypericin were detected in the cell fraction. The hydrophilic PVP-hypericin and AlPCS4 also diffused into the cells, but the quantities of PS adherence were considerably lower. Microplate-adhered Foscan appeared not to be redistributed. In contrast to the hydrophilic PSs, the cellular phototoxicity of microplate-adhered lipophilic PS was high, independent of whether the PS (i) was pre-loaded onto microplates or (ii) added simultaneously with the cells or (iii) one day after cell seeding. Based on these results, we suggest testing lipophilic PS dyes for their adherence to microplates. Furthermore, the ability of plastic materials to (reversibly) store PSs might represent a new approach for the PS delivery or the development of antimicrobial coatings.

Photosensitizer Adhered to Cell Culture Microplates Induces Phototoxicity in Carcinoma Cells

PubMed Central

Ziegler, Verena; Kiesslich, Tobias; Krammer, Barbara; Plaetzer, Kristjan

2013-01-01

In vitro experiments in plastic receptacles are the basis of characterization of new photosensitizers (PSs) for the photodynamic therapy. We recently reported that lipophilic PSs adhere to cell culture microplates in a kinetic-like manner (Engelhardt et al., 2011). In the current study, we examined the interaction and phototoxic effects of the microplate-adhered PS in cancer cells. Therefore, we preloaded microplates with hypericin, Foscan, PVP-hypericin, or aluminum (III) phthalocyanine tetrasulfonate chloride (AlPCS4) for 24 hours and measured the PS distribution after addition of A431 human carcinoma cells: following another 24 hours up to 68% of hypericin were detected in the cell fraction. The hydrophilic PVP-hypericin and AlPCS4 also diffused into the cells, but the quantities of PS adherence were considerably lower. Microplate-adhered Foscan appeared not to be redistributed. In contrast to the hydrophilic PSs, the cellular phototoxicity of microplate-adhered lipophilic PS was high, independent of whether the PS (i) was pre-loaded onto microplates or (ii) added simultaneously with the cells or (iii) one day after cell seeding. Based on these results, we suggest testing lipophilic PS dyes for their adherence to microplates. Furthermore, the ability of plastic materials to (reversibly) store PSs might represent a new approach for the PS delivery or the development of antimicrobial coatings. PMID:23509741

Neutron diffraction reveals hydrogen bonds critical for cGMP-selective activation: Insights for cGMP-dependent protein kinase agonist design

DOE PAGES

Huang, Gilbert Y.; Gerlits, Oksana O.; Blakeley, Matthew P.; ...

2014-10-01

High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). Finally, the XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted,more » explaining its low affinity for cAMP.« less

Plant species dispersed by Galapagos tortoises surf the wave of habitat suitability under anthropogenic climate change

PubMed Central

Blake, Stephen; Soultan, Alaaeldin; Guézou, Anne; Cabrera, Fredy; Lötters, Stefan

2017-01-01

Native biodiversity on the Galapagos Archipelago is severely threatened by invasive alien species. On Santa Cruz Island, the abundance of introduced plant species is low in the arid lowlands of the Galapagos National Park, but increases with elevation into unprotected humid highlands. Two common alien plant species, guava (Psidium guajava) and passion fruit (Passiflora edulis) occur at higher elevations yet their seeds are dispersed into the lowlands by migrating Galapagos tortoises (Chelonoidis spp.). Tortoises transport large quantities of seeds over long distances into environments in which they have little or no chance of germination and survival under current climate conditions. However, climate change is projected to modify environmental conditions on Galapagos with unknown consequences for the distribution of native and introduced biodiversity. We quantified seed dispersal of guava and passion fruit in tortoise dung piles and the distribution of adult plants along two elevation gradients on Santa Cruz to assess current levels of ‘wasted’ seed dispersal. We computed species distribution models for both taxa under current and predicted future climate conditions. Assuming that tortoise migratory behaviour continues, current levels of “wasted” seed dispersal in lowlands were projected to decline dramatically in the future for guava but not for passion fruit. Tortoises will facilitate rapid range expansion for guava into lowland areas within the Galapagos National Park where this species is currently absent. Coupled with putative reduction in arid habitat for native species caused by climate change, tortoise driven guava invasion will pose a serious threat to local plant communities. PMID:28727747

Plant species dispersed by Galapagos tortoises surf the wave of habitat suitability under anthropogenic climate change.

PubMed

Ellis-Soto, Diego; Blake, Stephen; Soultan, Alaaeldin; Guézou, Anne; Cabrera, Fredy; Lötters, Stefan

2017-01-01

Native biodiversity on the Galapagos Archipelago is severely threatened by invasive alien species. On Santa Cruz Island, the abundance of introduced plant species is low in the arid lowlands of the Galapagos National Park, but increases with elevation into unprotected humid highlands. Two common alien plant species, guava (Psidium guajava) and passion fruit (Passiflora edulis) occur at higher elevations yet their seeds are dispersed into the lowlands by migrating Galapagos tortoises (Chelonoidis spp.). Tortoises transport large quantities of seeds over long distances into environments in which they have little or no chance of germination and survival under current climate conditions. However, climate change is projected to modify environmental conditions on Galapagos with unknown consequences for the distribution of native and introduced biodiversity. We quantified seed dispersal of guava and passion fruit in tortoise dung piles and the distribution of adult plants along two elevation gradients on Santa Cruz to assess current levels of 'wasted' seed dispersal. We computed species distribution models for both taxa under current and predicted future climate conditions. Assuming that tortoise migratory behaviour continues, current levels of "wasted" seed dispersal in lowlands were projected to decline dramatically in the future for guava but not for passion fruit. Tortoises will facilitate rapid range expansion for guava into lowland areas within the Galapagos National Park where this species is currently absent. Coupled with putative reduction in arid habitat for native species caused by climate change, tortoise driven guava invasion will pose a serious threat to local plant communities.

The Galapagos Islands: Darwin and Modern Conservation Stories

ERIC Educational Resources Information Center

Burr, Susan

2010-01-01

The author visited the Galapagos Islands in 2009 and here looks at their biodiversity through pictures taken then. The diverse habitats of the Islands are reflected in the great diversity of flora and fauna found on them, with many species endemic to the Islands. The stories of the land iguanas, control of introduced species and the giant…

Genetic Diversity and Population Structure of Two Tomato Species from the Galapagos Islands

PubMed Central

Pailles, Yveline; Ho, Shwen; Pires, Inês S.; Tester, Mark; Negrão, Sónia; Schmöckel, Sandra M.

2017-01-01

Endemic flora of the Galapagos Islands has adapted to thrive in harsh environmental conditions. The wild tomato species from the Galapagos Islands, Solanum cheesmaniae and S. galapagense, are tolerant to various stresses, and can be crossed with cultivated tomato. However, information about genetic diversity and relationships within and between populations is necessary to use these resources efficiently in plant breeding. In this study, we analyzed 3,974 polymorphic SNP markers, obtained through the genotyping-by-sequencing technique, DArTseq, to elucidate the genetic diversity and population structure of 67 accessions of Galapagos tomatoes (compared to two S. lycopersicum varieties and one S. pimpinellifolium accession). Two clustering methods, Principal Component Analysis and STRUCTURE, showed clear distinction between the two species and a subdivision in the S. cheesmaniae group corresponding to geographical origin and age of the islands. High genetic variation among the accessions within each species was suggested by the AMOVA. High diversity in the S. cheesmaniae group and its correlation with the islands of origin were also suggested. This indicates a possible influence of the movement of the islands, from west to east, on the gene flow. Additionally, the absence of S. galapagense populations in the eastern islands points to the species divergence occurring after the eastern islands became isolated. Based on these results, it can be concluded that the population structure of the Galapagos tomatoes collection partially explains the evolutionary history of both species, knowledge that facilitates exploitation of their genetic potential for the identification of novel alleles contributing to stress tolerance. PMID:28261227

Enhanced dual-frequency operation of a polymerized liquid crystal microplate by liquid crystal infiltration

NASA Astrophysics Data System (ADS)

Kumagai, Takayuki; Yoshida, Hiroyuki; Ozaki, Masanori

2017-04-01

The electric-field-induced switching behavior of a polymer microplate is investigated. A microplate fabricated with a photopolymerizable dual-frequency liquid crystal was surrounded by an unpolymerized photopolymerizable dual-frequency liquid crystal in the isotropic phase. As an electric field was applied along the plane of the microplate, the microplate switched to set its interior molecular orientation to be either parallel or perpendicular to the field, depending on the frequency. Analysis of the rotational behavior, as well as numerical calculations, showed that the surrounding unpolymerized photopolymerizable dual-frequency liquid crystal infiltrated into the microplate, which enhanced the dielectric properties of the microplate. To the best of our knowledge, this is the first report of an enhanced dual-frequency dielectric response of a polymer microplate induced by liquid crystal infiltration.

Forecastable and Guidable Bubble-Propelled Microplate Motors for Cell Transport.

PubMed

Hu, Narisu; Zhang, Bin; Gai, Meiyu; Zheng, Ce; Frueh, Johannes; He, Qiang

2017-06-01

Cell transport is important to renew body functions and organs with stem cells, or to attack cancer cells with immune cells. The main hindrances of this method are the lack of understanding of cell motion as well as proper transport systems. In this publication, bubble-propelled polyelectrolyte microplates are used for controlled transport and guidance of HeLa cells. Cells survive attachment on the microplates and up to 22 min in 5% hydrogen peroxide solution. They can be guided by a magnetic field whereby increased friction of cells attached to microplates decreases the speed by 90% compared to pristine microplates. The motion direction of the cell-motor system is easier to predict due to the cell being opposite to the bubbles. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Competition influence in the segregation of the trophic niche of otariids: a case study using isotopic Bayesian mixing models in Galapagos pinnipeds.

PubMed

Páez-Rosas, Diego; Rodríguez-Pérez, Mónica; Riofrío-Lazo, Marjorie

2014-12-15

The feeding success of predators is associated with the competition level for resources, and, thus, sympatric species are exposed to a potential trophic overlap. Isotopic Bayesian mixing models should provide a better understanding of the contribution of preys to the diet of predators and the feeding behavior of a species over time. The carbon and nitrogen isotopic signatures from pup hair samples of 93 Galapagos sea lions and 48 Galapagos fur seals collected between 2003 and 2009 in different regions (east and west) of the archipelago were analyzed. A PDZ Europa ANCA-GSL elemental analyzer interfaced with a PDZ Europa 20-20 continuous flow gas source mass spectrometer was employed. Bayesian models, SIAR and SIBER, were used to estimate the contribution of prey to the diet of predators, the niche breadth, and the trophic overlap level between the populations. Statistical differences in the isotopic values of both predators were observed over the time. The mixing model determined that Galapagos fur seals had a primarily teutophagous diet, whereas the Galapagos sea lions fed exclusively on fish in both regions of the archipelago. The SIBER analysis showed differences in the trophic niche between the two sea lion populations, with the western rookery of the Galapagos sea lion being the population with the largest trophic niche area. A trophic niche partitioning between Galapagos fur seals and Galapagos sea lions in the west of the archipelago is suggested by our results. At intraspecific level, the western population of the Galapagos sea lion (ZwW) showed higher trophic breadth than the eastern population, a strategy adopted by the ZwW to decrease the interspecific competition levels in the western region. Copyright © 2014 John Wiley & Sons, Ltd.

Enzymatic Production of c-di-GMP Using a Thermophilic Diguanylate Cyclase.

PubMed

Venkataramani, Prabhadevi; Liang, Zhao-Xun

2017-01-01

C-di-GMP has emerged as a prevalent bacterial messenger that controls a multitude of bacterial behaviors. Having access to milligram or gram quantities of c-di-GMP is essential for the biochemical and structural characterization of enzymes and effectors involved in c-di-GMP signaling. Although c-di-GMP can be synthesized using chemical methods, diguanylate cyclases (DGC)-based enzymatic synthesis is the most efficient method of preparing c-di-GMP today. Many DGCs are not suitable for c-di-GMP production because of poor protein stability and the presence of a c-di-GMP-binding inhibitory site (I-site) in most DGCs. We have identified and engineered a thermophilic DGC for efficient production of c-di-GMP for characterizing c-di-GMP signaling proteins and riboswitches. Importantly, residue replacement in the inhibitory I-site of the thermophilic DGC drastically relieved product inhibition to enable the production of hundreds of milligrams of c-di-GMP using 5-10 mg of this robust biocatalyst.

Microplate-based method for high-throughput screening of microalgae growth potential.

PubMed

Van Wagenen, Jon; Holdt, Susan Løvstad; De Francisci, Davide; Valverde-Pérez, Borja; Plósz, Benedek Gy; Angelidaki, Irini

2014-10-01

Microalgae cultivation conditions in microplates will differ from large-scale photobioreactors in crucial parameters such as light profile, mixing and gas transfer. Hence volumetric productivity (P(v)) measurements made in microplates cannot be directly scaled up. Here we demonstrate that it is possible to use microplates to measure characteristic exponential growth rates and determine the specific growth rate light intensity dependency (μ-I curve), which is useful as the key input for several models that predict P(v). Nannochloropsis salina and Chlorella sorokiniana specific growth rates were measured by repeated batch culture in microplates supplied with continuous light at different intensities. Exponential growth unlimited by gas transfer or self-shading was observable for a period of several days using fluorescence, which is an order of magnitude more sensitive than optical density. The microplate datasets were comparable to similar datasets obtained in photobioreactors and were used an input for the Huesemann model to accurately predict P(v). Copyright © 2014 Elsevier Ltd. All rights reserved.

Absorbance and fluorometric sensing with capillary wells microplates.

PubMed

Tan, Han Yen; Cheong, Brandon Huey-Ping; Neild, Adrian; Liew, Oi Wah; Ng, Tuck Wah

2010-12-01

Detection and readout from small volume assays in microplates are a challenge. The capillary wells microplate approach [Ng et al., Appl. Phys. Lett. 93, 174105 (2008)] offers strong advantages in small liquid volume management. An adapted design is described and shown here to be able to detect, in a nonimaging manner, fluorescence and absorbance assays minus the error often associated with meniscus forming at the air-liquid interface. The presence of bubbles in liquid samples residing in microplate wells can cause inaccuracies. Pipetting errors, if not adequately managed, can result in misleading data and wrong interpretations of assay results; particularly in the context of high throughput screening. We show that the adapted design is also able to detect for bubbles and pipetting errors during actual assay runs to ensure accuracy in screening.

Interactions of social, terrestrial, and marine sub-systems in the Galapagos Islands, Ecuador.

PubMed

Walsh, Stephen J; Mena, Carlos F

2016-12-20

Galapagos is often cited as an example of the conflicts that are emerging between resource conservation and economic development in island ecosystems, as the pressures associated with tourism threaten nature, including the iconic and emblematic species, unique terrestrial landscapes, and special marine environments. In this paper, two projects are described that rely upon dynamic systems models and agent-based models to examine human-environment interactions. We use a theoretical context rooted in complexity theory to guide the development of our models that are linked to social-ecological dynamics. The goal of this paper is to describe key elements, relationships, and processes to inform and enhance our understanding of human-environment interactions in the Galapagos Islands of Ecuador. By formalizing our knowledge of how systems operate and the manner in which key elements are linked in coupled human-natural systems, we specify rules, relationships, and rates of exchange between social and ecological features derived through statistical functions and/or functions specified in theory or practice. The processes described in our models also have practical applications in that they emphasize how political policies generate different human responses and model outcomes, many detrimental to the social-ecological sustainability of the Galapagos Islands.

Crude and purified proteasome activity assays are affected by type of microplate.

PubMed

Cui, Ziyou; Gilda, Jennifer E; Gomes, Aldrin V

2014-02-01

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate. Copyright © 2013 Elsevier Inc. All rights reserved.

New Insights on Submarine Volcanism in the Western Galapagos Archipelago from High Resolution Sonar and Magnetic Surveys

NASA Astrophysics Data System (ADS)

Glass, J. B.; Fornari, D. J.; Tivey, M. A.; Hall, H. F.; Cougan, A. A.; Berkenbosch, H. A.; Holmes, M. L.; White, S. M.; de La Torre, G.

2006-12-01

We combine high-resolution MR-1 sidescan sonar and EM-300 bathymetric data collected on four cruises (AHA-Nemo2 in 2000 (R/V Melville), DRIFT4 in 2001 (R/V Revelle), TN188 and TN189 in January 2006 (R/V Thompson) to study volcanic platform-building processes on the submarine flanks of Fernandina, Isabela, Roca Redonda and Santiago volcanoes, in the western Galapagos. Three primary volcanic provinces were identified including: rift zones (16, ranging from 5 to 20 km in length), small submarine volcanic cones (<3 km in diameter and several 100 m high) and deep (>3000 m), long (>10 km), large-area submarine lava flows. Lengths of the Galapagos rift zones are comparable to western Canary Island rift zones, but significantly shorter than Hawaiian submarine rift zones, possibly reflecting lower magma supply. A surface-towed magnetic survey was conducted over the NW Fernandina rift on TN189 and Fourier inversions were performed to correct for topographic effects. Calculated magnetization was highest (up to +32 A/m) over the shallow southwest flank of the rift, coinciding with cone fields and suggesting most recent volcanism has focused at this portion of the rift. Small submarine volcanic cones with various morphologies (e.g., pointed, cratered and occasionally breached) are common in the submarine western Galapagos both on rift zones and on the island flanks where no rifts are present, such as the northern flank of Santiago Island. Preliminary study of these cones suggests that their morphologies and depth of occurrence may reflect a combination of petrogenetic and eruption processes. Deep, long large-area lava flow fields in regions of low bathymetric relief have been previously identified as a common seafloor feature in the western Galapagos by Geist et al. [in press], and new EM300 data show that a number of the deep lava flows originate from small cones along the mid-lower portion of the NW submarine rift of Fernandina. Our high-resolution sonar data suggest that

Absorbance and fluorometric sensing with capillary wells microplates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Han Yen; Cheong, Brandon Huey-Ping; Neild, Adrian

2010-12-15

Detection and readout from small volume assays in microplates are a challenge. The capillary wells microplate approach [Ng et al., Appl. Phys. Lett. 93, 174105 (2008)] offers strong advantages in small liquid volume management. An adapted design is described and shown here to be able to detect, in a nonimaging manner, fluorescence and absorbance assays minus the error often associated with meniscus forming at the air-liquid interface. The presence of bubbles in liquid samples residing in microplate wells can cause inaccuracies. Pipetting errors, if not adequately managed, can result in misleading data and wrong interpretations of assay results; particularly inmore » the context of high throughput screening. We show that the adapted design is also able to detect for bubbles and pipetting errors during actual assay runs to ensure accuracy in screening.« less

Perceptions of Body Size in Mothers and Their Young Children in the Galapagos Islands.

PubMed

Waldrop, Julee B; Page, Rachel A; Bentley, Margaret E

2016-10-01

Introduction Little specific information has been published about the health of people who live in the Galapagos Islands. As part of determining the status of the nutrition transition that may be occurring in the islands mothers of young children in the Galapagos perceptions of their child's body size and therefore health status was evaluated along with actual body size. Methods This paper presents data collected as part of a pilot study that used a mixed methods approach to identify and describe health and nutrition issues for mother-child pairs on Isla Isabela in Galapagos, Ecuador. It includes participant anthropometric assessment and self-perception of body size using silhouettes for themselves and one of their children along with open-ended questions to elicit further understanding of body size perceptions. Twenty mothers of children greater than 6 months of age but less than 6 years of age were interviewed. Results The women preferred a smaller body size for themselves but a larger body size for their children. Findings of different body size combinations between mothers and children in the same household demonstrated that the island is undergoing or may be post the nutrition transition. Discussion This dual burden of body weights (especially overweight or obese mothers) in the same household with underweight, normal and overweight or obese children and the potential nutrition related chronic disease burden in the future will require more educational resources and innovative health services than are currently available for the people of the Galapagos.

NASA Spacecraft Peers Into the Mouth of the Galapagos Wolf Volcano

NASA Image and Video Library

2015-06-12

On May 26, 2015, Wolf Volcano on Isabela Island in the Galapagos Islands erupted for the first time in 33 years. This image was acquired by NASA Terra spacecraft on June 11, 2015, after the eruption had quieted.

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay.

PubMed

Yoo, Gu; Park, Min; Lee, Eun-Hang; Jose, Joachim; Pyun, Jae-Chul

2011-11-30

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Cyclic Di-GMP modulates the disease progression of Erwinia amylovora.

PubMed

Edmunds, Adam C; Castiblanco, Luisa F; Sundin, George W; Waters, Christopher M

2013-05-01

The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.

Cyclic Di-GMP Modulates the Disease Progression of Erwinia amylovora

PubMed Central

Edmunds, Adam C.; Castiblanco, Luisa F.; Sundin, George W.

2013-01-01

The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis. PMID:23475975

Interactions of social, terrestrial, and marine sub-systems in the Galapagos Islands, Ecuador

PubMed Central

Walsh, Stephen J.; Mena, Carlos F.

2016-01-01

Galapagos is often cited as an example of the conflicts that are emerging between resource conservation and economic development in island ecosystems, as the pressures associated with tourism threaten nature, including the iconic and emblematic species, unique terrestrial landscapes, and special marine environments. In this paper, two projects are described that rely upon dynamic systems models and agent-based models to examine human–environment interactions. We use a theoretical context rooted in complexity theory to guide the development of our models that are linked to social–ecological dynamics. The goal of this paper is to describe key elements, relationships, and processes to inform and enhance our understanding of human–environment interactions in the Galapagos Islands of Ecuador. By formalizing our knowledge of how systems operate and the manner in which key elements are linked in coupled human–natural systems, we specify rules, relationships, and rates of exchange between social and ecological features derived through statistical functions and/or functions specified in theory or practice. The processes described in our models also have practical applications in that they emphasize how political policies generate different human responses and model outcomes, many detrimental to the social–ecological sustainability of the Galapagos Islands. PMID:27791072

cGMP accumulation causes photoreceptor degeneration in CNG channel deficiency: evidence of cGMP cytotoxicity independently of enhanced CNG channel function.

PubMed

Xu, Jianhua; Morris, Lynsie; Thapa, Arjun; Ma, Hongwei; Michalakis, Stylianos; Biel, Martin; Baehr, Wolfgang; Peshenko, Igor V; Dizhoor, Alexander M; Ding, Xi-Qin

2013-09-11

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca(2+) influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3(-/-);Nrl(-/-) mice with CNGA3 deficiency on a cone-dominant background was sharply increased at postnatal day 8 (P8), peaked around P10-P15, remained high through P30-P60, and returned to near control level at P90. This elevation pattern correlated with photoreceptor apoptotic death, which peaked around P15-P20. In Cnga3(-/-);Gucy2e(-/-) mice lacking retGC1, cone density and expression levels of cone-specific proteins were significantly increased compared with Cnga3(-/-), consistent with a role of cGMP accumulation as the major contributor to cone death caused by CNG channel deficiency. The activity and expression levels of cGMP-dependent protein kinase G (PKG) were significantly increased in Cnga3(-/-);Nrl(-/-) retina compared with Nrl(-/-), suggesting an involvement of PKG regulation in cell death. Our results indicate that cGMP accumulation in photoreceptors can itself exert cytotoxic effect in cones, independently of CNG channel activity and Ca(2+) influx.

Modulation of cGMP in Heart Failure

PubMed Central

Boerrigter, Guido; Lapp, Harald; Burnett, John C.

2009-01-01

Heart failure (HF) is a common disease that continues to be associated with high morbidity and mortality warranting novel therapeutic strategies. Cyclic guanosine monophosphate (cGMP) is the second messenger of several important signaling pathways based on distinct guanylate cyclases (GCs) in the cardiovascular system. Both the nitric oxide/soluble GC (NO/sGC) as well as the natriuretic peptide/GC-A (NP/GC-A) systems are disordered in HF, providing a rationale for their therapeutic augmentation. Soluble GC activation with conventional nitrovasodilators has been used for more than a century but is associated with cGMP-independent actions and the development of tolerance, actions which novel NO-independent sGC activators now in clinical development lack. Activation of GC-A by administration of naturally occurring or designer natriuretic peptides is an emerging field, as is the inhibition of enzymes that degrade endogenous NPs. Finally, inhibition of cGMP-degrading phosphodiesterases, particularly phosphodiesterase 5 provides an additional strategy to augment cGMP-signaling. PMID:19089342

The pattern of circumferential and radial eruptive fissures on the volcanoes of Fernandina and Isabela islands, Galapagos

USGS Publications Warehouse

Chadwick, W.W.; Howard, K.A.

1991-01-01

Maps of the eruptive vents on the active shield volcanoes of Fernandina and Isabela islands, Galapagos, made from aerial photographs, display a distinctive pattern that consists of circumferential eruptive fissures around the summit calderas and radial fissures lower on the flanks. On some volcano flanks either circumferential or radial eruptions have been dominant in recent time. The location of circumferential vents outside the calderas is independent of caldera-related normal faults. The eruptive fissures are the surface expression of dike emplacement, and the dike orientations are interpreted to be controlled by the state of stress in the volcano. Very few subaerial volcanoes display a pattern of fissures similar to that of the Galapagos volcanoes. Some seamounts and shield volcanoes on Mars morphologically resemble the Galapagos volcanoes, but more specific evidence is needed to determine if they also share common structure and eruptive style. ?? 1991 Springer-Verlag.

Enhancement of Fluorescence-Based Sandwich Immunoassay Using Multilayered Microplates Modified with Plasma-Polymerized Films

PubMed Central

Yano, Kazuyoshi; Iwasaki, Akira

2016-01-01

A functional modification of the surface of a 96-well microplate coupled with a thin layer deposition technique is demonstrated for enhanced fluorescence-based sandwich immunoassays. The plasma polymerization technique enabling the deposition of organic thin films was employed for the modification of the well surface of a microplate. A silver layer and a plasma-polymerized film were consecutively deposited on the microplate as a metal mirror and the optical interference layer, respectively. When Cy3-labeled antibody was applied to the wells of the resulting multilayered microplate without any immobilization step, greatly enhanced fluorescence was observed compared with that obtained with the unmodified one. The same effect could be also exhibited for an immunoassay targeting antigen directly adsorbed on the multilayered microplate. Furthermore, a sandwich immunoassay for the detection of interleukin 2 (IL-2) was performed with the multilayered microplates, resulting in specific and 88-fold–enhanced fluorescence detection. PMID:28029144

Magmatic evolution of the Easter microplate-Crough Seamount region (South East Pacific)

USGS Publications Warehouse

Hekinian, R.; Stoffers, P.; Akermand, D.; Binard, N.; Francheteau, Jean; Devey, C.; Garbe-Schonberg, D.

1995-01-01

The Easter microplate-Crough Seamount region located between 25?? S-116?? W and 25?? S-122?? W consists of a chain of seamounts forming isolated volcanoes and elongated (100-200 km in length) en echelon volcanic ridges oriented obliquely NE (N 065??), to the present day general spreading direction (N 100??) of the Pacific-Nazca plates. The extension of this seamount chain into the southwestern edge of the Easter microplate near 26??30??? S-115?? W was surveyed and sampled. The southern boundary including the Orongo fracture zone and other shallow ridges ( 0.25) MORBs which are similar in composition to other more recent basalts from the Southwest and East Rifts spreading axes of the Easter microplate. Incompatible element ratios normalized to chondrite values [(Ce/Yb)N = 1-2.5}, {(La/Sm)N = 0.4-1.2} and {(Zr/Y)N = 0.7-2.5} of the basalts are also similar to present day volcanism found in the Easter microplate. The volcanics from the Easter microplate-Crough region are unrelated to other known South Pacific intraplate magmatism (i.e. Society, Pitcairn, and Salas y Gomez Islands). Instead their range in incompatible element ratios is comparable to the submarine basalts from the recently investigated Ahu and Umu volcanic field (Easter hotspot) (Scientific Party SO80, 1993) and centered at about 80 km west of Easter Island. The oblique ridges and their associated seamounts are likely to represent ancient leaky transform faults created during the initial stage of the Easter microplate formation (??? 5 Ma). It appears that volcanic activity on seamounts overlying the oblique volcanic ridges has continued during their westward drift from the microplate as shown by the presence of relatively fresh lava observed on one of these structures, namely the first Oblique Volcanic Ridge near 25?? S-118?? W at about 160 km west of the Easter microplate West Rift. Based on a reconstruction of the Easter microplate, it is suggested that the Crough seamount (< 800 m depth) was formed

Crustal Accretion and Mantle Geodynamics at Microplates: Constraints from Gravity Analysis

NASA Astrophysics Data System (ADS)

Ames, K.; Georgen, J. E.; Dordevic, M. M.

2013-12-01

Oceanic crustal accretion occurs in a variety of locations, including mid-ocean ridges and back-arc spreading centers, and in unique settings within these systems, such as plate boundary triple junctions, intra-transform spreading centers, and microplates. This study focuses on crustal accretion and mantle geodynamics at microplates. The Easter and Juan Fernandez microplates are located in the South Pacific along the Pacific, Nazca and Antarctic plate boundaries. Both microplates formed 3-5 Ma and they are currently rotating clockwise at 15 deg/Ma and 9 deg/Ma respectively (e.g., Searle et al. J. Geol. Soc. Lond. 1993). The study area also encompasses the Easter/Sala y Gomez mantle plume and the Foundation seamount chain, both of which are located close to spreading centers. We calculate mantle Bouguer anomaly (MBA) from satellite gravity measurements and shipboard soundings in order to gain a better understanding of the thermal structure of these two oceanic microplates and to quantify the effect that melting anomalies may have on their boundaries. We assume a crustal thickness of 6.0 km, a 1.7 g/cm^3 density difference at the water/crust interface, and a 0.6 g/cm^3 density difference at the crust/mantle interface. The west rift of the Easter microplate has an MBA low ranging from approximately -50 to -100 mGal, while the east rift has slightly higher MBA values ranging from roughly 10 to -50 mGal. The west rift of the Juan Fernandez microplate has a maximum MBA low of about -100 mGal with a sharp increase to -20 mGal at -35 deg S. The east rift of the Juan Fernandez microplate is characterized by more variable MBA, ranging from 0 to -140 mGal. The MBA low associated with the Easter/Sala y Gomez mantle plume has a maximum amplitude about 150 mGal. Likewise, the Foundation seamounts show a gravity low of -140 to -150 mGal. These spatial variations in gravity, as well as published isotopic data and exploratory numerical models, are used to constrain upper mantle

Structural Basis of Ligand Binding by a C-di-GMP Riboswitch

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, K.; Lipchock, S; Ames, T

2009-01-01

The second messenger signaling molecule bis-(3{prime}-5{prime})-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 {angstrom} resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structuralmore » data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.« less

Customizable PCR-microplate array for differential identification of multiple pathogens

PubMed Central

Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

2014-01-01

Customizable PCR-microplate arrays were developed for the rapid identification of Francisella tularensis subsp. tularensis, Salmonella Typhi, Shigella dysenteriae, Yersinia pestis, Vibrio cholerae Escherichia coli O157:H7, Salmonella Typhimurium, Salmonella Saintpaul, Francisella tularensis subsp. novicida, Vibrio parahaemolyticus, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of the pathogens above. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers. A mixed aliquot of genomic DNA from 38 different strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Results show specific amplifications on all the three custom plates. In a preliminary test to evaluate the sensitivity of these assays in laboratory-inoculated samples, detection limits as low as 9 cfu/g/ml S. Typhimurium were obtained from beef hot dog, and 78 cfu/ml from milk. Such microplate arrays could serve as valuable tools for initial identification or secondary confirmation of these pathogens. PMID:24215700

Customizable PCR-microplate array for differential identification of multiple pathogens.

PubMed

Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

2013-11-01

Customizable PCR-microplate arrays were developed for the rapid identification of Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Typhi, Shigella dysenteriae, Escherichia coli O157:H7, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. novicida, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of these pathogens. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers identified. A mixed aliquot of genomic DNA from 38 strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Specific amplifications were obtained on all three custom plates. In preliminary tests performed to evaluate the sensitivity of these assays in samples inoculated in the laboratory with Salmonella Typhimurium, amplifications were obtained from 1 g of beef hot dog inoculated at as low as 9 CFU/ml or from milk inoculated at as low as 78 CFU/ml. Such microplate arrays could be valuable tools for initial identification or secondary confirmation of contamination by these pathogens.

Development of LEDs-based microplate reader for bioanalytical assay measurements

NASA Astrophysics Data System (ADS)

Alaruri, Sami D.; Katzlinger, Michael; Schinwald, Bernhard; Kronberger, Georg; Atzler, Joseph

2013-10-01

The optical design for an LEDs-based microplate reader that can perform fluorescence intensity (top and bottom), absorbance, luminescence and time-resolved fluorescence measurements is described. The microplate reader is the first microplate reader in the marketplace that incorporates LEDs as excitation light sources. Absorbance measurements over the 0-3.5 optical density range for caffeine solution are presented. Additionally, fluorescence intensity readings collected at 535 and 625 nm from a green and a red RediPlateTM are reported. Furthermore, fluorescence decay lifetime measurements obtained for Eu (europium) and Sm (samarium) standard solutions using 370 nm excitation are presented. The microplate reader detection limits for the fluorescence intensity top, fluorescence intensity bottom, fluorescence polarization and time-resolved fluorescence modes are 1.5 fmol 100 µL-1 fluorescein (384-well plate), 25 fmol 100 µL-1 fluorescein (384-well plate), 5 mP at 10 nM fluorescein (black 384-well plate) and 30 amol 100 µL-1 europium solution (white 384-well plate), respectively.

Enhanced Vascular Effects of Cyclic GMP in Septic Rat Aorta

DTIC Science & Technology

1988-01-01

enzyme in turn catalyzes Integrative Comp. Physiol. 23): R436-R442, 1988--The mod- the synthesis of 3’,5’-cyclic monophosp#* (cGMP), ulation of... synthesis of endogenous cGMP or after aug- significant disparity in cGMP content of tissue from mentation of intracellular cGMP concentration by treat...and a proposal. J. Vascular reactivity in endotoxin shock: effect of lidocaine or in- Surg. Res. 29: 189-201, 1980. UNCLASSIFIED SECURITY CLASSIFICATION

Saddle-nose deformity repair with microplate-adapted costal cartilage.

PubMed

Eren, Fikret; Öksüz, Sinan; Melikoğlu, Cenk; Karagöz, Hüseyin; Ülkür, Ersin

2014-08-01

Nasal deformities affecting the bone and lower two-thirds of the nose due to the loss of septal height and tip support are defined as "saddle-nose" deformity. Reconstruction of a saddle-nose deformity essentially necessitates structural grafting. This article presents an alternative approach for correction of saddle-nose deformity using a microplate and costal cartilage. The results are compared with those of the previously applied costal cartilage repair methods. Between 2004 and 2013, 16 patients were treated with costal cartilage autografts. Of these 16 patients, 7 were treated with a microplate and costal cartilage autograft combination, 4 were treated with a costal cartilage autograft and Kirschner (K)-wire, and 5 were treated with onlay costal cartilage grafts. The mean follow-up periods were 16 months for group treated with microplate-adapted autologous costal cartilage, 12 months for the group treated with K-wire and autologous costal cartilage, and 16 months for the group treated with onlay costal cartilage. The patients treated with K-wire inserted cartilages and the patients treated onlay dorsal costal cartilages encountered complications such as extrusion of the wire and warping, respectively. The seven patients treated with microplate and dorsal onlay costal cartilage graft did not experience any infection, warping, or extrusion complication. The warping tendency of the costal cartilage autograft can be efficiently prevented without a prominent complication risk by using microplate-adapted costal cartilage grafts. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Novel versatile smart phone based Microplate readers for on-site diagnoses.

PubMed

Fu, Qiangqiang; Wu, Ze; Li, Xiuqing; Yao, Cuize; Yu, Shiting; Xiao, Wei; Tang, Yong

2016-07-15

Microplate readers are important diagnostic instruments, used intensively for various readout test kits (biochemical analysis kits and ELISA kits). However, due to their expensive and non-portability, commercial microplate readers are unavailable for home testing, community and rural hospitals, especially in developing countries. In this study, to provide a field-portable, cost-effective and versatile diagnostic tool, we reported a novel smart phone based microplate reader. The basic principle of this devise relies on a smart phone's optical sensor that measures transmitted light intensities of liquid samples. To prove the validity of these devises, developed smart phone based microplate readers were applied to readout results of various analytical targets. These targets included analanine aminotransferase (ALT; limit of detection (LOD) was 17.54 U/L), alkaline phosphatase (AKP; LOD was 15.56 U/L), creatinine (LOD was 1.35μM), bovine serum albumin (BSA; LOD was 0.0041mg/mL), prostate specific antigen (PSA; LOD was 0.76pg/mL), and ractopamine (Rac; LOD was 0.31ng/mL). The developed smart phone based microplate readers are versatile, portable, and inexpensive; they are unique because of their ability to perform under circumstances where resources and expertize are limited. Copyright © 2016 Elsevier B.V. All rights reserved.

Allometric and temporal scaling of movement characteristics in Galapagos tortoises

USGS Publications Warehouse

Bastille-Rousseau, Guillaume; Yackulic, Charles B.; Frair, Jacqueline L.; Cabrera, Freddy; Blake, Stephen

2016-01-01

Understanding how individual movement scales with body size is of fundamental importance in predicting ecological relationships for diverse species. One-dimensional movement metrics scale consistently with body size yet vary over different temporal scales. Knowing how temporal scale influences the relationship between animal body size and movement would better inform hypotheses about the efficiency of foraging behaviour, the ontogeny of energy budgets, and numerous life-history trade-offs.We investigated how the temporal scaling of allometric patterns in movement varies over the course of a year, specifically during periods of motivated (directional and fast movement) and unmotivated (stationary and tortuous movement) behaviour. We focused on a recently diverged group of species that displays wide variation in movement behaviour – giant Galapagos tortoises (Chelonoidis spp.) – to test how movement metrics estimated on a monthly basis scaled with body size.We used state-space modelling to estimate seven different movement metrics of Galapagos tortoises. We used log-log regression of the power law to evaluate allometric scaling for these movement metrics and contrasted relationships by species and sex.Allometric scaling of movement was more apparent during motivated periods of movement. During this period, allometry was revealed at multiple temporal intervals (hourly, daily and monthly), with values observed at daily and monthly intervals corresponding most closely to the expected one-fourth scaling coefficient, albeit with wide credible intervals. We further detected differences in the magnitude of scaling among taxa uncoupled from observed differences in the temporal structuring of their movement rates.Our results indicate that the definition of temporal scales is fundamental to the detection of allometry of movement and should be given more attention in movement studies. Our approach not only provides new conceptual insights into temporal attributes in one

Measurement of filter paper activities of cellulase with microplate-based assay.

PubMed

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2016-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC.

Measurement of filter paper activities of cellulase with microplate-based assay

PubMed Central

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2015-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC. PMID:26858572

Euler-Vector Clustering of GPS Velocities Defines Microplate Geometry in Southwest Japan

NASA Astrophysics Data System (ADS)

Savage, J. C.

2018-02-01

I have used Euler-vector clustering to assign 469 GEONET stations in southwest Japan to k clusters (k = 2, 3,..., 9) so that, for any k, the velocities of stations within each cluster are most consistent with rigid-block motion on a sphere. That is, I attempt to explain the raw (i.e., uncorrected for strain accumulation), 1996-2006 velocities of those 469 Global Positioning System stations by rigid motion of k clusters on the surface of a spherical Earth. Because block geometry is maintained as strain accumulates, Euler-vector clustering may better approximate the block geometry than the values of the associated Euler vectors. The microplate solution for each k is constructed by merging contiguous clusters that have closely similar Euler vectors. The best solution consists of three microplates arranged along the Nankaido Trough-Ryukyu Trench between the Amurian and Philippine Sea Plates. One of these microplates, the South Kyushu Microplate (an extension of the Ryukyu forearc into the southeast corner of Kyushu), had previously been identified from paleomagnetic rotations. Relative to ITRF2000 the three microplates rotate at different rates about neighboring poles located close to the northwest corner of Shikoku. The microplate model is identical to that proposed in the block model of Wallace et al. (2009, https://doi.org/10.1130/G2522A.1) except in southernmost Kyushu. On Shikoku and Honshu, but not Kyushu, the microplate model is consistent with that proposed in the block models of Nishimura and Hashimoto (2006, https://doi.org/10.1016/j.tecto.2006.04.017) and Loveless and Meade (2010, https://doi.org/10.1029/2008JB006248) without the low-slip-rate boundaries proposed in the latter.

Detection of influenza A virus subtypes using a solid-phase PCR microplate chip assay.

PubMed

Sun, Xin-Cheng; Wang, YunLong; Yang, Liping; Zhang, HuiRu

2015-01-01

A rapid and sensitive microplate chip based on solid PCR was developed to identify influenza A subtypes. A simple ultraviolet cross-linking method was used to immobilize DNA probes on pretreated microplates. Solid-phase PCR was proven to be a convenient method for influenza A screening. The sensitivity of the microplate chip was 10(-3) μg/mL for the enzymatic colorimetric method and 10(-4) μg/mL for the fluorescence method. The 10 sets of primers and probes for the microplate chip were highly specific and did not interfere with each other. These results suggest that the microplate chip based on solid PCR can be used to rapidly detect universal influenza A and its subtypes. This platform can also be used to detect other pathogenic microorganisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of the Galapagos Hotspot on Seamount Formation along the Galapagos Spreading Center

NASA Astrophysics Data System (ADS)

Behn, M. D.; Sinton, J. M.; Detrick, R. S.

2002-12-01

Studies along the Mid-Atlantic Ridge (MAR) and East Pacific Rise (EPR) have shown seamount formation to be a strong function of spreading rate. At the MAR, seamounts are a dominant morphologic feature of the inner valley floor, while at the EPR seamounts are rarely observed within the neovolcanic zone. The Galapagos Spreading Center (GSC) provides an excellent location to test the influence of a hotspot on the process of seamount generation at a relatively constant spreading rate. In this study we use multi-beam bathymetry data acquired during the G-PRIME cruise in April-May, 2000 to examine the distribution of axial seamounts along the GSC with distance from the hotspot. We use a numerical algorithm to identify isolated volcanic edifices, by searching bathymetry for closed, concentric contours protruding above the surrounding seafloor. Seamount populations are fit with a maximum likelihood model to estimate the total number of seamounts per unit area, ν o, and the characteristic seamount height, β-1. The number of seamounts in the axial zone decreases significantly as the Galapagos hotspot is approached, suggesting a change from dominantly point-source to fissure-fed volcanism as magma supply increases. West of the 95.5°W propagator, the total number of seamounts per unit area (ν o = 279+/-16 per 103 km2) is similar to values observed at the MAR. In comparison, east of 92.7°W, where magma supply is higher, seamount density (50+/-9 per 103 km2) is similar to observations at the fast-spreading EPR. Our results show that the transition from point-source to fissure-fed eruptions occurs gradually, in contrast to the "threshold" effect observed in axial magma chamber depth and axial morphology in which small changes in magma supply result in large changes in these variables. In summary, the western GSC displays the same range in seamount density observed along the global mid-ocean ridge system suggesting that both spreading rate and magma supply are important

Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development.

PubMed

Nan, Wenbin; Wang, Xiaomin; Yang, Lei; Hu, Yanfeng; Wei, Yuantao; Liang, Xiaolei; Mao, Lina; Bi, Yurong

2014-04-01

The second messenger cyclic guanosine 3',5'-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCF(TIR1) ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation.

Seismic-refraction field experiments on Galapagos Islands: A quantitative tool for hydrogeology

NASA Astrophysics Data System (ADS)

Adelinet, M.; Domínguez, C.; Fortin, J.; Violette, S.

2018-01-01

Due to their complex structure and the difficulty of collecting data, the hydrogeology of basaltic islands remains misunderstood, and the Galapagos islands are not an exception. Geophysics allows the possibility to describe the subsurface of these islands and to quantify the hydrodynamical properties of its ground layers, which can be useful to build robust hydrogeological models. In this paper, we present seismic refraction data acquired on Santa Cruz and San Cristobal, the two main inhabited islands of Galapagos. We investigated sites with several hydrogeological contexts, located at different altitudes and at different distances to the coast. At each site, a 2D P-wave velocity profile is built, highlighting unsaturated and saturated volcanic layers. At the coastal sites, seawater intrusion is identified and basal aquifer is characterized in terms of variations in compressional sound wave velocities, according to saturation state. At highlands sites, the limits between soils and lava flows are identified. On San Cristobal Island, the 2D velocity profile obtained on a mid-slope site (altitude 150 m), indicates the presence of a near surface freshwater aquifer, which is in agreement with previous geophysical studies and the hydrogeological conceptual model developed for this island. The originality of our paper is the use of velocity data to compute field porosity based on poroelasticity theory and the Biot-Gassmann equations. Given that porosity is a key parameter in quantitative hydrogeological models, it is a step forward to a better understanding of shallow fluid flows within a complex structure, such as Galapagos volcanoes.

Development of Singlet Oxygen Absorption Capacity (SOAC) Assay Method Using a Microplate Reader.

PubMed

Takahashi, Shingo; Iwasaki-Kino, Yuko; Aizawa, Koichi; Terao, Junji; Mukai, Kazuo

2016-01-01

Recently, a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of natural antioxidants and food extracts was developed. The SOAC values were measured in ethanol-chloroform-D2O (50 + 50 + 1, v/v/v) solution at 35°C using a UV-Vis spectrophotometer equipped with a six-channel cell positioner and an electron-temperature control unit. In the present study, measurement of the SOAC values was performed for eight representative carotenoids and three vegetable extracts (tomato, carrot, and red paprika) using a versatile instrument, the microplate reader. A 24-well glass microplate was used for measurements because a plastic microplate, commonly used in the laboratory, dissolves in the ethanol-chloroform-D2O solution. The SOAC values of eight carotenoids and three vegetable extracts measured using a microplate reader were in good agreement with the corresponding values measured using a UV-Vis spectrophotometer, suggesting that the microplate reader is an applicable instrument for the measurement of reliable SOAC values for general antioxidants and food extracts in solution.

Transient cracks and triple junctions induced by Cocos-Nazca propagating rift

NASA Astrophysics Data System (ADS)

Schouten, H.; Smith, D. K.; Zhu, W.; Montesi, L. G.; Mitchell, G. A.; Cann, J. R.

2009-12-01

The Galapagos triple junction is a ridge-ridge-ridge triple junction where the Cocos, Nazca, and Pacific plates meet around the Galapagos microplate (GMP). On the Cocos plate, north of the large gore that marks the propagating Cocos-Nazca (C-N) Rift, a 250-km-long and 50-km-wide band of NW-SE-trending cracks crosscuts the N-S-trending abyssal hills of the East Pacific Rise (EPR). These appear as a succession of minor rifts, accommodating some NE-SW extension of EPR-generated seafloor. The rifts successively intersected the EPR in triple junctions at distances of 50-100 km north of the tip of the C-N Rift. We proposed a simple crack interaction model to explain the location of the transient rifts and their junction with the EPR. The model predicts that crack locations are controlled by the stress perturbation along the EPR, induced by the dominant C-N Rift, and scaled by the distance of its tip to the EPR (Schouten et al., 2008). The model also predicts that tensile stresses are symmetric about the C-N Rift and thus, similar cracks should have occurred south of the C-N Rift prior to formation of the GMP about 1 Ma. There were no data at the time to test this prediction. In early 2009 (AT 15-41), we mapped an area on the Nazca plate south of the C-N rift out to 4 Ma. The new bathymetric data confirm the existence of a distinctive pattern of cracks south of the southern C-N gore that mirrors the pattern on the Cocos plate until about 1 Ma, and lends support to the crack interaction model. The envelope of the symmetric cracking pattern indicates that the distance between the C-N Rift tip and the EPR varied between 40 and 65 km during this time (1-4 Ma). The breakdown of the symmetry at 1 Ma accurately dates the onset of a southern plate boundary of the GMP, now Dietz Deep Rift. At present, the southern rift boundary of the GMP joins the EPR with a steep-sided, 80 km long ridge. This ridge releases the stress perturbation otherwise induced along the EPR by elastic

A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA).

PubMed

Kai, Junhai; Puntambekar, Aniruddha; Santiago, Nelson; Lee, Se Hwan; Sehy, David W; Moore, Victor; Han, Jungyoup; Ahn, Chong H

2012-11-07

In this work we introduce a novel microfluidic enzyme linked immunoassays (ELISA) microplate as the next generation assay platform for unparalleled assay performances. A combination of microfluidic technology with standard SBS-configured 96-well microplate architecture, in the form of microfluidic microplate technology, allows for the improvement of ELISA workflows, conservation of samples and reagents, improved reaction kinetics, and the ability to improve the sensitivity of the assay by multiple analyte loading. This paper presents the design and characterization of the microfluidic microplate, and its application in ELISA.

Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction

PubMed Central

Steiner, Samuel; Lori, Christian; Boehm, Alex; Jenal, Urs

2013-01-01

In many bacterial pathogens, the second messenger c-di-GMP stimulates the production of an exopolysaccharide (EPS) matrix to shield bacteria from assaults of the immune system. How c-di-GMP induces EPS biogenesis is largely unknown. Here, we show that c-di-GMP allosterically activates the synthesis of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a major extracellular matrix component of Escherichia coli biofilms. C-di-GMP binds directly to both PgaC and PgaD, the two inner membrane components of the poly-GlcNAc synthesis machinery to stimulate their glycosyltransferase activity. We demonstrate that the PgaCD machinery is a novel type c-di-GMP receptor, where ligand binding to two proteins stabilizes their interaction and promotes enzyme activity. This is the first example of a c-di-GMP-mediated process that relies on protein–protein interaction. At low c-di-GMP concentrations, PgaD fails to interact with PgaC and is rapidly degraded. Thus, when cells experience a c-di-GMP trough, PgaD turnover facilitates the irreversible inactivation of the Pga machinery, thereby temporarily uncoupling it from c-di-GMP signalling. These data uncover a mechanism of c-di-GMP-mediated EPS control and provide a frame for c-di-GMP signalling specificity in pathogenic bacteria. PMID:23202856

Occurrence of Cyclic di-GMP-Modulating Output Domains in Cyanobacteria: an Illuminating Perspective

PubMed Central

Agostoni, Marco; Koestler, Benjamin J.; Waters, Christopher M.; Williams, Barry L.; Montgomery, Beronda L.

2013-01-01

ABSTRACT Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors—outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally, we compared intracellular levels of c-di-GMP in two cyanobacterial species under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo. PMID:23943760

Paleomagnetism of San Cristobal Island, Galapagos

USGS Publications Warehouse

Cox, A.

1971-01-01

Isla San Cristobal, the most easterly of the Galapagos Islands, consists of two parts: a large volcano constitutes the southwest half of the island and an irregular apron of small cones and flows makes up the northeast half. As some of the younger flows on the flanks of the large volcano are reversely magnetized, the minimum age of the volcano is 0.7 my, which is the age of the Brunhes-Matuyama reversal boundary. The true age is probably several times greater. The cones and flows to the northeast are all normally magnetized. The between-site angular dispersion of virtual poles is 11.3?? - a value consistent with mathematical models for the latitude dependence of geomagnetic secular variation. ?? 1971.

The Evolution of Floreana Island, Galapagos: Mantle Metasomatism as a Control of Structural and Geochemical Variations

NASA Astrophysics Data System (ADS)

Koleszar, A. M.; Rollins, N. A.; Harpp, K. S.; Geist, D. J.

2004-05-01

Floreana, the 6th largest island in the Galapagos Archipelago, is situated ESE of the current proposed location of the hotspot, believed to be near Fernandina Island. Floreana is the most distant Galapagos volcano from the Galapagos Spreading Center and is located on 12 Ma lithosphere. Both normally- and reversely-polarized flows are present on Floreana, which emerged more than 1 Ma. The emergent shield is constructed of lava flows and >80 cinder cones. In the final stage of island building, approximately 0.3 Ma, the eruptive activity on Floreana became more explosive and produced the largest cinder cones on the island. Spatter ramparts, cinder cones, vents, and pit craters are arranged in at least 3 major parallel to sub-parallel alignments oriented N40E. The basalts of Floreana are notably alkalic, primitive, and highly enriched in incompatible trace elements (ITE). MgO concentrations in the lavas range from <8 wt% to >13 wt%, and many of the magmas are likely related by fractional crystallization of olivine and clinopyroxene. The volcano has erupted ultramafic xenoliths, which are observed predominantly in the older, reversely-polarized flows and cones. Floreana lavas have the greatest light REE enrichment observed in the archipelago and the most radiogenic Sr- and Pb- isotopic ratios, indicative of an ITE-enriched source. Elevated ratios of alkali and alkaline earth contents to those of high-field strength elements indicate contributions from metasomatic fluids to Floreana melts. Although the effects of metasomatism are apparent in most Floreana basalts, normally-polarized lavas may have been affected to a greater extent by the metasomatism than the older flows. Temporal-compositional trends in trace element concentrations also suggest that the depth of melt generation may have decreased slightly over the course of the island's formation. Floreana is distinct from the rest of the Galapagos Archipelago in its explosive history, abundant mantle xenoliths

cGMP stimulates bile acid-independent bile formation and biliary bicarbonate excretion.

PubMed

Myers, N C; Grune, S; Jameson, H L; Sawkat-Anwer, M

1996-03-01

The effect of guanosine 3',5'-cyclic monophosphate (cGMP) on hepatic bile formation was studied in isolated perfused rat livers and rat hepatocytes. Studies in isolated perfused rat livers showed that infusion of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 3 micromol/min or 100 microM) 1) increased bile flow without affecting biliary excretion of simultaneously infused taurocholate, 2) increased biliary concentration and excretion of HCO3(-) but did not affect biliary excretion of glutathione, and 3) increased net perfusate H+ efflux without affecting hepatic O2 uptake. Studies in isolated rat hepatocytes showed that 1) 8-BrcGMP increased intracellular pH in the presence (but not in the absence) of extracellular HCO-3, and effect inhibited by 4,4' -diisothiocyanostilbene-2,2'-disulfonic acid and Na+ replacement, 2) 8-BrcGMP did not affect taurocholate uptake and intracellular [Ca2+], and 3) bile acids, like ursodeoxycholate and cholate, did not increase cellular cGMP. Taken together, these results indicate that cGMP stimulates bile acid-independent bile formation, in part by stimulating biliary HCO3- excretion. cGMP may increase HCO3- excretion by stimulating sinusoidal Na+ - HCO3- cotransport, but not Na+/H+ exchange. cGMP, unlike adenosine 3',5'-cyclic monophosphate, may not regulate hepatic taurocholate transport, and bile acid-induced HCO3- rich choleresis may not be mediated via cGMP.

Zoanthid diversity (Anthozoa: Hexacorallia) in the Galapagos Islands: a molecular examination

NASA Astrophysics Data System (ADS)

Reimer, J. D.; Sinniger, F.; Hickman, C. P.

2008-09-01

Although the Galapagos are famous for their unique biodiversity, many groups of marine invertebrates from this isolated archipelago remain understudied or not investigated. One such group is the zoanthids (Order Zoantharia, =Zoanthidea, =Zoanthiniaria), anthozoans (Cnidaria) found in marine ecosystems worldwide. Zoanthid taxonomy has been in a state of disorganization and neglect due in large part to the morphological plasticity within species and questions about the accuracy of traditionally used morphological and ecological characteristics. However, recent studies utilizing molecular methodology combined with morphology have proven to be very useful in understanding zoanthid diversity. The results of a survey of zoanthids from the Galapagos and the east Pacific are reported in this study. Shallow water (<35 m) zoanthid specimens were identified using the molecular markers mitochondrial 16S ribosomal DNA (mt 16S rDNA), cytochrome oxidase subunit I (COI) gene, and the internal transcribed spacer of ribosomal DNA (ITS-rDNA). From the collected specimens seven putative zoanthid species-level clades from three known genera ( Zoanthus, Palythoa, Parazoanthus) were identified at the molecular level. These identifications were further supported by morphological and ecological data. While almost all specimens belonged to known zoanthid genera, based on unique molecular and ecological data one group of specimens (designated unknown zoanthid sp. “03-103”) is potentially a novel undescribed genus. Additionally, the remaining three azooxanthellate Parazoanthus clades may also be undescribed new species, but due to the overall lack of zoanthid research and descriptions from neighboring areas (East Pacific, west coast of South America) further research is needed to clearly ascertain this. Additionally, notes on the four observed nominal azooxanthellate zoanthid species and a key to all eight nominal (seven from known genera, one from a potentially new genus) shallow water

Prolonged treatment of porcine pulmonary artery with nitric oxide decreases cGMP sensitivity and cGMP-dependent protein kinase specific activity

PubMed Central

Perkins, William J.; Warner, David O.; Jones, Keith A.

2009-01-01

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response to acutely applied NO, cGMP analog, or atrial natriuretic peptide (ANP). Twenty-four-hour treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) resulted in >10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, β-phenyl-1,N2-etheno-8-bromoguanosine-3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-PET-cGMPS), and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by 10−6 M ANP. Twenty-four hours in culture and treatment with DETA-NO decreased total cGMP-dependent protein kinase (cGKI) mRNA level compared with that in freshly prepared PA (1.05 ± 0.12, 0.42 ± 0.08, and 0.11 ± 0.01 amol/μg, respectively). Total cGKI protein levels were decreased to a lesser extent by 24 h in culture and further decreased by 24-h DETA-NO treatment compared with that in freshly prepared PA (361 ± 33, 272 ± 20, and 238 ± 25 ng/mg total protein, respectively). Maximal cGMP-stimulated phosphotransferase activity was reduced in 24-h cultured and DETA-NO-treated PA (986 ± 84, 815 ± 81, and 549 ± 78 pmol Pi·min−1·mg soluble protein−1), but the cGMP concentration resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24 h compared with freshly prepared and 24-h cultured PA (1.95 ± 0.22, 2.64 ± 0.25, and 2.85 ± 0.28 pmol Pi·min−1·ng cGKI−1, respectively). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity. PMID:18952758

[Comparative study on microplate and anchor fixation in open-door cervical expansive laminoplasty].

PubMed

Zeng, Yun; Xiong, Min; Yu, Hualong; He, Ning; Wang, Zhiyong; Liu, Zhigang; Han, Heng; Chen, Sen

2011-08-01

To evaluate the effectiveness of microplate fixation in open-door cervical expansive laminoplasty (ELP) by comparing with anchor fixation. Between January 2005 and October 2008, 35 patients with multi-segment cervical spondylotic myelopathy were treated. Of them, 15 patients underwent ELP by microplate fixation (microplate group) and 20 patients underwent ELP by anchor fixation (anchor group). In microplate group, there were 10 males and 5 females with the age of (51.2 +/- 11.5) years; the disease duration ranged from 6 to 60 months (mean, 14 months); and the preoperative Japanese Orthopaedic Association (JOA) score was 7.7 +/- 2.5. In anchor group, there were 13 males and 7 females with the age of (50.7 +/- 10.8) years; the disease duration ranged from 3 to 58 months (mean, 17 months); and the preoperative JOA score was 7.8 +/- 2.9. There was no significant difference in the general data, such as gender, age, and JOA score between 2 groups (P > 0.05). All incisions healed by first intention. Thirty-five cases were followed up 24-68 months (mean, 32 months). The operation time was (113 +/- 24) minutes in anchor group and (111 +/- 27) minutes in microplate group, showing no significant difference (t = 0.231 3, P = 0.818 5). The rate of spinal canal expansion in microplate group (60% +/- 24%) was significantly higher than that in anchor group (40% +/- 18%) (t = 2.820, P = 0.008). The JOA scores of 2 groups at 3 months and 24 months after operation were significantly higher than the preoperative scores (P < 0.01). There was no significant difference in JOA score between 2 groups at 3 months after operation (t = 1.620 5, P = 0.114 6), but the JOA score of microplate group was significantly higher than that of anchor group at 24 months after operation (t = 3.454 3, P = 0.001 5). X-ray film, MRI, and CT scan at 3-6 months after operation displayed that door spindle reached bony fusion. There was no occurrence of "re-close of door" in 2 groups. The rate of complication in

Allometric and temporal scaling of movement characteristics in Galapagos tortoises.

PubMed

Bastille-Rousseau, Guillaume; Yackulic, Charles B; Frair, Jacqueline L; Cabrera, Freddy; Blake, Stephen

2016-09-01

Understanding how individual movement scales with body size is of fundamental importance in predicting ecological relationships for diverse species. One-dimensional movement metrics scale consistently with body size yet vary over different temporal scales. Knowing how temporal scale influences the relationship between animal body size and movement would better inform hypotheses about the efficiency of foraging behaviour, the ontogeny of energy budgets, and numerous life-history trade-offs. We investigated how the temporal scaling of allometric patterns in movement varies over the course of a year, specifically during periods of motivated (directional and fast movement) and unmotivated (stationary and tortuous movement) behaviour. We focused on a recently diverged group of species that displays wide variation in movement behaviour - giant Galapagos tortoises (Chelonoidis spp.) - to test how movement metrics estimated on a monthly basis scaled with body size. We used state-space modelling to estimate seven different movement metrics of Galapagos tortoises. We used log-log regression of the power law to evaluate allometric scaling for these movement metrics and contrasted relationships by species and sex. Allometric scaling of movement was more apparent during motivated periods of movement. During this period, allometry was revealed at multiple temporal intervals (hourly, daily and monthly), with values observed at daily and monthly intervals corresponding most closely to the expected one-fourth scaling coefficient, albeit with wide credible intervals. We further detected differences in the magnitude of scaling among taxa uncoupled from observed differences in the temporal structuring of their movement rates. Our results indicate that the definition of temporal scales is fundamental to the detection of allometry of movement and should be given more attention in movement studies. Our approach not only provides new conceptual insights into temporal attributes in one

Examination of the constructional processes of submarine Cerro Azul and the Galapagos Platform

NASA Astrophysics Data System (ADS)

Lambert, M. K.; Harpp, K. S.; Geist, D. J.; Fornari, D. J.; Kurz, M. D.; Koleszar, A. M.; Rollins, N. A.

2004-05-01

One of the primary goals of the 2001 Drift04 cruise was to examine the constructional processes responsible for the Galapagos platform and to investigate the relationship between the platform and the overlying volcanoes. Cerro Azul volcano is located above the steep escarpment that marks the southwestern limit of the Galapagos platform, at the leading edge of the hotspot. This area is of particular interest in light of a recent seismic tomography experiment by Toomey, Hooft, et al., which suggests that the root of the Galapagos plume is centered between Cerro Azul and adjacent Fernandina Island. During the Drift04 cruise, detailed bathymetric and sidescan sonar studies were carried out across the submarine sector of Cerro Azul and 14 dredges were collected from the same area. Major element analyses of the submarine lavas indicate that the lavas from the platform edge and the subaerial Cerro Azul lavas constitute a suite of petrologically-related lavas. The dredged glasses of the Drift04 cruise have MgO contents of <7.5% and are indistinguishable from published data on Cerro Azul. Whole rock analyses include a highly primitive sample (20 wt% MgO), which probably contains accumulated olivine. All the submarine and subaerial lavas define coherent trends in major element space that are consistent with variable amounts of olivine and olivine+cpx fractionation. Incompatible trace element (ITE) ratios indicate that the mantle source for the submarine platform flows is intermediate in composition between the magmas supplying Fernandina and Cerro Azul. Previous researchers have proposed that two mantle endmembers are interacting across the leading edge of the plume, one focused at Fernandina and the other at Floreana Island. The intermediate ITE ratios of the submarine and subaerial Cerro Azul lavas are consistent both geographically and compositionally with this hypothesis. Naumann and co-workers concluded that the lavas erupted at Cerro Azul were stored in small

Correction of microplate location effects improves performance of the thrombin generation test

PubMed Central

2013-01-01

Background Microplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate. Methods In this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment. Results Location of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment. Conclusions Use of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field. PMID:23829491

Correction of microplate location effects improves performance of the thrombin generation test.

PubMed

Liang, Yideng; Woodle, Samuel A; Shibeko, Alexey M; Lee, Timothy K; Ovanesov, Mikhail V

2013-07-05

Microplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate. In this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment. Location of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment. Use of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field.

Dog overpopulation and burden of exposure to canine distemper virus and other pathogens on Santa Cruz Island, Galapagos.

PubMed

Diaz, Nicole M; Mendez, Gabriella S; Grijalva, C Jaime; Walden, Heather S; Cruz, Marilyn; Aragon, Eduardo; Hernandez, Jorge A

2016-01-01

Dog overpopulation and diseases are hazards to native island species and humans on the Galapagos. Vaccination and importation of dogs are prohibited on the Galapagos. Risk management of these hazards requires the use of science-based risk assessment and risk communication. The objectives of the study reported here were (i) to estimate the human:dog ratio and (ii) the prevalence of and identify exposure factors associated with positive antibody titers to canine distemper virus (CDV) and other pathogens, as well as infection with intestinal parasites in owned dogs on Santa Cruz Island, Galapagos in September 2014. The observed human:dog ratio was 6.148:1 which extrapolates to 2503 dogs (two times more than a recent dog count conducted by Galapagos Biosecurity Agency in March 2014). The proportion of spayed female dogs (50%) was higher, compared to neutered male dogs (30%) (p=0.04). Prevalence of dogs with positive antibody titers to CDV was 36% (95% CI=26, 46%), to canine parvovirus was 89% (95% CI=82, 95%), and to canine adenovirus was 40% (95% CI=30, 51%). The frequency of seropositive dogs to CDV was lower in urban dogs (26%), compared to rural dogs (53%) (p<0.05). A positive interaction effect between rural residence and spay/neuter status on seropositivity to CDV was observed, which we discuss in this report. Because vaccination is prohibited, the dog population on Santa Cruz is susceptible to an outbreak of CDV (particularly among urban dogs) with potential spill over to marine mammals. Dog's age (1-2 or 3-14 years old, compared to younger dogs), and residence (rural, urban) were associated with positive antibody titers to parvovirus, adenovirus, Ehrlichia spp., or Anaplasma spp., as well as infection with Ancylostoma spp., an intestinal parasite in dogs that can be transmitted to humans, particularly children. These results provide the most comprehensive assessment of dog overpopulation and exposure to CDV and other pathogens on the Galapagos to date

Tectonics and evolution of the Juan Fernandez microplate at the Pacific-Nazca-Antarctic triple junction

NASA Technical Reports Server (NTRS)

Anderson-Fontana, S.; Larson, R. L.; Engein, J. F.; Lundgren, P.; Stein, S.

1986-01-01

Magnetic and bathymetric profiles derived from the R/V Endeavor survey and focal mechanism studies for earthquakes on two of the Juan Fernandez microplate boundaries are analyzed. It is observed that the Nazca-Juan Fernandez pole is in the northern end of the microplate since the magnetic lineation along the East Ridge of the microplate fans to the south. The calculation of the relative motion of the Juan Fernandez-Pacific-Nazca-Antarctic four-plate system using the algorithm of Minster et al. (1974) is described. The development of tectonic and evolutionary models of the region is examined. The tectonic model reveals that the northern boundary of the Juan Fernandez microplate is a zone of compression and that the West Ridge and southwestern boundary are spreading obliquely; the evolutionary model relates the formation of the Juan Fernandez microplate to differential spreading rates at the triple junction.

Finite element analysis to determine the stress distribution, displacement and safety factor on a microplate for the fractured jaw case

NASA Astrophysics Data System (ADS)

Pratama, Juan; Mahardika, Muslim

2018-03-01

Microplate is a connecting plate that can be used for jaw bone fixation. In the last two decades, microplate has been used so many times to help reconstruction of fractured jaw bone which is called mandibular bone or mandible bone. The plate is used to provide stable fixation of the fractured bone tissue during healing and reconstruction process. In this study Finite Element Analysis was used to predict the stress concentration and distribution on a microplate, displacement on the microplate and also to determine the safety factor of the microplate based on maximum allowable stress value, and finally to ascertain whether microplate is safe to use or not. The microplate was produced from punching process using titanium grade 1 (pure titanium) as material with a thickness of 500 µm. The results of the research indicated that the microplate was safe to use according to the maximum stress around the hole, displacement around the hole and also the safety factor of the microplate.

Catch, bycatch and discards of the Galapagos Marine Reserve small-scale handline fishery.

PubMed

Zimmerhackel, Johanna S; Schuhbauer, Anna C; Usseglio, Paolo; Heel, Lena C; Salinas-de-León, Pelayo

2015-01-01

Fisheries bycatch is a significant marine conservation issue as valuable fish are wasted and protected species harmed with potential negative ecological and socio-economic consequences. Even though there are indications that the small-scale handline fishery of the Galapagos Marine Reserve has a low selectivity, information on its bycatch has never been published. We used onboard monitoring and interview data to assess the bycatch of the Galapagos handline fishery by estimating the bycatch ratio, determining species compositions of landings and bycatch, identifying fishers' reasons for discarding certain individuals, and revealing historical trends in the bycatch ratio. The estimated bycatch ratio as a function of biomass of 0.40 and a diverse species composition of target catch and bycatch confirmed the low selectivity of this fishery. Most individuals were not landed for economic motivations, either because species (77.4%) or sizes (17.7%) are unmarketable or for regulatory reasons (5.9%). We found that bycatch contributes to growth overfishing of some target species because they are discarded or used as bait before reaching their first maturity. Moreover, over half of interviewees perceived a historical decrease in bycatch ratios that was explained by a diversification of the target catch due to the reduction in abundance of the traditionally most important target species. As some target species show signs of overfishing and to date there are no specific regulations for the finfish fishery species in place, we recommend the implementation of a series of management measures to protect critical life stages of overexploited species and to improve the selectivity of the Galapagos handline fishery.

Catch, bycatch and discards of the Galapagos Marine Reserve small-scale handline fishery

PubMed Central

Schuhbauer, Anna C.; Usseglio, Paolo; Heel, Lena C.; Salinas-de-León, Pelayo

2015-01-01

Fisheries bycatch is a significant marine conservation issue as valuable fish are wasted and protected species harmed with potential negative ecological and socio-economic consequences. Even though there are indications that the small-scale handline fishery of the Galapagos Marine Reserve has a low selectivity, information on its bycatch has never been published. We used onboard monitoring and interview data to assess the bycatch of the Galapagos handline fishery by estimating the bycatch ratio, determining species compositions of landings and bycatch, identifying fishers’ reasons for discarding certain individuals, and revealing historical trends in the bycatch ratio. The estimated bycatch ratio as a function of biomass of 0.40 and a diverse species composition of target catch and bycatch confirmed the low selectivity of this fishery. Most individuals were not landed for economic motivations, either because species (77.4%) or sizes (17.7%) are unmarketable or for regulatory reasons (5.9%). We found that bycatch contributes to growth overfishing of some target species because they are discarded or used as bait before reaching their first maturity. Moreover, over half of interviewees perceived a historical decrease in bycatch ratios that was explained by a diversification of the target catch due to the reduction in abundance of the traditionally most important target species. As some target species show signs of overfishing and to date there are no specific regulations for the finfish fishery species in place, we recommend the implementation of a series of management measures to protect critical life stages of overexploited species and to improve the selectivity of the Galapagos handline fishery. PMID:26082874

Investigating Island Evolution: A Galapagos-Based Lesson Using the 5E Instructional Model.

ERIC Educational Resources Information Center

DeFina, Anthony V.

2002-01-01

Introduces an inquiry-based lesson plan on evolution and the Galapagos Islands. Uses the 5E instructional model which includes phases of engagement, exploration, explanation, elaboration, and evaluation. Includes information on species for exploration and elaboration purposes, and a general rubric for student evaluation. (YDS)

Specific Detection and Identification of Herpes B Virus by a PCR-Microplate Hybridization Assay

PubMed Central

Oya, Chika; Ochiai, Yoshitsugu; Taniuchi, Yojiro; Takano, Takashi; Ueda, Fukiko; Yoshikawa, Yasuhiro; Hondo, Ryo

2004-01-01

Herpes B virus DNA was specifically amplified by PCR, targeting the regions that did not cross-react with herpes simplex virus (HSV). The amplified products, which were shown to be highly genetic polymorphisms among herpes B virus isolates, were identified by microplate hybridization with probes generated by PCR. The products immobilized in microplate wells were hybridized with the biotin-labeled probes derived from the SMHV strain of herpes B virus. The amplified products derived from the SMHV and E2490 strains of herpes B virus were identified by microplate hybridization. PCR products amplified from the trigeminal ganglia of seropositive cynomolgus macaques were identified as herpes B virus DNA. The utility of the PCR-microplate hybridization assay for genetic detection and identification of the polymorphic region of herpes B virus was determined. PMID:15131142

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

PubMed Central

Galperin, Michael Y.; Gomelsky, Mark

2013-01-01

SUMMARY Twenty-five years have passed since the discovery of cyclic dimeric (3′→5′) GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger. PMID:23471616

Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis.

PubMed

Egbert, Jeremy R; Yee, Siu-Pok; Jaffe, Laurinda A

2018-03-01

Prior to birth, oocytes within mammalian ovarian follicles initiate meiosis, but then arrest in prophase until puberty, when with each reproductive cycle, one or more follicles are stimulated by luteinizing hormone (LH) to resume meiosis in preparation for fertilization. Within preovulatory follicles, granulosa cells produce high levels of cGMP, which diffuses into the oocyte to maintain meiotic arrest. LH signaling restarts meiosis by rapidly lowering the levels of cGMP in the follicle and oocyte. Part of this decrease is mediated by the dephosphorylation and inactivation the NPR2 guanylyl cyclase in response to LH, but the mechanism for the remainder of the cGMP decrease is unknown. At least one cGMP phosphodiesterase, PDE5, is activated by LH signaling, which would contribute to lowering cGMP. PDE5 exhibits increased cGMP-hydrolytic activity when phosphorylated on serine 92, and we recently demonstrated that LH signaling phosphorylates PDE5 on this serine and increases its activity in rat follicles. To test the extent to which this mechanism contributes to the cGMP decrease that restarts meiosis, we generated a mouse line in which serine 92 was mutated to alanine (Pde5-S92A), such that it cannot be phosphorylated. Here we show that PDE5 phosphorylation is required for the LH-induced increase in cGMP-hydrolytic activity, but that this increase has only a modest effect on the LH-induced cGMP decrease in mouse follicles, and does not affect the timing of meiotic resumption. Though we show that the activation of PDE5 is among the mechanisms contributing to the cGMP decrease, these results suggest that another cGMP phosphodiesterase is also activated by LH signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

c-di-GMP is an Effective Immunomodulator and Vaccine Adjuvant Against Pneumococcal Infection

PubMed Central

Ogunniyi, Abiodun D.; Paton, James C.; Kirby, Alun C.; McCullers, Jonathan A.; Cook, Jan; Hyodo, Mamoru; Hayakawa, Yoshihiro; Karaolis, David K. R.

2009-01-01

Cyclic diguanylate (c-di-GMP) is a unique bacterial intracellular signaling molecule capable of stimulating enhanced protective innate immunity against various bacterial infections. The effects of intranasal pretreatment with c-di-GMP, or intraperitoneal coadministration of c-di-GMP with the pneumolysin toxoid (PdB) or PspA before pneumococcal challenge, was investigated in mice. We found that c-di-GMP had no significant direct short-term effect on the growth rate of S. pneumoniae either in vitro or in vivo. However, intranasal pretreatment of mice with c-di-GMP resulted in significant decrease in bacterial load in lungs and blood after serotypes 2 and 3 challenge, and significant decrease in lung titers after serotype 4 challenge. Potential cellular mediators of these enhanced protective responses were identified in lungs and draining lymph nodes. Intraperitoneal coadministration of c-di-GMP with PdB or PspA before challenge resulted in significantly higher antigen-specific antibody titers and increased survival of mice, compared to that obtained with alum adjuvant. These findings demonstrate that local or systemic c-di-GMP administration stimulates innate and adaptive immunity against invasive pneumococcal disease. We propose that c-di-GMP can be used as an effective broad spectrum immunomodulator and vaccine adjuvant to prevent infectious diseases. PMID:18640167

Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, (p)ppGpp signaling in bacteria and implications in pathogenesis.

PubMed

Kalia, Dimpy; Merey, Gökçe; Nakayama, Shizuka; Zheng, Yue; Zhou, Jie; Luo, Yiling; Guo, Min; Roembke, Benjamin T; Sintim, Herman O

2013-01-07

For an organism to survive, it must be able to sense its environment and regulate physiological processes accordingly. Understanding how bacteria integrate signals from various environmental factors and quorum sensing autoinducers to regulate the metabolism of various nucleotide second messengers c-di-GMP, c-di-AMP, cGMP, cAMP and ppGpp, which control several key processes required for adaptation is key for efforts to develop agents to curb bacterial infections. In this review, we provide an update of nucleotide signaling in bacteria and show how these signals intersect or integrate to regulate the bacterial phenotype. The intracellular concentrations of nucleotide second messengers in bacteria are regulated by synthases and phosphodiesterases and a significant number of these metabolism enzymes had been biochemically characterized but it is only in the last few years that the effector proteins and RNA riboswitches, which regulate bacterial physiology upon binding to nucleotides, have been identified and characterized by biochemical and structural methods. C-di-GMP, in particular, has attracted immense interest because it is found in many bacteria and regulate both biofilm formation and virulence factors production. In this review, we discuss how the activities of various c-di-GMP effector proteins and riboswitches are modulated upon c-di-GMP binding. Using V. cholerae, E. coli and B. subtilis as models, we discuss how both environmental factors and quorum sensing autoinducers regulate the metabolism and/or processing of nucleotide second messengers. The chemical syntheses of the various nucleotide second messengers and the use of analogs thereof as antibiofilm or immune modulators are also discussed.

Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

NASA Astrophysics Data System (ADS)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

A calcium-permeable cGMP-activated cation conductance in hippocampal neurons

NASA Technical Reports Server (NTRS)

Leinders-Zufall, T.; Rosenboom, H.; Barnstable, C. J.; Shepherd, G. M.; Zufall, F.

1995-01-01

Whole-cell patch clamp recordings detected a previously unidentified cGMP-activated membrane conductance in cultured rat hippocampal neurons. This conductance is nonselectively permeable for cations and is completely but reversibly blocked by external Cd2+. The Ca2+ permeability of the hippocampal cGMP-activated conductance was examined in detail, indicating that the underlying ion channels display a high relative permeability for Ca2+. The results indicate that hippocampal neurons contain a cGMP-activated membrane conductance that has some properties similar to the cyclic nucleotide-gated channels previously shown in sensory receptor cells and retinal neurons. In hippocampal neurons this conductance similarly could mediate membrane depolarization and Ca2+ fluxes in response to intracellular cGMP elevation.

Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes

PubMed Central

Chen, Li-Hong; Köseoğlu, Volkan K.; Güvener, Zehra T.; Myers-Morales, Tanya; Reed, Joseph M.; D'Orazio, Sarah E. F.; Miller, Kurt W.; Gomelsky, Mark

2014-01-01

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence. PMID:25101646

Molecular mechanisms of gravity-dependent signaling in human melanocytic cells involve cyclic GMP

NASA Astrophysics Data System (ADS)

Ivanova, Krassimira; Lambers, Britta; Block, Ingrid; Bromeis, Birgit; Das, Pranab K.; Gerzer, Rupert

2005-08-01

Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) is an important messenger in melanocyte signaling we have compared the regulation of cGMP levels in human melanocytes and melanoma cells with different metastatic potential under hypergravity conditions. We were able to demonstrate that long-term exposure to hypergravity stimulates cGMP efflux in cultured human melanocytes and non- metastatic melanoma cells, whereas highly metastatic melanoma cells appear to be insensitive to hypergravity, most probably, due to an up-regulated cGMP efflux at 1g. Here we report that these effects are associated with the expression of the multidrug resistance proteins 4 and 5 known to act as selective export pumps for amphiphilic anions like cGMP. Thus, an altered gravity vector may induce cGMP-dependent signaling events in melanocytic cells that could be important for malignant transformation.

Development of a microplate coagulation assay for Factor V in human plasma.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2011-06-28

Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement

Development of a microplate coagulation assay for Factor V in human plasma

PubMed Central

2011-01-01

Background Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. Methods The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. Results The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. Conclusions The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and

Coordinated Cyclic-Di-GMP Repression of Salmonella Motility through YcgR and Cellulose

PubMed Central

Zorraquino, Violeta; García, Begoña; Latasa, Cristina; Echeverz, Maite; Toledo-Arana, Alejandro; Valle, Jaione

2013-01-01

Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition. PMID:23161026

Integration of the Second Messenger c-di-GMP into the Chemotactic Signaling Pathway

PubMed Central

Russell, Matthew H.; Bible, Amber N.; Fang, Xin; Gooding, Jessica R.; Campagna, Shawn R.; Gomelsky, Mark; Alexandre, Gladys

2013-01-01

ABSTRACT Elevated intracellular levels of the bacterial second messenger c-di-GMP are known to suppress motility and promote sessility. Bacterial chemotaxis guides motile cells in gradients of attractants and repellents over broad concentration ranges, thus allowing bacteria to quickly adapt to changes in their surroundings. Here, we describe a chemotaxis receptor that enhances, as opposed to suppresses, motility in response to temporary increases in intracellular c-di-GMP. Azospirillum brasilense’s preferred metabolism is adapted to microaerophily, and these motile cells quickly navigate to zones of low oxygen concentration by aerotaxis. We observed that changes in oxygen concentration result in rapid changes in intracellular c-di-GMP levels. The aerotaxis and chemotaxis receptor, Tlp1, binds c-di-GMP via its C-terminal PilZ domain and promotes persistent motility by increasing swimming velocity and decreasing swimming reversal frequency, which helps A. brasilense reach low-oxygen zones. If c-di-GMP levels remain high for extended periods, A. brasilense forms nonmotile clumps or biofilms on abiotic surfaces. These results suggest that association of increased c-di-GMP levels with sessility is correct on a long-term scale, while in the short-term c-di-GMP may actually promote, as opposed to suppress, motility. Our data suggest that sensing c-di-GMP by Tlp1 functions similar to methylation-based adaptation. Numerous chemotaxis receptors contain C-terminal PilZ domains or other sensory domains, suggesting that intracellular c-di-GMP as well as additional stimuli can be used to modulate adaptation of bacterial chemotaxis receptors. PMID:23512960

Regulation of the Na(+)-K(+)-2Cl(-) cotransporter by cGMP/cGMP-dependent protein kinase I after furosemide administration.

PubMed

Limmer, Franziska; Schinner, Elisabeth; Castrop, Hayo; Vitzthum, Helga; Hofmann, Franz; Schlossmann, Jens

2015-10-01

Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well-established effects of nitric oxide on NKCC2 activity, cGMP is likely involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) and cGKIα-rescue mice with furosemide. cGKIα-rescue mice expressed cGKIα only under the control of the smooth muscle-specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα-rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in cGKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2. © 2015 FEBS.

Application of a fluorometric microplate algal toxicity assay for riverine periphytic algal species.

PubMed

Nagai, Takashi; Taya, Kiyoshi; Annoh, Hirochica; Ishihara, Satoru

2013-08-01

Although riverine periphytic algae attached to riverbed gravel are dominant species in flowing rivers, there is limited toxicity data on them because of the difficulty in cell culture and assays. Moreover, it is well known that sensitivity to pesticides differ markedly among species, and therefore the toxicity data for multiple species need to be efficiently obtained. In this study, we investigated the use of fluorometric microplate toxicity assay for testing periphytic algal species. We selected five candidate test algal species Desmodesmus subspicatus, Achnanthidium minutissimum, Navicula pelliculosa, Nitzschia palea, and Pseudanabaena galeata. The selected species are dominant in the river, include a wide range of taxon, and represent actual species composition. Other additional species were also used to compare the sensitivity and suitability of the microplate assay. A 96-well microplate was used as a test chamber and algal growth was measured by in-vivo fluorescence. Assay conditions using microplate and fluorometric measurement were established, and sensitivities of 3,5-dichlorophenol as a reference substance were assayed. The 50 percent effect concentrations (EC50s) obtained by fluorometric microplate assay and those obtained by conventional Erlenmeyer flask assay conducted in this study were consistent. Moreover, the EC50 values of 3,5-dichlorophenol were within the reported confidence intervals in literature. These results supported the validity of our microplate assay. Species sensitivity distribution (SSD) analysis was conducted using the EC50s of five species. The SSD was found to be similar to the SSD obtained using additional tested species, suggesting that SSD using the five species largely represents algal sensitivity. Our results provide a useful and efficient method for high-tier probabilistic ecological risk assessment of pesticides. Copyright © 2013 Elsevier Inc. All rights reserved.

Rigid and non-rigid micro-plates: Philippines and Myanmar-Andaman case studies

NASA Astrophysics Data System (ADS)

Rangin, Claude

2016-01-01

Generally, tectonic plates are considered as rigid. Oblique plate convergence favors the development of micro-plates along the converging boundaries. The north-south-trending Philippines archipelago (here named Philippine Mobile Belt, PMB), a few hundreds kilometers wide, is one of such complex tectonic zones. We show here that it is composed of rigid rotating crustal blocks (here called platelets). In Myanmar, the northernmost tip of the Sumatra-Andaman subduction system is another complex zone made of various crustal blocks in-between convergent plates. Yet, contrary to PMB, it sustains internal deformation with platelet buckling, altogether indicative of a non-rigid behavior. Therefore, the two case studies, Philippine Mobile Belt and Myanmar-Andaman micro-plate (MAS), illustrate the complexity of micro-plate tectonics and kinematics at convergent plate boundaries.

Galapagos: Darwin, evolution, and ENT.

PubMed

Bluestone, Charles D

2009-10-01

This year is especially important in the history of the theory of evolution; 2009 is the bicentennial anniversary of the birth of Charles Darwin and the sesquicentennial anniversary of his publication, The Origin of Species. Darwin visited the Galapagos Islands as a young man, which greatly influenced his thinking. My son Jim and I had the good fortune to visit these islands in January 2009 and see firsthand what led Darwin to arrive at his monumental insights into the origins of life on this planet. I have described my observations and related some of this experience to the ear, nose, and throat, albeit with whimsy in several instances. Nonetheless, some of the adaptations in the animals on these unique islands may have bearing on my hypotheses related to the incidence and pathogenesis of otitis media in humans. It is hoped the reader will share my enthusiasm for the experience we had on these fantastic islands and tour them in the future.

Megachile timberlakei Cockerell (Hymenoptera: Megachilidae): Yet another adventive bee species to the Galapagos Archipelago

USDA-ARS?s Scientific Manuscript database

The Galapagos Archipelago has been thought to be extremely depauperate in bees, with only one species known, Xylocopa darwini. Recently a second species, Anthidium vigintiduopunctatum, was detected. Here we document a third species, Megachile timberlakei. We provide floral records as well as a dia...

Bioreactor expansion of human mesenchymal stem cells according to GMP requirements.

PubMed

Elseberg, Christiane L; Salzig, Denise; Czermak, Peter

2015-01-01

In cell therapy, the use of autologous and allogenic human mesenchymal stem cells is rising. Accordingly, the supply of cells for clinical applications in highest quality is required. As hMSCs are considered as an advanced therapy medicinal products (ATMP), they underlie the requirements of GMP and PAT according to the authorities (FDA and EMA). The production process of these cells must therefore be documented according to GMP, which is usually performed via a GMP protocol based on standard operating procedures. This chapter provides an example of such a GMP protocol for hMSC, here a genetically modified allogenic cell line, based on a production process in a microcarrier-based stirred tank reactor including process monitoring according to PAT and final product quality assurance.

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Chundawat, Shishir P S; Balan, Venkatesh; Dale, Bruce E

2008-04-15

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover. Copyright 2008 Wiley Periodicals, Inc.

Mantle viscosity beneath the Galapagos 95.5 deg W propagating rift

NASA Technical Reports Server (NTRS)

Schubert, G.; Hey, R. N.

1986-01-01

Detailed geophysical surveys in the vicinity of the Galapagos 95.5 deg W propagating rift tip establish the opening history of the rift and its velocity of propagation. These data together with a theory for mantle upwelling into slowly widening lithospheric cracks constrain the viscosity of the asthenosphere beneath the propagating rift to be less than about 10 to the 17th to 10 to the 18th Pa s.

A new adjuvant delivery system 'cyclic di-GMP/YSK05 liposome' for cancer immunotherapy.

PubMed

Miyabe, Hiroko; Hyodo, Mamoru; Nakamura, Takashi; Sato, Yusuke; Hayakawa, Yoshihiro; Harashima, Hideyoshi

2014-06-28

Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm. Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol=40/25/35 was found to induce IFN-β in Raw264.7 cells. The induction of IFN-β from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-β caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo. Copyright �

High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP.

PubMed

Petrova, Olga E; Sauer, Karin

2017-01-01

The modulation of c-di-GMP levels plays a vital role in the regulation of various processes in a wide array of bacterial species. Thus, investigation of c-di-GMP regulation requires reliable methods for the assessment of c-di-GMP levels and turnover. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis has become a commonly used approach to accomplish these goals. The following describes the extraction and HPLC-based detection and quantification of c-di-GMP from Pseudomonas aeruginosa samples, a procedure that is amenable to modifications for the analysis of c-di-GMP in other bacterial species.

A Capstone Course in Ecuador: The Andes/Galapagos Volcanology Field Camp Program

ERIC Educational Resources Information Center

Kelley, Daniel F.; Uzunlar, Nuri; Lisenbee, Alvis; Beate, Bernardo; Turner, Hope E.

2017-01-01

We developed and implemented the Galapagos Volcanology Field Camp, a 3 week, 3 credit hour course for upper-level university students with a major course of study in geology. The course is offered by the South Dakota School of Mines and Technology, is open to any student, and is usually populated by students from many universities across the U.S.…

Quantitative Microplate-Based Respirometry with Correction for Oxygen Diffusion

PubMed Central

2009-01-01

Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O2 is consumed by the specimen, atmospheric O2 leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O2 but also store substantial amounts of gas. O2 flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O2] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O2]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems. PMID:19555051

Modulation by cyclic GMP of the odour sensitivity of vertebrate olfactory receptor cells

NASA Technical Reports Server (NTRS)

Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

1996-01-01

Recent evidence has indicated a significant role for the cGMP second messenger system in vertebrate olfactory transduction but no clear functions have been identified for cGMP so far. Here, we have examined the effects of 8-Br-cGMP and carbon monoxide (CO) on odour responses of salamander olfactory receptor neurons using perforated patch recordings. We report that 8-Br-cGMP strongly down-regulates the odour sensitivity of the cells, with a K1/2 of 460 nM. This adaptation-like effect can be mimicked by CO, an activator of soluble guanylyl cyclase, with a K1/2 of 1 microM. Sensitivity modulation is achieved through a regulatory chain of events in which cGMP stimulates a persistent background current due to the activation of cyclic nucleotide-gated channels. This in turn leads to sustained Ca2+ entry providing a negative feedback signal. One consequence of the Ca2+ entry is a shift to the right of the stimulus-response curve and a reduction in saturating odour currents. Together, these two effects can reduce the sensory generator current by up to twenty-fold. Thus, cGMP functions to control the gain of the G-protein coupled cAMP pathway. Another consequence of the action of cGMP is a marked prolongation of the odour response kinetics. The effects of CO/cGMP are long-lasting and can continue for minutes. Hence, we propose that cGMP helps to prevent saturation of the cell's response by adjusting the operational range of the cAMP cascade and contributes to olfactory adaptation by decreasing the sensitivity of olfactory receptor cells to repeated odour stimuli.

The cyclic-di-GMP signaling pathway in the Lyme disease spirochete, Borrelia burgdorferi

PubMed Central

Novak, Elizabeth A.; Sultan, Syed Z.; Motaleb, Md. A.

2014-01-01

In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease. PMID:24822172

Structural Analysis of the GGDEF-EAL Domain-Containing c-di-GMP Receptor FimX

DOE Office of Scientific and Technical Information (OSTI.GOV)

Navarro, M.; De, N; Bae, N

2009-01-01

Bacterial pathogenesis involves social behavior including biofilm formation and swarming, processes that are regulated by the bacterially unique second messenger cyclic di-GMP (c-di-GMP). Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding signal transmission and the targets of c-di-GMP. FimX, a protein from Pseudomonas aeruginosa that governs twitching motility, belongs to a large subfamily containing both GGDEF and EAL domains. Biochemical and structural analyses reveals its function as a high-affinity receptor for c-di-GMP. A model for full-length FimX was generated combining solution scattering data andmore » crystal structures of the degenerate GGDEF and EAL domains. Although FimX forms a dimer in solution via the N-terminal domains, a crystallographic EAL domain dimer suggests modes for the regulation of FimX by c-di-GMP binding. The results provide the structural basis for c-di-GMP sensing via degenerate phosphodiesterases.« less

Nitric Oxide Mediates Glutamate-Linked Enhancement of cGMP Levels in the Cerebellum

NASA Astrophysics Data System (ADS)

Bredt, David S.; Snyder, Solomon H.

1989-11-01

Nitric oxide, which mediates influences of numerous neurotransmitters and modulators on vascular smooth muscle and leukocytes, can be formed in the brain from arginine by an enzymatic activity that stoichiometrically generates citrulline. We show that glutamate and related amino acids, such as N-methyl-D-aspartate, markedly stimulate arginine-citrulline transformation in cerebellar slices stoichiometrically with enhancement of cGMP levels. Nω-monomethyl-L-arginine blocks the augmentation both of citrulline and cGMP with identical potencies. Arginine competitively reverses both effects of Nω-monomethyl-L-arginine with the same potencies. Hemoglobin, which complexes nitric oxide, prevents the stimulation by N-methyl-D-aspartate of cGMP levels, and superoxide dismutase, which elevates nitric oxide levels, increases cGMP formation. These data establish that nitric oxide mediates the stimulation by glutamate of cGMP formation.

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

DOE PAGES

Campbell, James C.; Kim, Jeong Joo; Li, Kevin Y.; ...

2016-01-14

Membrane-bound cGMP-dependent protein kinase (PKG) II is an important regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKGII binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415more » of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.« less

Posttranscriptional regulation of human iNOS by the NO/cGMP pathway.

PubMed

Pérez-Sala, D; Cernuda-Morollón, E; Díaz-Cazorla, M; Rodríguez-Pascual, F; Lamas, S

2001-03-01

Nitric oxide (NO) and cGMP may exert positive or negative effects on inducible NO synthase (iNOS) expression. We have explored the influence of the NO/cGMP pathway on iNOS levels in human mesangial cells. Inhibition of NOS activity during an 8-h stimulation with IL-1beta plus tumor necrosis factor (TNF)-alpha reduced iNOS levels, while NO donors amplified iNOS induction threefold. However, time-course studies revealed a subsequent inhibitory effect of NO donors on iNOS protein and mRNA levels. This suggests that NO may contribute both to iNOS induction and downregulation. Soluble guanylyl cyclase (sGC) activation may be involved in these effects. Inhibition of sGC attenuated IL-1beta/TNF-alpha-elicited iNOS induction and reduced NO-driven amplification. Interestingly, cGMP analogs also modulated iNOS protein and mRNA levels in a biphasic manner. Inhibition of transcription unveiled a negative posttranscriptional modulation of the iNOS transcript by NO and cGMP at late times of induction. Supplementation with 8-bromo-cGMP (8-BrcGMP) reduced iNOS mRNA stability by 50%. These observations evidence a complex feedback regulation of iNOS expression, in which posttranscriptional mechanisms may play an important role.

Volcanic Evolution in the Galapagos: The Geochemistry and Petrology of Espanola Island

NASA Astrophysics Data System (ADS)

McGuire, M.; Varga, K. C.; Harpp, K. S.; Geist, D.; Hall, M. L.

2015-12-01

The Galapagos Archipelago consists of a series of volcanic islands located ~1,000 km west of South America that are thought to be the result of a mantle plume. The southeasternmost island, Espanola, is one of the smallest of the major islands, measuring only 7 by 14 km and reaching an elevation of 200 m. Espanola is also the oldest island in the chain, with K-Ar dates from 3.01 ± 0.11 to 3.31 ± 0.36 million years (Hall et al. 1983; White et al., 1993). The southern coast is defined by cliffs that exceed 100 m in height, made up of nearly flat-lying lavas that are each several meters thick. The northern coastline consists of lavas that dip gently toward the ocean from the highlands, as well as remnants of eroded cinder cones. Paleomagnetic measurements made in the field indicate that the western half of the island is reversely polarized, whereas most lavas measured across the eastern half are normally polarized. Major element analyses of samples from across the island indicate that fractional crystallization is the dominant process controlling chemical variations in Espanola lavas, suggesting a relatively long-lived magmatic plumbing system. Stratigraphically constrained chemical variations suggest the magma chamber may have experienced periodic replenishment by compositionally homogeneous primitive melts. Variable fluid-mobile trace element concentrations provide some evidence for contributions from ancient, recycled oceanic crust to the parental melts. Espanola lavas have more depleted Sr and Pb radiogenic isotope ratios than either Floreana or Fernandina, and lie on a mixing curve between the composition of the plume and that of the depleted upper mantle. Between ~3 and 8 Ma, the Galapagos Spreading Center was closer to the Galapagos plume than it is today. Given that Espanola was constructed during the same period, the depleted isotopic signatures suggest that plume-ridge interaction may have been a strong influence on the island's geochemical composition.

Robust Microplate-Based Methods for Culturing and in Vivo Phenotypic Screening of Chlamydomonas reinhardtii.

PubMed

Haire, Timothy C; Bell, Cody; Cutshaw, Kirstin; Swiger, Brendan; Winkelmann, Kurt; Palmer, Andrew G

2018-01-01

Chlamydomonas reinhardtii (Cr), a unicellular alga, is routinely utilized to study photosynthetic biochemistry, ciliary motility, and cellular reproduction. Its minimal culture requirements, unicellular morphology, and ease of transformation have made it a popular model system. Despite its relatively slow doubling time, compared with many bacteria, it is an ideal eukaryotic system for microplate-based studies utilizing either, or both, absorbance as well as fluorescence assays. Such microplate assays are powerful tools for researchers in the areas of toxicology, pharmacology, chemical genetics, biotechnology, and more. However, while microplate-based assays are valuable tools for screening biological systems, these methodologies can significantly alter the conditions in which the organisms are cultured and their subsequent physiology or morphology. Herein we describe a novel method for the microplate culture and in vivo phenotypic analysis of growth, viability, and photosynthetic pigments of C. reinhardtii . We evaluated the utility of our assay by screening silver nanoparticles for their effects on growth and viability. These methods are amenable to a wide assortment of studies and present a significant advancement in the methodologies available for research involving this model organism.

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles.

PubMed

Shuhaibar, Leia C; Egbert, Jeremy R; Norris, Rachael P; Lampe, Paul D; Nikolaev, Viacheslav O; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A

2015-04-28

Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.

Microplate based optical biosensor for L-Dopa using tyrosinase from Amorphophallus campanulatus.

PubMed

Saini, Amardeep Singh; Kumar, Jitendra; Melo, Jose Savio

2014-11-07

Developing a biosensor which is capable of simultaneously monitoring l-Dopa levels in multiple samples besides requiring small reaction volume is of great value. The present study describes the detection of l-Dopa using tyrosinase enzyme extracted from Amorphophallus campanulatus and immobilized on the surface of the microplate wells. Among the different approaches used for immobilizing tyrosinase onto the microplate wells, glutaraldehyde treatment was found to be most effective. Besides enzyme activity, ESEM-EDS (environmental scanning electron microscope-energy dispersive system) and Atomic Force Microscopy (AFM) were also carried out to confirm the immobilization of tyrosinase enzyme onto the microplate well surface. This immobilized biocomponent was then integrated with an optical transducer for l-Dopa detection and it showed good reproducibility. The sensing property of the system was studied by measuring the initial rate of dopachrome formation at 475 nm. The calibration plot gave a linear range of detection from 10-1000 μM and the detection limit was calculated to be 3 μM. The immobilized biocomponent was stable for 41 days and was reused up to nine times. Spiked samples (blood plasma) were also analyzed using this biocomponent. This microplate based biosensor thus provides a convenient system for detection of multiple samples in a single run. Copyright © 2014 Elsevier B.V. All rights reserved.

From bedside to bench--meeting report of the 7th International Conference on cGMP "cGMP: generators, effectors and therapeutic implications" in Trier, Germany, from June 19th to 21st 2015.

PubMed

Friebe, Andreas; Sandner, Peter; Seifert, Roland

2015-12-01

During the past decade, our knowledge on the physiology, pathophysiology, basic pharmacology, and clinical pharmacology of the second messenger (cGMP) has increased tremendously. It is now well-established that cGMP, generated by soluble and particulate guanylate cyclases, is highly compartmentalized in cells and regulates numerous body functions. New cGMP-regulated physiological functions include meiosis and temperature perception. cGMP is involved in the genesis of numerous pathologies including cardiovascular, pulmonary, endocrine, metabolic, neuropsychiatric, eye, and tumor diseases. Several new clinical uses of stimulators and activators of soluble guanylate cyclase and of phosphodiesterase inhibitors such as heart failure, kidney failure, cognitive disorders, obesity bronchial asthma, and osteoporosis are emerging. The combination of neprilysin inhibitors-enhancing stimulation of the particulate guanylate cyclase pathway by preventing natriuretic peptide degradation-with angiotensin AT1 receptor antagonists constitutes a novel promising strategy for heart failure treatment. The role of oxidative stress in cGMP signaling, application of cGMP sensors, and gene therapy for degenerative eye diseases are emerging topics. It is anticipated that cGMP research will further prosper over the next years and reach out into more and more basic and clinical disciplines.

Volcanism on the fossil Galapagos Rise spreading centre, SE Pacific

NASA Astrophysics Data System (ADS)

Haase, K. M.; Stroncik, N. A.

2002-12-01

A part of the fossil spreading centre of the Galapagos Rise at 10° S, 95° W in the SE Pacific Ocean was mapped and sampled. This spreading centre was active for about 12 Ma and was abandoned about 6.5 Ma ago when the spreading rate of the East Pacific Rise (EPR) increased. The aim of this study is to understand the tectonic and petrological implications of the ridge jump for the spreading centre and to gain insights into the processes in its melting column. Bathymetric swath mapping of a part of the Galapagos Rise revealed an elongated structure with a NNE-SSW strike direction which is bounded by a large fracture zone in the north. The mapped area can be divided into three segments, each of about 50 km length. The northernmost segment consists of an ~4400 m deep rift which shows similarities to a slow-spreading centre, e.g. the Mid-Atlantic Ridge. The southern two segments are volcanic ridges with numerous volcanic flank cones which reach water depths up to 490 m. This volcanic ridge is interpreted as the continuation of the fossil spreading axis. While the northernmost segment is magmatically starved, the volcanic ridges of the southern two segments apparently formed after cessation of spreading. The rock samples from the rift flanks in the north are incompatible element-depleted (K/Ti 0.08-0.28) and plagioclase-phyric basalts resembling typical mid-ocean ridge basalts (MORB). In contrast, the lavas from the two volcanic ridge segments in the south are highly vesicular incompatible element-enriched alkali basalts with K/Ti of 0.65-1.4. The depleted rift basalts have Sr isotope ratios below 0.7027 while the alkali basalts from the ridge range between 0.7029 and 0.7031. The rift basalts have significantly lower sodium contents than the alkali basalts and thus the southern lavas are probably derived by smaller degrees of partial melting. The relatively low Si contents of the alkali basalts also indicates formation deeper in the melting column than the northern MORB

Three dimensional perspective view of portion of western Galapagos Islands

NASA Image and Video Library

1994-04-18

STS059-S-085 (18 April 1994) --- This is a three-dimensional perspective view of part of Isla Isabela in the western Galapagos Islands. It was taken by the L-Band radar in HH polarization from the Spaceborne Imaging Radar-C and X-Band Synthetic Aperture Radar (SIR-C/X-SAR) on the 40th orbit of the Space Shuttle Endeavour. This view was constructed by overlaying a SIR-C radar image on a U.S. Geological Survey digital elevation map. The image is centered at about .5 degrees south latitude and 91 degrees west longitude, and covers an area of 75 by 60 kilometers. The radar incidence angle at the center of the image is about 20 degrees. The western Galapagos Islands, which lie about 1200 kilometers west of Ecuador in the eastern Pacific, have six active volcanoes similar to the volcanoes found in Hawaii. Since the time of Charles Darwin's visit to the area in 1835, there have been over 60 recorded eruptions on these volcanoes. This SIR-C/X-SAR image of Alcedo and Sierra Negra volcanoes shows the rougher lava flows as bright features, while ash deposits and smooth pahoehoe lava flows appear dark. The Galapagos Islands are one of the SIR-C/X-SAR supersites and data of this area will be taken several times during the flight to allow scientists to conduct topographic change studies and to search for different lava flow types, ash deposits and fault lines. SIR-C/X-SAR is part of NASA's Mission to Planet Earth (MTPE). SIR-C/X-SAR radars illuminate Earth with microwaves allowing detailed observations at any time, regardless of weather or sunlight conditions. SIR-C/X-SAR uses three microwave wavelengths: L-Band (24 cm), C-Band (6 cm), and X-Band (3 cm). The multi-frequency data will be used by the international scientific community to better understand the global environment and how it is changing. The SIR-C/X-SAR data, complemented by aircraft and ground studies, will give scientists clearer insights into those environmental changes which are caused by nature and those changes

Bacterial Signal Transduction by Cyclic Di-GMP and Other Nucleotide Second Messengers

PubMed Central

Gründling, Angelika; Jenal, Urs; Ryan, Robert; Yildiz, Fitnat

2015-01-01

The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany) brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i) c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology, with major impacts in research fields ranging from human health to microbial ecology. PMID:26055111

A case study of human migration and the sea cucumber crisis in the Galapagos Islands.

PubMed

Bremner, Jason; Perez, Jaime

2002-06-01

The sea cucumber fishing crisis in Galapagos is an example of the potential consequences of rapid migration, growing economic competition, and weak regulatory mechanisms. In a short period of time sea cucumber fishing has become the most inflammatory issue in the Galapagos. The key factors that allowed for the efficient exploitation of the new resource were not the fishermen themselves but rather the new fishing techniques and access to credit and markets. This suggests that the annual sea cucumber crisis is due to factors more complex than simply more fishermen generating greater sea cucumber catches. This paper examines census data and fisher registries to analyze population growth in the islands. A public opinion survey is used to determine the population's attitudes toward sea cucumber fishing and regulations. Qualitative interviews explore the history of the sea cucumber boom. Information from the sea cucumber monitoring program provides estimates for the annual sea cucumber catches.

Conformation changes, N-terminal involvement and cGMP signal relay in phosphodiesterase-5 GAF domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, H.; Robinson, H.; Ke, H.

2010-12-03

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

Importance of NO/cGMP signalling via cGMP-dependent protein kinase II for controlling emotionality and neurobehavioural effects of alcohol.

PubMed

Werner, Claudia; Raivich, Gennadij; Cowen, Michael; Strekalova, Tatyana; Sillaber, Inge; Buters, Jeroen T; Spanagel, Rainer; Hofmann, Franz

2004-12-01

Cyclic GMP is a second messenger for nitric oxide (NO) that acts as a mediator for many different physiological functions. The cGMP-dependent protein kinases (cGKs) mediate cellular signalling induced by NO and cGMP. Here, we explored the localization of cGMP-dependent protein kinase type II (cGKII) in the mouse brain. In situ hybridization revealed high levels of cGKII mRNA in cerebral cortex, thalamic nuclei, hypothalamic nuclei, and in several basal forebrain regions including medial septum, striatum and amygdala. The close link to NO and the distribution pattern of cGKII suggested that this enzyme might be involved in emotional reactions and responses to drugs of abuse. Therefore, cGKII knockout animals (cGKII-/-) were compared with littermate controls in behavioural tests (i) for emotion-linked and (ii) for acute and chronic ethanol responses. Deletion of cGKII did not influence aggressive behaviour but led to enhanced anxiety-like behaviour. In terms of acute responses to ethanol, cGKII-/- mice were hyposensitive to hypnotic doses of ethanol as measured by the loss of righting reflex, without an alteration in their blood alcohol elimination. In a two-bottle free choice test, cGKII-/- mice showed elevated alcohol consumption. No taste differences to sweet solutions were observed compared to control animals. In summary, our data show that cGKII activity modulates anxiety-like behaviour and neurobehavioural effects of alcohol.

The Leading Edge of the Galapagos Hotspot: Geochemistry and Geochronology of Submarine Glasses Coupled to New Sidescan Sonar Imagery

NASA Astrophysics Data System (ADS)

Kurz, M. D.; Fornari, D. J.; Geist, D. J.; Johnson, P. D.; Curtice, J. M.; Lott, D. E.; Harpp, K.; Saal, A. E.; Peckman, U. G.

2001-12-01

Fernandina, the western-most volcano in the Galapagos archipelago, is at the leading edge of the hotspot with respect to plate motion. Recent mapping of the ocean floor west of Fernandina (on R/V Revelle, using the HMRG towed sidescan sonar MR1, and Simrad EM120 multibeam) provides a dramatic new view of the volcanic constructional processes that have created the islands. The western flank of the volcano is characterized by the prominent Northwest, West, and Southwest rift zones, which are constructed of hummocky pillow lavas. Older lava flow terrain is distinguished by weaker acoustic return, whereas extensive younger flows are characterized by strong backscatter patterns with distinctive flow-like margins. MR1 sidescan sonar mapping provides an important new geologic and stratigraphic context for understanding the submarine Galapagos platform, particularly from a geochemical perspective. Fernandina lavas have high 3He/4He ratios, up to 29 times atmospheric, and solar-like neon isotopic compositions, characteristics which suggest they are derived from the deep mantle. The high 3He/4He ratios, and rapid eruption rates at Fernandina also indicate that it lies directly above the center of the Galapagos hotspot. In order to place these geochemical data into a chronological framework, we have determined ages for Fernandina submarine glasses using the Th-U-He crushing/melting disequilibrium method. Preliminary Th-U-He ages (from the 2000 R/V Melville AHA-Nemo expedition), combined with the new MR1 sonar mapping, shows that the rift zones are characterized by extremely young ages (0 to 30 Ka) while older submarine lava flows with lower acoustic backscatter have significantly older ages ( ~ 100 Ka). The geochronological data, and the geological context from the side-scan sonar, provide new evidence for volcano growth rates in oceanic hotspot provinces, and will be used to determine the growth rate of the Galapagos platform.

High-throughput measurements of the optical redox ratio using a commercial microplate reader.

PubMed

Cannon, Taylor M; Shah, Amy T; Walsh, Alex J; Skala, Melissa C

2015-01-01

There is a need for accurate, high-throughput, functional measures to gauge the efficacy of potential drugs in living cells. As an early marker of drug response in cells, cellular metabolism provides an attractive platform for high-throughput drug testing. Optical techniques can noninvasively monitor NADH and FAD, two autofluorescent metabolic coenzymes. The autofluorescent redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD, quantifies relative rates of cellular glycolysis and oxidative phosphorylation. However, current microscopy methods for redox ratio quantification are time-intensive and low-throughput, limiting their practicality in drug screening. Alternatively, high-throughput commercial microplate readers quickly measure fluorescence intensities for hundreds of wells. This study found that a commercial microplate reader can differentiate the receptor status of breast cancer cell lines (p < 0.05) based on redox ratio measurements without extrinsic contrast agents. Furthermore, microplate reader redox ratio measurements resolve response (p < 0.05) and lack of response (p > 0.05) in cell lines that are responsive and nonresponsive, respectively, to the breast cancer drug trastuzumab. These studies indicate that the microplate readers can be used to measure the redox ratio in a high-throughput manner and are sensitive enough to detect differences in cellular metabolism that are consistent with microscopy results.

8-Nitro-cGMP promotes bone growth through expansion of growth plate cartilage.

PubMed

Hoshino, Marie; Kaneko, Kotaro; Miyamoto, Yoichi; Yoshimura, Kentaro; Suzuki, Dai; Akaike, Takaaki; Sawa, Tomohiro; Ida, Tomoaki; Fujii, Shigemoto; Ihara, Hideshi; Tanaka, Junichi; Tsukuura, Risa; Chikazu, Daichi; Mishima, Kenji; Baba, Kazuyoshi; Kamijo, Ryutaro

2017-09-01

In endochondral ossification, growth of bones occurs at their growth plate cartilage. While it is known that nitric oxide (NO) synthases are required for proliferation of chondrocytes in growth plate cartilage and growth of bones, the precise mechanism by which NO facilitates these process has not been clarified yet. C-type natriuretic peptide (CNP) also positively regulate elongation of bones through expansion of the growth plate cartilage. Both NO and CNP are known to use cGMP as the second messenger. Recently, 8-nitro-cGMP was identified as a signaling molecule produced in the presence of NO in various types of cells. Here, we found that 8-nitro-cGMP is produced in proliferating chondrocytes in the growth plates, which was enhanced by CNP, in bones cultured ex vivo. In addition, 8-nitro-cGMP promoted bone growth with expansion of the proliferating zone as well as increase in the number of proliferating cells in the growth plates. 8-Nitro-cGMP also promoted the proliferation of chondrocytes in vitro. On the other hand, 8-bromo-cGMP enhanced the growth of bones with expansion of hypertrophic zone of the growth plates without affecting either the width of proliferating zone or proliferation of chondrocytes. These results indicate that 8-nitro-cGMP formed in growth plate cartilage accelerates chondrocyte proliferation and bone growth as a downstream molecule of NO. Copyright © 2017. Published by Elsevier Inc.

Role of nitric oxide and cyclic GMP signaling in melanocyte response to hypergravity

NASA Astrophysics Data System (ADS)

Ivanova, Krassimira; Lambers, Britta; Tsiockas, Wasiliki; Block, Ingrid; Gerzer, Rupert

Nitric oxide (NO) has a prominent role in many (patho)physiological processes in the skin including erythema, inflammation, and cancerogenesis. The soluble guanylyl cyclase (sGC), a key transducer in NO signaling, catalyzes the formation of the second messenger guanosine 3´,5´-cyclic monophosphate (cyclic cGMP or cGMP). For human melanocytes, which are responsible for skin pigmentation by synthesizing the pigment melanin, it has been reported that the NO/sGC/cGMP pathway is involved in UVB-induced melanogenesis. Melanin acts as a scavenger for free radicals that may arise during metabolic stress. It may also act as a photosensitizer that generates active oxygen species upon UV irradiation, which may initiate hypopigmentary disorders (e.g., vitiligo) as well as UV-induced oncogene cell transformation. In addition, melanoma, a deadly skin cancer, which arises from transformed melanocytes, is characterized by a resistance to chemotherapy. In our studies we have shown that NO can induce perturbation of melanocyte-extracellular matrix component interactions, which may contribute to loss of melanocytes or melanoma metastasis. Such NO effects appear to be modulated partly via cGMP. Moreover, we found that different guanylyl cyclase isoforms are responsible for cGMP synthesis in melanocytic cells. Normal human melanocytes and nonmetastatic melanoma cells predominantly express sGC, which appears to be associated with melanogenesis, whereas absence of NO-sensitive GC, but up-regulated activities of the natriuretic peptide-sensitive membrane guanylyl cyclase isoforms were found in highly metastatic phenotypes. Due to the growing interest in the regulation of signaling activities in normal and transformed cells under altered gravity conditions, we have further investigated whether the NO/cGMP signaling is involved in melanocyte response to gravitational stress. We found that normal human melanocytes and non-metastatic melanoma cell lines, but not highly metastatic cells

New Insights into the Morphology of the Galapagos Platform from Lower Crustal Flow Models

NASA Astrophysics Data System (ADS)

Orellana Rovirosa, F.; Richards, M. A.

2014-12-01

The volcanically-active Galapagos Islands are constructed upon a broad platform, with the westernmost islands being the most active, marking the present-day position of the Galapagos hotspot (mantle plume). This volcanic platform overlies relatively young oceanic lithosphere (<15 Myr) and exhibits unique morphologic features along its boundaries. The most spectacular of these features is a system of stepped terraces on the southwestern escarpment, with very large vertical relief (>3 km), and contrasting with relatively gentle slopes off the eastern platform edge toward the Carnegie ridge. Considering the horizontal lithostatic pressure differences associated with this bathymetric relief, along with the high temperatures within this young, hotspot-affected oceanic lithosphere and crust; it is likely that lower crustal flow contributes significantly, perhaps even dominantly, to lithospheric and crustal deformation within the Galapagos Platform. Using 2D numerical models that invoke a thin-sheet approximation for the Stokes' equation for a Newtonian fluid with space- and time-dependent viscosity, and assuming isostatic conditions, we show that: (1) the pronounced bathymetric rim along the Eastern platform region (where gravimetric studies indicate Airy isostasy) near Española Island may be the expression of a mature stage of a lower crustal flow front evolving asymptotically during the last ~3 Myr; (2) the spectacular system of stepped terraces along the southwestern edge of the platform may be explained by lower crustal flow-associated backward tilting of the bathymetric surface that, even with small amounts of angular change (~0.1 deg) and potentially occurring in non-isostatic regimes, effectively hinders the horizontal flow of lava. This process of backward-tilting may have been largely restricted to the last ~1 Myr of platform growth, and hence may be a unique event that may involve horizontal extrusion of large lava sheets from within the southwestward

Amyloid-β Peptide Is Needed for cGMP-Induced Long-Term Potentiation and Memory.

PubMed

Palmeri, Agostino; Ricciarelli, Roberta; Gulisano, Walter; Rivera, Daniela; Rebosio, Claudia; Calcagno, Elisa; Tropea, Maria Rosaria; Conti, Silvia; Das, Utpal; Roy, Subhojit; Pronzato, Maria Adelaide; Arancio, Ottavio; Fedele, Ernesto; Puzzo, Daniela

2017-07-19

High levels of amyloid-β peptide (Aβ) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aβ are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aβ levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aβ due to a change in the approximation of amyloid precursor protein (APP) and the β-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aβ function, by using anti-murine Aβ antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aβ levels in the healthy brain which, in turn, boosts synaptic plasticity and memory. SIGNIFICANCE STATEMENT Amyloid-β (Aβ) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aβ, mimicking levels of the peptide in the healthy brain, enhance hippocampal long-term potentiation (LTP) and memory. Because the second messenger cGMP exerts a central role in LTP mechanisms, here we studied whether cGMP affects Aβ levels and function during LTP. We show that cGMP enhances Aβ production by increasing the APP/BACE-1 convergence in endolysosomal compartments. Moreover, the cGMP-induced enhancement of LTP and memory was disrupted by blockade of Aβ, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aβ. Copyright © 2017 the authors 0270-6474/17/376926-12$15.00/0.

Bovine anaplasmosis and tick-borne pathogens in cattle of the Galapagos Islands.

PubMed

Gioia, G V; Vinueza, R L; Marsot, M; Devillers, E; Cruz, M; Petit, E; Boulouis, H J; Moutailler, S; Monroy, F; Coello, M A; Gondard, M; Bournez, L; Haddad, N; Zanella, G

2018-03-22

A cross-sectional study was conducted to determine the species of Anaplasma spp. and estimate its prevalence in cattle of the three main cattle-producing Galapagos Islands (Santa Cruz, San Cristóbal and Isabela) using indirect PCR assays, genetic sequencing and ELISA. Ticks were also collected from cattle and scanned for 47 tick-borne pathogens in a 48 × 48 real-time PCR chip. A mixed effects logistic regression was performed to identify potential risk factors explaining Anaplasma infection in cattle. A. phagocytophilum was not detected in any of the tested animals. Genetic sequencing allowed detection of A. platys-like strains in 11 (36.7%) of the 30 Anaplasma spp.-positive samples analysed. A. marginale was widespread in the three islands with a global between-herd prevalence of 100% [89; 100] 95% CI and a median within-herd prevalence of 93%. A significant association was found between A. marginale infection and age with higher odds of being positive for adults (OR = 3.3 [1.2; 9.9] 95% Bootstrap CI ). All collected ticks were identified as Rhipicephalus microplus. A. marginale, Babesia bigemina, Borrelia theileri and Francisella-like endosymbiont were detected in tick pools. These results show that the Galapagos Islands are endemic for A. marginale. © 2018 Blackwell Verlag GmbH.

cGMP in ozone and NO dependent responses

PubMed Central

Ederli, Luisa; Meier, Stuart; Borgogni, Andrea; Reale, Lara; Ferranti, Francesco; Gehring, Chris

2008-01-01

We have recently reported that ozone (O3) can inhibit mitochondrial respiration and induce activation of the alternative oxidase (AOX) pathway and in particular AOX1a in tobacco. While O3 causes mitochondrial H2O2, early leaf nitric oxide (NO) as well as transient ethylene (ET) accumulation, the levels of jasmonic acid and 12-oxo-phytodienoic acid remained unchanged. It was shown that both, NO and ET dependent pathways can induce AOX1a transcription by O3. AOX plays a role in reducing reactive oxygen species (ROS) which in turn are linked to biotic and abiotic plant stresses, much like the second messengers guanosine 3′, 5′-cyclic monophosphate (cGMP). The goal is to unravel specific cGMP signatures and induction pathways downstream from O3 and NO, including transcription of AOX1a. Here we propose that some late (>3 h) responses to NO, e.g., the accumulation of phenylalanine lyase (PAL) transcripts, are critically cGMP dependent, while the early (<2 h) responses, including AOX1a induction are not. PMID:19704720

Robust Microplate-Based Methods for Culturing and in Vivo Phenotypic Screening of Chlamydomonas reinhardtii

PubMed Central

Haire, Timothy C.; Bell, Cody; Cutshaw, Kirstin; Swiger, Brendan; Winkelmann, Kurt; Palmer, Andrew G.

2018-01-01

Chlamydomonas reinhardtii (Cr), a unicellular alga, is routinely utilized to study photosynthetic biochemistry, ciliary motility, and cellular reproduction. Its minimal culture requirements, unicellular morphology, and ease of transformation have made it a popular model system. Despite its relatively slow doubling time, compared with many bacteria, it is an ideal eukaryotic system for microplate-based studies utilizing either, or both, absorbance as well as fluorescence assays. Such microplate assays are powerful tools for researchers in the areas of toxicology, pharmacology, chemical genetics, biotechnology, and more. However, while microplate-based assays are valuable tools for screening biological systems, these methodologies can significantly alter the conditions in which the organisms are cultured and their subsequent physiology or morphology. Herein we describe a novel method for the microplate culture and in vivo phenotypic analysis of growth, viability, and photosynthetic pigments of C. reinhardtii. We evaluated the utility of our assay by screening silver nanoparticles for their effects on growth and viability. These methods are amenable to a wide assortment of studies and present a significant advancement in the methodologies available for research involving this model organism. PMID:29623083

76 FR 51395 - Draft Environmental Impact Statement for the General Management Plan (DEIS/GMP), Canaveral...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-18

... Statement for the General Management Plan (DEIS/GMP), Canaveral National Seashore, FL AGENCY: National Park... General Management Plan (DEIS/GMP), Canaveral National Seashore (Seashore). SUMMARY: Pursuant to 42 U.S.C... DEIS/GMP for Canaveral National Seashore, Florida. The document provides a framework for management...

Urban life of Galapagos sea lions (Zalophus wollebaeki) on San Cristobal Island, Ecuador: colony trends and threats

NASA Astrophysics Data System (ADS)

Denkinger, Judith; Gordillo, Luis; Montero-Serra, Ignasi; Murillo, Juan Carlos; Guevara, Nataly; Hirschfeld, Maximilian; Fietz, Katharina; Rubianes, Francisco; Dan, Michael

2015-11-01

Worldwide, pristine environments are influenced by human societies. In the Galapagos Islands, the endangered, endemic Galapagos sea lion (Zalophus wollebaeki) has formed one of the biggest colonies within the town center of Puerto Baquerizo Moreno. About 8,000 people live there and human wildlife interactions occur daily. With colony counts and direct observations from 2008 to 2012, we analyze cause of death, injuries and disease of urban sea lion colonies at Wreck Bay. Population increase since 2008 can be attributed to an immigration of adult sea lions in 2010, resulting in an increase in the pup and juvenile production in 2011 and 2012. Pup mortality increased drastically to 2009 and again in 2011 and 2012. Besides pup mortality, most of the deaths are caused by increased disease incidences and human activity. Our observations suggest that overall 65% of the injuries observed are produced by human interaction. The increase in threats leading to death, injuries or disease can have long-term effects on the population. Although threats that cause physical injuries can be managed locally, sea lions range movements contributes to the spread of infectious pathogens, which may affect neighbor colonies and potentially have an impact on the survival of the species. Our study reveals the need of stronger efforts towards a more complete understanding of threats and especially disease spread among Galapagos Sea lions in urban environments and the establishment of more effective management measures.

Establishment and validation of a method for multi-dose irradiation of cells in 96-well microplates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abatzoglou, Ioannis; Zois, Christos E.; Pouliliou, Stamatia

2013-02-15

Highlights: ► We established a method for multi-dose irradiation of cell cultures within a 96-well plate. ► Equations to adjust to preferable dose levels are produced and provided. ► Up to eight different dose levels can be tested in one microplate. ► This method results in fast and reliable estimation of radiation dose–response curves. -- Abstract: Microplates are useful tools in chemistry, biotechnology and molecular biology. In radiobiology research, these can be also applied to assess the effect of a certain radiation dose delivered to the whole microplate, to test radio-sensitivity, radio-sensitization or radio-protection. Whether different radiation doses can bemore » accurately applied to a single 96-well plate to further facilitate and accelerated research by one hand and spare funds on the other, is a question dealt in the current paper. Following repeated ion-chamber, TLD and radiotherapy planning dosimetry we established a method for multi-dose irradiation of cell cultures within a 96-well plate, which allows an accurate delivery of desired doses in sequential columns of the microplate. Up to eight different dose levels can be tested in one microplate. This method results in fast and reliable estimation of radiation dose–response curves.« less

Structural and Biochemical Determinants of Ligand Binding by the c-di-GMP Riboswitch

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, K.; Lipchock, S; Livingston,

2010-01-01

The bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that themore » most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high-resolution (2.3 {angstrom}) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3{prime}-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.« less

Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria.

PubMed

Arora, Naveen Kumar; Verma, Maya

2017-12-01

In this study, siderophore production by various bacteria amongst the plant-growth-promoting rhizobacteria was quantified by a rapid and efficient method. In total, 23 siderophore-producing bacterial isolates/strains were taken to estimate their siderophore-producing ability by the standard method (chrome azurol sulphonate assay) as well as 96 well microplate method. Production of siderophore was estimated in percent siderophore unit by both the methods. It was observed that data obtained by both methods correlated positively with each other proving the correctness of microplate method. By the modified microplate method, siderophore production by several bacterial strains can be estimated both qualitatively and quantitatively at one go, saving time, chemicals, making it very less tedious, and also being cheaper in comparison with the method currently in use. The modified microtiter plate method as proposed here makes it far easier to screen the plant-growth-promoting character of plant-associated bacteria.

A microplate assay for measuring cell death in C2C12 cells.

PubMed

Lima, Tanes; Silveira, Leonardo

2018-03-22

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method proves to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 minutes, and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 hours to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and reactive oxygen species production validated the deleterious effects of palmitate treatment. Also, the microplate PI assay demonstrated high sensitivity as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method to be used for in vitro studies.

Inhibition of Cyclic GMP Export by Multidrug Resistance Protein 4: A New Strategy to Treat Erectile Dysfunction?

PubMed

Boydens, Charlotte; Pauwels, Bart; Vanden Daele, Laura; Van de Voorde, Johan

2017-04-01

Intracellular cyclic guanosine monophosphate (cGMP) concentrations are regulated by degradation enzymes (phosphodiesterases) and by active transport across the plasma membrane by multidrug resistance proteins (MRPs) 4 and 5. To evaluate the functional effect of MRP-4 inhibition and the role of MRP-4-mediated cGMP export in mouse corpora cavernosa. Isometric tension of mouse corpora cavernosa was measured after cumulative addition of MK-571, an inhibitor of MRP-4, or sildenafil, a phosphodiesterase type 5 inhibitor. In addition, the effect of MRP-4 inhibition on cGMP-independent and cGMP-dependent relaxations was studied. In vivo intracavernosal pressure and mean arterial pressure measurements were performed after intracavernosal injection of MK-571. The effect of MRP-4 inhibition on cGMP content was determined using an enzyme immunoassay kit. Measurement of the effect of MK-571 on cGMP content, relaxant responses of mouse corpora cavernosa to cGMP-independent and cGMP-dependent vasodilating substances, and determination of the ratio of intracavernosal pressure to mean arterial pressure after intracavernosal injection of MK-571. MK-571 and sildenafil relaxed the corpora cavernosa concentration dependently, with sildenafil being the more potent relaxing compound. Furthermore, MK-571 enhanced relaxing responses to cGMP-dependent substances, such as sodium nitroprusside, sildenafil, acetylcholine, and electrical field stimulation, with the latter even under in vitro diabetic conditions. In contrast, cGMP-independent relaxations were not altered by MRP-4 inhibition. Intracavernosal administration of MK-571 significantly increased intracavernosal pressure, with minimal effect on mean arterial pressure. The cGMP analysis showed that MRP-4 inhibition was accompanied by increased cGMP levels. MRP-4, at least when targeted locally in the penis or when combined with a phosphodiesterase type 5 inhibitor, might be a valuable alternative strategy for the treatment of

Isla Isabela in the western Galapagos Islands

NASA Technical Reports Server (NTRS)

1994-01-01

This is an image showing part of Isla Isabela in the western Galapagos Islands. It was taken by the L-band radar in HH polarization from the Spaceborne Imaging Radar-C/X-Band Synthetic Aperature Radar on the 40th orbit of the Shuttle Endeavour. The image is centered at about .5 degrees south latitude and 91 degrees West longitude and covers an area of 75 km by 60 km. The radar incidence angle at the center of the image is about 20 degrees. This SIR-C/X-SAR image of Alcedo and Sierra Negra volcanoes shows the rougher lava flows as bright features, while ash deposits and smooth Pahoehoe lava flows appear dark. A small portion of Isla Fernandina is visible in the extreme upper left corner of the image. The Jet Propulsion Laboratory alternative photo number is P-43899.

Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma.

PubMed

Nakamura, Takashi; Miyabe, Hiroko; Hyodo, Mamoru; Sato, Yusuke; Hayakawa, Yoshihiro; Harashima, Hideyoshi

2015-10-28

Malignant melanomas escape immunosurveillance via the loss/down-regulation of MHC-I expression. Natural killer (NK) cells have the potential to function as essential effector cells for eliminating melanomas. Cyclic di-GMP (c-di-GMP), a ligand of the stimulator of interferon genes (STING) signal pathway, can be thought of as a new class of adjuvant against cancer. However, it is yet to be tested, because technologies for delivering c-di-GMP to the cytosol are required. Herein, we report that c-di-GMP efficiently activates NK cells and induces antitumor effects against malignant melanomas when loaded in YSK05 lipid containing liposomes, by assisting in the efficient delivery of c-di-GMP to the cytosol. The intravenous administration of c-di-GMP encapsulated within YSK05-liposomes (c-di-GMP/YSK05-Lip) into mice efficiently induced the production of type I interferon (IFN) as well as the activation of NK cells, resulting in a significant antitumor effect in a lung metastasis mouse model using B16-F10. This antitumor effect was dominated by NK cells. The infiltration of NK cells was observed in the lungs with B16-F10 melanomas. These findings indicate that the c-di-GMP/YSK05-Lip induces MHC-I non-restricted antitumor immunity mediated by NK cells. Consequently, c-di-GMP/YSK05-Lip represents a potentially new adjuvant system for use in immunotherapy against malignant melanomas. Copyright © 2015 Elsevier B.V. All rights reserved.

Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain*

PubMed Central

Wang, Huanchen; Robinson, Howard; Ke, Hengming

2010-01-01

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98–147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes. PMID:20861010

Ratiometric fluorescence detection of superoxide anion based on AuNPs-BSA@Tb/GMP nanoscale coordination polymers.

PubMed

Liu, Nan; Hao, Juan; Cai, Keying; Zeng, Mulan; Huang, Zhenzhong; Chen, Lili; Peng, Bingxian; Li, Ping; Wang, Li; Song, Yonghai

2018-02-01

A novel ratiometric fluorescence nanosensor for superoxide anion (O 2 •- ) detection was designed with gold nanoparticles-bovine serum albumin (AuNPs-BSA)@terbium/guanosine monophosphate disodium (Tb/GMP) nanoscale coordination polymers (NCPs) (AuNPs-BSA@Tb/GMP NCPs). The abundant hydroxyl and amino groups of AuNPs-BSA acted as binding points for the self-assembly of Tb 3+ and GMP to form core-shell AuNPs-BSA@Tb/GMP NCP nanosensors. The obtained probe exhibited the characteristic fluorescence emission of both AuNPs-BSA and Tb/GMP NCPs. The AuNPs-BSA not only acted as a template to accelerate the growth of Tb/GMP NCPs, but also could be used as the reference fluorescence for the detection of O 2 •- . The resulting AuNPs-BSA@Tb/GMP NCP ratiometric fluorescence nanosensor for the detection of O 2 •- demonstrated high sensitivity and selectivity with a wide linear response range (14 nM-10 μM) and a low detection limit (4.7 nM). Copyright © 2017 John Wiley & Sons, Ltd.

Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Wang; H Robinson; H Ke

2011-12-31

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

The effect of an East Pacific Rise offset on the formation of secondary cracks ahead of the Cocos-Nazca Rift at the Galapagos Triple Junction

NASA Astrophysics Data System (ADS)

Smith, D. K.; Montesi, L. G.; Schouten, H.; Zhu, W.

2011-12-01

A succession of short-lived, E-W trending cracks at the Galapagos Triple Junction north and south of the Cocos-Nazca (C-N) Rift, has been explained by a simple crack interaction model. The locations of where the cracks initiate are controlled by tensile stresses generated at the East Pacific Rise (EPR) by two interacting cracks: One representing the north-south trending EPR, and the other the large, westward propagating C-N Rift, whose tip is separated from the EPR by a distance D. The model predicts symmetric cracking at the EPR north and south of the C-N Rift tip. Symmetry in the distribution of cracks north and south of the C-N Rift is observed and especially remarkable between 2.5 and 1.5 Ma when the rapid jumping of cracks toward the C-N Rift appears synchronous. The rapid jumping can be explained by decreasing D, which means that the tip of the C-N Rift was moving closer to the EPR. Symmetry of cracking breaks down at 1.5 Ma, however, with the establishment of the Dietz Deep Rift, the southern boundary of the Galapagos microplate. Symmetry of cracking also breaks down on older crust to the east between about 100 35'W and 100 45'W (about 2.6 Ma) where a rapid jumping of cracks toward the C-N Rift is observed in the south cracking region. There is no evidence of similar rapid jumping in the north cracking region. It could be simply that the response to changing the value of D is not always as predicted. It could also be that the shape of the EPR has not always been symmetric about the C-N Rift, as assumed in the model. Currently, an overlapping spreading center with a 15 km east-west offset between the limbs of the EPR has formed at 1 50'N. We assess the importance of the geometry of the EPR on the crack interaction model. The model has been modified to include a ridge offset similar to what is observed today. We find that the region of stress enhancement at the EPR (where cracks initiate) is subdued south of the C-N Rift tip because of the EPR offset. It is

Adapted to change: Low energy requirements in a low and unpredictable productivity environment, the case of the Galapagos sea lion

NASA Astrophysics Data System (ADS)

Villegas-Amtmann, Stella; McDonald, Birgitte I.; Páez-Rosas, Diego; Aurioles-Gamboa, David; Costa, Daniel P.

2017-06-01

The rate of energy expenditure and acquisition are fundamental components of an animals' life history. Within mammals, the otariids (sea lions and fur seals) exhibit energetically expensive life styles, which can be challenging in equatorial regions where resources are particularly limited and unpredictable. To better understand how this energetically expensive life history pattern functions in an energetically challenging equatorial system, we concurrently measured the field metabolic rate (FMR) and foraging behavior of lactating Galapagos sea lions (GSL) rearing pups and yearlings. Females with pups tended to forage to the north, diving deeper, epi and mesopelagically compared to females with yearlings, which foraged to the west and performed dives to the sea bed that were shallower. FMR did not differ between females with pups or yearlings but, increased significantly with % time spent at-sea. Females with yearlings had higher water influx, suggesting greater food intake, but had lower body condition. The FMR (4.08±0.6 W/kg) of GSL is the lowest measured for any otariid, but is consistent with Galapagos fur seals which also exhibit low FMR. The observation that these two otariids have reduced energy requirements is consistent with an adaptation to the reduced prey availability of the Galapagos marine environment compared to other more productive marine systems.

Desensitization of atriopeptin stimulated accumulation and extrusion of cyclic GMP from a kidney epithelial cell line (MDCK).

PubMed

Woods, M; Houslay, M D

1991-02-01

Atriopeptin caused dose- (EC50 ca. 2 x 10(-8) M) and time-dependent increases in the intracellular concentration of cyclic GMP in the MDCK kidney epithelial cell line; an effect potentiated by the phosphodiesterase inhibitor, IBMX. The atriopeptin-catalysed increase in cyclic GMP was transient and reached a maximum some 10-20 min after challenge of cells with atriopeptin. The basis for the transience of this increase was shown to be due to the desensitization of guanylate cyclase coupled with extrusion of cyclic GMP from the cells and the degradation of cyclic GMP by phosphodiesterase activity. Atriopeptin-catalysed extrusion of cyclic GMP was time- and dose-(EC50 ca. 1.5 x 10(-8) M) dependent and was inhibited by probenecid but not by high external cyclic GMP concentrations. The extrusion process underwent apparent desensitization as did guanylate cyclase with similar half lives (T1/2 of ca. 20 min). Desensitization was dose-dependent upon atriopeptin and did not appear to be mediated by elevated cyclic GMP concentrations as pre-incubation with 8-bromo cyclic GMP did not cause desensitization and the half-times for desensitization were similar whether or not IBMX was present. The majority of the cyclic nucleotide phosphodiesterase activity was found in the cytosol fraction of the cells and could be separated into two cyclic AMP specific forms and two cyclic GMP preferring forms.

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

PubMed

Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

2002-11-01

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

An atypical CNG channel activated by a single cGMP molecule controls sperm chemotaxis.

PubMed

Bönigk, Wolfgang; Loogen, Astrid; Seifert, Reinhard; Kashikar, Nachiket; Klemm, Clementine; Krause, Eberhard; Hagen, Volker; Kremmer, Elisabeth; Strünker, Timo; Kaupp, U Benjamin

2009-10-27

Sperm of the sea urchin Arbacia punctulata can respond to a single molecule of chemoattractant released by an egg. The mechanism underlying this extreme sensitivity is unknown. Crucial signaling events in the response of A. punctulata sperm to chemoattractant include the rapid synthesis of the intracellular messenger guanosine 3',5'-monophosphate (cGMP) and the ensuing membrane hyperpolarization that results from the opening of potassium-selective cyclic nucleotide-gated (CNGK) channels. Here, we use calibrated photolysis of caged cGMP to show that approximately 45 cGMP molecules are generated during the response to a single molecule of chemoattractant. The CNGK channel can respond to such small cGMP changes because it is exquisitely sensitive to cGMP and activated in a noncooperative fashion. Like voltage-activated Ca(v) and Na(v) channels, the CNGK polypeptide consists of four homologous repeat sequences. Disabling each of the four cyclic nucleotide-binding sites through mutagenesis revealed that binding of a single cGMP molecule to repeat 3 is necessary and sufficient to activate the CNGK channel. Thus, CNGK has developed a mechanism of activation that is different from the activation of other CNG channels, which requires the cooperative binding of several ligands and operates in the micromolar rather than the nanomolar range.

Preventing Establishment: An Inventory of Introduced Plants in Puerto Villamil, Isabela Island, Galapagos

PubMed Central

Guézou, Anne; Pozo, Paola; Buddenhagen, Christopher

2007-01-01

As part of an island-wide project to identify and eradicate potentially invasive plant species before they become established, a program of inventories is being carried out in the urban and agricultural zones of the four inhabited islands in Galapagos. This study reports the results of the inventory from Puerto Villamil, a coastal village representing the urban zone of Isabela Island. We visited all 1193 village properties to record the presence of the introduced plants. In addition, information was collected from half of the properties to determine evidence for potential invasiveness of the plant species. We recorded 261 vascular taxa, 13 of which were new records for Galapagos. Most of the species were intentionally grown (cultivated) (73.3%) and used principally as ornamentals. The most frequent taxa we encountered were Cocos nucifera (coconut tree) (22.1%) as a cultivated plant and Paspalum vaginatum (salt water couch) (13.2%) as a non cultivated plant. In addition 39 taxa were naturalized. On the basis of the invasiveness study, we recommend five species for eradication (Abutilon dianthum, Datura inoxia, Datura metel, Senna alata and Solanum capsicoides), one species for hybridization studies (Opuntia ficus-indica) and three species for control (Furcraea hexapetala, Leucaena leucocephala and Paspalum vaginatum). PMID:17940606

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

PubMed Central

Jackson, Colin R.; Tyler, Heather L.; Millar, Justin J.

2013-01-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample. PMID:24121617

Determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays.

PubMed

Jackson, Colin R; Tyler, Heather L; Millar, Justin J

2013-10-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism

NASA Astrophysics Data System (ADS)

Cai, Weiming; Hu, Liwei; Hu, Xiangyang; Cui, Dayong; Cai, Weiming

Gravitropism is the asymmetric growth or curvature of plant organs in response to gravistimulation. There is a complex signal transduction cascade which involved in the differential growth of plants in response to changes in the gravity vector. The role of auxin in gravitropism has been demonstrated by many experiments, but little is known regarding the molecular details of such effects. In our studies before, mediation of the gravitropic bending of soybean roots and rice leaf sheath bases by nitric oxide, cGMP and gibberellins, are induced by auxin. The asymmetrical distribution of nitric oxide, cGMP and gibberellins resulted from the asymmetrical synthesis of them in bending sites. In soybean roots, inhibitions of NO and cGMP synthesis reduced differential NO and cGMP accumulation respectively, which both of these effects can lead to the reduction of gravitropic bending. Gibberellin-induced OsXET, OsEXPA4 and OsRWC3 were also found involved in the gravitropic bending. These data indicated that auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism. More experiments need to prove the more detailed mechanism of them.

Feeding Habits of Introduced Black Rats, Rattus rattus, in Nesting Colonies of Galapagos Petrel on San Cristóbal Island, Galapagos

PubMed Central

Riofrío-Lazo, Marjorie; Páez-Rosas, Diego

2015-01-01

Introduced rodents are responsible for ecosystem changes in islands around the world. In the Galapagos archipelago, their effects on the native flora and fauna are adverse, including the extinction of endemic rodents in some islands and the reduction in the reproductive success of the Galapagos petrel (Pterodroma phaeopygia) in its nesting zones. Understanding the feeding behavior of introduced rodents and their trophic interactions with native and non-native species on islands, can assist in the design of management strategies and conservation plans of invasive and endemic species respectively. Four petrel nesting colonies were monitored during June 2013 on San Cristóbal Island (El Plátano, El Junco, San Joaquín, and La Comuna). The feeding habits of black rats were evaluated by analyzing stomach contents and stable isotopes in hair. Three species of introduced rodents were captured. R. rattus was the most abundant at all sites (n=43, capture success (CS) = 55.8%), followed by the house mouse, Mus musculus (n = 17, CS = 37.8%), and the Norwegian rat, R. norvegicus (n = 4, CS = 4.5%), captured only at La Comuna. The omnivorous black rat ate mostly plants (98%) and arthropods (2%). Intact seeds of Miconia robinsoniana were the main food at all sites (relative abundance=72.1%, present in 95% of the analyzed stomachs), showing the black rats’ possible role in the archipelago as endemic seed dispersers. There was no evidence of petrel’s intake; however, its possible consumption is not discarded at all. The δ15N and δ13C analysis corroborated the primarily herbivorous diet of black rats. The isotopic signatures of the three rodent species reflect the inter- and intra-specific differential use of food resources. Black rat showed a wider diet in La Comuna, which was related to a lower availability of its primary prey and its ability to adapt to the available resources in its habitat. PMID:25984724

Gating by Cyclic Gmp and Voltage in the α Subunit of the Cyclic Gmp–Gated Channel from Rod Photoreceptors

PubMed Central

Benndorf, Klaus; Koopmann, Rolf; Eismann, Elisabeth; Kaupp, U. Benjamin

1999-01-01

Gating by cGMP and voltage of the α subunit of the cGMP-gated channel from rod photoreceptor was examined with a patch-clamp technique. The channels were expressed in Xenopus oocytes. At low [cGMP] (<20 μM), the current displayed strong outward rectification. At low and high (700 μM) [cGMP], the channel activity was dominated by only one conductance level. Therefore, the outward rectification at low [cGMP] results solely from an increase in the open probability, P o. Kinetic analysis of single-channel openings revealed two exponential distributions. At low [cGMP], the larger P o at positive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, depolarizing voltage steps, starting from −100 mV, generated a time-dependent current that increased with the step size (activation). At low [cGMP] (20 μM), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective changes were small and fast. The dose–response relation at −100 mV was shifted to the right and saturated at significantly lower P o values with respect to that at +100 mV (0.77 vs. 0.96). P o was determined as function of the [cGMP] (at +100 and −100 mV) and voltage (at 20, 70, and 700 μM, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation time course was monoexponential, which allowed us to determine the individual rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is concluded that an allosteric model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric reaction, describes the cGMP- and voltage-dependent gating of cGMP-gated channels

75 FR 17756 - Availability of the Final General Management Plan and Environmental Impact Statement (GMP/EIS...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-07

... and Environmental Impact Statement (GMP/EIS) for Chattahoochee River National Recreation Area (CRNRA...) announces the availability of a Final GMP/EIS for the CRNRA, Georgia. Consistent with NPS laws, regulations, and policies, and the purpose of the CRNRA, the Final GMP/EIS describes Alternative F at the NPS...

The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

PubMed

Khan, Mike; Harms, Jerome S; Marim, Fernanda M; Armon, Leah; Hall, Cherisse L; Liu, Yi-Ping; Banai, Menachem; Oliveira, Sergio C; Splitter, Gary A; Smith, Judith A

2016-12-01

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

PubMed

Cohen-Haguenauer, O; Creff, N; Cruz, P; Tunc, C; Aïuti, A; Baum, C; Bosch, F; Blomberg, P; Cichutek, K; Collins, M; Danos, O; Dehaut, F; Federspiel, M; Galun, E; Garritsen, H; Hauser, H; Hildebrandt, M; Klatzmann, D; Merten, O W; Montini, E; O'Brien, T; Panet, A; Rasooly, L; Scherman, D; Schmidt, M; Schweitzer, M; Tiberghien, P; Vandendriessche, T; Ziehr, H; Ylä-Herttuala, S; von Kalle, C; Gahrton, G; Carrondo, M

2010-12-01

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

PubMed

Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

2018-06-01

Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

An automated image-collection system for crystallization experiments using SBS standard microplates.

PubMed

Brostromer, Erik; Nan, Jie; Su, Xiao Dong

2007-02-01

As part of a structural genomics platform in a university laboratory, a low-cost in-house-developed automated imaging system for SBS microplate experiments has been designed and constructed. The imaging system can scan a microplate in 2-6 min for a 96-well plate depending on the plate layout and scanning options. A web-based crystallization database system has been developed, enabling users to follow their crystallization experiments from a web browser. As the system has been designed and built by students and crystallographers using commercially available parts, this report is aimed to serve as a do-it-yourself example for laboratory robotics.

Structural Insight into the Mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tchigvintsev, A.; Xu, X.; Singer, A.

2010-08-01

Cyclic diguanylate (or bis-(3'-5') cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 {angstrom}) and in complex with c-di-GMP (2.35 {angstrom}), andmore » unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.« less

A minimalist biosensor: Quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer.

PubMed

Kellenberger, Colleen A; Sales-Lee, Jade; Pan, Yuchen; Gassaway, Madalee M; Herr, Amy E; Hammond, Ming C

2015-01-01

Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.

Cryoalgotox: Use of cryopreserved alga in a semistatic microplate test

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benhra, A.; Radetski, C.M.; Ferard, J.F.

1997-03-01

Use of cryopreserved alga Selenastrum capricornutum has been evaluated as a simple and cost-efficient procedure in a new semistatic algal ecotoxicity test. Experiments have been conducted to compare performance criteria of this method, named Cryoalgotox, versus the classic microplate test using fresh algae. Cryoalgotox 72-h 50% effective concentrations (EC50s) determined with Cd{sup 2+}, Cu{sup 2+}, Cr{sup 6+}, and atrazine were more sensitive, repeatable (low coefficients of variation), and reproducible (low time effect) than the results obtained with the classical microplate tests. The effect of storage time at {minus}80 C on the sensitivity of the algae was assessed using cadmium asmore » a toxic reference; it was shown that algae stored at {minus}80 C over a 3-month period gave comparable toxicity results to those found with fresh algae.« less

A microplate reader-based method to quantify NADH-cytochrome b5 reductase activity for diagnosis of recessive congenital methaemoglobinemia.

PubMed

Kedar, Prabhakar; Desai, Anand; Warang, Prashant; Colah, Roshan

2017-05-01

Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiencies is an autosomal recessive disorder that occurs sporadically worldwide, A sensitive, accurate, and rapid analysis of NADH-CYB5R enzyme concentrations is necessary for the diagnosis of RCM. Here we present an alternative microplate method that is based on a standard 96-well microplate format and microplate reader that simplify the quantification of NADH-CYB5R activity. TECAN (Infinite 200 PRO series) microplate reader with Tecan's proven Magellan™ software measured the NADH-CYB5R enzyme activity in 250 normal controls and previously diagnosed 25 cases of RCM due to NADH-CYB5R deficiency in the Indian population using 96-well microplates using 200 μl of total reaction mixture and also compared with standard spectrophotometric assay. We have also studied stability of the hemolysate stored at 4 and -20°C temperature. Enzyme activity in all 25 samples ranged from 6.09 to 10.07 IU/g Hb (mean ± SD: 8.08 ± 1.99 IU/g Hb) where as normal control ranged (n = 250) between 13.42 and 21.58 IU/g Hb) (mean ± SD: 17.5 ± 4.08 IU/g of Hb). Data obtained from the microplate reader were compared with standard spectrophotometer method and found 100% concordance using both methods. Microplate method allows differentiating between normal, deficient and intermediate enzyme activity. It was observed that samples had significant loss of activity when stored at 4°C and retained stable activity at -20°C for 1 week time. Our new method, incorporating a whole process of enzyme assay into a microplate format is readily applicable and allows rapid monitoring of enzyme assay. It is readily applicable to quantitative assay on pediatric sample as well as large number of samples for population screening.

Chemical and isotopic diversity in basalts dredged from the East Pacific Rise at 10°S, the fossil Galapagos Rise and the Nazca plate

USGS Publications Warehouse

Batiza, Rodey; Oestrike, Richard; Futa, Kiyoto

1982-01-01

The dredges from the East Pacific Rise at about 10°S recovered unusual transitional, light rare-earth element (LREE) enriched basalts which show a range of fractionation. On the basis of their chemical and isotopic abundances, it is unlikely that the lavas are related by a single simple process of magmatic differentiation. We suggest that the mantle source region of these basalts was chemically and isotopically heterogeneous. The chemistry of LREE-depleted tholeiitic basalt dredged from near the axis of the extinct Galapagos Rise indicates complex petrogenesis and differentiation. The presence of tholeiitic basalts here indicates that unlike the Guadalupe and Mathematician fossil ridges, the Galapagos Rise has not been the site of voluminous post-abandonment alkalic volcanism. Alkalic basalts of picritic bulk composition dredged from an elongate seamount near the Galapagos Rise do not represent liquid compositions. Instead, we suggest that these alkalic liquids contain added olivine and plagioclase xenocrysts. Although most of the samples analyzed are very fresh, a few have been altered. The latter exhibit characteristic chemical and isotopic effects of seawater alteration.

Functional and radiologic outcome of open reduction and internal fixation of condylar head and neck fractures using miniplate or microplate system.

PubMed

Xie, Si-Tian; Singhal, Dhruv; Chen, Chien-Tzung; Chen, Yu-Ray

2013-12-01

Although the appropriate management of condylar process fractures after miniplate or microplate fixation has been described, there has been no comparative analysis of these plating systems. A retrospective review of patients who underwent open reduction and internal fixation (ORIF) of condylar head or neck fractures at our institution from January 2000 through August 2010 identified 70 patients. Of these, 38 were treated with microplates and 32 with miniplates. The primary functional and radiographic results were the maximal mouth opening and condylar bone resorption, respectively. The rates of complications, including malocclusion, chin deviation, temporomandibular joint complaints, and facial nerve palsy, were recorded. The maximal mouth opening was larger in the microplate group than in the miniplate group throughout the follow-up period; this difference was statistically significant 12 (P = 0.020), 18 (P = 0.026), and 24 (P = 0.032) months after ORIF. Similarly, the radiographic scores for bone resorption and condyle morphology were significantly better in the microplate group than in the miniplate group throughout the follow-up period [6 (P = 0.011), 12 (P = 0.035), 24 (P = 0.026), and 48 (P = 0.040) months after ORIF]. Moreover, patients who underwent miniplate fixation experienced a significantly higher incidence of temporomandibular joint click than those who underwent microplate fixation (P = 0.014). Microplates limit dissection, providing excellent fixation for intracapsular condylar head fractures, and also provide adequate rigidity for fixation of condylar neck fractures. Microplate fixation of condylar head and neck fractures yielded excellent functional and radiographic results. The rates of complications after microplate fixation were equal to or less than those in the miniplate group. Prospective studies are needed to confirm these findings.

Cross regulation between cGMP-dependent protein kinase and Akt in vasodilatation of porcine pulmonary artery.

PubMed

Liu, Juan; Liu, Huixia; Li, Yanjing; Xu, Xiaojian; Chen, Zhengju; Liu, Limei; Yu, Xiaoxing; Gao, Yuansheng; Dou, Dou

2014-11-01

cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Magnetization measurements of Sr2RuO4-Ru eutectic microplates using dc-SQUIDs

NASA Astrophysics Data System (ADS)

Nago, Y.; Sakuma, D.; Ishiguro, R.; Kashiwaya, S.; Nomura, S.; Kono, K.; Maeno, Y.; Takayanagi, H.

2018-03-01

We report magnetization measurements of Sr2RuO4-Ru eutectic microplates using micro-dc-SQUIDs. Sr2RuO4 is considered as a chiral p-wave superconductor and hence Sr2RuO4-Ru eutectic becomes in an unstable state with a superconducting phase frustration between a chiral p-wave state of Sr2RuO4 and a s-wave state of Ru. To compensate the frustration, a single quantum vortex is spontaneously formed at the center of the Ru inclusion at sufficiently low temperatures. However, such a spontaneous vortex state has not been experimentally observed yet. In this study, we prepared a micro-dc-SQUID and a Sr2RuO4-Ru eutectic microplate containing a single Ru-inclusion at the center of the microplate. We performed magnetization measurements down below the superconducting transition temperature of the Ru inclusion to investigate the spontaneous Ru-center vortex state.

Platelet aggregation inhibitors from Philippine marine invertebrate samples screened in a new microplate assay.

PubMed

Pimentel, Sheila Marie V; Bojo, Zenaida P; Roberto, Amy V D; Lazaro, Jose Enrico H; Mangalindan, Gina C; Florentino, Leila M; Lim-Navarro, Pilar; Tasdemir, Deniz; Ireland, Chris M; Concepcion, Gisela P

2003-01-01

A new microplate assay for Ca(2+)-induced platelet aggregation as detected by Giemsa dye was used to screen marine invertebrate samples from the Philippines for inhibitors of human platelet aggregation. Out of 261 crude methanol extracts of marine sponges and tunicates, 25 inhibited aggregation at 2 mg/ml. Inhibition of agonist-induced aggregation in an aggregometer was used to confirm results of the microplate assay and to determine the specific mode of inhibition of 2 samples. The marine sponge Xestospongia sp. yielded a xestospongin/araguspongine-type molecule that inhibited collagen-induced aggregation by 87% at 2 micro g/ml, and epinephrine-induced aggregation by 78% at 20 micro g/ml, while the marine sponge Aplysina sp. yielded 5,6-dibromotryptamine, which inhibited epinephrine-induced aggregation by 51% at 20 micro g/ml. In this study we have found that the microplate assay is a simple, inexpensive, yet useful preliminary tool to qualitatively screen a large number of marine samples for antiplatelet aggregation activity.

White - cGMP Interaction Promotes Fast Locomotor Recovery from Anoxia in Adult Drosophila

PubMed Central

2017-01-01

Increasing evidence indicates that the white (w) gene in Drosophila possesses extra-retinal functions in addition to its classical role in eye pigmentation. We have previously shown that w+ promotes fast and consistent locomotor recovery from anoxia, but how w+ modulates locomotor recovery is largely unknown. Here we show that in the absence of w+, several PDE mutants, especially cyclic guanosine monophosphate (cGMP)-specific PDE mutants, display wildtype-like fast locomotor recovery from anoxia, and that during the night time, locomotor recovery was light-sensitive in white-eyed mutant w1118, and light-insensitive in PDE mutants under w1118 background. Data indicate the involvement of cGMP in the modulation of recovery timing and presumably, light-evoked cGMP fluctuation is associated with light sensitivity of locomotor recovery. This was further supported by the observations that w-RNAi-induced delay of locomotor recovery was completely eliminated by upregulation of cGMP through multiple approaches, including PDE mutation, simultaneous overexpression of an atypical soluble guanylyl cyclase Gyc88E, or sildenafil feeding. Lastly, prolonged sildenafil feeding promoted fast locomotor recovery from anoxia in w1118. Taken together, these data suggest that a White-cGMP interaction modulates the timing of locomotor recovery from anoxia. PMID:28060942

Low Temperature Extends the Lifespan of Bursaphelenchus xylophilus through the cGMP Pathway

PubMed Central

Wang, Bowen; Ma, Ling; Wang, Feng; Wang, Buyong; Hao, Xin; Xu, Jiayao; Ma, Yan

2017-01-01

The causal agent of pine wilt disease, pine wood nematode (PWN) (Bursaphelenchus xylophilus), revealed extended lifespan at low temperature. To discover the molecular mechanism of this phenomenon, we attempted to study the molecular characterization, transcript abundance, and functions of three genes of the cyclic guanosine monophosphate (cGMP) pathway from B. xylophilus. Three cGMP pathway genes were identified from B. xylophilus. Bioinformatic software was utilized to analyze the characteristics of the three putative proteins. Function of the three genes in cold tolerance was studied with RNA interference (RNAi). The results showed that the deduced protein of Bx-DAF-11 has an adenylate and guanylate cyclase catalytic domain, indicating an ability to bind to extracellular ligands and synthesizing cGMP. Both Bx-TAX-2 and Bx-TAX-4 have cyclic nucleotide-binding domains and ion transport protein domains, illustrating that they are cGMP-gated ion channels. The transcript level of Bx-daf-11, Bx-tax-2, and Bx-tax-4 increased at low temperature. The survival rates of three gene silenced B. xylophilus revealed a significant decrease at low temperature. This study illustrated that the cGMP pathway plays a key role in low-temperature-induced lifespan extension in B. xylophilus. PMID:29099744

Influence of electrical double-layer dispersion forces and size dependency on pull-in instability of clamped microplate immersed in ionic liquid electrolytes

NASA Astrophysics Data System (ADS)

Karimipour, I.; Beni, Yaghoub Tadi; Taheri, N.

2017-10-01

Plate-type clamped microplate is of the most common constructive elements for developing in-liquid-operating devices. While the electromechanical behavior of clamped microplate in non-liquid environments has exclusively been addressed in the literature, no theoretical studies have yet been conducted on precise modeling of the clamped microplate in electrolyte liquid. Herein, the electromechanical response and instability of the clamped microplate immersed in ionic electrolyte media are investigated. The electrochemical force field is determined using double layer theory and linearized Poisson-Boltzmann equation. The presence of dispersion forces, i.e., Casimir and van der Waals attractions, are included in the theoretical model considering the correction due to the presence of liquid media between the interacting surfaces (three-layer model). To this end, a kind of microplate has been designed, i.e., a square microplate with all edges clamped supported. The strain gradient elasticity is employed to model the size-dependent structural behavior of the clamped microplate. To solve the nonlinear constitutive equation of the system, Extended Kantorovich Method, is employed and the pull-in parameter of the microplate are extracted. Impacts of the dispersion forces and size effect on the instability characteristics are discussed as well as the effect of ion concentration and potential ratio. It is found that the significant difference between the pull-in instability parameters in the modified strain gradient theory and the classical theory for thin microplates is merely due to the consideration of size effect parameter in the modified strain gradient theory. To confirm the validity of formulations, the numerical values of the results are compared. The results predicted via the aforementioned approach are in excellent agreement with those in the literature. Some new examples are solved to demonstrate the applicability of the procedure.

Stable Isotopes Reveal Long-Term Fidelity to Foraging Grounds in the Galapagos Sea Lion (Zalophus wollebaeki)

PubMed Central

Drago, Massimiliano; Franco-Trecu, Valentina; Cardona, Luis; Inchausti, Pablo; Tapia, Washington; Páez-Rosas, Diego

2016-01-01

Most otariids have colony-specific foraging areas during the breeding season, when they behave as central place foragers. However, they may disperse over broad areas after the breeding season and individuals from different colonies may share foraging grounds at that time. Here, stable isotope ratios in the skull bone of adult Galapagos sea lions (Zalophus wollebaeki) were used to assess the long-term fidelity of both sexes to foraging grounds across the different regions of the Galapagos archipelago. Results indicated that the stable isotope ratios (δ13C and δ15N) of sea lion bone significantly differed among regions of the archipelago, without any significant difference between sexes and with a non significant interaction between sex and region. Moreover, standard ellipses, estimated by Bayesian inference and used as a measure of the isotopic resource use area at the population level, overlapped widely for the sea lions from the southern and central regions, whereas the overlap of the ellipses for sea lions from the central and western regions was small and non-existing for those from the western and southern regions. These results suggest that males and females from the same region within the archipelago use similar foraging grounds and have similar diets. Furthermore, they indicate that the exchange of adults between regions is limited, thus revealing a certain degree of foraging philopatry at a regional scale within the archipelago. The constraints imposed on males by an expanded reproductive season (~ 6 months), resulting from the weak reproductive synchrony among females, and those imposed on females by a very long lactation period (at least one year but up to three years), may explain the limited mobility of adult Galapagos sea lions of both sexes across the archipelago. PMID:26808381

Involvement of the cGMP pathway in the osthole-facilitated glutamate release in rat hippocampal nerve endings.

PubMed

Lin, Tzu Yu; Lu, Cheng Wei; Huang, Wei-Jan; Wang, Su-Jane

2012-03-01

Osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has previously been shown to have the capacity to increase depolarization-evoked glutamate release in rat hippocampal nerve terminals. As cGMP-dependent signaling cascade has been found to modulate glutamate release at the presynaptic level, the aim of this study was to further examine the role of cGMP signaling pathway in the regulation of osthole on glutamate release in hippocampal synaptosomes. Results showed that osthole dose-dependently increased intrasynaptosomal cGMP levels. The elevation of cGMP levels by osthole was prevented by the phosphodiesterase 5 inhibitor sildenafil but was insensitive to the guanylyl cyclase inhibitor ODQ. In addition, osthole-induced facilitation of 4-aminopyridine (4-AP)-evoked glutamate release was completely prevented by the cGMP-dependent protein kinase (PKG) inhibitors, KT5823, and Rp-8-Br-PET-cGMPS. Direct activation of PKG with 8-Br-cGMP or 8-pCPT-cGMP also occluded the osthole-mediated facilitation of 4-AP-evoked glutamate release. Furthermore, sildenafil exhibited a dose-dependent facilitation of 4-AP-evoked release of glutamate and occluded the effect of osthole on the 4-AP-evoked glutamate release. Collectively, our findings suggest that osthole-mediated facilitation of glutamate release involves the activation of cGMP/PKG-dependent pathway. Copyright © 2011 Wiley Periodicals, Inc.

Philopatry drives genetic differentiation in an island archipelago: comparative population genetics of Galapagos Nazca boobies (Sula granti) and great frigatebirds (Fregata minor)

PubMed Central

Levin, Iris I; Parker, Patricia G

2012-01-01

Seabirds are considered highly mobile, able to fly great distances with few apparent barriers to dispersal. However, it is often the case that seabird populations exhibit strong population genetic structure despite their potential vagility. Here we show that Galapagos Nazca booby (Sula granti) populations are substantially differentiated, even within the small geographic scale of this archipelago. On the other hand, Galapagos great frigatebird (Fregata minor) populations do not show any genetic structure. We characterized the genetic differentiation by sampling five colonies of both species in the Galapagos archipelago and analyzing eight microsatellite loci and three mitochondrial genes. Using an F-statistic approach on the multilocus data, we found significant differentiation between nearly all island pairs of Nazca booby populations and a Bayesian clustering analysis provided support for three distinct genetic clusters. Mitochondrial DNA showed less differentiation of Nazca booby colonies; only Nazca boobies from the island of Darwin were significantly differentiated from individuals throughout the rest of the archipelago. Great frigatebird populations showed little to no evidence for genetic differentiation at the same scale. Only two island pairs (Darwin – Wolf, N. Seymour – Wolf) were significantly differentiated using the multilocus data, and only two island pairs had statistically significant φST values (N. Seymour – Darwin, N. Seymour – Wolf) according to the mitochondrial data. There was no significant pattern of isolation by distance for either species calculated using both markers. Seven of the ten Nazca booby migration rates calculated between island pairs were in the south or southeast to north or northwest direction. The population differentiation found among Galapagos Nazca booby colonies, but not great frigatebird colonies, is most likely due to differences in natal and breeding philopatry. PMID:23170212

Cyclic GMP-dependent protein kinase-I in the guinea pig cochlea.

PubMed

Tian, F; Fessenden, J D; Schacht, J

1999-05-01

Recent studies have begun to characterize the nitric oxide/cyclic GMP/protein kinase G pathway in the mammalian cochlea by demonstrating the presence of both the enzyme that produces nitric oxide (NO), nitric oxide synthase, and the NO receptor, soluble guanylate cyclase. The present study investigated protein kinase G (cyclic GMP-dependent protein kinase-I, cGK-I), the downstream enzyme of this pathway that frequently mediates its physiological effects. A commercial antibody to a human cGK-I sequence recognized a protein of appropriate molecular weight in Western blots of guinea pig aorta. Immunostaining of guinea pig aorta was consistent with the expected distribution of cGK-I. In lateral wall tissues of the cochlea, pericytes lining the blood vessels of the spiral ligament were strongly immunoreactive. In the organ of Corti, cGK-I was detected in Hensen's, Deiters', and pillar cells, but not in inner and outer hair cells. This distribution coincides with the localization of soluble guanylate cyclase activity and suggests that cGK-I mediates the effects of the NO/cyclic GMP pathway in the cochlea. It reinforces the hypothesis that the NO/cyclic GMP/cGK-I pathway is involved in regulation of cochlear blood flow and supporting cell physiology.

Atrial natriuretic peptide receptor heterogeneity and effects on cyclic GMP accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leitman, D.C.

1988-01-01

The effects of atrial natriuretic peptide (ANP), oxytocin (OT) and vasopressin (AVP) on guanylate cyclase activity and cyclic GMP accumulation were examined, since these hormones appear to be intimately associated with blood pressure and intravascular volume homeostasis. ANP was found to increase cyclic GMP accumulation in ten cell culture systems, which were derived from blood vessels, adrenal cortex, kidney, lung, testes and mammary gland. ANP receptors were characterized in intact cultured cells using {sup 125}I-ANP{sub 8-33}. Specific {sup 125}I-ANP binding was saturable and of high affinity. Scratchard analysis of the binding data for all cell types exhibited a straight line,more » indicating that these cells possessed a single class of binding sites. Despite the presence of linear Scatchard plots, these studies demonstrated that cultured cells possess two functionally and physically distinct ANP-binding sites. Most of the ANP-binding sites in cultured cells have a molecular size of 66,000 daltons under reducing conditions. The identification of cultured cell types in which hormones (ANP and oxytocin) regulate guanylate cyclase activity and increase cyclic GMP synthesis will provide valuable systems to determine the mechanisms of hormone-receptor coupling to guanylate cyclase and the cellular processes regulated by cyclic GMP.« less

Fluorescein Diacetate Microplate Assay in Cell Viability Detection.

PubMed

Chen, Xi; Yang, Xiu-Ying; Fang, Lian-Hua; DU, Guan-Hua

2016-12-20

Objective To investigate the application of the fluorescein diacetate (FDA) microplate assay in cell viability detection. Methods Cells were seeded in a 96-well culture plate until detection. After incubated with FDA,the plate was detected by fluorescence microplate analyzer. The effects of FDA incubation duration,concentration,and other factors on the assay's accuracy and stability were assessed. We also compared the results of FDA with methyl thiazolyl(MTT) in terms of cell numbers and H 2 O 2 injury. Results Within 0-30 minutes,the fluorescence-cell number coefficient of FDA assay increased with duration and reached 0.99 in 27-30 minutes. The optimum concentration of final FDA in this study was 10-30 μg/ml. On cell viability detection,the result of FDA method was equivalent to MTT method. As to H 2 O 2 injury assay,the sensitivity of FDA method was superior to MTT on the higher concentration H 2 O 2 treatment due to a relative shorter duration for detection. Conclusion As a stable and reliable method,FDA is feasible for cell variability detection under varied conditions.

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

PubMed Central

Tilley, Derek; Levit, Irina; Samis, John A.

2012-01-01

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre

Measurement of factor v activity in human plasma using a microplate coagulation assay.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2012-09-09

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging

PubMed Central

Bhargava, Yogesh; Hampden-Smith, Kathryn; Chachlaki, Konstantina; Wood, Katherine C.; Vernon, Jeffrey; Allerston, Charles K.; Batchelor, Andrew M.; Garthwaite, John

2013-01-01

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations. PMID:24068983

Biofilm Formation by the Acidophile Bacterium Acidithiobacillus thiooxidans Involves c-di-GMP Pathway and Pel exopolysaccharide.

PubMed

Díaz, Mauricio; Castro, Matias; Copaja, Sylvia; Guiliani, Nicolas

2018-02-21

Acidophile bacteria belonging to the Acidithiobacillus genus are pivotal players for the bioleaching of metallic values such as copper. Cell adherence to ores and biofilm formation, mediated by the production of extracellular polymeric substances, strongly favors bioleaching activity. In recent years, the second messenger cyclic diguanylate (c-di-GMP) has emerged as a central regulator for biofilm formation in bacteria. C-di-GMP pathways have been reported in different Acidithiobacillus species; however, c-di-GMP effectors and signal transduction networks are still largely uncharacterized in these extremophile species. Here we investigated Pel exopolysaccharide and its role in biofilm formation by sulfur-oxidizing species Acidithiobacillus thiooxidans . We identified 39 open reading frames (ORFs) encoding proteins involved in c-di-GMP metabolism and signal transduction, including the c-di-GMP effector protein PelD, a structural component of the biosynthesis apparatus for Pel exopolysaccharide production. We found that intracellular c-di-GMP concentrations and transcription levels of pel genes were higher in At . thiooxidans biofilm cells compared to planktonic ones. By developing an At . thiooxidans Δ pelD null-mutant strain we revealed that Pel exopolysaccharide is involved in biofilm structure and development. Further studies are still necessary to understand how Pel biosynthesis is regulated in Acidithiobacillus species, nevertheless these results represent the first characterization of a c-di-GMP effector protein involved in biofilm formation by acidophile species.

Fast and Accurate Microplate Method (Biolog MT2) for Detection of Fusarium Fungicides Resistance/Sensitivity.

PubMed

Frąc, Magdalena; Gryta, Agata; Oszust, Karolina; Kotowicz, Natalia

2016-01-01

The need for finding fungicides against Fusarium is a key step in the chemical plant protection and using appropriate chemical agents. Existing, conventional methods of evaluation of Fusarium isolates resistance to fungicides are costly, time-consuming and potentially environmentally harmful due to usage of high amounts of potentially toxic chemicals. Therefore, the development of fast, accurate and effective detection methods for Fusarium resistance to fungicides is urgently required. MT2 microplates (Biolog(TM)) method is traditionally used for bacteria identification and the evaluation of their ability to utilize different carbon substrates. However, to the best of our knowledge, there is no reports concerning the use of this technical tool to determine fungicides resistance of the Fusarium isolates. For this reason, the objectives of this study are to develop a fast method for Fusarium resistance to fungicides detection and to validate the effectiveness approach between both traditional hole-plate and MT2 microplates assays. In presented study MT2 microplate-based assay was evaluated for potential use as an alternative resistance detection method. This was carried out using three commercially available fungicides, containing following active substances: triazoles (tebuconazole), benzimidazoles (carbendazim) and strobilurins (azoxystrobin), in six concentrations (0, 0.0005, 0.005, 0.05, 0.1, 0.2%), for nine selected Fusarium isolates. In this study, the particular concentrations of each fungicides was loaded into MT2 microplate wells. The wells were inoculated with the Fusarium mycelium suspended in PM4-IF inoculating fluid. Before inoculation the suspension was standardized for each isolates into 75% of transmittance. Traditional hole-plate method was used as a control assay. The fungicides concentrations in control method were the following: 0, 0.0005, 0.005, 0.05, 0.5, 1, 2, 5, 10, 25, and 50%. Strong relationships between MT2 microplate and traditional hole

Fast and Accurate Microplate Method (Biolog MT2) for Detection of Fusarium Fungicides Resistance/Sensitivity

PubMed Central

Frąc, Magdalena; Gryta, Agata; Oszust, Karolina; Kotowicz, Natalia

2016-01-01

The need for finding fungicides against Fusarium is a key step in the chemical plant protection and using appropriate chemical agents. Existing, conventional methods of evaluation of Fusarium isolates resistance to fungicides are costly, time-consuming and potentially environmentally harmful due to usage of high amounts of potentially toxic chemicals. Therefore, the development of fast, accurate and effective detection methods for Fusarium resistance to fungicides is urgently required. MT2 microplates (BiologTM) method is traditionally used for bacteria identification and the evaluation of their ability to utilize different carbon substrates. However, to the best of our knowledge, there is no reports concerning the use of this technical tool to determine fungicides resistance of the Fusarium isolates. For this reason, the objectives of this study are to develop a fast method for Fusarium resistance to fungicides detection and to validate the effectiveness approach between both traditional hole-plate and MT2 microplates assays. In presented study MT2 microplate-based assay was evaluated for potential use as an alternative resistance detection method. This was carried out using three commercially available fungicides, containing following active substances: triazoles (tebuconazole), benzimidazoles (carbendazim) and strobilurins (azoxystrobin), in six concentrations (0, 0.0005, 0.005, 0.05, 0.1, 0.2%), for nine selected Fusarium isolates. In this study, the particular concentrations of each fungicides was loaded into MT2 microplate wells. The wells were inoculated with the Fusarium mycelium suspended in PM4-IF inoculating fluid. Before inoculation the suspension was standardized for each isolates into 75% of transmittance. Traditional hole-plate method was used as a control assay. The fungicides concentrations in control method were the following: 0, 0.0005, 0.005, 0.05, 0.5, 1, 2, 5, 10, 25, and 50%. Strong relationships between MT2 microplate and traditional hole

[Change in soil enzymes activities after adding biochar or straw by fluorescent microplate method].

PubMed

Zhang, Yu-Lan; Chen, Li-Jun; Duan, Zheng-Hu; Wu, Zhi-Jie; Sun, Cai-Xia; Wang, Jun-Yu

2014-02-01

The present work was aimed to study soil a-glucosidase and beta-glucosidase activities of and red soils based on fluorescence detection method combined with 96 microplates with TECAN Infinite 200 Multi-Mode Microplate Reader. We added biochar or straw (2.5 g air dry sample/50g air dry soil sample) into and red soils and the test was carried under fixed temperature and humidity condition (25 degrees C, 20% soil moisture content). The results showed that straw addition enhances soil alpha-glucosidase and beta-glucosidase activities, beta-glucosidase activity stimulated by rice straw treatment was higher than that of corn straw treatment, and activity still maintains strong after 40 days, accounting for increasing soil carbon transformation with straw inputting. Straw inputting increased soil nutrients contents and may promote microbial activity, which also lead to the increase oin enzyme Straw inputting increased soil nutrients contents and may promote microbial activity, which also lead to the increase oin enzyme activities. Different effects of straw kinds may be related to material source that needs further research. However, biochar inputting has little effect on soil alpha-glucosidase and beta-glucosidase activity. Biochar contains less available nutrients than straw and have degradation-resistant characteristics. Compared with the conventional spectrophotometric method, fluorescence microplate method is more sensitive to soil enzyme activities in suspension liquid, which can be used in a large number of samples. In brief, fluorescence microplate method is fast, accurate, and simple to determine soil enzymes activities.

Npom-Protected NONOate Enables Light-Triggered NO/cGMP Signalling in Primary Vascular Smooth Muscle Cells.

PubMed

Stroppel, Anna S; Paolillo, Michael; Ziegler, Thomas; Feil, Robert; Stafforst, Thorsten

2018-06-18

Diazeniumdiolates (NONOates) are a class of nitric-oxide-releasing substances widely used in studies of NO/cGMP signalling. Because spatiotemporal control is highly desirable for such purposes, we have synthesised a new Npom-caged pyrrolidine NONOate. A kinetic analysis together with a Griess assay showed the photodependent release of NO with high quantum yield (UV light). In primary vascular smooth muscle cells (VSMCs), our compound was reliably able to induce fast increases in cGMP, as measured with a genetically encoded FRET-based cGMP sensor and further validated by the phosphorylation of the downstream target vasodilator-stimulated phosphoprotein (VASP). Thanks to their facile synthesis, good decaging kinetics and capability to activate cGMP signalling in a fast and efficient manner, Npom-protected NONOates allow for improved spatiotemporal control of NO/cGMP signalling. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Microplate luminometry for toxicity bioassay of chemicals on luciferase].

PubMed

Ge, Hui-Lin; Liu, Shu-Shen; Chen, Fu; Luo, Jin-Hui; Lü, Dai-Zhu; Su, Bing-Xia

2013-10-01

A new microplate luminometry for the toxicity bioassay of chemicals on firefly luciferase, was developed using the multifunctional microplate reader (SpectraMax M5) to measure the luminous intensity of luciferase. Efects of luciferase concentration, luciferin concentration, ATP concentration, pH, temperature, and reaction time on the luminescence were systematically investigated. It was found that ATP exerted a biphasic response on the luciferase luminescence and the maximum relative light units (RLU) occurred at an ATP concentration of 1.1 x 10(-4) mol x L(-1). The method was successfully employed in the toxic effect test of NaF, NaCl, KBr and NaBF4 on luciferase. Using nonlinear least square technique, the dose-response curves (DRC) of the 4 chemicals were accurately fitted with the coefficient of determination (R2) between the fitted and observed responses being greater than 0.99. The median effective concentration (EC50) of the 4 chemicals were accurately measured from the DRC models. Compared with some literatures, the bioassay is a fast easy-operate and cost-effective method with high accuracy.

The crucial role of cyclic GMP in the eclosion hormone mediated signal transduction in the silkworm metamorphoses.

PubMed

Shibanaka, Y; Hayashi, H; Okada, N; Fujita, N

1991-10-31

The signal transduction of the peptide, eclosion hormone, in the silkworm Bombyx mori appears to be mediated via the second messenger cyclic GMP throughout their life cycle. Injection of 8-bromo-cGMP induced the ecdysis behavior in pharate adults with similar latency to eclosion hormone-induced ecdysis; the moulting occurred 50-70 min after the injection. The potency of 8Br-cGMP was 10(2) fold higher than that of cGMP and the efficacy was increased by the co-injection of the phosphodiesterase inhibitor IBMX. On the other hand, in the silkworm pupal ecdysis the eclosion hormone and also 8Br-cGMP induced the moulting behavior in a dose-dependent manner. The adult development of the ability to respond to 8Br-cGMP took place concomitantly with the response to the eclosion hormone. Both the developmental time courses were shifted by a shift of light and dark cycles. Accordingly, the sensitivities to the peptide and cyclic nucleotide developed correspondently under the light and dark circadian rhythm. Thus throughout the silkworm life cycle, eclosion hormone is effective to trigger the ecdysis behavior and cGMP plays a crucial role as the second messenger in the eclosion hormone-mediated signal transduction.

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

PubMed

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

2012-12-04

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

PubMed Central

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

2014-01-01

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021

Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

DOE PAGES

Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; ...

2015-05-15

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

PubMed Central

Whitney, John C.; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Robinson, Howard; Ohman, Dennis E.; Howell, P. Lynne

2015-01-01

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa. PMID:25817996

Characterization of particulate cyclic nucleotide phosphodiesterases from bovine brain: Purification of a distinct cGMP-stimulated isoenzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murashima, Seiko; Tanaka, Takayuki; Hockman, S.

1990-06-05

In the absence of detergent, {approx}80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; {approx}85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity {approx}100% and calmodulin-stimulated {approx}400-500%. Although 1% Lubrol readily solubilized these PDE activities, {approx}75% of the cAMP PDE activity (0.5 {mu}M ({sup 3}H)cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide.more » Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. The brain enzyme exhibited a slightly greater subunit M{sub r} than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V{sub 8} protease produced several peptides of similar size, as well as at least two distinct fragments of {approx}27 kDa from the brain and {approx}23 kDa from the liver enzyme. After photolabeling in the presence of ({sup 32}P)cGMP and digestion with V{sub 8} protease, ({sup 32}P)cGMP in each PDE was predominantly recovered with a peptide of {approx}14 kDa. All of these observations are consistent with the existence of at least two discrete forms (isoenzymes) of cGMP-stimulated PDE.« less

75 FR 17761 - Termination of an Environmental Impact Statement (EIS) for the General Management Plan (GMP) for...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-07

... (EIS) for the General Management Plan (GMP) for Kings Mountain National Military Park (Park), South... Service (NPS) is terminating preparation of an EIS for the GMP for the Park, South Carolina. A Notice of Intent to prepare an EIS for the Park GMP was published in the Federal Register on October 10, 2006 (71...

Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

PubMed

Miner, Kyle D; Kurtz, Donald M

2016-02-16

HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

[The Contribution of GMP-grade Hospital Preparation to Translational Research].

PubMed

Yonezawa, Atsushi; Kajiwara, Moto; Minami, Ikuko; Omura, Tomohiro; Nakagawa, Shunsaku; Matsubara, Kazuo

2015-01-01

Translational research is important for applying the outcomes of basic research studies to practical medical treatments. In exploratory early-phase clinical trials for an innovative therapy, researchers should generally manufacture investigational agents by themselves. To provide investigational agents with safety and high quality in clinical studies, appropriate production management and quality control are essential. In the Department of Pharmacy of Kyoto University Hospital, a manufacturing facility for sterile drugs was established, independent of existing manufacturing facilities. Manuals on production management and quality control were developed according to Good Manufacturing Practices (GMP) for Investigational New Drugs (INDs). Advanced clinical research has been carried out using investigational agents manufactured in our facility. These achievements contribute to both the safety of patients and the reliability of clinical studies. In addition, we are able to do licensing-out of our technique for the manufacture of investigational drugs. In this symposium, we will introduce our GMP grade manufacturing facility for sterile drugs and discuss the role of GMP grade hospital preparation in translational research.

c-di-GMP enhances protective innate immunity in a murine model of pertussis.

PubMed

Elahi, Shokrollah; Van Kessel, Jill; Kiros, Tedele G; Strom, Stacy; Hayakawa, Yoshihiro; Hyodo, Mamoru; Babiuk, Lorne A; Gerdts, Volker

2014-01-01

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

PubMed Central

Elahi, Shokrollah; Van Kessel, Jill; Kiros, Tedele G.; Strom, Stacy; Hayakawa, Yoshihiro; Hyodo, Mamoru; Babiuk, Lorne A.; Gerdts, Volker

2014-01-01

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required. PMID:25333720

Surface derivatization with spacer molecules on glutaraldehyde-activated amino-microplates for covalent immobilization of β-glucosidase

NASA Astrophysics Data System (ADS)

Zhang, Yaodong; Zhang, Yun; Jiang, Juanjuan; Li, Li; Yu, Caihong; Hei, Tingting

2011-01-01

Protein molecules immobilized on a hydrophobic polystyrene microplate by passive adsorption lose their activity and suffer considerable denaturation. In this paper, we report a thorough evaluation of a protocol for enzyme immobilization on a microplate with relatively inexpensive reagents, involving glutaraldehyde coupling and spacer molecules, and employing β-glucosidase as a model enzyme. The recommended conditions for the developed method include 2.5% glutaraldehyde to activate the reaction, 1% chitosan in an HAc solution to increase the binding capacity, 2% bovine serum albumin to block non-specific binding sites, and 0.1 M NaBH4 to stabilize Schiff's base intermediates. Using this method, the amount of β-glucosidase immobilized on amino-microplate was 24-fold with chitosan than without spacer molecules. The procedure is efficient and quite simple, and may thus have potential applications in biosensing and bioreactor systems.

Interactions in the aqueous phase and adsorption at the air-water interface of caseinoglycomacropeptide (GMP) and beta-lactoglobulin mixed systems.

PubMed

Martinez, María J; Sánchez, Cecilio Carrera; Patino, Juan M Rodríguez; Pilosof, Ana M R

2009-01-01

The aim of this work was to study the interactions and adsorption of caseinoglycomacropeptide (GMP) and GMP:beta-lactoglobulin (beta-lg) mixed system in the aqueous phase and at the air-water interface. The existence of associative interactions between GMP and beta-lg in the aqueous phase was investigated by dynamic light scattering, differential scanning calorimetry (DSC), fluorometry and native PAGE-electrophoresis. The surface pressure isotherm and the static and dynamic surface pressure were determined by tensiometry and surface dilatational properties. The results showed that GMP presented higher surface activity than beta-lg at a concentration of 4%wt but beta-lg showed higher film forming ability. In the mixed systems beta-lg dominated the static and dynamic surface pressure and the rheological properties of interfacial films suggesting that beta-lg hinders GMP adsorption because, in simple competition, GMP should dominate because of its higher surface activity. The surface predominance of beta-lg can be attributed to binding of GMP to beta-lg in the aqueous phase that prevents GMP adsorption on its own.

Improved high-throughput quantification of luminescent microplate assays using a common Western-blot imaging system.

PubMed

Hawkins, Liam J; Storey, Kenneth B

2017-01-01

Common Western-blot imaging systems have previously been adapted to measure signals from luminescent microplate assays. This can be a cost saving measure as Western-blot imaging systems are common laboratory equipment and could substitute a dedicated luminometer if one is not otherwise available. One previously unrecognized limitation is that the signals captured by the cameras in these systems are not equal for all wells. Signals are dependent on the angle of incidence to the camera, and thus the location of the well on the microplate. Here we show that: •The position of a well on a microplate significantly affects the signal captured by a common Western-blot imaging system from a luminescent assay.•The effect of well position can easily be corrected for.•This method can be applied to commercially available luminescent assays, allowing for high-throughput quantification of a wide range of biological processes and biochemical reactions.

Endogenous cGMP regulates adult longevity via the insulin signaling pathway in Caenorhabditis elegans.

PubMed

Hahm, Jeong-Hoon; Kim, Sunhee; Paik, Young-Ki

2009-08-01

G-proteins, including GPA-3, play an important role in regulating physiological responses in Caenorhabditis elegans. When confronted with an environmental stimulus such as dauer pheromone, or poor nutrients, C. elegans receives and integrates external signals through its nervous system (i.e. amphid neurons), which interprets and translates them into biological action. Here it is shown that a suppressed neuronal cGMP level caused by GPA-3 activation leads to a significant increase (47.3%) in the mean lifespan of adult C. elegans through forkhead transcription factor family O (FOXO)-mediated signal. A reduced neuronal cGMP level was found to be caused by an increased cGMP-specific phosphodiesterase activity at the transcriptional level. Our results using C. elegans mutants with specific deficits in TGF-beta and FOXO RNAi system suggest a mechanism in that cGMP, TGF-beta, and FOXO signaling interact to differentially produce the insulin-like molecules, ins-7 and daf-28, causing suppression of the insulin/IGF-1 pathway and promoting lifespan extension. Our findings provide not only a new mechanism of cGMP-mediated induction of longevity in adult C. elegans but also a possible therapeutic strategy for neuronal disease, which has been likened to brain diabetes.

Intra-annual variability of the radiocarbon content of corals from the Galapagos Islands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, T.A.; Farwell, G.W.; Schmidt, F.H.

1993-01-01

The authors report AMS [sup 14]C measurements on sub annual samples of coral from the Galapagos Islands that span the period, 1970-1973. Both the major 1972 El Nino/Southern Oscillation event and intra-annual changes in regional upwelling of [sup 14]C-depleted waters associated with alternation of surface-ocean current patterns are evident in the record. These data show that the corals preserve a detailed record of past intra-annual variations of the [sup 14]C content of surface ocean water.

Cellphone-based hand-held microplate reader for point-of-care ELISA testing (Conference Presentation)

NASA Astrophysics Data System (ADS)

Berg, Brandon; Cortazar, Bingen; Tseng, Derek; Ozkan, Haydar; Feng, Steve; Wei, Qingshan; Chan, Raymond Y.; Burbano, Jordi; Farooqui, Qamar; Lewinski, Michael; Di Carlo, Dino; Garner, Omai B.; Ozcan, Aydogan

2016-03-01

Enzyme-linked immunosorbent assay (ELISA) in a microplate format has been a gold standard first-line clinical test for diagnosis of various diseases including infectious diseases. However, this technology requires a relatively large and expensive multi-well scanning spectrophotometer to read and quantify the signal from each well, hindering its implementation in resource-limited-settings. Here, we demonstrate a cost-effective and handheld smartphone-based colorimetric microplate reader for rapid digitization and quantification of immunoserology-related ELISA tests in a conventional 96-well plate format at the point of care (POC). This device consists of a bundle of 96 optical fibers to collect the transmitted light from each well of the microplate and direct all the transmission signals from the wells onto the camera of the mobile-phone. Captured images are then transmitted to a remote server through a custom-designed app, and both quantitative and qualitative diagnostic results are returned back to the user within ~1 minute per 96-well plate by using a machine learning algorithm. We tested this mobile-phone based micro-plate reader in a clinical microbiology lab using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests on 1138 remnant patient samples (roughly 50% training and 50% testing), and achieved an overall accuracy of ~99% or higher for each ELISA test. This handheld and cost-effective platform could be immediately useful for large-scale vaccination monitoring in low-infrastructure settings, and also for other high-throughput disease screening applications at POC.

Supercritical fluid chromatography for GMP analysis in support of pharmaceutical development and manufacturing activities.

PubMed

Hicks, Michael B; Regalado, Erik L; Tan, Feng; Gong, Xiaoyi; Welch, Christopher J

2016-01-05

Supercritical fluid chromatography (SFC) has long been a preferred method for enantiopurity analysis in support of pharmaceutical discovery and development, but implementation of the technique in regulated GMP laboratories has been somewhat slow, owing to limitations in instrument sensitivity, reproducibility, accuracy and robustness. In recent years, commercialization of next generation analytical SFC instrumentation has addressed previous shortcomings, making the technique better suited for GMP analysis. In this study we investigate the use of modern SFC for enantiopurity analysis of several pharmaceutical intermediates and compare the results with the conventional HPLC approaches historically used for analysis in a GMP setting. The findings clearly illustrate that modern SFC now exhibits improved precision, reproducibility, accuracy and robustness; also providing superior resolution and peak capacity compared to HPLC. Based on these findings, the use of modern chiral SFC is recommended for GMP studies of stereochemistry in pharmaceutical development and manufacturing. Copyright © 2015 Elsevier B.V. All rights reserved.

cGMP Signaling in the Cardiovascular System—The Role of Compartmentation and Its Live Cell Imaging

PubMed Central

Bork, Nadja I.; Nikolaev, Viacheslav O.

2018-01-01

The ubiquitous second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) regulates multiple physiologic processes in the cardiovascular system. Its intracellular effects are mediated by stringently controlled subcellular microdomains. In this review, we will illustrate the current techniques available for real-time cGMP measurements with a specific focus on live cell imaging methods. We will also discuss currently accepted and emerging mechanisms of cGMP compartmentation in the cardiovascular system. PMID:29534460

A C-di-GMP-proflavine-hemin supramolecular complex has peroxidase activity--implication for a simple colorimetric detection.

PubMed

Nakayama, Shizuka; Roelofs, Kevin; Lee, Vincent T; Sintim, Herman O

2012-03-01

Herein, we demonstrate that the bacterial signaling molecule, c-di-GMP, can enhance the peroxidation of hemin when proflavine is present. The c-di-GMP-proflavine-hemin nucleotidezyme can oxidize the colorless compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, to the colored radical cation ABTS˙(+) and hence provides simple colorimetric detection of c-di-GMP at low micromolar concentrations.

Seismicity of the Adriatic microplate

USGS Publications Warehouse

Console, R.; Di, Giovambattista R.; Favali, P.; Presgrave, B.W.; Smriglio, G.

1993-01-01

The Adriatic microplate was previously considered to be a unique block, tectonically active only along its margins. The seismic sequences that took place in the basin from 1986 to 1990 give new information about the geodynamics of this area. Three subsets of well recorded events were relocated by the joint hypocentre determination technique. On the whole, this seismic activity was concentrated in a belt crossing the southern Adriatic sea around latitude 42??, in connection with regional E-W fault systems. Some features of this seismicity, similar to those observed in other well known active margins of the Adriatic plate, support a model of a southern Adriatic lithospheric block, detached from the Northern one. Other geophysical information provides evidence of a transitional zone at the same latitude. ?? 1993.

Microplate Bioassay for Determining Substrate Selectivity of "Candida rugosa" Lipase

ERIC Educational Resources Information Center

Wang, Shi-zhen; Fang, Bai-shan

2012-01-01

Substrate selectivity of "Candida rugosa" lipase was tested using "p"-nitrophenyl esters of increasing chain length (C[subscript 1], C[subscript 7], C[subscript 15]) using the high-throughput screening method. A fast and easy 96-well microplate bioassay was developed to help students learn and practice biotechnological specificity screen. The…

Quality Risk Management: Putting GMP Controls First.

PubMed

O'Donnell, Kevin; Greene, Anne; Zwitkovits, Michael; Calnan, Nuala

2012-01-01

This paper presents a practical way in which current approaches to quality risk management (QRM) may be improved, such that they better support qualification, validation programs, and change control proposals at manufacturing sites. The paper is focused on the treatment of good manufacturing practice (GMP) controls during QRM exercises. It specifically addresses why it is important to evaluate and classify such controls in terms of how they affect the severity, probability of occurrence, and detection ratings that may be assigned to potential failure modes or negative events. It also presents a QRM process that is designed to directly link the outputs of risk assessments and risk control activities with qualification and validation protocols in the GMP environment. This paper concerns the need for improvement in the use of risk-based principles and tools when working to ensure that the manufacturing processes used to produce medicines, and their related equipment, are appropriate. Manufacturing processes need to be validated (or proven) to demonstrate that they can produce a medicine of the required quality. The items of equipment used in such processes need to be qualified, in order to prove that they are fit for their intended use. Quality risk management (QRM) tools can be used to support such qualification and validation activities, but their use should be science-based and subject to as little subjectivity and uncertainty as possible. When changes are proposed to manufacturing processes, equipment, or related activities, they also need careful evaluation to ensure that any risks present are managed effectively. This paper presents a practical approach to how QRM may be improved so that it better supports qualification, validation programs, and change control proposals in a more scientific way. This improved approach is based on the treatment of what are called good manufacturing process (GMP) controls during those QRM exercises. A GMP control can be considered

BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation

PubMed Central

Dressaire, Clémentine; Barahona, Susana; Galego, Lisete; Kaever, Volkhard; Jenal, Urs

2017-01-01

ABSTRACT The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli. This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health. PMID:28928205

Ejection of small droplet from microplate using focused ultrasound

NASA Astrophysics Data System (ADS)

Tanaka, Hiroki; Mizuno, Yosuke; Nakamura, Kentaro

2017-08-01

We discussed an ultrasonic system for single-droplet ejection from a microplate, which is one of the basic and important procedures in the noncontact handling of droplets in air. In this system, a 1.5 MHz concave transducer located below the microplate is used for chasing the liquid surface through a pulse echo method, and also for the ejection of a 1 µL single droplet by the burst of focused ultrasound. We investigated the relationship between the droplet ejection characteristics, the distance from the transducer to the surface of liquid, the material property, and the excitation condition of the focused ultrasonic transducer. It was verified that the optimal position of the transducer was off the focal point of sound pressure by ±1 mm, because the sound intensity had to be controlled to eject a single droplet. Subsequently, we confirmed experimentally that the ejected droplet volume linearly depended on the surface tension of the liquid, and that the droplet volume and ejection velocity were determined by the Webber number, Reynolds number, and Ohnesolge number. In addition, by optimizing the duration of the burst ultrasound, the droplet volume and ejection velocity were controlled.

A microplate assay for DNA damage determination (fast micromethod).

PubMed

Batel, R; Jaksić, Z; Bihari, N; Hamer, B; Fafandel, M; Chauvin, C; Schröder, H C; Müller, W E; Zahn, R K

1999-06-01

A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 microg tissue per well), or in environmental samples for genotoxicity assessment. Copyright 1999 Academic Press.

The HD-GYP domain, cyclic di-GMP signaling, and bacterial virulence to plants.

PubMed

Dow, J Maxwell; Fouhy, Yvonne; Lucey, Jean F; Ryan, Robert P

2006-12-01

Cyclic di-GMP is an almost ubiquitous second messenger in bacteria that was first described as an allosteric activator of cellulose synthase but is now known to regulate a range of functions, including virulence in human and animal pathogens. Two protein domains, GGDEF and EAL, are implicated in the synthesis and degradation, respectively, of cyclic di-GMP. These domains are widely distributed in bacteria, including plant pathogens. The majority of proteins with GGDEF and EAL domains contain additional signal input domains, suggesting that their activities are responsive to environmental cues. Recent studies have demonstrated that a third domain, HD-GYP, is also active in cyclic di-GMP degradation. In the plant pathogen Xanthomonas campestris pv. campestris, a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC positively controls virulence. The signals recognized by RpfC may include the cell-cell signal DSF, which also acts to regulate virulence in X. campestris pv. campestris. Here, we review these recent advances in our understanding of cyclic di-GMP signaling with particular reference to one or more roles in the bacterial pathogenesis of plants.

Molecule counting with alkanethiol and DNA immobilized on gold microplates for extended gate FET.

PubMed

Cao, Zhong; Xiao, Zhong-Liang; Zhang, Ling; Luo, Dong-Mei; Kamahori, Masao; Shimoda, Maki

2013-04-01

Several molecule counting methods based on electrochemical characterization of alkanethiol and thiolated single-stranded oligonucleotide (HS-ssDNA) immobilized on gold microplates, which were used as extended gates of field effect transistors (FETs), have been investigated in this paper. The surface density of alkanethiol and DNA monolayers on gold microplates were quantitatively evaluated from the reductive desorption charge by using cyclic voltammetry (CV) and fast CV (FCV) methods in strong alkali solution. Typically, the surface density of 6-hydroxy-1-hexanethiol (6-HHT) was evaluated to be 4.639 molecules/nm(2), and the 28 base-pair dsDNA about 1.226-4.849 molecules/100 nm(2) on Au microplates after post-treatment with 6-HHT. The behaviors on surface potential and capacitance of different aminoalkanethiols on Au microplates were measured in 0.1 mol/L Na2SO4 and 10 mmol/L Tris-HCl (pH=7.4) solutions, indicating that the surface potential increases and the double-layer capacitance decreases with the length of carbon chain increased for the thiol monolayers, which obey a physics relationship for a capacitor. Comparably, a simple sensing method based on the electronic signals of biochemical reaction events on DNA immobilization and hybridization at the Au surface of the extended gate FET (EGFET) was developed, with which the surface density of the hybridized dsDNA on the gold surface of the EGFET was evaluated to be 1.36 molecules per 100 nm(2), showing that the EGFET is a promising sensing biochip for DNA molecule counting. Copyright © 2012 Elsevier B.V. All rights reserved.

8-pCPT-cGMP prevents mitochondrial depolarization and improves the outcome of steatotic partial liver transplantation

PubMed Central

Liu, Qinlong; Rehman, Hasibur; Krishnasamy, Yasodha; Lemasters, John J; Zhong, Zhi

2017-01-01

Permeant cGMP analogs prevent the mitochondria permeability transition (MPT) in vitro. In this study, we explored whether 8-pCPT-cGMP prevents the MPT and decreases post-transplant damage to fatty partial liver grafts (FPG) in vivo. Rats were fed a control or high-fat, high-fructose diet for 2-week. Lean and fatty liver explants were reduced in size ex vivo to ~35% and stored in the University of Wisconsin solution with and without 8-pCPT-cGMP (300 µM) for 2 h. After transplantation, alanine aminotransferase release (indicator of hepatocellular injury), hyperbilirubinemia (indicator of poor liver function), and cell death were all higher in FPG than in lean partial grafts (LPG). Liver regeneration increased in LPG but was suppressed in FPG. 8-pCPT-cGMP blunted graft injury, improved liver regeneration and function, and increased survival of FPG. Hepatic mitochondrial depolarization detected by intravital multiphoton microscopy of rhodamine 123 in living rats was ~3.5-fold higher in FPG than in LPG. 8-pCPT-cGMP decreased mitochondrial depolarization in FPG almost to the level of LPG. Activation of mammalian target of rapamycin (mTOR), an energy sensitive kinase that stimulates cell proliferation and growth, and p70S6 kinase, a downstream signaling molecule of mTOR, was increased in LPG but suppressed in FPG. 8-pCPT-cGMP restored the activity of mTOR and p70S6 kinase in FPG. 8-pCPT-cGMP also increased activation of cAMP response element-binding protein (CREB) and expression of cyclins D1 and E in FPG. Non-alcoholic steatosis increases injury and suppresses regeneration after partial liver transplantation, at least in part, due to more severe mitochondrial dysfunction. Protection of mitochondria with a cGMP analog effectively improves outcomes of FPG transplantation. PMID:28694919

Blood gases, biochemistry, and hematology of Galapagos green turtles (Chelonia mydas).

PubMed

Lewbart, Gregory A; Hirschfeld, Maximilian; Denkinger, Judith; Vasco, Karla; Guevara, Nataly; García, Juan; Muñoz, Juanpablo; Lohmann, Kenneth J

2014-01-01

The green turtle, Chelonia mydas, is an endangered marine chelonian with a circum-global distribution. Reference blood parameter intervals have been published for some chelonian species, but baseline hematology, biochemical, and blood gas values are lacking from the Galapagos sea turtles. Analyses were done on blood samples drawn from 28 green turtles captured in two foraging locations on San Cristóbal Island (14 from each site). Of these turtles, 20 were immature and of unknown sex; the other eight were males (five mature, three immature). A portable blood analyzer (iSTAT) was used to obtain near immediate field results for pH, lactate, pO2, pCO2, HCO3-, Hct, Hb, Na, K, iCa, and Glu. Parameter values affected by temperature were corrected in two ways: (1) with standard formulas; and (2) with auto-corrections made by the iSTAT. The two methods yielded clinically equivalent results. Standard laboratory hematology techniques were employed for the red and white blood cell counts and the hematocrit determination, which was also compared to the hematocrit values generated by the iSTAT. Of all blood analytes, only lactate concentrations were positively correlated with body size. All other values showed no significant difference between the two sample locations nor were they correlated with body size or internal temperature. For hematocrit count, the iSTAT blood analyzer yielded results indistinguishable from those obtained with high-speed centrifugation. The values reported in this study provide baseline data that may be useful in comparisons among populations and in detecting changes in health status among Galapagos sea turtles. The findings might also be helpful in future efforts to demonstrate associations between specific biochemical parameters and disease.

Blood Gases, Biochemistry, and Hematology of Galapagos Green Turtles (Chelonia Mydas)

PubMed Central

Lewbart, Gregory A.; Hirschfeld, Maximilian; Denkinger, Judith; Vasco, Karla; Guevara, Nataly; García, Juan; Muñoz, Juanpablo; Lohmann, Kenneth J.

2014-01-01

The green turtle, Chelonia mydas, is an endangered marine chelonian with a circum-global distribution. Reference blood parameter intervals have been published for some chelonian species, but baseline hematology, biochemical, and blood gas values are lacking from the Galapagos sea turtles. Analyses were done on blood samples drawn from 28 green turtles captured in two foraging locations on San Cristóbal Island (14 from each site). Of these turtles, 20 were immature and of unknown sex; the other eight were males (five mature, three immature). A portable blood analyzer (iSTAT) was used to obtain near immediate field results for pH, lactate, pO2, pCO2, HCO3 −, Hct, Hb, Na, K, iCa, and Glu. Parameter values affected by temperature were corrected in two ways: (1) with standard formulas; and (2) with auto-corrections made by the iSTAT. The two methods yielded clinically equivalent results. Standard laboratory hematology techniques were employed for the red and white blood cell counts and the hematocrit determination, which was also compared to the hematocrit values generated by the iSTAT. Of all blood analytes, only lactate concentrations were positively correlated with body size. All other values showed no significant difference between the two sample locations nor were they correlated with body size or internal temperature. For hematocrit count, the iSTAT blood analyzer yielded results indistinguishable from those obtained with high-speed centrifugation. The values reported in this study provide baseline data that may be useful in comparisons among populations and in detecting changes in health status among Galapagos sea turtles. The findings might also be helpful in future efforts to demonstrate associations between specific biochemical parameters and disease. PMID:24824065

Improved estimates of age, growth and reproduction for the regionally endemic Galapagos sailfin grouper Mycteroperca olfax (Jenyns, 1840)

PubMed Central

Friedlander, Alan M.; DeMartini, Edward E.; Schuhbauer, Anna; Schemmel, Eva; Salinas de Léon, Pelayo

2015-01-01

The Galapagos Sailfin grouper, Mycteroperca olfax, locally known as bacalao and listed as vulnerable by the IUCN, is culturally, economically, and ecologically important to the Galapagos archipelago and its people. It is regionally endemic to the Eastern Tropical Pacific, and, while an important fishery resource that has shown substantial declines in recent years, to date no effective management regulations are in place to ensure the sustainability of the Galapagos fishery for this species. Previous estimates of longevity and size at maturity for bacalao are inconsistent with estimates for congeners, which brings into question the accuracy of prior estimates. We set out to assess the age, growth, and reproductive biology of bacalao in order to provide more accurate life history information to inform more effective fisheries management for this species. The oldest fish in our sample was 21 years old, which is 2–3 times greater than previously reported estimates of longevity. Parameter estimates for the von Bertalanffy growth function (k = 0.11, L∞ = 110 cm TL, and to = − 1.7 years) show bacalao to grow much slower and attain substantially larger asymptotic maximum length than previous studies. Mean size at maturity (as female) was estimated at 65.3 cm TL, corresponding to a mean age of 6.5 years. We found that sex ratios were extremely female biased (0.009 M:1F), with a large majority of the individuals in our experimental catch being immature (79%). Our results show that bacalao grow slower, live longer, and mature at a much larger size and greater age than previously thought, with very few mature males in the population. These findings have important implications for the fishery of this valuable species and provide the impetus for a long-overdue species management plan to ensure its long-term sustainability. PMID:26401463

Improved estimates of age, growth and reproduction for the regionally endemic Galapagos sailfin grouper Mycteroperca olfax (Jenyns, 1840).

PubMed

Usseglio, Paolo; Friedlander, Alan M; DeMartini, Edward E; Schuhbauer, Anna; Schemmel, Eva; Salinas de Léon, Pelayo

2015-01-01

The Galapagos Sailfin grouper, Mycteroperca olfax, locally known as bacalao and listed as vulnerable by the IUCN, is culturally, economically, and ecologically important to the Galapagos archipelago and its people. It is regionally endemic to the Eastern Tropical Pacific, and, while an important fishery resource that has shown substantial declines in recent years, to date no effective management regulations are in place to ensure the sustainability of the Galapagos fishery for this species. Previous estimates of longevity and size at maturity for bacalao are inconsistent with estimates for congeners, which brings into question the accuracy of prior estimates. We set out to assess the age, growth, and reproductive biology of bacalao in order to provide more accurate life history information to inform more effective fisheries management for this species. The oldest fish in our sample was 21 years old, which is 2-3 times greater than previously reported estimates of longevity. Parameter estimates for the von Bertalanffy growth function (k = 0.11, L ∞ = 110 cm TL, and to = - 1.7 years) show bacalao to grow much slower and attain substantially larger asymptotic maximum length than previous studies. Mean size at maturity (as female) was estimated at 65.3 cm TL, corresponding to a mean age of 6.5 years. We found that sex ratios were extremely female biased (0.009 M:1F), with a large majority of the individuals in our experimental catch being immature (79%). Our results show that bacalao grow slower, live longer, and mature at a much larger size and greater age than previously thought, with very few mature males in the population. These findings have important implications for the fishery of this valuable species and provide the impetus for a long-overdue species management plan to ensure its long-term sustainability.

Intra-laboratory validation of microplate methods for total phenolic content and antioxidant activity on polyphenolic extracts, and comparison with conventional spectrophotometric methods.

PubMed

Bobo-García, Gloria; Davidov-Pardo, Gabriel; Arroqui, Cristina; Vírseda, Paloma; Marín-Arroyo, María R; Navarro, Montserrat

2015-01-01

Total phenolic content (TPC) and antioxidant activity (AA) assays in microplates save resources and time, therefore they can be useful to overcome the fact that the conventional methods are time-consuming, labour intensive and use large amounts of reagents. An intra-laboratory validation of the Folin-Ciocalteu microplate method to measure TPC and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) microplate method to measure AA was performed and compared with conventional spectrophotometric methods. To compare the TPC methods, the confidence intervals of a linear regression were used. In the range of 10-70 mg L(-1) of gallic acid equivalents (GAE), both methods were equivalent. To compare the AA methodologies, the F-test and t-test were used in a range from 220 to 320 µmol L(-1) of Trolox equivalents. Both methods had homogeneous variances, and the means were not significantively different. The limits of detection and quantification for the TPC microplate method were 0.74 and 2.24 mg L(-1) GAE and for the DPPH 12.07 and 36.58 µmol L(-1) of Trolox equivalents. The relative standard deviation of the repeatability and reproducibility for both microplate methods were ≤ 6.1%. The accuracy ranged from 88% to 100%. The microplate and the conventional methods are equals in a 95% confidence level. © 2014 Society of Chemical Industry.

Thymocyte cyclic AMP and cyclic GMP response to treatment with metabolites issued from the lipoxygenase pathway.

PubMed

Mexmain, S; Cook, J; Aldigier, J C; Gualde, N; Rigaud, M

1985-08-01

Evidence has been presented that cGMP is the second messenger for the lipoxygenase metabolites 15-HETE and LTB4 in the mouse splenocyte and thymocyte. Incubation of splenocytes with 10(-7) to 10(-9) M 15-HETE caused a slight decrease in cAMP levels and an increase in cGMP levels after 10 to 20 min. Mature PNA-, immature PNA+, and whole thymocytes treated with 10(-7) to 10(-10) M 15-HETE and 10(-11) M LTB4 showed an approximately 100% increase in cGMP production. In mixed lymphocyte reactions, 15-HETE- and LTB4-treated PNA+, PNA-, and whole thymocyte populations inhibited thymidine uptake by fresh allostimulated splenocytes. These results demonstrate that the eicosanoid-induced generation of suppressor cells follows a rise in lymphocyte cGMP levels.

Purified anthocyanin supplementation improves endothelial function via NO-cGMP activation in hypercholesterolemic individuals.

PubMed

Zhu, Yanna; Xia, Min; Yang, Yan; Liu, Fengqiong; Li, Zhongxia; Hao, Yuantao; Mi, Mantian; Jin, Tianru; Ling, Wenhua

2011-11-01

Anthocyanins have been shown to improve endothelial function in animal models. However, whether these compounds have similar beneficial effects in humans is largely unknown. In a short-term crossover study, 12 hypercholesterolemic individuals were given oral anthocyanins (320 mg) isolated from berries or placebo. Brachial artery flow-mediated dilation (FMD) was assessed before and after the intervention. In a long-term intervention trial (12 weeks), 150 hypercholesterolemic individuals were given anthocyanins (320 mg/day, n = 75) or placebo (n = 75), after which we measured FMD, plasma cGMP, and other serum biomarkers. Another short-term intervention was conducted in the presence of NO-cGMP inhibitors in 6 people and in a rat aortic ring model (n = 8). Significant increases of FMD from 8.3% (0.6%) at baseline to 11.0% (0.8%) at 1 h and 10.1% (0.9%) at 2 h were observed after short-term anthocyanin consumption, concomitantly with increases of plasma anthocyanin concentrations (P < 0.05). In the study participants who received long-term anthocyanin intervention, compared with the control group, we observed significant increases in the FMD (28.4% vs 2.2%), cGMP (12.6% vs -1.2%), and HDL-cholesterol concentrations, but decreases in the serum soluble vascular adhesion molecule-1 and LDL cholesterol concentrations (P < 0.05). The changes in the cGMP and HDL cholesterol concentrations positively correlated with FMD in the anthocyanin group (P < 0.05). In the presence of NO-cGMP inhibitors, the effects of anthocyanin on endothelial function were abolished in human participants and in a rat aortic ring model. Anthocyanin supplementation improves endothelium-dependent vasodilation in hypercholesterolemic individuals. This effect involves activation of the NO-cGMP signaling pathway, improvements in the serum lipid profile, and decreased inflammation.

A Cenozoic tectonic model for Southeast Asia - microplates and basins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maher, K.A.

1995-04-01

A computer-assisted Cenozoic tectonic model was built for Southeast Asia and used to construct 23 base maps, 2 to 6 million years apart. This close temporal spacing was necessary to constrain all the local geometric shifts in a consistent and geologically feasible fashion. More than a hundred individual blocks were required to adequately treat Cenozoic microplate processes at a basic level. The reconstructions show tectonic evolution to be characterized by long periods of gradual evolution, interrupted by brief, widespread episodes of reorganization in fundamental plate geometries and kinematics. These episodes are triggered by major collisions, or by accumulation of smallermore » changes. The model takes into account difficulties inherent in the region. The Pacific and Indo-Australian plates and their predecessors have driven westward and northward since the late Paleozoic, towards each other and the relatively stationary backstop of Asia. Southeast Asia is therefore the result of a long-lived, complex process of convergent tectonics, making it difficult to reconstruct tectonic evolution as much of the continental margin and sea floor spreading record was erased. In addition, the region has been dominated by small-scale microplate processes with short time scales and internal deformation, taking place in rapidly evolving and more ductile buffer zones between the major rigid plate systems. These plate interaction zones have taken up much of the relative motion between the major plates. Relatively ephemeral crustal blocks appear and die within the buffer zones, or accrete to and disperse from the margins of the major plate systems. However, such microplate evolution is the dominant factor in Cenozoic basin evolution. This detailed testonic model aids in comprehension and prediction of basin development, regional hydrocarbon habitat, and petroleum systems.« less

Wafer-scale growth of large arrays of perovskite microplate crystals for functional electronics and optoelectronics.

PubMed

Wang, Gongming; Li, Dehui; Cheng, Hung-Chieh; Li, Yongjia; Chen, Chih-Yen; Yin, Anxiang; Zhao, Zipeng; Lin, Zhaoyang; Wu, Hao; He, Qiyuan; Ding, Mengning; Liu, Yuan; Huang, Yu; Duan, Xiangfeng

2015-10-01

Methylammonium lead iodide perovskite has attracted intensive interest for its diverse optoelectronic applications. However, most studies to date have been limited to bulk thin films that are difficult to implement for integrated device arrays because of their incompatibility with typical lithography processes. We report the first patterned growth of regular arrays of perovskite microplate crystals for functional electronics and optoelectronics. We show that large arrays of lead iodide microplates can be grown from an aqueous solution through a seeded growth process and can be further intercalated with methylammonium iodide to produce perovskite crystals. Structural and optical characterizations demonstrate that the resulting materials display excellent crystalline quality and optical properties. We further show that perovskite crystals can be selectively grown on prepatterned electrode arrays to create independently addressable photodetector arrays and functional field effect transistors. The ability to grow perovskite microplates and to precisely place them at specific locations offers a new material platform for the fundamental investigation of the electronic and optical properties of perovskite materials and opens a pathway for integrated electronic and optoelectronic systems.

Microplate-based method to screen inhibitors of isozymes of cyclic nucleotide phosphodiesterase fused to SUMO.

PubMed

Chen, Chunyan; Liu, Miaomiao; Wu, Jing; Yang, Xiaolan; Hu, Xiaolei; Pu, Jun; Long, Gaobo; Xie, Yanling; Jiang, Hairong; Yuan, Yonghua; Liao, Fei

2014-12-01

The feasibility for microplate-based screening of inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE) was tested via the coupled action of a phosphatase on adenosine-5'-monophosphate and an improved malachite green assay of phosphate. Human full-length PDE4B2 and truncated mutant (152-528aa) were expressed in Escherichia coli via fusion to SUMO, which after purification through Ni-NTA column exhibited specific activities >0.017 U mg(-1). In the presence of proteins <30 mg L(-1), absorbance for 10 µΜ phosphate was measurable; a PDE isozyme of specific activity over 0.008 U mg(-1) after reaction for 20 min thus suited for microplate-based screening of inhibitors. By using Biotek ELX 800 microplate reader, affinities of two forms of PEDE4B2 for cAMP, rolipram and papaverine varied over three magnitudes and were consistent with those by routine assay, respectively. Hence, the proposed method was promising for high-throughput-screening of inhibitors of phosphate-releasing enzymes bearing specific activities over 0.008 U mg(-1).

Wafer-scale growth of large arrays of perovskite microplate crystals for functional electronics and optoelectronics

PubMed Central

Wang, Gongming; Li, Dehui; Cheng, Hung-Chieh; Li, Yongjia; Chen, Chih-Yen; Yin, Anxiang; Zhao, Zipeng; Lin, Zhaoyang; Wu, Hao; He, Qiyuan; Ding, Mengning; Liu, Yuan; Huang, Yu; Duan, Xiangfeng

2015-01-01

Methylammonium lead iodide perovskite has attracted intensive interest for its diverse optoelectronic applications. However, most studies to date have been limited to bulk thin films that are difficult to implement for integrated device arrays because of their incompatibility with typical lithography processes. We report the first patterned growth of regular arrays of perovskite microplate crystals for functional electronics and optoelectronics. We show that large arrays of lead iodide microplates can be grown from an aqueous solution through a seeded growth process and can be further intercalated with methylammonium iodide to produce perovskite crystals. Structural and optical characterizations demonstrate that the resulting materials display excellent crystalline quality and optical properties. We further show that perovskite crystals can be selectively grown on prepatterned electrode arrays to create independently addressable photodetector arrays and functional field effect transistors. The ability to grow perovskite microplates and to precisely place them at specific locations offers a new material platform for the fundamental investigation of the electronic and optical properties of perovskite materials and opens a pathway for integrated electronic and optoelectronic systems. PMID:26601297

Wafer-scale growth of large arrays of perovskite microplate crystals for functional electronics and optoelectronics

DOE PAGES

Wang, Gongming; Li, Dehui; Cheng, Hung -Chieh; ...

2015-10-02

Methylammonium lead iodide perovskite has attracted intensive interest for its diverse optoelectronic applications. However, most studies to date have been limited to bulk thin films that are difficult to implement for integrated device arrays because of their incompatibility with typical lithography processes. We report the first patterned growth of regular arrays of perovskite microplate crystals for functional electronics and optoelectronics. We show that large arrays of lead iodide microplates can be grown from an aqueous solution through a seeded growth process and can be further intercalated with methylammonium iodide to produce perovskite crystals. Structural and optical characterizations demonstrate that themore » resulting materials display excellent crystalline quality and optical properties. We further show that perovskite crystals can be selectively grown on prepatterned electrode arrays to create independently addressable photodetector arrays and functional field effect transistors. Furthermore, the ability to grow perovskite microplates and to precisely place them at specific locations offers a new material platform for the fundamental investigation of the electronic and optical properties of perovskite materials and opens a pathway for integrated electronic and optoelectronic systems.« less

Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

NASA Technical Reports Server (NTRS)

Kandasamy, S. B.; Williaes, B. A.

1983-01-01

It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

Imaging of the Galapagos Plume Using a Network of Mermaids

NASA Astrophysics Data System (ADS)

Nolet, G.; Hello, Y.; Chen, J.; Pazmino, A.; Van der Lee, S.; Bonnieux, S.; Deschamps, A.; Regnier, M. M.; Font, Y.; Simons, F.

2017-12-01

A network of nine submarine seismographs (Mermaids) has been floating freely from 2014 to 2016 around the Galapagos islands, with the aim to enhance the resolving power of deep tomographic images of the mantle plume in this region (see poster by Hello et al. in session S002 for technical details).Analysing a total of 1329 triggered signals transmitted by satellite, we were able to pick the onset times of 434 P waves, 95 PKP and 26 pP arrivals. For the events recorded by at least one Mermaid, these data were complemented with hand-picked onsets from stations on the islands, or on the continent nearby, for a total of 3892 onset times of rays crossing the mantle beneath the Galapagos, many of them with a small standard error estimated at 0.3s. These data are used in a local inversion using ray theory, as is appropriate for onset times. To compensate for delays acquired in the rest of the Earth, the local model is embedded in a global inversion of P delays from the EHB data set most recently published by the ISC for 2000-2003. By selecting a strongly redundant subset of more than one million EHB P wave arrivals, we determined an objective standard error for these delays of 0.51s using the method of Voronin et al. (GJI, 2014). Using a combination of (strong) smoothing and (weak) damping, we force the tomographic model to fit the data close to the level of the estimated standard errors.Preliminary images obtained at the time of writing of this abstract indicate a deep reaching plume that is stronger in the lower mantle than near the surface.Most importantly, the experiment shows how even a limited number of Mermaids can contribute a significant gain in resolution. This is a direct consequence of the fact that they float with abyssal currents, thus avoiding redundancy in raypaths even for aftershocks.The final tomographic images and an analysis of their significance will be subject of the presentation.

Size-dependent phase transition in methylammonium lead iodide perovskite microplate crystals

PubMed Central

Li, Dehui; Wang, Gongming; Cheng, Hung-Chieh; Chen, Chih-Yen; Wu, Hao; Liu, Yuan; Huang, Yu; Duan, Xiangfeng

2016-01-01

Methylammonium lead iodide perovskite has attracted considerable recent interest for solution processable solar cells and other optoelectronic applications. The orthorhombic-to-tetragonal phase transition in perovskite can significantly alter its optical, electrical properties and impact the corresponding applications. Here, we report a systematic investigation of the size-dependent orthorhombic-to-tetragonal phase transition using a combined temperature-dependent optical, electrical transport and transmission electron microscopy study. Our studies of individual perovskite microplates with variable thicknesses demonstrate that the phase transition temperature decreases with reducing microplate thickness. The sudden decrease of mobility around phase transition temperature and the presence of hysteresis loops in the temperature-dependent mobility confirm that the orthorhombic-to-tetragonal phase transition is a first-order phase transition. Our findings offer significant fundamental insight on the temperature- and size-dependent structural, optical and charge transport properties of perovskite materials, and can greatly impact future exploration of novel electronic and optoelectronic devices from these materials. PMID:27098114

Size-dependent phase transition in methylammonium lead iodide perovskite microplate crystals

DOE PAGES

Li, Dehui; Wang, Gongming; Cheng, Hung -Chieh; ...

2016-04-21

Methylammonium lead iodide perovskite has attracted considerable recent interest for solution processable solar cells and other optoelectronic applications. The orthorhombic-to-tetragonal phase transition in perovskite can significantly alter its optical, electrical properties and impact the corresponding applications. Here, we report a systematic investigation of the size-dependent orthorhombic-to-tetragonal phase transition using a combined temperature-dependent optical, electrical transport and transmission electron microscopy study. Our studies of individual perovskite microplates with variable thicknesses demonstrate that the phase transition temperature decreases with reducing microplate thickness. The sudden decrease of mobility around phase transition temperature and the presence of hysteresis loops in the temperature-dependent mobility confirmmore » that the orthorhombic-to-tetragonal phase transition is a first-order phase transition. Lastly, our findings offer significant fundamental insight on the temperature-and size-dependent structural, optical and charge transport properties of perovskite materials, and can greatly impact future exploration of novel electronic and optoelectronic devices from these materials.« less

Possible Impacts of the Invasive Plant Rubus niveus on the Native Vegetation of the Scalesia Forest in the Galapagos Islands

PubMed Central

Rentería, Jorge Luis; Gardener, Mark R.; Panetta, F. Dane; Atkinson, Rachel; Crawley, Mick J.

2012-01-01

Originally from Asia, Rubus niveus has become one of the most widespread invasive plant species in the Galapagos Islands. It has invaded open vegetation, shrubland and forest alike. It forms dense thickets up to 4 m high, appearing to displace native vegetation, and threaten the integrity of several native communities. This study used correlation analysis between a R. niveus cover gradient and a number of biotic (vascular plant species richness, cover and vegetation structure) and abiotic (light and soil properties) parameters to help understand possible impacts in one of the last remaining fragments of the Scalesia forest in Santa Cruz Island, Galapagos. Higher cover of R. niveus was associated with significantly lower native species richness and cover, and a different forest structure. Results illustrated that 60% R. niveus cover could be considered a threshold for these impacts. We suggest that a maximum of 40% R. niveus cover could be a suitable management target. PMID:23118934

Three dimensional perspective view of portion of western Galapagos Islands

NASA Technical Reports Server (NTRS)

1994-01-01

This is a three dimensional perspective view of Isla Isabela in the western Galapagos Islands. It was taken by the L-band radar in HH polarization from the Spaceborne Imaging Radar-C/X-Band Synthetic Aperature Radar on the 40th orbit of the Shuttle Endeavour. This view was constructed by overlaying a SIR-C radar image on a U.S. Geological Survey digital elevation map. The image is centered at about .5 degrees south latitude and 91 degrees West longitude and covers an area of 75 km by 60 km. This SIR-C/X-SAR image of Alcedo and Sierra Negra volcanoes shows the rougher lava flows as bright features, while ash deposits and smooth Pahoehoe lava flows dark. The Jet Propulsion Laboratory alternative photo number is P-43938.

High glucose inhibits the aspirin-induced activation of the nitric oxide/cGMP/cGMP-dependent protein kinase pathway and does not affect the aspirin-induced inhibition of thromboxane synthesis in human platelets.

PubMed

Russo, Isabella; Viretto, Michela; Barale, Cristina; Mattiello, Luigi; Doronzo, Gabriella; Pagliarino, Andrea; Cavalot, Franco; Trovati, Mariella; Anfossi, Giovanni

2012-11-01

Since hyperglycemia is involved in the "aspirin resistance" occurring in diabetes, we aimed at evaluating whether high glucose interferes with the aspirin-induced inhibition of thromboxane synthesis and/or activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway in platelets. For this purpose, in platelets from 60 healthy volunteers incubated for 60 min with 5-25 mmol/L d-glucose or iso-osmolar mannitol, we evaluated the influence of a 30-min incubation with lysine acetylsalicylate (L-ASA; 1-300 μmol/L) on 1) platelet function under shear stress; 2) aggregation induced by sodium arachidonate or ADP; 3) agonist-induced thromboxane production; and 4) NO production, cGMP synthesis, and PKG-induced vasodilator-stimulated phosphoprotein phosphorylation. Experiments were repeated in the presence of the antioxidant agent amifostine. We observed that platelet exposure to 25 mmol/L d-glucose, but not to iso-osmolar mannitol, 1) reduced the ability of L-ASA to inhibit platelet responses to agonists; 2) did not modify the L-ASA-induced inhibition of thromboxane synthesis; and 3) prevented the L-ASA-induced activation of the NO/cGMP/PKG pathway. Preincubation with amifostine reversed the high-glucose effects. Thus, high glucose acutely reduces the antiaggregating effect of aspirin, does not modify the aspirin-induced inhibition of thromboxane synthesis, and inhibits the aspirin-induced activation of the NO/cGMP/PKG pathway. These results identify a mechanism by which high glucose interferes with the aspirin action.

Ultrasensitive and high-throughput analysis of chlorophyll a in marine phytoplankton extracts using a fluorescence microplate reader.

PubMed

Mandalakis, Manolis; Stravinskaitė, Austėja; Lagaria, Anna; Psarra, Stella; Polymenakou, Paraskevi

2017-07-01

Chlorophyll a (Chl a) is the predominant pigment in every single photosynthesizing organism including phytoplankton and one of the most commonly measured water quality parameters. Various methods are available for Chl a analysis, but the majority of them are of limited throughput and require considerable effort and time from the operator. The present study describes a high-throughput, microplate-based fluorometric assay for rapid quantification of Chl a in phytoplankton extracts. Microplate sealing combined with ice cooling was proved an effective means for diminishing solvent evaporation during sample loading and minimized the analytical errors involved in Chl a measurements with a fluorescence microplate reader. A set of operating parameters (settling time, detector gain, sample volume) were also optimized to further improve the intensity and reproducibility of Chl a fluorescence signal. A quadratic regression model provided the best fit (r 2  = 0.9998) across the entire calibration range (0.05-240 pg μL -1 ). The method offered excellent intra- and interday precision (% RSD 2.2 to 11.2%) and accuracy (% relative error -3.8 to 13.8%), while it presented particularly low limits of detection (0.044 pg μL -1 ) and quantification (0.132 pg μL -1 ). The present assay was successfully applied on marine phytoplankton extracts, and the overall results were consistent (average % relative error -14.8%) with Chl a concentrations (including divinyl Chl a) measured by high-performance liquid chromatography (HPLC). More importantly, the microplate-based method allowed the analysis of 96 samples/standards within a few minutes, instead of hours or days, when using a traditional cuvette-based fluorometer or an HPLC system. Graphical abstract TChl a concentrations (i.e. sum of Chl a and divinyl Chl a in ng L -1 ) measured in seawater samples by HPLC and fluorescence microplate reader.

Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade.

PubMed

Dumoulin, Alexandre; Ter-Avetisyan, Gohar; Schmidt, Hannes; Rathjen, Fritz G

2018-04-24

Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation.

PubMed

Moreira, Ricardo N; Dressaire, Clémentine; Barahona, Susana; Galego, Lisete; Kaever, Volkhard; Jenal, Urs; Arraiano, Cecília M

2017-09-19

The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health. IMPORTANCE Bacterial cells have evolved several

The use of a single titanium microplate in displaced pediatric parasymphysial mandibular fractures.

PubMed

Abdullah, Walid A

2009-07-01

The objective of this study was to evaluate the use of one titanium microplate in the fixation of displaced pediatric parasymphysial mandibular fractures. The study was conducted on 7 children in the mixed dentition stage with displaced parasymphysial fracture. Patients' age ranged between 5 years 9 months and 8 years 4 months with an average of 7 years 1 month. Fractured bone segments were exposed, reduced and then fixed using 1.5 linear microplates at the inferior border of the mandible using monocortical screws, with 1.5 mm in diameter and 5 mm in length. Stainless steel wire was used as a tension band by ligating the teeth around the fracture line. Patients were followed up for occlusion and stability clinically and radiographically (panoramic X-ray and CT). According to clinical and radiographic post-operative follow-up, none of the patients showed displacement of the fixed bony segments. The present study concluded that using one microplate with 1.5 monocortical microscrews and dental tension band by a stainless steel wire could be adequate for fixing displaced pediatric parasymphysial mandibular fractures. This technique has the following advantages: decreases the amount of titanium used, decreases the risk of injury of the roots and teeth buds, and decreases the cost and time of surgery.

The use of a single titanium microplate in displaced pediatric parasymphysial mandibular fractures

PubMed Central

Abdullah, Walid A.

2009-01-01

Objective The objective of this study was to evaluate the use of one titanium microplate in the fixation of displaced pediatric parasymphysial mandibular fractures. Materials and methods The study was conducted on 7 children in the mixed dentition stage with displaced parasymphysial fracture. Patients’ age ranged between 5 years 9 months and 8 years 4 months with an average of 7 years 1 month. Fractured bone segments were exposed, reduced and then fixed using 1.5 linear microplates at the inferior border of the mandible using monocortical screws, with 1.5 mm in diameter and 5 mm in length. Stainless steel wire was used as a tension band by ligating the teeth around the fracture line. Patients were followed up for occlusion and stability clinically and radiographically (panoramic X-ray and CT). Results According to clinical and radiographic post-operative follow-up, none of the patients showed displacement of the fixed bony segments. Conclusion The present study concluded that using one microplate with 1.5 monocortical microscrews and dental tension band by a stainless steel wire could be adequate for fixing displaced pediatric parasymphysial mandibular fractures. This technique has the following advantages: decreases the amount of titanium used, decreases the risk of injury of the roots and teeth buds, and decreases the cost and time of surgery. PMID:23960466

Triple Junctions, Boninites, and a New Microplate in the Western Pacific

NASA Astrophysics Data System (ADS)

Flores, J. A.; Casey, J.

2017-12-01

A new microplate has been discovered while trying to correlate melting processes in subduction zones that are forming boninites along the southern Mariana Plate. The westward boundary between the Mariana plate and the Philippine Sea plate is along a well-defined back-arc spreading center. The southern extension of this spreading center to the intersection with the Mariana Trench does not have a recognized morphological boundary. Previous work has hypothesized that subduction beneath a spreading center provides conditions required for boninite petrogenesis. Therefore, the exact location of the trench-trench-ridge triple junction needs to be found and correlated with known boninite locations. The triple junction was found using fault plane solutions to constrain the southern boundary of the two plates as it transects across the forearc. Normal faults suggest the triple junction to be at approximately 11.9N 144.1W; slip direction of reverse faults associated with the subducting plate are dominantly north-south west of this junction and northwest-southeast on the east side. While locating the southern boundary, the nucleation of a new spreading center that creates a ridge-ridge-ridge triple junction was found. The main spreading center trends mostly north-south until about 12.5N 143W, where two other spreading centers meet. The western spreading zone trends mostly east-west and seems to be in its infancy whereas there is another spreading center trending northwest-southeast. It is this last spreading center that forms the trench-ridge-trench triple junction. Discovery of these triple junctions isolates a piece of lithosphere that we interpret to be a new microplate that we name the Challenger Microplate.

The dominance of introduced plant species in the diets of migratory Galapagos tortoises increases with elevation on a human-occupied island

USGS Publications Warehouse

Blake, Stephen; Guézou, Anne; Deem, Sharon L.; Yackulic, Charles B.; Cabrera, Fredy

2015-01-01

The distribution of resources and food selection are fundamental to the ecology, life history, physiology, population dynamics, and conservation of animals. Introduced plants are changing foraging dynamics of herbivores in many ecosystems often with unknown consequences. Galapagos tortoises, like many herbivores, undertake migrations along elevation gradients driven by variability in vegetation productivity which take them into upland areas dominated by introduced plants. We sought to characterize diet composition of two species of Galapagos tortoises, focussing on how the role of introduced forage species changes over space and the implications for tortoise conservation. We quantified the distribution of tortoises with elevation using GPS telemetry. Along the elevation gradient, we quantified the abundance of introduced and native plant species, estimated diet composition by recording foods consumed by tortoises, and assessed tortoise physical condition from body weights and blood parameter values. Tortoises ranged between 0 and 429 m in elevation over which they consumed at least 64 plant species from 26 families, 44 percent of which were introduced species. Cover of introduced species and the proportion of introduced species in tortoise diets increased with elevation. Introduced species were positively selected for by tortoises at all elevations. Tortoise physical condition was either consistent or increased with elevation at the least biologically productive season on Galapagos. Santa Cruz tortoises are generalist herbivores that have adapted their feeding behavior to consume many introduced plant species that has likely made a positive contribution to tortoise nutrition. Some transformed habitats that contain an abundance of introduced forage species are compatible with tortoise conservation.

Regulate axon branching by the cyclic GMP pathway via inhibition of glycogen synthase kinase 3 in dorsal root ganglion sensory neurons.

PubMed

Zhao, Zhen; Wang, Zheng; Gu, Ying; Feil, Robert; Hofmann, Franz; Ma, Le

2009-02-04

Cyclic GMP has been proposed to regulate axonal development, but the molecular and cellular mechanisms underlying the formation of axon branches are not well understood. Here, we report the use of rodent embryonic sensory neurons from the dorsal root ganglion (DRG) to demonstrate the role of cGMP signaling in axon branching and to identify the downstream molecular pathway mediating this novel regulation. Pharmacologically, a specific cGMP analog promotes DRG axon branching in culture, and this activity can be achieved by activating the endogenous soluble guanylyl cyclase that produces cGMP. At the molecular level, the cGMP-dependent protein kinase 1 (PrkG1) mediates this activity, as DRG neurons isolated from the kinase-deficient mouse fail to respond to cGMP activation to make branches, whereas overexpression of a PrkG1 mutant with a higher-than-normal basal kinase activity is sufficient to induce branching. In addition, cGMP activation in DRG neurons leads to phosphorylation of glycogen synthase kinase 3 (GSK3), a protein that normally suppresses branching. This interaction is direct, because PrkG1 binds GSK3 in heterologous cells and the purified kinase can phosphorylate GSK3 in vitro. More importantly, overexpression of a dominant active form of GSK3 suppresses cGMP-dependent branching in DRG neurons. Thus, our study establishes an intrinsic signaling cascade that links cGMP activation to GSK3 inhibition in controlling axon branching during sensory axon development.

The cGMP/PKG pathway as a common mediator of cardioprotection: translatability and mechanism

PubMed Central

Inserte, Javier; Garcia-Dorado, David

2015-01-01

Cardiomyocyte cell death occurring during myocardial reperfusion (reperfusion injury) contributes to final infarct size after transient coronary occlusion. Different interrelated mechanisms of reperfusion injury have been identified, including alterations in cytosolic Ca2+ handling, sarcoplasmic reticulum-mediated Ca2+ oscillations and hypercontracture, proteolysis secondary to calpain activation and mitochondrial permeability transition. All these mechanisms occur during the initial minutes of reperfusion and are inhibited by intracellular acidosis. The cGMP/PKG pathway modulates the rate of recovery of intracellular pH, but has also direct effect on Ca2+ oscillations and mitochondrial permeability transition. The cGMP/PKG pathway is depressed in cardiomyocytes by ischaemia/reperfusion and preserved by ischaemic postconditioning, which importantly contributes to postconditioning protection. The present article reviews the mechanisms and consequences of the effect of ischaemic postconditioning on the cGMP/PKG pathway, the different pharmacological strategies aimed to stimulate it during myocardial reperfusion and the evidence, limitations and promise of translation of these strategies to the clinical practice. Overall, the preclinical and clinical evidence suggests that modulation of the cGMP/PKG pathway may be a therapeutic target in the context of myocardial infarction. PMID:25297462

Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme-Mg 2+-8-oxo-dGMP complex

NASA Astrophysics Data System (ADS)

Massiah, M. A.; Saraswat, V.; Azurmendi, H. F.; Mildvan, A. S.

2004-08-01

The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a KD=52 nM, (Δ G°=-9.8 kcal/mol) which is 6.1 kcal/mol tighter than the binding of dGMP (Δ G°=-3.7 kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable Δ Hbinding (-32 kcal/mol) despite an unfavorable - TΔ S° binding (+22 kcal/mol). The solution structure of the MutT-Mg 2+-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in 1H- 15N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3 kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1 kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position -119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3-0.4 kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT-Mg 2+-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1 kcal/mol, respectively, indicating a lesser role of Arg-78 than of

c-di-GMP can form remarkably stable G-quadruplexes at physiological conditions in the presence of some planar intercalators.

PubMed

Nakayama, Shizuka; Kelsey, Ilana; Wang, Jingxin; Sintim, Herman O

2011-04-28

The ubiquitous bacterial biofilm regulator, c-di-GMP can form G-quadruplexes at physiological conditions in the presence of some aromatic compounds, such as acriflavine and proflavine. The fluorescence of these compounds is quenched upon c-di-GMP binding and some of the formed c-di-GMP G-quadruplexes are stable even at 75 °C. © The Royal Society of Chemistry 2011

Nanoshell-Enhanced Raman Spectroscopy on a Microplate for Staphylococcal Enterotoxin B Sensing.

PubMed

Wang, Wenbin; Wang, Weiwei; Liu, Liqiang; Xu, Liguang; Kuang, Hua; Zhu, Jianping; Xu, Chuanlai

2016-06-22

A sensitive surface-enhanced Raman scattering (SERS) immunosensor based on the Au nanoparticle (Au NP) shell structure was developed to detect staphylococcal enterotoxin B (SEB) on a microplate. Au NPs modified with 4-nitrothiophenol (4-NTP) and coated with Ag shell of controlled thickness at 6.6 nm exhibited excellent SERS intensity and were used as signal reporters in the detection of SEB. The engaged 4-NTP allowed the significant electromagnetic enhancement between Au NPs and the Ag shell and prevented the dissociation of the Raman reporter. More importantly, 4-NTP-differentiated SERS signals between the sample and microplate. The SERS-based immunosensor had a limit of detection of 1.3 pg/mL SEB. Analysis of SEB-spiked milk samples revealed that the developed method had high accuracy. Therefore, the SERS-encoded Au@Ag core-shell structure-based immunosensor is promising for the detection of biotoxins, pathogens, and environmental pollutants.

Effects of van der Waals Force and Thermal Stresses on Pull-in Instability of Clamped Rectangular Microplates.

PubMed

Batra, Romesh C; Porfiri, Maurizio; Spinello, Davide

2008-02-15

We study the influence of von Karman nonlinearity, van der Waals force, and a athermal stresses on pull-in instability and small vibrations of electrostatically actuated mi-croplates. We use the Galerkin method to develop a tractable reduced-order model for elec-trostatically actuated clamped rectangular microplates in the presence of van der Waals forcesand thermal stresses. More specifically, we reduce the governing two-dimensional nonlineartransient boundary-value problem to a single nonlinear ordinary differential equation. For thestatic problem, the pull-in voltage and the pull-in displacement are determined by solving apair of nonlinear algebraic equations. The fundamental vibration frequency corresponding toa deflected configuration of the microplate is determined by solving a linear algebraic equa-tion. The proposed reduced-order model allows for accurately estimating the combined effectsof van der Waals force and thermal stresses on the pull-in voltage and the pull-in deflectionprofile with an extremely limited computational effort.

Effects of van der Waals Force and Thermal Stresses on Pull-in Instability of Clamped Rectangular Microplates

PubMed Central

Batra, Romesh C.; Porfiri, Maurizio; Spinello, Davide

2008-01-01

We study the influence of von Kármán nonlinearity, van der Waals force, and thermal stresses on pull-in instability and small vibrations of electrostatically actuated microplates. We use the Galerkin method to develop a tractable reduced-order model for electrostatically actuated clamped rectangular microplates in the presence of van der Waals forces and thermal stresses. More specifically, we reduce the governing two-dimensional nonlinear transient boundary-value problem to a single nonlinear ordinary differential equation. For the static problem, the pull-in voltage and the pull-in displacement are determined by solving a pair of nonlinear algebraic equations. The fundamental vibration frequency corresponding to a deflected configuration of the microplate is determined by solving a linear algebraic equation. The proposed reduced-order model allows for accurately estimating the combined effects of van der Waals force and thermal stresses on the pull-in voltage and the pull-in deflection profile with an extremely limited computational effort. PMID:27879752

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements.

PubMed

Boutagy, Nabil E; Rogers, George W; Pyne, Emily S; Ali, Mostafa M; Hulver, Matthew W; Frisard, Madlyn I

2015-10-30

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

Anxiolytic effects of phosphodiesterase-2 inhibitors associated with increased cGMP signaling.

PubMed

Masood, Anbrin; Huang, Ying; Hajjhussein, Hassan; Xiao, Lan; Li, Hao; Wang, Wei; Hamza, Adel; Zhan, Chang-Guo; O'Donnell, James M

2009-11-01

Phosphodiesterase (PDE)-2 is a component of the nitric-oxide synthase (NOS)/guanylyl cyclase signaling pathway in the brain. Given recent evidence that pharmacologically induced changes in NO-cGMP signaling can affect anxiety-related behaviors, the effects of the PDE2 inhibitors (2-(3,4-dimethoxybenzyl)-7-det-5-methylimidazo-[5,1-f][1,2,4]triazin-4(3H)-one) (Bay 60-7550) and 3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-5-yl)benzamide (ND7001), as well as modulators of NO, were assessed on cGMP signaling in neurons and on the behavior of mice in the elevated plus-maze, hole-board, and open-field tests, well established procedures for the evaluation of anxiolytics. Bay 60-7550 (1 microM) and ND7001 (10 microM) increased basal and N-methyl-d-aspartate- or detanonoate-stimulated cGMP in primary cultures of rat cerebral cortical neurons; Bay 60-7550, but not ND7001, also increased cAMP. Increased cGMP signaling, either by administration of the PDE2 inhibitors Bay 60-7550 (0.5, 1, and 3 mg/kg) or ND7001 (1 mg/kg), or the NO donor detanonoate (0.5 mg/kg), antagonized the anxiogenic effects of restraint stress on behavior in the three tests. These drugs also produced anxiolytic effects on behavior in nonstressed mice in the elevated plus-maze and hole-board tests; these effects were antagonized by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (20 mg/kg). By contrast, the NOS inhibitor N(omega)-nitro-l-arginine methyl ester (50 mg/kg), which reduces cGMP signaling, produced anxiogenic effects similar to restraint stress. Overall, the present behavioral and neurochemical data suggest that PDE2 may be a novel pharmacological target for the development of drugs for the treatment of anxiety disorders.

HNO₃-assisted polyol synthesis of ultralarge single-crystalline Ag microplates and their far propagation length of surface plasmon polariton.

PubMed

Chang, Cheng-Wei; Lin, Fan-Cheng; Chiu, Chun-Ya; Su, Chung-Yi; Huang, Jer-Shing; Perng, Tsong-Pyng; Yen, Ta-Jen

2014-07-23

We developed a HNO3-assisted polyol reduction method to synthesize ultralarge single-crystalline Ag microplates routinely. The edge length of the synthesized Ag microplates reaches 50 μm, and their top facets are (111). The mechanism for dramatically enlarging single-crystalline Ag structure stems from a series of competitive anisotropic growths, primarily governed by carefully tuning the adsorption of Ag(0) by ethylene glycol and the desorption of Ag(0) by a cyanide ion on Ag(100). Finally, we measured the propagation length of surface plasmon polaritons along the air/Ag interface under 534 nm laser excitation. Our single-crystalline Ag microplate exhibited a propagation length (11.22 μm) considerably greater than that of the conventional E-gun deposited Ag thin film (5.27 μm).

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway

PubMed Central

Sandoval, Alejandro; Duran, Paz; Gandini, María A.; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

2018-01-01

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca2+ macroscopic currents and impair insulin release stimulated with high K+. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion. PMID:28807144

Ibudilast attenuates astrocyte apoptosis via cyclic GMP signalling pathway in an in vitro reperfusion model

PubMed Central

Takuma, K; Lee, E; Enomoto, R; Mori, K; Baba, A; Matsuda, T

2001-01-01

We examined the effect of 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast), which has been clinically used for bronchial asthma and cerebrovascular disorders, on cell viability induced in a model of reperfusion injury. Ibudilast at 10 – 100 μM significantly attenuated the H2O2-induced decrease in cell viability. Ibudilast inhibited the H2O2-induced cytochrome c release, caspase-3 activation, DNA ladder formation and nuclear condensation, suggesting its anti-apoptotic effect. Phosphodiesterase inhibitors such as theophylline, pentoxyfylline, vinpocetine, dipyridamole and zaprinast, which increased the guanosine-3′,5′-cyclic monophosphate (cyclic GMP) level, and dibutyryl cyclic GMP attenuated the H2O2-induced injury in astrocytes. Ibudilast increased the cyclic GMP level in astrocytes. The cyclic GMP-dependent protein kinase inhibitor KT5823 blocked the protective effects of ibudilast and dipyridamole on the H2O2-induced decrease in cell viability, while the cyclic AMP-dependent protein kinase inhibitor KT5720, the cyclic AMP antagonist Rp-cyclic AMPS, the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059 and the leukotriene D4 antagonist LY 171883 did not. KT5823 also blocked the effect of ibudilast on the H2O2-induced cytochrome c release and caspase-3-like protease activation. These findings suggest that ibudilast prevents the H2O2-induced delayed apoptosis of astrocytes via a cyclic GMP, but not cyclic AMP, signalling pathway. PMID:11454657

Correction of Microplate Data from High-Throughput Screening.

PubMed

Wang, Yuhong; Huang, Ruili

2016-01-01

High-throughput screening (HTS) makes it possible to collect cellular response data from a large number of cell lines and small molecules in a timely and cost-effective manner. The errors and noises in the microplate-formatted data from HTS have unique characteristics, and they can be generally grouped into three categories: run-wise (temporal, multiple plates), plate-wise (background pattern, single plate), and well-wise (single well). In this chapter, we describe a systematic solution for identifying and correcting such errors and noises, mainly basing on pattern recognition and digital signal processing technologies.

Evaluation of the Alexon-Trend ProSpecT Campylobacter Microplate Assay

PubMed Central

Tolcin, Rita; LaSalvia, Margaret M.; Kirkley, Barbara A.; Vetter, Emily A.; Cockerill, Franklin R.; Procop, Gary W.

2000-01-01

We evaluated stool specimens known to contain or be free of Campylobacter by traditional culture, using the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of Campylobacter-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of Campylobacter detection. PMID:11015419

Modeling of mixing in 96-well microplates observed with fluorescence indicators.

PubMed

Weiss, Svenja; John, Gernot T; Klimant, Ingo; Heinzle, Elmar

2002-01-01

Mixing in 96-well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double-disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 microL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition.

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

PubMed

Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

2009-05-07

Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMvarphi), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMvarphi. Resultant MDMvarphi were treated for 24 h with DETA/NO (100 - 1000 muM) or GEA-3162 (10 - 300 muM) in the presence or absence of BAY 41-2272 (1 muM), isobutylmethylxanthine (IBMX; 1 muM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 muM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMvarphi was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMvarphi. Preconditioning of MDMvarphi with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41-2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. These results

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

PubMed Central

Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

2009-01-01

Background Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM) or GEA-3162 (10 – 300 μM) in the presence or absence of BAY 41–2272 (1 μM), isobutylmethylxanthine (IBMX; 1 μM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner

Involvement of the cGMP pathway in mediating the insulin-inhibitory effect of melatonin in pancreatic beta-cells.

PubMed

Stumpf, Ina; Mühlbauer, Eckhard; Peschke, Elmar

2008-10-01

Recent investigations have demonstrated an influence of melatonin on insulin secretion in pancreatic beta-cells. The effects are receptor-mediated via two parallel signaling pathways. The aim of this study was to examine the relevance of a second melatonin receptor (MT2) as well as the involvement of a third signaling cascade in mediating melatonin effects, i.e. the cyclic guanosine monophosphate (cGMP) pathway. Our results demonstrate that the insulin-inhibiting effect of melatonin could be partly reversed by preincubation with the unspecific melatonin receptor antagonist luzindole as well as by the MT2-receptor-specific antagonist 4P-PDOT (4-phenyl-2-propionamidotetraline). As melatonin is known to modulate cGMP concentration via the MT2 receptor, these data indicate transmission of the melatonin effects via the cGMP transduction cascade. Molecular investigations established the presence of different types of guanylate cyclases, cGMP-specific phosphodiesterases and cyclic nucleotide-gated channels in rat insulinoma beta-cells (INS1). Moreover, variations in mRNA expression were found when comparing day and night values as well as different states of glucose metabolism. Incubation experiments provided evidence that 3-isobutyl-1-methylxanthine (IBMX)-stimulated cGMP concentrations were significantly decreased in INS1 cells exposed to melatonin for 1 hr in a dose- and time-dependent manner. This effect could also be reversed by application of luzindole and 4P-PDOT. Stimulation with 8-Br-cGMP resulted in significantly increased insulin production. In conclusion, it could be demonstrated that the melatonin receptor subtype MT2 as well as the cGMP signaling pathway are involved in mediating the insulin-inhibiting effect of melatonin.

Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krasteva, P.; Fong, J; Shikuma, N

2010-01-01

Microorganisms can switch from a planktonic, free-swimming life-style to a sessile, colonial state, called a biofilm, which confers resistance to environmental stress. Conversion between the motile and biofilm life-styles has been attributed to increased levels of the prokaryotic second messenger cyclic di-guanosine monophosphate (c-di-GMP), yet the signaling mechanisms mediating such a global switch are poorly understood. Here we show that the transcriptional regulator VpsT from Vibrio cholerae directly senses c-di-GMP to inversely control extracellular matrix production and motility, which identifies VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerizationmore » on c-di-GMP binding.« less

Temporal change in megafauna at the Rose Garden hydrothermal vent (Galapagos Rift; eastern tropical Pacific)

NASA Astrophysics Data System (ADS)

Hessler, Robert R.; Smithey, William M.; Boudrias, Michel A.; Keller, Clifford H.; Lutz, Richard A.; Childress, James J.

1988-10-01

Hydrothermal vent communities must undergo substantial temporal change because of their dynamic physical milieu. This was verified in March 1985, when the Rose Garden hydrothermal vent on the Galapagos Rift was revisited after 5 1/4 years' absence. Comparison of photographs from the two visits revealed considerable faunal change. Among the hosts to chemoautotrophic bacteria, vestimentiferans were reduced from dominance to very low numbers. The mytilid was now extremely abundant and dominated vent openings. Vesicomyids also were more abundant. In general, vent-field suspension feeders had declined; anemones were distinctly less abundant, and siphonophores and enteropneusts were virtually absent. The decline of serpulids was likely, but less obvious. Of the mobile scavengers and carnivores, both galatheids and whelks were distincly more common. These community changes appear to result from both continuing recruitment and changes in the physical milieu. While the growth of some populations could have resulted from expanding opportunities, the population of at least one, the vesicomyid, had not achieved carrying capacity in 1979, and this could have pertained to others as well. The decrease of vestimentiferans may have been caused by declining vent-water flux, a process that would favor mytilids, or more complete vent-water filtration by the mytilids themselves. The same factors also could explain the reduction of vent-field filter feeders. These observations suggest that early stages in the cycle of Galapagos-type vent communities are likely to be dominated by vestimentiferans, but that bivalves will replace them with time.

A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin.

PubMed

Popova, Dina; Jacobsson, Stig O P

2014-04-01

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity. In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vitro toxicity testing with microplate cell cultures: Impact of cell binding.

PubMed

Gülden, Michael; Schreiner, Jeannine; Seibert, Hasso

2015-06-05

In vitro generated data on toxic potencies are generally based on nominal concentrations. However, cellular and extracellular binding and elimination processes may reduce the available free fraction of a compound. Then, nominal effective concentrations do not represent appropriate measures of toxic exposure in vitro and underestimate toxic potencies. In this study it was investigated whether cell binding can affect the availability of chemicals in microplate based toxicity assays. To this end the cytotoxicity of compounds like mercury chloride, digitonin and alcohol ethoxylates, accumulated by cells via different modes, was investigated in 96-well microplate cultures with varying concentrations of Balb/c 3T3 cells. The median effective nominal concentrations of all but one of the tested compounds depended linearly from the cell concentration. Applying a previously developed equilibrium distribution model cell concentration-independent median effective extracellular concentrations and cell burdens, respectively, could be calculated. The compounds were accumulated by the cells with bioconcentration factors, BCF, between 480 and ≥ 25,000. Cell binding of the alcohol ethoxylates was correlated with their lipophilicity. The results show that significant cell binding can occur even at the small cell volume fractions (∼ 1 × 10(-5) to 3 × 10(-3) L/L) encountered in microplate assays. To what extent cell binding affects the bioavailability depends on the BCF and the cell volume fraction. EC50 measurements in the presence of at least two different cell concentrations allow for excluding or detecting significant cell binding and for determining more appropriate measures of toxic exposure in vitro like median effective extracellular (free) concentrations or cell burdens. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Good Manufacturing Practices (GMP) manufacturing of advanced therapy medicinal products: a novel tailored model for optimizing performance and estimating costs.

PubMed

Abou-El-Enein, Mohamed; Römhild, Andy; Kaiser, Daniel; Beier, Carola; Bauer, Gerhard; Volk, Hans-Dieter; Reinke, Petra

2013-03-01

Advanced therapy medicinal products (ATMP) have gained considerable attention in academia due to their therapeutic potential. Good Manufacturing Practice (GMP) principles ensure the quality and sterility of manufacturing these products. We developed a model for estimating the manufacturing costs of cell therapy products and optimizing the performance of academic GMP-facilities. The "Clean-Room Technology Assessment Technique" (CTAT) was tested prospectively in the GMP facility of BCRT, Berlin, Germany, then retrospectively in the GMP facility of the University of California-Davis, California, USA. CTAT is a two-level model: level one identifies operational (core) processes and measures their fixed costs; level two identifies production (supporting) processes and measures their variable costs. The model comprises several tools to measure and optimize performance of these processes. Manufacturing costs were itemized using adjusted micro-costing system. CTAT identified GMP activities with strong correlation to the manufacturing process of cell-based products. Building best practice standards allowed for performance improvement and elimination of human errors. The model also demonstrated the unidirectional dependencies that may exist among the core GMP activities. When compared to traditional business models, the CTAT assessment resulted in a more accurate allocation of annual expenses. The estimated expenses were used to set a fee structure for both GMP facilities. A mathematical equation was also developed to provide the final product cost. CTAT can be a useful tool in estimating accurate costs for the ATMPs manufactured in an optimized GMP process. These estimates are useful when analyzing the cost-effectiveness of these novel interventions. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

DoGMP1 from Dendrobium officinale contributes to mannose content of water-soluble polysaccharides and plays a role in salt stress response

PubMed Central

He, Chunmei; Yu, Zhenming; Teixeira da Silva, Jaime A.; Zhang, Jianxia; Liu, Xuncheng; Wang, Xiaojuan; Zhang, Xinhua; Zeng, Songjun; Wu, Kunlin; Tan, Jianwen; Ma, Guohua; Luo, Jianping; Duan, Jun

2017-01-01

GDP-mannose pyrophosphorylase (GMP) catalyzed the formation of GDP-mannose, which serves as a donor for the biosynthesis of mannose-containing polysaccharides. In this study, three GMP genes from Dendrobium officinale (i.e., DoGMPs) were cloned and analyzed. The putative 1000 bp upstream regulatory region of these DoGMPs was isolated and cis-elements were identified, which indicates their possible role in responses to abiotic stresses. The DoGMP1 protein was shown to be localized in the cytoplasm. To further study the function of the DoGMP1 gene, 35S:DoGMP1 transgenic A. thaliana plants with an enhanced expression level of DoGMP1 were generated. Transgenic plants were indistinguishable from wild-type (WT) plants in tissue culture or in soil. However, the mannose content of the extracted water-soluble polysaccharides increased 67%, 96% and 92% in transgenic lines #1, #2 and #3, respectively more than WT levels. Germination percentage of seeds from transgenic lines was higher than WT seeds and the growth of seedlings from transgenic lines was better than WT seedlings under salinity stress (150 mM NaCl). Our results provide genetic evidence for the involvement of GMP genes in the biosynthesis of mannose-containing polysaccharides and the mediation of GMP genes in the response to salt stress during seed germination and seedling growth. PMID:28176760

Differential Regulation of c-di-GMP Metabolic Enzymes by Environmental Signals Modulates Biofilm Formation in Yersinia pestis.

PubMed

Ren, Gai-Xian; Fan, Sai; Guo, Xiao-Peng; Chen, Shiyun; Sun, Yi-Cheng

2016-01-01

Cyclic diguanylate (c-di-GMP) is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs), HmsT and HmsD and one phosphodiesterase (PDE), HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD, and HmsP in Y. pestis. Biofilm formation was higher in the presence of non-lethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulate activity of DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments.

Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn

PubMed Central

Lakshminrusimha, Satyan; Wedgwood, Stephen; Czech, Lyubov; Gugino, Sylvia F.; Russell, James A.; Farrow, Kathryn N.; Steinhorn, Robin H.

2012-01-01

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O2 for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg−1·dose−1 times two doses (PPHN-loHC), and six lambs were ventilated with O2 alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O2 ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O2. We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress. PMID:22198909

Portable Microplate Analyzer with a Thermostatic Chamber Based on a Smartphone for On-site Rapid Detection.

PubMed

Wan, Zijian; Zhong, Longjie; Pan, Yuxiang; Li, Hongbo; Zou, Quchao; Su, Kaiqi; Wang, Ping

2017-01-01

A microplate method provides an efficient way to use modern detection technology. However, there are some difficulties concerning on-site detection, such as being non-portable and time-consuming. In this work, a novel portable microplate analyzer with a thermostatic chamber based on a smartphone was designed for rapid on-site detection. An analyzer with a wide-angle lens and an optical filter provides a proper environment for the microplate. A smartphone app-iPlate Monitor was used for RGB analyze of image. After a consistency experiment with a microtiter plate reader (MTPR), the normalized calibration curves were y = 0.7276x + 0.0243 (R 2 = 0.9906) and y = 0.3207x + 0.0094 (R 2 = 0.9917) with a BCA protein kit as well as y = 0.182x + 0.0134 (R 2 = 0.994) and y = 0.0674x + 0.0003 (R 2 = 0.9988) with a glucose kit. The times for obtaining the detection requirement were 15 and 10 min for the BCA protein kit and the glucose kit at 37°C; in contrast, it required more than 30 and 20 min at ambient temperature. Meanwhile, it also showed good repeatability for detections.

Phytoplankton Biomass Distribution and Identification of Productive Habitats Within the Galapagos Marine Reserve by MODIS, a Surface Acquisition System, and In-Situ Measurements

EPA Science Inventory

The Galapagos Marine Reserve (GMR) is one of the most diverse ecosystems in the world. Phytoplankton are the base of the ecosystem food chain for many higher trophic organisms, so identifying phytoplankton biomass distribution is the first step in understanding the dynamic envir...

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nygaard, Gyrid; Department of Biomedicine, University of Bergen, Bergen; Herfindal, Lars

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigatedmore » whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.« less

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

PubMed Central

Schäper, Simon; Steinchen, Wieland; Krol, Elizaveta; Altegoer, Florian; Skotnicka, Dorota; Bange, Gert; Becker, Anke

2017-01-01

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal “RxxxR” motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP–responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP–responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins. PMID:28559336

One microplate - three orogens: Alps, Dinarides, Apennines and the role of the Adriatic plate

NASA Astrophysics Data System (ADS)

Ustaszewski, Kamil; Le Breton, Eline; Balling, Philipp; Handy, Mark R.; Molli, Giancarlo; Tomljenović, Bruno

2017-04-01

The motion of the Adriatic microplate with respect to the Eurasian and African plates is responsible for the Mesozoic to present tectonic evolution of the Alps, Carpathians, the Dinarides and Hellenides as well as the Apennines. The classical approach for reconstructing plate motions is to assume that tectonic plates are rigid, then apply Euler's theorem to describe their rotation on an ideally spherical Earth by stepwise restorations of magnetic anomalies and fracture zones in oceanic basins. However, this approach is inadequate for reconstructing the motion of Mediterranean microplates like Adria, which, at present, is surrounded by convergent margins and whose oceanic portions have by now been entirely subducted. Most constraints on the motion of the Adriatic microplate come either from palaeomagnetics or from shortening estimates in the Alps, i.e., its northern margin. This approach renders plate tectonic reconstructions prone to numerous errors, yielding inadmissible misfits in the Ionian Sea between southern Italy and northern Greece. At the same time, Adria's western and eastern margins in the Apennines and in the Dinarides have hitherto not been appropriately considered for improving constraints on the motion of Adria. This presentation presents new results of ongoing collaborative research that aims at improving the relative motion path for the Adriatic microplate for the Cenozoic by additionally quantifying and restoring the amount of shortening and extension in a set of geophysical-geological transects from the Tyrrhenian Sea, the Apennines and the Dinarides. Already now, our approach yields an improved motion path for the Adriatic microplate for the last 20 Ma, which minimizes misfits in previous reconstructions. The currently largest challenge in our reconstructions is to reconcile amount and age of shortening in the Dinarides fold-and-thrust belt. For one thing, we see good agreement between the cross-sectional length of subducted material (c. 135 km

Mangiferin Prevents Guinea Pig Tracheal Contraction via Activation of the Nitric Oxide-Cyclic GMP Pathway

PubMed Central

Vieira, Aline B.; Coelho, Luciana P.; Insuela, Daniella B. R.; Carvalho, Vinicius F.; dos Santos, Marcelo H.; Silva, Patricia MR.; Martins, Marco A.

2013-01-01

Previous studies have described the antispasmodic effect of mangiferin, a natural glucoside xanthone (2-C-β-Dgluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) that is present in mango trees and other plants, but its mechanism of action remains unknown. The aim of this study was to examine the potential contribution of the nitric oxide-cyclic GMP pathway to the antispasmodic effect of mangiferin on isolated tracheal rings preparations. The functional effect of mangiferin on allergic and non-allergic contraction of guinea pig tracheal rings was assessed in conventional organ baths. Cultured tracheal rings were exposed to mangiferin or vehicle, and nitric oxide synthase (NOS) 3 and cyclic GMP (cGMP) levels were quantified using western blotting and enzyme immunoassays, respectively. Mangiferin (0.1–10 µM) inhibited tracheal contractions induced by distinct stimuli, such as allergen, histamine, 5-hydroxytryptamine or carbachol, in a concentration-dependent manner. Mangiferin also caused marked relaxation of tracheal rings that were precontracted by carbachol, suggesting that it has both anti-contraction and relaxant properties that are prevented by removing the epithelium. The effect of mangiferin was inhibited by the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (100 µM), and the soluble guanylate cyclase inhibitor, 1H-[1], [2], [4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM), but not the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536) (100 µM). The antispasmodic effect of mangiferin was also sensitive to K+ channel blockers, such as tetraethylammonium (TEA), glibenclamide and apamin. Furthermore, mangiferin inhibited Ca2+-induced contractions in K+ (60 mM)-depolarised tracheal rings preparations. In addition, mangiferin increased NOS3 protein levels and cGMP intracellular levels in cultured tracheal rings. Finally, mangiferin-induced increase in cGMP levels was abrogated by co-incubation with either ODQ or L

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway.

PubMed

Sandoval, Alejandro; Duran, Paz; Gandini, María A; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

2017-09-01

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca 2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca V 1.3L-type Ca 2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca V 1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Ca V α 1 ion-conducting subunit of the Ca V 1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca 2+ macroscopic currents and impair insulin release stimulated with high K + . In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca V 1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca V α 1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca V 1.3 channels and contribute to regulate insulin secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of a GMP training of milkers in dairy goat farms in São Paulo, Brazil.

PubMed

Tavolaro, Paula; Oliveira, Carlos A F

2006-02-01

A good manufacturing practices (GMP) training course was applied for goat milkers and evaluated using microbiological analysis in milk before and after training. Milkers from three dairy goat farms located in São Paulo, Brazil, were submitted to a one-hour course on GMP and recommended guidelines for milking. Samples of raw milk were collected before and one to two months after training, and analysed for aerobic mesophilic, psychrotrophic, coliform, Staphylococcus aureus and Salmonella spp counts. Only mesophilic counts decreased (p < 0.05) after training in two of three farms studied. Although important to assess the overall quality of milk, microbiological parameters should not be used alone for the evaluation of GMP effectiveness for goat milkers.

LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis

PubMed Central

Li, Weihui; He, Zheng-Guo

2012-01-01

In a bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP)/transcription factor binding screen, we identified Mycobacterium smegmatis Ms6479 as the first c-di-GMP-responsive transcriptional factor in mycobacteria. Ms6479 could specifically bind with c-di-GMP and recognize the promoters of 37 lipid transport and metabolism genes. c-di-GMP could enhance the ability of Ms6479 to bind to its target DNA. Furthermore, our results establish Ms6479 as a global activator that positively regulates the expression of diverse target genes. Overexpression of Ms6479 in M. smegmatis significantly reduced the permeability of the cell wall to crystal violet and increased mycobacterial resistance to anti-tuberculosis antibiotics. Interestingly, Ms6479 lacks the previously reported c-di-GMP binding motifs. Our findings introduce Ms6479 (here designated LtmA for lipid transport and metabolism activator) as a new c-di-GMP-responsive regulator. PMID:23047950

Sources of volatiles in basalts from the Galapagos Archipelago: deep and shallow evidence

NASA Astrophysics Data System (ADS)

Peterson, M. E.; Saal, A. E.; Hauri, E. H.; Werner, R.; Hauff, S. F.; Kurz, M. D.; Geist, D.; Harpp, K. S.

2010-12-01

The study of volatiles (H2O, CO2, F, S, and Cl) is important because volatiles assert a strong influence on mantle melting and magma crystallization, as well as on the viscosity and rheology of the mantle. Despite this importance, there have been a minimal number of volatile studies done on magmas from the four main mantle sources that define the end member compositions of the Galapagos lavas. For this reason, we here present new volatile concentrations of 89 submarine glass chips from dredges collected across the archipelago during the SONNE SO158, PLUM02, AHA-NEMO, and DRIFT04 cruises. All samples, with the exception of six, were collected at depths greater than 1000m. Major elements (E-probe), and volatile and trace elements (SIMS), are analyzed on the same glass chip, using 4 chips per sample, to better represent natural and analytical variation. Trace element contents reveal three main compositional groups: an enriched group typical of OIB, a group with intermediate compositions, and a group with a depleted trace element composition similar to MORB. The absolute ranges of volatile contents for all three compositional groups are .098-1.15wt% for H2O, 10.7-193.7 ppm for CO2, 61.4-806.5 ppm for F, 715.8-1599.2 ppm for S and 3.8-493.3 for Cl. The effect of degassing, sulfide saturation and assimilation of hydrothermally altered material must be understood before using the volatile content of submarine glasses to establish the primary volatile concentration of basalts and their mantle sources. CO2 has a low solubility in basaltic melts causing it to extensively degas. Based on the CO2/Nb ratio, we estimate the extent of degassing for the Galapagos lavas to range from approximately undegassed to 90% degassed. We demonstrate that 98% of the samples are sulfur undersaturated. Therefore, sulfur will behave as a moderately incompatible element during magmatic processes. Finally, we evaluate the effect of assimilation of hydrothermally altered material on the volatile

The role of cGMP signalling in regulating life cycle progression of Plasmodium.

PubMed

Hopp, Christine S; Bowyer, Paul W; Baker, David A

2012-08-01

The 3'-5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regulate cyclic nucleotide levels are presented. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A simple and rapid microplate assay for glycoprotein-processing glycosidases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, M.S.; Zwolshen, J.H.; Harry, B.S.

1989-08-15

A simple and convenient microplate assay for glycosidases involved in the glycoprotein-processing reactions is described. The assay is based on specific binding of high-mannose-type oligosaccharide substrates to concanavalin A-Sepharose, while monosaccharides liberated by enzymatic hydrolysis do not bind to concanavalin A-Sepharose. By the use of radiolabeled substrates (( 3H)glucose for glucosidases and (3H)mannose for mannosidases), the radioactivity in the liberated monosaccharides can be determined as a measure of the enzymatic activity. This principle was employed earlier for developing assays for glycosidases previously reported. These authors have reported the separation of substrate from the product by concanavalin A-Sepharose column chromatography. Thismore » procedure is handicapped by the fact that it cannot be used for a large number of samples and is time consuming. We have simplified this procedure and adapted it to the use of a microplate (96-well plate). This would help in processing a large number of samples in a short time. In this report we show that the assay is comparable to the column assay previously reported. It is linear with time and enzyme concentration and shows expected kinetics with castanospermine, a known inhibitor of alpha-glucosidase I.« less

Microbial Populations in Extreme Environments: Investigations and Characterizations of the Microbiology and Geochemistry of Galapagos Island Fumaroles

NASA Astrophysics Data System (ADS)

Mayhew, L. E.; Childers, S. E.; Geist, D.

2005-12-01

The extreme physiochemical conditions, insularity, and wide range in ages of fumaroles of the Galapagos Islands provide an excellent opportunity to explore for novel microorganisms and to study life in extreme environments. This is the first study that measures microbial diversity of Galapagos fumaroles. Forty-seven samples were collected from six distinct fumarole fields on Sierra Negra and Alcedo volcanoes. Vulcan Chico, on Sierra Negra, was activated during the last eruption in 1979. Two of the other fumarole fields on Sierra Negra are associated with a long-lived fault system on the caldera floor and are therefore likely to be significantly older. The fault-associated fumaroles have widespread alteration haloes (up to 100 m in diameter) and thick deposits of native sulfur. The most vigorous of the fumarole fields on Alcedo activated in late 1993 to early 1994. The second fumarole field on Alcedo is associated with a recently extinct geyser and the third is located on a rhyolite vent. A diversity of colors was observed in the substrates at all of the fumarole fields and some may be the result of microbial activity. Collection sites were chosen on the basis of temperature and the variations in the substrate in order to obtain samples from a variety of environments. Temperatures at sample sites range from 25.0 to 178.5° C, and pH from 0 to 6. The material collected varies between sites and includes crystalline sulfur deposits, clay, sandy and rocky soils, and microbial mats. Substrate material is characterized by powder x-ray diffractometry and scanning electron microscopy and gases collected from five of the fumarole fields are being analyzed to test for chemical controls on the microbial populations. Genomic DNA is being extracted from all of the samples. Primers for Bacteria and Archaea are used for PCR amplification of the 16S rRNA gene. To date, 22 of 37 processed samples have amplifiable DNA. Microbial diversity of samples possessing amplifiable DNA is

Aging has the opposite effect on cAMP and cGMP circadian variations in rat Leydig cells.

PubMed

Baburski, Aleksandar Z; Sokanovic, Srdjan J; Andric, Silvana A; Kostic, Tatjana S

2017-05-01

The Leydig cell physiology displays a circadian rhythm driven by a complex interaction of the reproductive axis hormones and circadian system. The final output of this regulatory process is circadian pattern of steroidogenic genes expression and testosterone production. Aging gradually decreases robustness of rhythmic testosterone secretion without change in pattern of LH secretion. Here, we analyzed effect of aging on circadian variation of cAMP and cGMP signaling in Leydig cells. Results showed opposite effect of aging on cAMP and cGMP daily variation. Reduced amplitude of cAMP circadian oscillation was probably associated with changed expression of genes involved in cAMP production (increased circadian pattern of Adcy7, Adcy9, Adcy10 and decreased Adcy3); cAMP degradation (increased Pde4a, decreased Pde8b, canceled rhythm of Pde4d, completely reversed circadian pattern of Pde7b and Pde8a); and circadian expression of protein kinase A subunits (Prkac/PRKAC and Prkar2a). Aging stimulates expression of genes responsible for cGMP production (Nos2, Gucy1a3 and Gucy1b3/GUCYB3) and degradation (Pde5a, Pde6a and Pde6h) but the overall net effect is elevation of cGMP circadian oscillations in Leydig cells. In addition, the expression of cGMP-dependent kinase, Prkg1/PRKG1 is up-regulated. It seems that aging potentiate cGMP- and reduce cAMP-signaling in Leydig cells. Since both signaling pathways affect testosterone production and clockwork in the cells, further insights into these signaling pathways will help to unravel disorders linked to the circadian timing system, aging and reproduction.

Hypergravity differentially modulates cGMP efflux in human melanocytic cells stimulated by nitric oxide and natriuretic peptides

NASA Astrophysics Data System (ADS)

Ivanova, K.; Stieber, C.; Lambers, B.; Block, I.; Krieg, R.; Wellmann, A.; Gerzer, R.

Nitric oxide NO plays a key role in many patho physiologic processes including inflammation and skin cancer The diverse cellular effects of NO are mainly mediated by activation of the soluble guanylyl cyclase sGC isoform that leads to increases in intracellular cGMP levels whereas the membrane-bound isoforms serve as receptors for natriuretic peptides e g ANP In human skin epidermal melanocytes represent the principal cells for skin pigmentation by synthesizing the pigment melanin Melanin acts as a scavenger for free radicals that may arise during metabolic stress as a result of potentially harmful effects of the environment In previous studies we found that long-term exposure to hypergravity stimulated cGMP efflux in normal human melanocytes NHMs and non-metastatic melanoma cells at least partly by an enhanced expression of the multidrug resistance proteins MRP and cGMP transporters MRP4 5 The present study investigated whether hypergravity generated by centrifugal acceleration may modulate the cGMP efflux in NO-stimulated NHMs and melanoma cells MCs with different metastatic potential The NONOates PAPA-NO and DETA-NO were used as direct NO donors for cell stimulation In the presence of 0 1 mM DETA-NO t 1 2 sim 20 h long-term application of hypergravity up to 5 g for 24 h reduced intracellular cGMP levels by stimulating cGMP efflux in NHMs and non-metastatic MCs in comparison to 1 g whereas exposure to 5 g for 6 h in the presence of 0 1 mM PAPA-NO t 1 2 sim 30 min was not effective The hypergravity-stimulated

Renal Integrin-Linked Kinase Depletion Induces Kidney cGMP-Axis Upregulation: Consequences on Basal and Acutely Damaged Renal Function

PubMed Central

Cano-Peñalver, José Luis; Griera, Mercedes; García-Jerez, Andrea; Hatem-Vaquero, Marco; Ruiz-Torres, María Piedad; Rodríguez-Puyol, Diego; de Frutos, Sergio; Rodríguez-Puyol, Manuel

2015-01-01

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and produces cGMP, which activates cGMP-dependent protein kinases (PKG) and is hydrolyzed by specific phosphodiesterases (PDE). The vasodilatory and cytoprotective capacity of cGMP-axis activation results in a therapeutic strategy for several pathologies. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix and intracellular signaling pathways, may modulate the expression and functionality of the cGMP-axis–related proteins. We introduce ILK as a novel modulator in renal homeostasis as well as a potential target for cisplatin (CIS)-induced acute kidney injury (AKI) improvement. We used an adult mice model of depletion of ILK (cKD-ILK), which showed basal increase of sGC and PKG expressions and activities in renal cortex when compared with wildtype (WT) littermates. Twenty-four h activation of sGC activation with NO enhanced the filtration rate in cKD-ILK. During AKI, cKD-ILK maintained the cGMP-axis upregulation with consequent filtration rates enhancement and ameliorated CIS-dependent tubular epithelial-to-mesenchymal transition and inflammation and markers. To emphasize the role of cGMP-axis upregulation due to ILK depletion, we modulated the cGMP axis under AKI in vivo and in renal cultured cells. A suboptimal dose of the PDE inhibitor ZAP enhanced the beneficial effects of the ILK depletion in AKI mice. On the other hand, CIS increased contractility-related events in cultured glomerular mesangial cells and necrosis rates in cultured tubular cells; ILK depletion protected the cells while sGC blockade with ODQ fully recovered the damage. PMID:26562149

Fumarole/plume and diffuse CO2 emission from Sierra Negra caldera, Galapagos archipelago

NASA Astrophysics Data System (ADS)

Padrón, Eleazar; Hernández, Pedro A.; Pérez, Nemesio M.; Toulkeridis, Theofilos; Melián, Gladys; Barrancos, José; Virgili, Giorgio; Sumino, Hirochika; Notsu, Kenji

2012-08-01

Measurements of visible and diffuse gas emission were conducted in 2006 at the summit of Sierra Negra volcano, Galapagos, with the aim to better characterize degassing after the 2005 eruption. A total SO2 emission of 11 ± 2 t day-1 was derived from miniature differential optical absorption spectrometer (mini-DOAS) ground-based measurements of the plume emanating from the Mini Azufral fumarolic area, the most important site of visible degassing at Sierra Negra volcano. Using a portable multigas system, the H2S/SO2, CO2/SO2, and H2O/SO2 molar ratios in the Mina Azufral plume emissions were found to be 0.41, 52.2, and 867.9, respectively. The corresponding H2O, CO2, and H2S emission rates were 562, 394, and 3 t day-1, respectively. The total output of diffuse CO2 emissions from the summit of Sierra Negra volcano was 990 ± 85 t day-1, with 605 t day-1 being released by a deep source. The diffuse-to-plume CO2 emission ratio was about 1.5. Mina Azufral fumaroles released gasses containing 73.6 mol% of H2O; the main noncondensable components amounted to 97.4 mol% CO2, 1.5 mol% SO2, 0.6 mol% H2S, and 0.35 mol% N2. The higher H2S/SO2 ratio values found in 2006 as compared to those reported before the 2005 eruption reveal a significant hydrothermal contribution to the fumarolic emissions. 3He/4He ratios measured at Mina Azufral fumarolic discharges showed values of 17.88 ± 0.25 R A , indicating a mid-ocean ridge basalts (MORB) and a Galapagos plume contribution of 53 and 47 %, respectively.

Now that you want to take your HIV/AIDS vaccine/biological product research concept into the clinic: what are the "cGMP"?

PubMed

Sheets, Rebecca L; Rangavajhula, Vijaya; Pullen, Jeffrey K; Butler, Chris; Mehra, Vijay; Shapiro, Stuart; Pensiero, Michael

2015-04-08

The Division of AIDS Vaccine Research Program funds the discovery and development of HIV/AIDS vaccine candidates. Basic researchers, having discovered a potential vaccine in the laboratory, next want to take that candidate into the clinic to test the concept in humans, to see if it translates. Many of them have heard of "cGMP" and know that they are supposed to make a "GMP product" to take into the clinic, but often they are not very familiar with what "cGMP" means and why these good practices are so important. As members of the Vaccine Translational Research Branch, we frequently get asked "can't we use the material we made in the lab in the clinic?" or "aren't Phase 1 studies exempt from cGMP?" Over the years, we have had many experiences where researchers or their selected contract manufacturing organizations have not applied an appropriate degree of compliance with cGMP suitable for the clinical phase of development. We share some of these experiences and the lessons learned, along with explaining the importance of cGMP, just what cGMP means, and what they can assure, in an effort to de-mystify this subject and facilitate the rapid and safe translational development of HIV vaccines. Published by Elsevier Ltd.

Alps to Apennines zircon roller coaster along the Adria microplate margin.

PubMed

Jacobs, J; Paoli, G; Rocchi, S; Ksienzyk, A K; Sirevaag, H; Elburg, M A

2018-02-09

We have traced the particle path of high-pressure metasedimentary rocks on Elba Island, Northern Apennines, with the help of a U-Pb-Hf detrital zircon study. One quarter of the analysed zircons are surprisingly young, 41-30 Ma, with a main age peak at ca. 32 Ma, indicating an unexpected early Oligocene maximum deposition age. These Oligocene ages with negative εHf indicate a volcanic source region in the central-southern Alps. Though young by geological means, these zircons record an extraordinary geodynamic history. They originated in a volcanic arc, during the convergence/collision of the the Adria microplate with Europe from ca. 65 to 30 Ma. Thereafter, the Oligocene zircons travelled ca. 400 km southward along the Adria margin and the accretionary prism to present-day Tuscany, where they were subducted to depths of at least 40 km. Shortly thereafter, they were brought to the surface again in the wake of hinge roll back of the Apennine subduction zone and the resulting rapid extensional exhumation. Such a zircon roller coaster requires a microplate that has back-to-back subduction zones with opposing polarities on two sides.

Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDE6alpha '.

PubMed

Granovsky, A E; Artemyev, N O

2000-12-29

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (Pgamma). Chimeric proteins between cone PDE6alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pgamma. Substitution of the segment PDE5-(773-820) by the corresponding PDE6alpha'-(737-784) sequence in the wild-type PDE5 or in a PDE5/PDE6alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pgamma. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pgamma-binding region, PDE6alpha'-(750-760), revealed PDE6alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pgamma. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met(758) and Gln(752) directly bind Pgamma. A model of the PDE6 catalytic site shows that PDE6alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pgamma to Met(758) would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

PubMed

Schwarz, Kátia R L; de Castro, Fernanda C; Schefer, Letícia; Botigelli, Ramon C; Paschoal, Daniela M; Fernandes, Hugo; Leal, Cláudia L V

2018-01-01

This study aimed to determine the influence of cyclic guanosine 3'5'-monophosphate (cGMP) and cGMP-dependent kinase (PKG) during in vitro maturation (IVM) on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs), and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII), cGMP levels, lipid content in oocytes (OO), transcript abundance for genes involved in lipolysis (ATGL) and lipid droplets (PLIN2) in cumulus cells (CC) and OO, and presence of phosphorylated (active) hormone sensitive lipase (HSLser563) in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme) with 10-5M sildenafil (SDF) during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05) and did not affect on maturation rate (84.3±6.4% MII). Fetal calf serum (FCS) in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA) + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05). FCS increased lipid content in OO (40.1 FI, p<0.05) compared to BSA (34.6 FI), while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05). PKG inhibitor (KT5823) reversed this effect (38.9 FI, p<0.05). ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI) compared to its use in either or none of the culture periods (34.2 FI, p<0.05). Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

Fabrication of single phase 2D homologous perovskite microplates by mechanical exfoliation

NASA Astrophysics Data System (ADS)

Li, Junze; Wang, Jun; Zhang, Yingjun; Wang, Haizhen; Lin, Gaoming; Xiong, Xuan; Zhou, Weihang; Luo, Hongmei; Li, Dehui

2018-04-01

The two-dimensional (2D) Ruddlesden-Popper type perovskites have attracted intensive interest for their great environmental stability and various potential optoelectronic applications. Fundamental understanding of the photophysical and electronic properties of the 2D perovskites with pure single phase is essential for improving the performance of the optoelectronic devices and designing devices with new architectures. Investigating the optical and electronic properties of these materials with pure single phase is required to obtain pure single phase 2D perovskites. Here, we report on an alternative approach to fabricate (C4H9NH3)2(CH3NH3) n-1Pb n I3n+1 microplates with pure single n-number perovskite phase for n  >  2 by mechanical exfoliation. Micro-photoluminescence and absorption spectroscopy studies reveal that the as-synthesized 2D perovskite plates for n  >  2 are comprised by dominant n-number phase and small inclusions of hybrid perovskite phases with different n values, which is supported by excitation power dependent photoluminescence. By mechanical exfoliation method, 2D perovskite microplates with the thickness of around 20 nm are obtained, which surprisingly have single n-number perovskite phase for n  =  2-5. In addition, we have demonstrated that the exfoliated 2D perovskite microplates can be integrated with other 2D layered materials such as boron nitride, and are able to be transferred to prefabricated electrodes for photodetections. Our studies not only provide a strategy to prepare 2D perovskites with a single n-number perovskite phase allowing us to extract the basic optical and electronic parameters of pure phase perovskites, but also demonstrate the possibility to integrate the 2D perovskites with other 2D layered materials to extend the device’s functionalities.

Immunocytology on microwave-fixed cells reveals rapid and agonist-specific changes in subcellular accumulation patterns for cAMP or cGMP.

PubMed Central

Barsony, J; Marx, S J

1990-01-01

We developed a method for cAMP and cGMP immunocytology based upon fixation by microwave irradiation. Fixation by microwave irradiation prevented three problems found with other fixation methods: nucleotide loss from cells, nucleotide diffusion within cells, and chemical modification of immunologic epitopes. Six agonists (four that stimulate adenylate cyclase and two that stimulate guanylate cyclase) produced cAMP or cGMP accumulation patterns that were agonist-specific, dose-dependent, detectable at physiologic concentrations of hormone, and time-dependent within 15 sec to 30 min. cAMP accumulation after 1 mM forskolin was greatest in the nucleus. Isoproterenol, prostaglandin E2, or calcitonin caused initial accumulation of cAMP along the plasma membrane, but later accumulation was greater in the cytoplasm. With calcitonin the later accumulation of cAMP was selectively perinuclear and along the nuclear membrane. Sodium nitroprusside stimulated cGMP accumulation diffusely throughout the cytoplasm. Atrial natriuretic peptide initiated cGMP accumulation near the plasma membrane, and cGMP accumulation moved from there into the cytoplasm. In conclusion, microwave irradiation preserved cell structure and allowed visualization of expected as well as unsuspected changes in intracellular accumulation patterns of cAMP and cGMP. Images PMID:2153973

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum.

PubMed

Oguntimein, Gbekeloluwa B; Rodriguez, Miguel; Dumitrache, Alexandru; Shollenberger, Todd; Decker, Stephen R; Davison, Brian H; Brown, Steven D

2018-02-01

To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum

DOE PAGES

Oguntimein, Gbekeloluwa B.; Rodriguez, Jr., Miguel; Dumitrache, Alexandru; ...

2017-11-09

Here, to develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δ hpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentationsmore » when compared to the Δ hpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.« less

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oguntimein, Gbekeloluwa B.; Rodriguez, Jr., Miguel; Dumitrache, Alexandru

Here, to develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δ hpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentationsmore » when compared to the Δ hpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.« less

Molecular Rotors for Universal Quantitation of Nanoscale Hydrophobic Interfaces in Microplate Format.

PubMed

Bisso, Paul W; Tai, Michelle; Katepalli, Hari; Bertrand, Nicolas; Blankschtein, Daniel; Langer, Robert

2018-01-10

Hydrophobic self-assembly pairs diverse chemical precursors and simple formulation processes to access a vast array of functional colloids. Exploration of this design space, however, is stymied by lack of broadly general, high-throughput colloid characterization tools. Here, we show that a narrow structural subset of fluorescent, zwitterionic molecular rotors, dialkylaminostilbazolium sulfonates [DASS] with intermediate-length alkyl tails, fills this major analytical void by quantitatively sensing hydrophobic interfaces in microplate format. DASS dyes supersede existing interfacial probes by avoiding off-target fluorogenic interactions and dye aggregation while preserving hydrophobic partitioning strength. To illustrate the generality of this approach, we demonstrate (i) a microplate-based technique for measuring mass concentration of small (20-200 nm), dilute (submicrogram sensitivity) drug delivery nanoparticles; (ii) elimination of particle size, surfactant chemistry, and throughput constraints on quantifying the complex surfactant/metal oxide adsorption isotherms critical for environmental remediation and enhanced oil recovery; and (iii) more reliable self-assembly onset quantitation for chemically and structurally distinct amphiphiles. These methods could streamline the development of nanotechnologies for a broad range of applications.

Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitation.

PubMed

Hu, Junjie; Liu, Fei; Ju, Huangxian

2015-04-21

A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

PubMed Central

2011-01-01

Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic

Reduced cGMP levels in CSF of AD patients correlate with severity of dementia and current depression.

PubMed

Hesse, Raphael; Lausser, Ludwig; Gummert, Pauline; Schmid, Florian; Wahler, Anke; Schnack, Cathrin; Kroker, Katja S; Otto, Markus; Tumani, Hayrettin; Kestler, Hans A; Rosenbrock, Holger; von Arnim, Christine A F

2017-03-09

Alzheimer's disease (AD) is a neurodegenerative disorder, primarily affecting memory. That disorder is thought to be a consequence of neuronal network disturbances and synapse loss. Decline in cognitive function is associated with a high burden of neuropsychiatric symptoms (NPSs) such as depression. The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are essential second messengers that play a crucial role in memory processing as well as synaptic plasticity and are potential therapeutic targets. Biomarkers that are able to monitor potential treatment effects and that reflect the underlying pathology are of crucial interest. In this study, we measured cGMP and cAMP in cerebrospinal fluid (CSF) in a cohort of 133 subjects including 68 AD patients and 65 control subjects. To address the association with disease progression we correlated cognitive status with cyclic nucleotide levels. Because a high burden of NPSs is associated with decrease in cognitive function, we performed an exhaustive evaluation of AD-relevant marker combinations in a depressive subgroup. We show that cGMP, but not cAMP, levels in the CSF of AD patients are significantly reduced compared with the control group. Reduced cGMP levels in AD patients correlate with memory impairment based on Mini-Mental State Examination score (r = 0.17, p = 0.048) and tau as a marker of neurodegeneration (r = -0.28, p = 0.001). Moreover, we were able to show that AD patients suffering from current depression show reduced cGMP levels (p = 0.07) and exhibit a higher degree of cognitive impairment than non-depressed AD patients. These results provide further evidence for an involvement of cGMP in AD pathogenesis and accompanying co-morbidities, and may contribute to elucidating synaptic plasticity alterations during disease progression.

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

PubMed Central

Solano, Cristina; García, Begoña; Latasa, Cristina; Toledo-Arana, Alejandro; Zorraquino, Violeta; Valle, Jaione; Casals, Joan; Pedroso, Enrique; Lasa, Iñigo

2009-01-01

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3′-5′-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable. PMID:19416883

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

PubMed Central

Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

2015-01-01

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

PubMed

Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

2015-09-08

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Development, optimization and validation of a rapid colorimetric microplate bioassay for neomycin sulfate in pharmaceutical drug products.

PubMed

Francisco, Fabiane Lacerda; Saviano, Alessandro Morais; Pinto, Terezinha de Jesus Andreoli; Lourenço, Felipe Rebello

2014-08-01

Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite their limitations, microbiological assays are widely employed to determine antibiotic potency of pharmaceutical dosage forms, since they provide a measure of biological activity. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and neomycin concentration. The optimized bioassay method was validated by the assessment of linearity (range 3.0 to 5.0μg/mL, r=0.998 and 0.994 for standard and sample curves, respectively), precision (relative standard deviation (RSD) of 2.8% and 4.0 for repeatability and intermediate precision, respectively), accuracy (mean recovery=100.2%) and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assay and rapid colorimetric microplate bioassay. In addition, microplate bioassay had advantages concerning the sensitivity of response, time of incubation, and amount of culture medium and solutions required. Copyright © 2014 Elsevier B.V. All rights reserved.

Derivation of vascular endothelial cells from human embryonic stem cells under GMP-compliant conditions: towards clinical studies in ischaemic disease.

PubMed

Kaupisch, A; Kennedy, L; Stelmanis, V; Tye, B; Kane, N M; Mountford, J C; Courtney, A; Baker, A H

2012-10-01

Revascularisation of ischaemic tissue remains an area of substantial unmet clinical need in cardiovascular disease. Strategies to induce therapeutic angiogenesis are therefore attractive. Our recent focus has been on human embryonic stem cell (hESC) strategies since hESC can be maintained in a pluripotent state or differentiated into any desired cell type, including endothelial cells (EC), under defined differentiation culture conditions. We recently published a protocol for non-good manufacturing practice (GMP) feeder- and serum-free hESC-EC-directed monolayer differentiation to vascular EC demonstrating the potential to generate hESC-derived EC in a GMP-compliant manner suitable for use in clinical trials. In this study we modified that laboratory protocol to GMP compliance. EC production was confirmed by flow cytometry, qRT-PCR and production of vascular structures in Matrigel®, yielding approximately 30 % mature VE-cadherin(+)/PECAM-1(+) cells using the GMP-compliant hESC line RC13. In conclusion, we have successfully demonstrated the production of vascular EC under GMP-compliant conditions suitable for clinical evaluation.

The role of high-pressure metamorphic rocks in collisional processes with a microplate involved

NASA Astrophysics Data System (ADS)

Massonne, Hans-Joachim

2013-04-01

High-pressure (HP: p >10 kbar) rocks, especially easily recognizable eclogite lenses, are markers of a collision of major continental plates in Phanerozoic times, but how about a collison with a microplate? To answer this question, petrological studies were undertaken on garnet-bearing metamorphic rocks. Especially pressure (P) - temperature (T) pseudosections were calculated to reconstruct the P-T path of such rocks. Areas in the Andes and their eastern foreland (Argentina, Ecuador) and the Carboniferous realm of western and central Europe were studied, where microplates either had collided with the South American plate or had been located between colliding Laurussia and Gondwana. The following features resulted: (1) laterally extended zones of HP rocks mark collisional sutures, but eclogite lenses are virtually absent; (2) these HP rocks are dominantly metapelites which have experienced pressures as high as 1.2 to 1.5 GPa; (3) these pressures were reached between 450 to 550°C, followed by a P release at increasing T; (4) the T peak in the range of 500-650°C at P between 0.5 and 0.8 GPa is often characterized by the blastesis of plagioclase occasionally together with staurolite; (5) this event was pre-dated by two deformational events. The second deformation occurred during the early exhumation. Considering the regional geology of the study areas, these features are interpreted as follows: Prior to the collision a basin, extending over at most some hundred kilometers perpendicular to the basin axis, existed between the colliding microplate and the major plate. Eventually, a rifting event had caused the separation of the microplate from the major continental plate. The basin, which was opened by this event and filled with sediments, could have developed further to a back-arc basin. Then, the extensional regime turned to a compressional one. During compression the basin sediments on top of thinned continental crust were buried beneath the overriding major plate

A synthetic cGMP-sensitive gene switch providing Viagra(®)-controlled gene expression in mammalian cells and mice.

PubMed

Kim, Taeuk; Folcher, Marc; Charpin-El Hamri, Ghislaine; Fussenegger, Martin

2015-05-01

Cyclic guanosine monophosphate (cGMP) is a universal second messenger that is synthesized from guanosine triphosphate (GTP) by guanylyl cyclases (GCs) and hydrolyzed into guanosine monophosphate (GMP) by phosphodiesterases (PDEs). Small-molecule drugs that induce high cGMP levels in specialized tissues by boosting GC activity or inhibiting PDE activity have become the predominant treatment strategy for a wide range of medical conditions, including congestive heart failure, pulmonary hypertension, atherosclerosis-based claudication and erectile dysfunction. By fusing the cGMP receptor protein (CRP) of Rhodospirillum centenum to the Herpes simplex-derived transactivation domain VP16, we created a novel synthetic mammalian cGMP-sensing transcription factor (GTA) that activates synthetic promoters (PGTA) containing newly identified GTA-specific operator sites in a concentration-dependent manner. In cell lines expressing endogenous natriuretic peptide receptor A (NPR-A) (HeLa), GTA/PGTA-driven transgene expression was induced by B-type natriuretic peptide (BNP; Nesiritide(®)) in a concentration-dependent manner, which activated NPR-A׳s intracellular GC domain and triggered a corresponding cGMP surge. Ectopic expression of NPR-A in NPR-A-negative cell lines (HEK-293T) produced high cGMP levels and mediated maximum GTA/PGTA-driven transgene expression, which was suppressed by co-expression of PDEs (PDE-3A, PDE-5A and PDE-9A) and was re-triggered by the corresponding PDE inhibitor drugs (Pletal(®), Perfan(®), Primacor(®) (PDE-3A), Viagra(®), Levitra(®), Cialis(®) (PDE-5A) and BAY73-6691 (PDE-9A)). Mice implanted with microencapsulated designer cells co-expressing the GTA/PGTA device with NPR-A and PDE-5A showed control of blood SEAP levels through administration of sildenafil (Viagra(®)). Designer cells engineered for PDE inhibitor-modulated transgene expression may provide a cell-based PDE-targeting drug discovery platform and enable drug-adjusted gene- and cell

Distinct phosphodiesterase 5A-containing compartments allow selective regulation of cGMP-dependent signalling in human arterial smooth muscle cells.

PubMed

Wilson, Lindsay S; Guo, Manhong; Umana, M Bibiana; Maurice, Donald H

2017-08-01

Cyclic GMP (cGMP) translates and integrates much of the information encoded by nitric oxide (NO · ) and several natriuretic peptides, including the atrial natriuretic peptide (ANP). Previously, we reported that integration of a cGMP-specific cyclic nucleotide phosphodiesterase, namely phosphodiesterase 5A (PDE5A), into a protein kinase G (PKG)- and inositol-1,4,5-trisphosphate receptor (IP 3 R)-containing endoplasmic reticulum (ER) signalosome allows localized control of PDE5A activity and of PKG-dependent inhibition of IP 3 -mediated release of ER Ca 2+ in human platelets. Herein, we report that PDE5A integrates into an analogous signalosome in human arterial smooth muscle cells (HASMC), wherein it regulates muscarinic agonist-dependent Ca 2+ release and is activated selectively by PKG-dependent phosphorylation. In addition, we report that PDE5A also regulates HASMC functions via events independent of PKG, but rather through actions coordinated by competitive cGMP-mediated inhibition of cAMP hydrolysis by the so-called cGMP-inhibited cAMP PDE, namely phosphodiesterase 3A (PDE3A). Indeed, we show that ANP increases both cGMP and cAMP levels in HASMC and promotes phosphorylation of vasodilator-stimulated phospho-protein (VASP) at each the PKG and PKA phospho-acceptor sites. Since selective inhibition of PDE5 decreased DNA synthesis and chemotaxis of HASMC, and that PDE3A knockdown obviated these effects, our findings are consistent with a role for a PDE5A-PDE3A-PKA axis in their regulation. Our findings provide insight into the existence of distinct "pools" of PDE5A in HASMC and support the idea that these discrete compartments regulate distinct cGMP-dependent events. As a corollary, we suggest that it may be possible to target these distinct PDE5A-regulated pools and in so-doing differentially impact selected cGMP-regulated functions in these cells. Copyright © 2017. Published by Elsevier Inc.

The extremophile Acidithiobacillus ferrooxidans possesses a c-di-GMP signalling pathway that could play a significant role during bioleaching of minerals.

PubMed

Ruiz, L M; Castro, M; Barriga, A; Jerez, C A; Guiliani, N

2012-02-01

  The primary goal of this study was to characterize the existence of a functional c-di-GMP pathway in the bioleaching bacterium Acidithiobacillus ferrooxidans.   A bioinformatic search revealed that the genome sequence of At. ferrooxidans ATCC 23270 codes for several proteins involved in the c-di-GMP pathway, including diguanylate cyclases (DGC), phosphodiesterases and PilZ effector proteins. Overexpression in Escherichia coli demonstrated that four At. ferrooxidans genes code for proteins containing GGDEF/EAL domains with functional DGC activity. MS/MS analysis allowed the identification of c-di-GMP in nucleotide preparations obtained from At. ferrooxidans cells. In addition, c-di-GMP levels in cells grown on the surface of solid energetic substrates such as sulfur prills or pyrite were higher than those measured in ferrous iron planktonic cells.   At. ferrooxidans possesses a functional c-di-GMP pathway that could play a key role in At. ferrooxidans biofilm formation during bioleaching processes.   This is the first global study about the c-di-GMP pathway in an acidophilic bacterium of great interest for the biomining industry. It opens a new way to explore the regulation of biofilm formation by biomining micro-organisms during the bioleaching process. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

PubMed

Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

2015-06-01

cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. Copyright © 2015 the American Physiological Society.

Micro-Photoluminescence (micro-PL) Study of Core-Shell GaAs/GaAsSb Nanowires Grown by Self-Assisted Molecular Beam Epitaxy

DTIC Science & Technology

2015-06-18

public release; distribution is unlimited. Micro-Photoluminescence (micro-PL) Study of Core-Shell GaAs/GaAsSb Nanowires grown by Self-Assisted Molecular...U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 GaAsSb, Core Shell Nanowires , Micro Photoluminescence...University 1601 East Market Street Greensboro, NC 27411 -0001 ABSTRACT Micro-Photoluminescence (micro-PL) Study of Core-Shell GaAs/GaAsSb Nanowires grown by

Interaction between macrocyclic nickel complexes and the nucleotides GMP, AMP and ApG.

PubMed

Liu, Yangzhong; Sletten, Einar

2003-01-15

Reactions between the nucleotides GMP, AMP and ApG and the complexes Ni(tren), Ni(cyclam) and NiCR in aqueous solution have been monitored by (1)H, (15)N NMR and UV spectroscopy. The three nickel complexes display different properties in reactions with nucleotides. Ni(tren) which has a pseudo-octahedral coordination geometry was shown to bind to all three nucleotides. Ni(cyclam) and NiCR, both with four nitrogen atoms in a square planar arrangement are not able to bind to nucleotides efficiently because of steric hindrance. Oxidation of Ni(cyclam) by KHSO(5) to produce trivalent Ni(III)(cyclam) improves the coordination capacity, while oxidation of NiCR does not produce a similar effect. The nucleotides interact with trivalent nickel complexes to different extent. Ni(III)CR is seen to oxidize GMP gradually but does not affect AMP significantly. Ni(III)(cyclam), on the other hand, does not oxidize either GMP or AMP at the 1:1 concentration of oxidant used. This result is probably due to the lower redox potential of Ni(cyclam). ApG binds less efficiently to the Ni complexes but is easier oxidized than the mononucleotides.

GMP Synthase Is Required for Virulence Factor Production and Infection by Cryptococcus neoformans*

PubMed Central

Chitty, Jessica L.; Tatzenko, Tayla L.; Williams, Simon J.; Koh, Y. Q. Andre E.; Corfield, Elizabeth C.; Butler, Mark S.; Robertson, Avril A. B.; Cooper, Matthew A.; Kappler, Ulrike; Kobe, Bostjan; Fraser, James A.

2017-01-01

Over the last four decades the HIV pandemic and advances in medical treatments that also cause immunosuppression have produced an ever-growing cohort of individuals susceptible to opportunistic pathogens. Of these, AIDS patients are particularly vulnerable to infection by the encapsulated yeast Cryptococcus neoformans. Most commonly found in the environment in purine-rich bird guano, C. neoformans experiences a drastic change in nutrient availability during host infection, ultimately disseminating to colonize the purine-poor central nervous system. Investigating the consequences of this challenge, we have characterized C. neoformans GMP synthase, the second enzyme in the guanylate branch of de novo purine biosynthesis. We show that in the absence of GMP synthase, C. neoformans becomes a guanine auxotroph, the production of key virulence factors is compromised, and the ability to infect nematodes and mice is abolished. Activity assays performed using recombinant protein unveiled differences in substrate binding between the C. neoformans and human enzymes, with structural insights into these kinetic differences acquired via homology modeling. Collectively, these data highlight the potential of GMP synthase to be exploited in the development of new therapeutic agents for the treatment of disseminated, life-threatening fungal infections. PMID:28062578

In Vivo Biochemistry: Single-Cell Dynamics of Cyclic Di-GMP in Escherichia coli in Response to Zinc Overload.

PubMed

Yeo, Jongchan; Dippel, Andrew B; Wang, Xin C; Hammond, Ming C

2018-01-09

Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.

Importance and globalization status of good manufacturing practice (GMP) requirements for pharmaceutical excipients

PubMed Central

Abdellah, Abubaker; Noordin, Mohamed Ibrahim; Wan Ismail, Wan Azman

2013-01-01

Pharmaceutical excipients are no longer inert materials but it is effective and able to improve the characteristics of the products’ quality, stability, functionality, safety, solubility and acceptance of patients. It can interact with the active ingredients and alter the medicament characteristics. The globalization of medicines’ supply enhances the importance of globalized good manufacturing practice (GMP) requirements for pharmaceutical excipients. This review was intended to assess the globalization status of good manufacturing practice (GMP) requirements for pharmaceutical excipients. The review outcomes demonstrate that there is a lack of accurately defined methods to evaluate and measure excipients’ safety. Furthermore good manufacturing practice requirements for excipients are not effectively globalized. PMID:25685037

Characterization of growth inhibition of oral bacteria by sophorolipid using a microplate-format assay

USDA-ARS?s Scientific Manuscript database

Sophorolipid (SL) is a class of glycolipid biosurfactant produced by yeast and has potent antimicrobial activity against many microorganisms. In this paper, a microplate-based method was developed to characterize the growth inhibition by SL on five representative species of caries-causing oral bact...

cGMP-Phosphodiesterase Inhibition Enhances Photic Responses and Synchronization of the Biological Circadian Clock in Rodents

PubMed Central

Plano, Santiago A.; Agostino, Patricia V.; de la Iglesia, Horacio O.; Golombek, Diego A.

2012-01-01

The master circadian clock in mammals is located in the hypothalamic suprachiasmatic nuclei (SCN) and is synchronized by several environmental stimuli, mainly the light-dark (LD) cycle. Light pulses in the late subjective night induce phase advances in locomotor circadian rhythms and the expression of clock genes (such as Per1-2). The mechanism responsible for light-induced phase advances involves the activation of guanylyl cyclase (GC), cGMP and its related protein kinase (PKG). Pharmacological manipulation of cGMP by phosphodiesterase (PDE) inhibition (e.g., sildenafil) increases low-intensity light-induced circadian responses, which could reflect the ability of the cGMP-dependent pathway to directly affect the photic sensitivity of the master circadian clock within the SCN. Indeed, sildenafil is also able to increase the phase-shifting effect of saturating (1200 lux) light pulses leading to phase advances of about 9 hours, as well as in C57 a mouse strain that shows reduced phase advances. In addition, sildenafil was effective in both male and female hamsters, as well as after oral administration. Other PDE inhibitors (such as vardenafil and tadalafil) also increased light-induced phase advances of locomotor activity rhythms and accelerated reentrainment after a phase advance in the LD cycle. Pharmacological inhibition of the main downstream target of cGMP, PKG, blocked light-induced expression of Per1. Our results indicate that the cGMP-dependent pathway can directly modulate the light-induced expression of clock-genes within the SCN and the magnitude of light-induced phase advances of overt rhythms, and provide promising tools to design treatments for human circadian disruptions. PMID:22590651

Good manufacturing practice (GMP) compliance in the biologics sector: plasma fractionation.

PubMed

Ways, J P; Preston, M S; Baker, D; Huxsoll, J; Bablak, J

1999-12-01

The U.S. blood supply is the safest it has ever been. Due to blood safety and the introduction of viral inactivation/clearance technologies, protein therapies derived from human blood have also in recent years had a history of product safety. Nevertheless, since 1995, the plasma-fractionation industry has experienced increased compliance-related actions by the Food and Drug Administration (FDA), as shown by a substantive increase in the number of FDA 483 inspectional observations, FDA warning letters and other FDA regulatory action. An evaluation of these trends shows that they reflect the implementation by the FDA of increased inspectional interest in the plasma-fractionation industry and an evolution of inspectional practices and standards of current good manufacturing practice (cGMP). Plasma fractionators have responded to FDA actions by carefully evaluating and addressing each inspectional observation, assessing impact to product and taking appropriate actions, including corrective actions to prevent future occurrence. They have made major investments in facilities, quality systems, personnel and training to meet the evolving standards of cGMP and in an effort to implement these standards systemically. Through industry associations, manufacturers have further enhanced product safety by adopting additional voluntary standards for plasma to prevent the entry of potentially unsuitable plasma into the production process. The industry remains committed to application of cGMP and to working with the FDA in further evolution of these standards while striving to assure a continued supply of safe, pure and effective plasma-derived therapies.

Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device.

PubMed

Reinholt, Sarah J; Ozer, Abdullah; Lis, John T; Craighead, Harold G

2016-07-19

We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA's reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO's exoribonuclease activity, and in studies monitoring DXO's degradation of a 30-nucleotide substrate, less than 1 μM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation.

GMP-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation.

PubMed

Larijani, Bagher; Aghayan, Hamid-Reza; Goodarzi, Parisa; Arjmand, Babak

2015-01-01

Stem cell therapy seems a promising avenue in regenerative medicine. Within various stem cells, mesenchymal stem cells have progressively used for cellular therapy. Because of the age-related decreasing in the frequency and differentiating capacity of adult MSCs, fetal tissues such as fetal liver, lung, pancreas, spleen, etc. have been introduced as an alternative source of MSCs for cellular therapy. On the other hand, using stem cells as advanced therapy medicinal products, must be performed in compliance with cGMP as a quality assurance system to ensure the safety, quality, and identity of cell products during translation from the basic stem cell sciences into clinical cell transplantation. In this chapter the authors have demonstrated the manufacturing of GMP-grade human fetal liver-derived mesenchymal stem cells.

Feeding behavior and trophic interaction of three shark species in the Galapagos Marine Reserve

PubMed Central

Insuasti-Zarate, Paul; Riofrío-Lazo, Marjorie; Galván-Magaña, Felipe

2018-01-01

There is great concern about the future of sharks in Ecuador because of the lack of biological knowledge of most species that inhabit the region. This paper analyzes the feeding behavior of the pelagic thresher shark (Alopias pelagicus), the blue shark (Prionace glauca) and the silky shark (Carcharhinus falciformis) through the use of stable isotopes of carbon and nitrogen (δ13C and δ15N), with the aim of determining the degree of interaction between these species in the Galapagos Marine Reserve. No interspecific differences were found in use of oceanic vs. inshore feeding areas (δ13C: Kruskal–Wallis test, p = 0.09). The position in the hierarchy of the food web where A. pelagicus feeds differed from that of the other species (δ15N: Kruskal–Wallis test, p = 0.01). There were no significant differences in δ13C and δ15N values between males and females of the three species (Student’s t-test, p > 0.05), which suggests that both sexes have a similar feeding behavior. A specialist strategy was observed in P. glauca (trophic niche breadth TNB = 0.69), while the other species were found to be generalist (A. pelagicus TNB = 1.50 and C. falciformis TNB = 1.09). The estimated trophic level (TL) varied between the three species. C. falciformis occupied the highest trophic level (TL = 4.4), making it a quaternary predator in the region. The results of this study coincide with the identified behavior in these predators in other areas of the tropical Pacific (Colombia and Mexico), and suggest a pelagic foraging strategy with differential consumption of prey between the three species. These ecological aspects can provide timely information when implementing in conservation measures for these shark species in the Tropical Pacific and Galapagos Marine Reserve. PMID:29844971

Feeding behavior and trophic interaction of three shark species in the Galapagos Marine Reserve.

PubMed

Páez-Rosas, Diego; Insuasti-Zarate, Paul; Riofrío-Lazo, Marjorie; Galván-Magaña, Felipe

2018-01-01

There is great concern about the future of sharks in Ecuador because of the lack of biological knowledge of most species that inhabit the region. This paper analyzes the feeding behavior of the pelagic thresher shark ( Alopias pelagicus ), the blue shark ( Prionace glauca ) and the silky shark ( Carcharhinus falciformis ) through the use of stable isotopes of carbon and nitrogen ( δ 13 C and δ 15 N), with the aim of determining the degree of interaction between these species in the Galapagos Marine Reserve. No interspecific differences were found in use of oceanic vs. inshore feeding areas ( δ 13 C: Kruskal-Wallis test, p = 0.09). The position in the hierarchy of the food web where A. pelagicus feeds differed from that of the other species ( δ 15 N: Kruskal-Wallis test, p = 0.01). There were no significant differences in δ 13 C and δ 15 N values between males and females of the three species (Student's t -test, p  > 0.05), which suggests that both sexes have a similar feeding behavior. A specialist strategy was observed in P. glauca (trophic niche breadth TNB = 0.69), while the other species were found to be generalist ( A. pelagicus TNB = 1.50 and C. falciformis TNB = 1.09). The estimated trophic level (TL) varied between the three species. C. falciformis occupied the highest trophic level (TL = 4.4), making it a quaternary predator in the region. The results of this study coincide with the identified behavior in these predators in other areas of the tropical Pacific (Colombia and Mexico), and suggest a pelagic foraging strategy with differential consumption of prey between the three species. These ecological aspects can provide timely information when implementing in conservation measures for these shark species in the Tropical Pacific and Galapagos Marine Reserve.

The Status of Implementation of Good Manufacturing Practices (GMP) Shredded Fish Production in UMKM Az-Zahrah, Makassar

NASA Astrophysics Data System (ADS)

Latif, Rindam; Dirpan, Andi; Indriani, Sylvia

2017-12-01

Good manufacturing practices (GMP) describes the requirements that an industry should follow throughout the production process starting from sorting raw materials to handling final products. The purpose of this research is to disclose each phase of GMP in making shredded fish and to assess the status of the practices implemented by the UMKM Az-zahrah in producing its product. This descriptive study is conducted by doing interview with owner followed by observation and documentation of all activities related to the production process in order to assess the status of GMP applied by the UMKM Az-zahrah. In assessing the status, we referred to Permenperin RI No.75 / M-Ind / Per / 7/2010 and KBPOM regulation no. HK 03.1.23.04.12.22007 Year 2012. The findings indicate some deviation occurred when UMKM Az-zahrah implemented it, those are (1) there is a pet in production room (2) production employees are not yet wearing work uniform (3) production room has directly access to toilet (4) there is no health label about health and nutritional claims. The status of applied GMP in the UMKM Az-zahrah is at D level (less good) with rating IV.

Seismic evidence for rotating mantle flow around subducting slab edge associated with oceanic microplate capture

NASA Astrophysics Data System (ADS)

Mosher, Stephen G.; Audet, Pascal; L'Heureux, Ivan

2014-07-01

Tectonic plate reorganization at a subduction zone edge is a fundamental process that controls oceanic plate fragmentation and capture. However, the various factors responsible for these processes remain elusive. We characterize seismic anisotropy of the upper mantle in the Explorer region at the northern limit of the Cascadia subduction zone from teleseismic shear wave splitting measurements. Our results show that the mantle flow field beneath the Explorer slab is rotating anticlockwise from the convergence-parallel motion between the Juan de Fuca and the North America plates, re-aligning itself with the transcurrent motion between the Pacific and North America plates. We propose that oceanic microplate fragmentation is driven by slab stretching, thus reorganizing the mantle flow around the slab edge and further contributing to slab weakening and increase in buoyancy, eventually leading to cessation of subduction and microplate capture.

c-di-GMP Turn-Over in Clostridium difficile Is Controlled by a Plethora of Diguanylate Cyclases and Phosphodiesterases

PubMed Central

Bordeleau, Eric; Fortier, Louis-Charles; Malouin, François; Burrus, Vincent

2011-01-01

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen. PMID:21483756

The Bolivar Channel Ecosystem of the Galapagos Marine Reserve: Energy flow structure and role of keystone groups

NASA Astrophysics Data System (ADS)

Ruiz, Diego J.; Wolff, Matthias

2011-08-01

The Bolivar Channel Ecosystem (BCE) is among the most productive zones in the Galapagos Marine Reserve (GMR). It is exposed to relatively cool, nutrient-rich waters of the Cromwell current, which are brought to the photic zone through topographic upwelling. The BCE is characterized by a heterogeneous rocky reef habitat covered by dense algae beds and inhabited by numerous invertebrate and fish species, which represent the food for higher predators including seals and sharks and exploited fish species. In addition, plankton and detritus based food chains channel large amounts of energy through the complex food web. Important emblematic species of the Galapagos archipelagos reside in this area such as the flightless cormorant, the Galapagos penguin and the marine iguanas. A trophic model of BCE was constructed for the habitats < 30 m depth that fringe the west coast of Isabela and east coast of Fernandina islands covering 14% of the total BCE area (44 km 2). The model integrates data sets from sub tidal ecological monitoring and marine vertebrate population monitoring (2004 to 2008) programs of the Charles Darwin Foundation and consists of 30 compartments, which are trophically linked through a diet matrix. Results reveal that the BCE is a large system in terms of flows (38 695 t km - 2 yr - 1 ) comparable to Peruvian Bay Systems of the Humboldt upwelling system. A very large proportion of energy flows from the primary producers (phytoplankton and macro-algae) to the second level and to the detritus pool. Catches are high (54.3 t km - 2 yr - 1 ) and are mainly derived from the second and third trophic levels (mean TL of catch = 2.45) making the fisheries gross efficiency high (0.3%). The system's degree of development seems rather low as indicated by a P/R ratio of 4.19, a low ascendency (37.4%) and a very low Finn's cycling index (1.29%). This is explained by the system's exposure to irregular changes in oceanographic conditions as related to the EL Niño Southern

Angiotensin II increases phosphodiesterase 5A expression in vascular smooth muscle cells: A mechanism by which angiotensin II antagonizes cGMP signaling

PubMed Central

Kim, Dongsoo; Aizawa, Toru; Wei, Heng; Pi, Xinchun; Rybalkin, Sergei D.; Berk, Bradford C.; Yan, Chen

2014-01-01

Angiotensin II (Ang II) and nitric oxide (NO)/natriuretic peptide (NP) signaling pathways mutually regulate each other. Imbalance of Ang II and NO/NP has been implicated in the pathophysiology of many vascular diseases. cGMP functions as a key mediator in the interaction between Ang II and NO/NP. Cyclic nucleotide phosphodiesterase 5A (PDE5A) is important in modulating cGMP signaling by hydrolyzing cGMP in vascular smooth muscle cells (VSMC). Therefore, we examined whether Ang II negatively modulates intracellular cGMP signaling in VSMC by regulating PDE5A. Ang II rapidly and transiently increased PDE5A mRNA levels in rat aortic VSMC. Upregulation of PDE5A mRNA was associated with a time-dependent increase of both PDE5 protein expression and activity. Increased PDE5A mRNA level was transcription-dependent and mediated by the Ang II type 1 receptor. Ang II-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was essential for Ang II-induced PDE5A upregulation. Pretreatment of VSMC with Ang II inhibited C-type NP (CNP) stimulated cGMP signaling, such as cGMP dependent protein kinase (PKG)-mediated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP). Ang II-mediated inhibition of PKG was blocked when PDE5 activity was decreased by selective PDE5 inhibitors, suggesting that upregulation of PDE5A expression is an important mechanism for Ang II to attenuate cGMP signaling. PDE5A may also play a critical role in the growth promoting effects of Ang II because inhibition of PDE5A activity significantly decreased Ang II-stimulated VSMC growth. These observations establish a new mechanism by which Ang II antagonizes cGMP signaling and stimulates VSMC growth. PMID:15623434

Discovery and Distribution of Black Smokers on the Western Galapagos Spreading Center: Implications for Spatial and Temporal Controls on High Temperature Venting at Ridge/Hotspot Intersections

NASA Astrophysics Data System (ADS)

Haymon, R. M.; Anderson, P. G.; Baker, E. T.; Resing, J. A.; White, S. M.; MacDonald, K. C.

2006-12-01

Though nearly one-fifth of the mid-ocean ridge (MOR) lies on or near hotspots, it has been debated whether hotspots increase or decrease MOR hydrothermal flux, or affect vent biota. Despite hotspot enhancement of melt supply, high-temperature vent plumes are enigmatically sparse along two previously-surveyed ridge- hotspot intersections [Reykjanes Ridge (RR), Southeast Indian Ridge (SEIR)]. This has been attributed to crustal thickening by excess volcanism. During the 2005-06 GalAPAGoS expedition, we conducted nested sonar, plume, and camera surveys along a 540 km-long portion of the Galapagos Spreading Center (GSC) where the ridge intersects the Galapagos hotspot at lon. 94.5 -89.5 deg. W. Although MOR hydrothermal springs were first found along the eastern GSC crest in 1977 near lon. 86 deg. W, the GalAPAGoS smokers are the first active high-temperature vents to be found anywhere along the Cocos-Nazca plate boundary. Active and/or recently-inactive smokers were located beneath plumes at 5 sites on the seafloor between lon. 91 deg. W and 94.5 deg. W (see Anderson et al., this session) during near-bottom, real-time fiber-optic Medea camera surveys. Smokers occur along eruptive seafloor fissures atop axial volcanic ridges near the middles of ridge segments, mainly in areas underlain by relatively shallow, continuous axial magma chamber (AMC) seismic reflectors. These findings (1) support magmatic, rather than tectonic, control of GSC smoker distribution; (2) demonstrate that thick crust at MOR-hotspot intersections does not prevent high-temperature hydrothermal vents from forming; and, (3) appear to be inconsistent with models suggesting that enhanced hydrothermal cooling causes abrupt deepening of the AMC and transition from non-rifted to rifted GSC morphology near lon. 92.7 deg. W. The widely-spaced smoker sites located on different GSC segments exhibit remarkably similar characteristics and seafloor settings. Most sites are mature or extinct, and are on lava

Glucocorticoid receptor ligand binding in monocytic cells using a microplate assay.

PubMed

Jansen, J; Uitdehaag, B; Koper, J W; van Den Berg, T K

1999-01-01

Glucocorticoids have profound effects on macrophage function and are widely used as anti-inflammatory drugs. Glucocorticoids receptor (GR) ligand binding capacity is a major determinant of cellular glucocorticoid sensitivity. The number and affinity of GR can be measured in a whole cell binding assay using (3)H-dexamethasone. Here, we describe a rapid and simple microplate assay for GR measurement using the human promonocytic cell line THP-1. Copyright 2000 S. Karger AG, Basel.

A modified method for determining tannin-protein precipitation capacity using accelerated solvent extraction (ASE) and microplate gel filtration.

PubMed

McArt, Scott H; Spalinger, Donald E; Kennish, John M; Collins, William B

2006-06-01

The protein precipitation assay used by Robbins et al., (1987) Ecology 68:98-107 has been shown to predict successfully the reduction in protein availability to some ruminants due to tannins. The procedure, however, is expensive and laborious, which limits its utility, especially for quantitative ecological or nutritional applications where large numbers of assays may be required. We have modified the method to decrease its cost and increase laboratory efficiency by: (1) automating the extraction by using Accelerated Solvent Extraction (ASE); and (2) by scaling and automating the precipitation reaction, chromatography, and spectrometry with microplate gel filtration and an automated UV-VIS microplate spectrometer. ASE extraction is shown to be as effective at extracting tannins as the hot methanol technique. Additionally, the microplate assay is sensitive and precise. We show that the results from the new technique correspond in a nearly 1:1 relationship to the results of the previous technique. Hence, this method could reliably replace the older method with no loss in relevance to herbivore protein digestion. Moreover, the ASE extraction technique should be applicable to other tannin-protein precipitation assays and possibly other phenolic assays.

Dispersal barriers and isolation among deep-sea mussel populations (Mytilidae: Bathymodiolus) from eastern Pacific hydrothermal vents.

PubMed

Won, Y; Young, C R; Lutz, R A; Vrijenhoek, R C

2003-01-01

Deep-sea hydrothermal vent species are widely dispersed among habitat islands found along the global mid-ocean ridge system. We examine factors that affect population structure, gene flow and isolation in vent-endemic mussels of the genus Bathymodiolus from the eastern Pacific Ocean. Mussels were sampled from localities including the Galapagos Rift (GAR, 0 degrees 48' N; 86 degrees 10' W) and the East Pacific Rise (EPR, 13 degrees N to 32 degrees S latitude) across a maximum distance of 4900 km. The sampled range crossed a series of topographical features that interrupt linear aspects of the ridge system, and it encompassed regions of strong cross-axis currents that could impede along-axis dispersal of mussel larvae. Examinations of mitochondrial DNA sequences and allozyme variation revealed significant barriers to gene flow along the ridge axis. All populations from the GAR and EPR from 13 degrees N to 11 degrees S were homogeneous genetically and appeared to experience unimpeded high levels of interpopulational gene flow. In contrast, mussels from north and south of the Easter Microplate were highly divergent (4.4%), possibly comprising sister-species that diverged after formation of the microplate approximately 4.5 Ma. Strong cross-axis currents associated with inflated bathymetry of the microplate region may reinforce isolation across this region.

Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

PubMed

Castro, Matías; Deane, Shelly M; Ruiz, Lina; Rawlings, Douglas E; Guiliani, Nicolas

2015-01-01

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

Diguanylate Cyclase Null Mutant Reveals That C-Di-GMP Pathway Regulates the Motility and Adherence of the Extremophile Bacterium Acidithiobacillus caldus

PubMed Central

Castro, Matías; Deane, Shelly M.; Ruiz, Lina; Rawlings, Douglas E.; Guiliani, Nicolas

2015-01-01

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process. PMID:25689133

cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

PubMed Central

De Jonge, Hugo R.; Tilly, Ben C.; Hogema, Boris M.; Pfau, Daniel J.; Kelley, Catherine A.; Kelley, Megan H.; Melita, August M.; Morris, Montana T.; Viola, Ryan M.

2013-01-01

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG. PMID:24259420

A concise discussion of the regulatory role of cGMP kinase I in cardiac physiology and pathology.

PubMed

Hofmann, Franz

2018-06-22

The underlying cause of cardiac hypertrophy, fibrosis, and heart failure has been investigated in great detail using different mouse models. These studies indicated that cGMP and cGMP-dependent protein kinase type I (cGKI) may ameliorate these negative phenotypes in the adult heart. Recently, evidence has been published that cardiac mitochondrial BKCa channels are a target for cGKI and that activation of mitoBKCa channels may cause some of the positive effects of conditioning in ischemia/reperfusion injury. It will be pointed out that most studies could not present convincing evidence that it is the cGMP level and the activity cGKI in specific cardiac cells that reduces hypertrophy or heart failure. However, anti-fibrotic compounds stimulating nitric oxide-sensitive guanylyl cyclase may be an upcoming therapy for abnormal cardiac remodeling.

Involvement of NO-cGMP pathway in anti-hyperalgesic effect of PDE5 inhibitor tadalafil in experimental hyperalgesia.

PubMed

Otari, K V; Upasani, C D

2015-08-01

The association of elevated level of cyclic guanosine monophosphate (cGMP) with inhibition of hyperalgesia and involvement of nitric oxide (NO)-cGMP pathway in the modulation of pain perception was previously reported. Phosphodiesterases 5 (PDE5) inhibitors, sildenafil and tadalafil (TAD) used in erectile dysfunction, are known to act via the NO-cGMP pathway. TAD exerts its action by increasing the levels of intracellular cGMP. Hence, the present study investigated the effect of TAD 5, 10, or 20 mg/kg, per os (p.o.) or L-NAME 20 mg/kg, intraperitoneally (i.p.) and TAD (20 mg/kg, p.o.) in carrageenan- and diabetes-induced hyperalgesia in rats using hot plate test at 55 ± 2 °C. In carrageenan- and diabetes-induced hyperalgesia, TAD (10 and 20 mg/kg, p.o.) significantly increased paw withdrawal latencies (PWLs) as compared to the control group. L-NAME significantly decreased PWLs as compared to the normal group and aggravated the hyperalgesia. Moreover, significant difference in PWLs of L-NAME and TAD 20 was evident. Co-administration of L-NAME (20 mg/kg) with TAD (20 mg/kg) showed significant difference in PWLs as compared to the TAD (20 mg/kg), indicating L-NAME reversed and antagonized TAD-induced anti-hyperalgesia. This suggested an important role of NO-cGMP pathway in TAD-induced anti-hyperalgesic effect.

NMDA inhibits oxotremorine-induced acid secretion via the NO-dependent cyclic GMP system in rat stomach.

PubMed

Tsai, L H; Lee, Y J

2001-12-31

The mechanism of N-methyl-D-aspartate (NMDA) inhibits oxotremorine-induced acid secretion was examined in rat stomach, in relation to the cyclic GMP system. NMDA (10(-7) M) did not affect the spontaneous acid secretion from the everted preparations of isolated rat stomach, but inhibited the acid secretion stimulated by oxotremorine, and this effect of NMDA was antagonized by 2-amino-5-phosphonovaleric acid (AP-5), (+/-)3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) or N(G)-nitro-L-arginine (L-NNA). NMDA also elevated the cyclic GMP content of mucosal slices from rat stomach, and this effect of NMDA was antagonized by L-NNA. These results indicate that NMDA receptors are present in the rat stomach and regulate the gastric acid secretion. The mechanism underlying the effect of NMDA inhibits oxotremorine-induced acid secretion may be mediated by the NO-dependent cyclic GMP system.

GMP Synthase Is Required for Virulence Factor Production and Infection by Cryptococcus neoformans.

PubMed

Chitty, Jessica L; Tatzenko, Tayla L; Williams, Simon J; Koh, Y Q Andre E; Corfield, Elizabeth C; Butler, Mark S; Robertson, Avril A B; Cooper, Matthew A; Kappler, Ulrike; Kobe, Bostjan; Fraser, James A

2017-02-17

Over the last four decades the HIV pandemic and advances in medical treatments that also cause immunosuppression have produced an ever-growing cohort of individuals susceptible to opportunistic pathogens. Of these, AIDS patients are particularly vulnerable to infection by the encapsulated yeast Cryptococcus neoformans Most commonly found in the environment in purine-rich bird guano, C. neoformans experiences a drastic change in nutrient availability during host infection, ultimately disseminating to colonize the purine-poor central nervous system. Investigating the consequences of this challenge, we have characterized C. neoformans GMP synthase, the second enzyme in the guanylate branch of de novo purine biosynthesis. We show that in the absence of GMP synthase, C. neoformans becomes a guanine auxotroph, the production of key virulence factors is compromised, and the ability to infect nematodes and mice is abolished. Activity assays performed using recombinant protein unveiled differences in substrate binding between the C. neoformans and human enzymes, with structural insights into these kinetic differences acquired via homology modeling. Collectively, these data highlight the potential of GMP synthase to be exploited in the development of new therapeutic agents for the treatment of disseminated, life-threatening fungal infections. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Congo Red Stain Identifies Matrix Overproduction and Is an Indirect Measurement for c-di-GMP in Many Species of Bacteria.

PubMed

Jones, Christopher J; Wozniak, Daniel J

2017-01-01

Congo red is a diazo textile dye that has been used to visualize the production of amyloid fibers for nearly a century. Microbiological applications were later developed, especially in identifying strains that produce amyloid appendages called curli and overexpressing polysaccharides in the biofilm matrix. The second messenger cyclic diguanylate (c-di-GMP) regulates the production of biofilm matrix polysaccharides, and therefore Congo red staining of samples can be utilized as an indirect measurement of elevated c-di-GMP production in bacteria. Congo red allows the identification of strains producing high c-di-GMP in an inexpensive, quantitative, and high-throughput manner.

GMP-based CD133+ cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy

PubMed Central

Gaipa, Giuseppe; Tilenni, Manuela; Straino, Stefania; Burba, Ilaria; Zaccagnini, Germana; Belotti, Daniela; Biagi, Ettore; Valentini, Marco; Perseghin, Paolo; Parma, Matteo; Campli, Cristiana Di; Biondi, Andrea; Capogrossi, Maurizio C; Pompilio, Giulio; Pesce, Maurizio

2010-01-01

Abstract The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133+ cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133+ cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133+ cells. Validation of CD133+ cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as ‘proofs of principle’ of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133+ cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133+ cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies. PMID:19627397

Nitric oxide attenuates matrix metalloproteinase-9 production by endothelial cells independent of cGMP- or NFκB-mediated mechanisms.

PubMed

Meschiari, Cesar A; Izidoro-Toledo, Tatiane; Gerlach, Raquel F; Tanus-Santos, Jose E

2013-06-01

Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10-400 μM) or SNAP (50-400 μM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs' (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases.

Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: Role of protein kinase G nitration

PubMed Central

Aggarwal, Saurabh; Gross, Christine M.; Kumar, Sanjiv; Datar, Sanjeev; Oishi, Peter; Kalka, Gokhan; Schreiber, Christian; Fratz, Sohrab; Fineman, Jeffrey R.; Black, Stephen M.

2012-01-01

Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO−) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO− donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24h (with exogenous activation of cGMP) revealed increased ONOO− levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO− and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity. PMID:21351102

Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery

USDA-ARS?s Scientific Manuscript database

We present here a whole-cell and permeabilized E. coli cell 1' active/inactive microplate screen for ß-D-xylosidase, xylanase, endocellulase, and ferulic acid esterase enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic...

Revision of the genera Microplitis and Snellenius (Hymenoptera, Braconidae, Microgastrinae) from Area de Conservacion Guanacaste, Costa Rica, with a key to all species previously described from Mesoamerica

USDA-ARS?s Scientific Manuscript database

The genera Microplitis and Snellenius (Hymenoptera: Braconidae, Microgastrinae) from Area de Conservacion Guanacaste (ACG), Costa Rica, are revised. A total of 28 new species are described: 23 of Snellenius (the first record for Mesoamerica) and five of Microplitis. A key is provided to all new spec...

A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide

PubMed Central

Klaiber, Michael; Dankworth, Beatrice; Kruse, Martin; Hartmann, Michael; Nikolaev, Viacheslav O.; Yang, Ruey-Bing; Völker, Katharina; Gaßner, Birgit; Oberwinkler, Heike; Feil, Robert; Freichel, Marc; Groschner, Klaus; Skryabin, Boris V.; Frantz, Stefan; Birnbaumer, Lutz; Pongs, Olaf; Kuhn, Michaela

2011-01-01

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca2+]i increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+ levels. This pathway involves the activation of Ca2+‐permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca2+ channels and ultimately increases myocyte Ca2+i levels. These observations reveal a dual role of the ANP/GC-A–signaling pathway in the regulation of cardiac myocyte Ca2+i homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+]i might increase the propensity to cardiac hypertrophy and arrhythmias. PMID:22027011

Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

ERIC Educational Resources Information Center

Aonuma, Hitoshi; Mizunami, Makoto; Matsumoto, Yukihisa; Unoki, Sae

2006-01-01

Cyclic AMP pathway plays an essential role in formation of long-term memory (LTM). In some species, the nitric oxide (NO)-cyclic GMP pathway has been found to act in parallel and complementary to the cAMP pathway for LTM formation. Here we describe a new role of the NO-cGMP pathway, namely, stimulation of the cAMP pathway to induce LTM. We have…

Cost benefit of investment on quality in pharmaceutical manufacturing: WHO GMP pre- and post-certification of a Nigerian pharmaceutical manufacturer.

PubMed

Anyakora, Chimezie; Ekwunife, Obinna; Alozie, Faith; Esuga, Mopa; Ukwuru, Jonathan; Onya, Steve; Nwokike, Jude

2017-09-18

Pharmaceutical companies in Africa need to invest in both facilities and quality management systems to achieve good manufacturing practice (GMP) compliance. Compliance to international GMP standards is important to the attainment of World Health Organization (WHO) prequalification. However, most of the local pharmaceutical manufacturing companies may be deterred from investing in quality because of many reasons, ranging from financial constraints to technical capacity. This paper primarily evaluates benefits against the cost of investing in GMP, using a Nigerian pharmaceutical company, Chi Pharmaceuticals Limited, as a case study. This paper also discusses how to drive more local manufacturers to invest in quality to attain GMP compliance; and proffers practical recommendations for local manufacturers who would want to invest in quality to meet ethical and regulatory obligations. The cost benefit of improving the quality of Chi Pharmaceuticals Limited's facilities and system to attain WHO GMP certification for the production of zinc sulfate 20-mg dispersible tablets was calculated by dividing the annual benefits derived from quality improvement interventions by the annual costs of implementing quality improvement interventions, referred to as a benefit-cost ratio (BCR). Cost benefit of obtaining WHO GMP certification for the production of zinc sulfate 20-mg dispersible tablets was 5.3 (95% confidence interval of 5.0-5.5). Investment in quality improvement intervention is cost-beneficial for local manufacturing companies. Governments and regulators in African countries should support pharmaceutical companies striving to invest in quality. Collaboration of local manufacturing companies with global companies will further improve quality. Local pharmaceutical companies should be encouraged to key into development opportunities available for pharmaceutical companies in Africa.

Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence ▿

PubMed Central

Pitzer, Joshua E.; Sultan, Syed Z.; Hayakawa, Yoshihiro; Hobbs, Gerry; Miller, Michael R.; Motaleb, Md A.

2011-01-01

The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein in Borrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [Kd], 1.25 μM), consistent with Kd values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed plzA resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role of plzA in motility. Furthermore, the plzA mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID50]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently, plzA mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating plzA is essential for the enzootic life cycle of B. burgdorferi. All of these defects were corrected when the mutant was complemented in cis. We propose that failure of plzA mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of the B. burgdorferi enzootic life cycle cannot yet be excluded. PMID:21357718

Hydrothermal alteration in oceanic ridge volcanics: A detailed study at the Galapagos Fossil Hydrothermal Field

USGS Publications Warehouse

Ridley, W.I.; Perfit, M.R.; Josnasson, I.R.; Smith, M.F.

1994-01-01

The Galapagos Fossil Hydrothermal Field is composed of altered oceanic crust and extinct hydrothermal vents within the eastern Galapagos Rift between 85??49???W and 85??55???W. The discharge zone of the hydrothermal system is revealed along scarps, thus providing an opportunity to examine the uppermost mineralized, and highly altered interior parts of the crust. Altered rocks collected in situ by the submersible ALVIN show complex concentric alteration zones. Microsamples of individual zones have been analysed for major/minor, trace elements, and strontium isotopes in order to describe the complex compositional details of the hydrothermal alteration. Interlayered chlorite-smectite and chlorite with disequilibrium compositions dominate the secondary mineralogy as replacement phases of primary glass and acicular pyroxene. Phenocrysts and matrix grains of plagioclase are unaffected during alteration. Using a modification of the Gresens' equation we demonstrate that the trivalent rare earth elements (REEs) are relatively immobile, and calculate degrees of enrichment and depletion in other elements. Strontium isotopic ratios increase as Sr concentrations decrease from least-altered cores to most-altered rims and cross-cutting veins in individual samples, and can be modeled by open system behaviour under low fluid-rock ratio (< 10) conditions following a period of lower-temperature weathering of volcanics within the rift zone. The complex patterns of element enrichment and depletion and strontium isotope variations indicate mixing between pristine seawater and ascending hot fluids to produce a compositional spectrum of fluids. The precipitation of base-metal sulfides beneath the seafloor is probably a result of fluid mixing and cooling. If, as suggested here, the discharge zone alteration occurred under relatively low fluid-rock ratios, then this shallow region must play an important role in determining the exit composition of vent fluids in marine hydrothermal systems

Seafloor Volcanic and Structural Features Adjacent to the 90deg 50'N Transform - Galapagos Spreading Center: Clues for Understanding Plate Boundary Kinematics and Lithospheric Melting Processes (Invited)

NASA Astrophysics Data System (ADS)

Fornari, D. J.; Soule, S.; Harpp, K. S.; Mittelstaedt, E. L.; Geist, D.; Kurz, M. D.; R/v Melville Mv1007 Cruise Scientific Party

2010-12-01

High-resolution EM122 multibeam and MR-1 sidescan sonar data collected over a wide area of seafloor west and east of the 90deg 50’N transform along the Galapagos Spreading Center (GSC) reveal seafloor morpho-structural fabric created along this intermediate spreading plate boundary. In concert with geochemical and geophysical data collected during the cruise, these data will be used to help unravel the kinematics of hotspot-ridge interactions in the northern Galapagos. West of the transform, the seafloor is dominated by three prominent NW-SE trending seamount lineaments, each ~20-30 km wide, including the prominent Wolf-Darwin Lineament (WDL) as well as two other smaller volcanic chains east of the WDL, which are oriented along intermediate trends that become more subparallel to the N-S trend of the transform from west to east. This suggests a possible strong controlling influence of the transform on the orientation of lithospheric fractures involved in supplying magmas to the volcanic centers. Interestingly, each seamount lineament west of the transform appears to have nascent volcanoes nucleating immediately south of the GSC axis at locations that mark along-axis discontinuities of the spreading center, suggesting ridge-related magmatic focusing is also influencing crustal generation on the Nazca plate in this region. The tectonized terrain associated with the transform is 60 km wide, whereas the transform valley is only 20 km wide. The northern 40 km of the transform has a well-defined linear shear zone and bounding faults. The southern 50 km of the transform are characterized by a wide zone of extensive oblique shear structures that trend NW-SE. Within this zone are numerous small volcanic cones and ridges that decorate the margins and axis of the transform domain. The structural evolution of the transform appears to be undergoing a transition along its length with intra-transform volcanism in the south and more normal shear in the north, however the tectonic

cGMP and cyclic nucleotide-gated channels participate in mouse sperm capacitation.

PubMed

Cisneros-Mejorado, Abraham; Sánchez Herrera, Daniel P

2012-01-20

During capacitation of mammalian sperm intracellular [Ca(2+)] and cyclic nucleotides increase, suggesting that CNG channels play a role in the physiology of sperm. Here we study the effect of capacitation, 8Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate) and 8Br-cGMP (8-bromoguanosine 3',5'-cyclic monophosphate) on the macroscopic ionic currents of mouse sperm, finding the existence of different populations of sperm, in terms of the recorded current and its response to cyclic nucleotides. Our results show that capacitation and cyclic nucleotides increase the ionic current, having a differential sensitivity to cGMP (cyclic guanosine monophosphate) and cAMP (cyclic adenosine monophosphate). Using a specific inhibitor we determine the contribution of CNG channels to macroscopic current and capacitation. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Characterizing human-environment interactions in the Galapagos Islands: A case study of land use/land cover dynamics in Isabela Island

NASA Astrophysics Data System (ADS)

McCleary, Amy L.

This dissertation examines contemporary land use and land cover (LULC) change in the communities and protected areas of Isabela Island to provide insights into human-environment interactions in the Galapagos Islands of Ecuador. The growing human presence in Galapagos over the last four decades has been accompanied by significant changes in LULC on inhabited islands in the archipelago. Local stakeholders and decision-makers have recently called for a more integrative approach to understanding interactions between people and the environment in the archipelago. This study is guided by two complementary bodies of work situated within the human-environment tradition of Geography---land change science and landscape ecology. First, support Vector Machine (SVM) and Object Based Image Analysis (OBIA) classifiers are evaluated for mapping LULC from high spatial resolution satellite images. The results show that thematic LULC classifications produced by OBIA are more accurate overall than those generated by SVM. However, important tradeoffs exist between improvements in classification accuracy and processing requirements. The composition and spatial configuration of LULC change are then mapped and quantified from a time series of QuickBird and WorldView-2 satellite images from 2003 to 2010. The pattern metric and change detection analyses reveal that land use change is extensive within the communities due to the expansion and consolidation of built-up areas, and fragmentation of and declines in agriculture. The Galapagos National Park is primarily transformed by exotic plant invasion, forests expansion, and shrinking coastal lagoons. Patterns of agricultural land abandonment, plant invasion, and forest expansion over the same period are described from pattern metric and overlay analyses. Potential drivers of these LULC transitions are identified from logistic regression models, descriptive statistics of agricultural surveys and population censuses, and interviews with

Melatonin protects diabetic heart against ischemia-reperfusion injury, role of membrane receptor-dependent cGMP-PKG activation.

PubMed

Yu, Li-Ming; Di, Wen-Cheng; Dong, Xue; Li, Zhi; Zhang, Yong; Xue, Xiao-Dong; Xu, Yin-Li; Zhang, Jian; Xiao, Xiong; Han, Jin-Song; Liu, Yu; Yang, Yang; Wang, Hui-Shan

2018-02-01

It has been demonstrated that the anti-oxidative and cardioprotective effects of melatonin are, at least in part, mediated by its membrane receptors. However, the direct downstream signaling remains unknown. We previously found that melatonin ameliorated myocardial ischemia-reperfusion (MI/R) injury in diabetic animals, although the underlying mechanisms are also incompletely understood. This study was designed to determine the role of melatonin membrane receptors in melatonin's cardioprotective actions against diabetic MI/R injury with a focus on cGMP and its downstream effector PKG. Streptozotocin-induced diabetic Sprague-Dawley rats and high-glucose medium-incubated H9c2 cardiomyoblasts were utilized to determine the effects of melatonin against MI/R injury. Melatonin treatment preserved cardiac function and reduced oxidative damage and apoptosis. Additionally, melatonin increased intracellular cGMP level, PKGIα expression, p-VASP/VASP ratio and further modulated myocardial Nrf-2-HO-1 and MAPK signaling. However, these effects were blunted by KT5823 (a selective inhibitor of PKG) or PKGIα siRNA except that intracellular cGMP level did not changed significantly. Additionally, our in vitro study showed that luzindole (a nonselective melatonin membrane receptor antagonist) or 4P-PDOT (a selective MT 2 receptor antagonist) not only blocked the cytoprotective effect of melatonin, but also attenuated the stimulatory effect of melatonin on cGMP-PKGIα signaling and its modulatory effect on Nrf-2-HO-1 and MAPK signaling. This study showed that melatonin ameliorated diabetic MI/R injury by modulating Nrf-2-HO-1 and MAPK signaling, thus reducing myocardial apoptosis and oxidative stress and preserving cardiac function. Importantly, melatonin membrane receptors (especially MT 2 receptor)-dependent cGMP-PKGIα signaling played a critical role in this process. Copyright © 2017 Elsevier B.V. All rights reserved.

A microplate assay to measure classical and alternative complement activity.

PubMed

Puissant-Lubrano, Bénédicte; Fortenfant, Françoise; Winterton, Peter; Blancher, Antoine

2017-05-01

We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

Analytical parameters of the microplate-based ORAC-pyrogallol red assay.

PubMed

Ortiz, Rocío; Antilén, Mónica; Speisky, Hernán; Aliaga, Margarita E; López-Alarcón, Camilo

2011-01-01

The analytical parameters of the microplate-based oxygen radicals absorbance capacity (ORAC) method using pyrogallol red (PGR) as probe (ORAC-PGR) are presented. In addition, the antioxidant capacity of commercial beverages, such as wines, fruit juices, and iced teas, is estimated. A good linearity of the area under the curve (AUC) versus Trolox concentration plots was obtained [AUC = (845 +/- 110) + (23 +/- 2) [Trolox, microM], R = 0.9961, n = 19]. QC experiments showed better precision and accuracy at the highest Trolox concentration (40 microM) with RSD and REC (recuperation) values of 1.7 and 101.0%, respectively. When red wine was used as sample, the method also showed good linearity [AUC = (787 +/- 77) + (690 +/- 60) [red wine, microL/mL]; R = 0.9926, n = 17], precision and accuracy with RSD values from 1.4 to 8.3%, and REC values that ranged from 89.7 to 103.8%. Additivity assays using solutions containing gallic acid and Trolox (or red wine) showed an additive protection of PGR given by the samples. Red wines showed higher ORAC-PGR values than white wines, while the ORAC-PGR index of fruit juices and iced teas presented a great variability, ranging from 0.6 to 21.6 mM of Trolox equivalents. This variability was also observed for juices of the same fruit, showing the influence of the brand on the ORAC-PGR index. The ORAC-PGR methodology can be applied in a microplate reader with good linearity, precision, and accuracy.

Establishing a cGMP pancreatic islet processing facility: the first experience in Iran.

PubMed

Larijani, Bagher; Arjmand, Babak; Amoli, Mahsa M; Ao, Ziliang; Jafarian, Ali; Mahdavi-Mazdah, Mitra; Ghanaati, Hossein; Baradar-Jalili, Reza; Sharghi, Sasan; Norouzi-Javidan, Abbas; Aghayan, Hamid Reza

2012-12-01

It has been predicted that one of the greatest increase in prevalence of diabetes will happen in the Middle East bear in the next decades. The aim of standard therapeutic strategies for diabetes is better control of complications. In contrast, some new strategies like cell and gene therapy have aimed to cure the disease. In recent years, significant progress has occurred in beta-cell replacement therapies with a progressive improvement of short-term and long term outcomes. In year 2005, considering the impact of the disease in Iran and the promising results of the Edmonton protocol, the funding for establishing a current Good Manufacturing Practice (cGMP) islet processing facility by Endocrinology and Metabolism Research Center was approved by Tehran University of Medical Sciences. Several islet isolations were performed following establishment of cGMP facility and recruitment of all required equipments for process validation and experimental purpose. Finally the first successful clinical islet isolation and transplantation was performed in September 2010. In spite of a high cost of the procedure it is considered beneficial and may prevent long term complications and the costs associated with secondary cares. In this article we will briefly describe our experience in setting up a cGMP islet processing facility which can provide valuable information for regional countries interested to establish similar facilities.

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms.

PubMed

Moon, Younghye; Balke, Jordan E; Madorma, Derik; Siegel, Michael P; Knowels, Gary; Brouckaert, Peter; Buys, Emmanuel S; Marcinek, David J; Percival, Justin M

2017-06-10

Skeletal muscle nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSμ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSμ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1 -/- muscle. Functional analyses of GC1 -/- muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA-IIX fiber balance. Force deficits in GC1 -/- muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency. Antioxid. Redox Signal. 26, 966-985.

The western transverse ranges microplate as a native terrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campbell, M.D.; Reed, W.E.

1994-04-01

Palocurrent measurements from the entire Cretaceous section of the western Transverse Ranges microplate (WTRM) yield a northerly flow direction. Point count data indicate a mixed provenance for both conglomerates and associated sandstones. The dominant provenance was mixed magmatic arc/recycled orogen and disected/transitional arc terranes. Petrographic, quantitative SEM and microprobe analysis also indicate the presence of diagnostic Franciscan mineralogy in these sediments, including glaucophane, riebeckite, lawsonite, and serpentine, suggesting derivation from a subduction complex. Olistoclasts of chert, jadeitic graywacke, serpentine and blueschist are found intermixed within the arc-derived sediments. Olistoclasts range in size from sub-millimeter to centimeter scale and olistoliths rangemore » up to 150 m. Well preserved internal bedding in some of the olistoliths suggest emplacement by landsliding indicating very short transport distance. This Franciscan material represents the oldest melange-derived material reported from this part of California and documents uplift and erosion of the subduction complex earlier than previously suggested. These data are consistent with deposition in a Cretaceous fore-arc basin located west or south of the San Diego area. The allochthonous WTRM of southern California can be reconstructed to an originally north-south oriented fore-arc basin. After deposition of the Sespe Formation (22 Ma [+-]) the microplate was slivered by strike-slip faults and rotated clockwise approximately 90[degrees], after which, the block again accreted against the continental margin. Our reconstruction suggest that depositional and structural trends for Eocene and Cretaceous sediments is likely to be different from that in the Miocene Monterey pay zones in the Santa Barbara channel region. If our reconstruction is correct, exploration strategy for Eocene and Cretaceous petroleum in the southern California Bight should take this tectonic model into

Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development

PubMed Central

Rodesney, Christopher A.; Roman, Brian; Dhamani, Numa; Cooley, Benjamin J.; Katira, Parag; Touhami, Ahmed; Gordon, Vernita D.

2017-01-01

Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For Pseudomonas aeruginosa, it has long been known that intracellular levels of the signal cyclic-di-GMP increase upon surface adhesion and that this is required to begin biofilm development. However, what cue is sensed to notify bacteria that they are attached to the surface has not been known. Here, we show that mechanical shear acts as a cue for surface adhesion and activates cyclic-di-GMP signaling. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We show that the envelope protein PilY1 and functional type IV pili are required mechanosensory elements. An analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides describes our data well. PMID:28533383

The cyclic-di-GMP phosphodiesterase BinA negatively regulates cellulose-containing biofilms in Vibrio fischeri.

PubMed

Bassis, Christine M; Visick, Karen L

2010-03-01

Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the DeltabinA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of the syp regulator sypG induced binA expression. Thus, this work reveals a mechanism by which V. fischeri inhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.

Soluble guanylate cyclase generation of cGMP regulates migration of MGE neurons.

PubMed

Mandal, Shyamali; Stanco, Amelia; Buys, Emmanuel S; Enikolopov, Grigori; Rubenstein, John L R

2013-10-23

Here we have provided evidence that nitric oxide-cyclic GMP (NO-cGMP) signaling regulates neurite length and migration of immature neurons derived from the medial ganglionic eminence (MGE). Dlx1/2(-/-) and Lhx6(-/-) mouse mutants, which exhibit MGE interneuron migration defects, have reduced expression of the gene encoding the α subunit of a soluble guanylate cyclase (Gucy1A3). Furthermore, Dlx1/2(-/-) mouse mutants have reduced expression of NO synthase 1 (NOS1). Gucy1A3(-/-) mice have a transient reduction in cortical interneuron number. Pharmacological inhibition of soluble guanylate cyclase and NOS activity rapidly induces neurite retraction of MGE cells in vitro and in slice culture and robustly inhibits cell migration from the MGE and caudal ganglionic eminence. We provide evidence that these cellular phenotypes are mediated by activation of the Rho signaling pathway and inhibition of myosin light chain phosphatase activity.

The implementation of tissue banking experiences for setting up a cGMP cell manufacturing facility.

PubMed

Arjmand, Babak; Emami-Razavi, Seyed Hassan; Larijani, Bagher; Norouzi-Javidan, Abbas; Aghayan, Hamid Reza

2012-12-01

Cell manufacturing for clinical applications is a unique form of biologics manufacturing that relies on maintenance of stringent work practices designed to ensure product consistency and prevent contamination by microorganisms or by another patient's cells. More extensive, prolonged laboratory processes involve greater risk of complications and possibly adverse events for the recipient, and so the need for control is correspondingly greater. To minimize the associate risks of cell manufacturing adhering to international quality standards is critical. Current good tissue practice (cGTP) and current good manufacturing practice (cGMP) are examples of general standards that draw a baseline for cell manufacturing facilities. In recent years, stem cell researches have found great public interest in Iran and different cell therapy projects have been started in country. In this review we described the role of our tissue banking experiences in establishing a new cGMP cell manufacturing facility. The authors concluded that, tissue banks and tissue banking experts can broaden their roles from preparing tissue grafts to manufacturing cell and tissue engineered products for translational researches and phase I clinical trials. Also they can collaborate with cell processing laboratories to develop SOPs, implement quality management system, and design cGMP facilities.

A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa.

PubMed

Paquet-Durand, François; Beck, Susanne; Michalakis, Stylianos; Goldmann, Tobias; Huber, Gesine; Mühlfriedel, Regine; Trifunović, Dragana; Fischer, M Dominik; Fahl, Edda; Duetsch, Gabriele; Becirovic, Elvir; Wolfrum, Uwe; van Veen, Theo; Biel, Martin; Tanimoto, Naoyuki; Seeliger, Mathias W

2011-03-01

The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1(-/-) × rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca(2+))-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca(2+)-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP.

cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

PubMed Central

Wiley, Luke A.; Burnight, Erin R.; DeLuca, Adam P.; Anfinson, Kristin R.; Cranston, Cathryn M.; Kaalberg, Emily E.; Penticoff, Jessica A.; Affatigato, Louisa M.; Mullins, Robert F.; Stone, Edwin M.; Tucker, Budd A.

2016-01-01

Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans. PMID:27471043

Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains.

PubMed

Muradov, Khakim G; Granovsky, Alexey E; Schey, Kevin L; Artemyev, Nikolai O

2002-03-26

Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.

The characteristics of hydrothermal plumes observed in the Precious Stone Mountain hydrothermal field, the Galapagos spreading center

NASA Astrophysics Data System (ADS)

Chen, S.; Tao, C.; Li, H.; Zhou, J.; Deng, X.; Tao, W.; Zhang, G.; Liu, W.; He, Y.

2014-12-01

The Precious Stone Mountain hydrothermal field (PSMHF) is located on the southern rim of the Galapagos Microplate. It was found at the 3rd leg of the 2009 Chinese DY115-21 expedition on board R/V Dayangyihao. It is efficient to learn the distribution of hydrothermal plumes and locate the hydrothermal vents by detecting the anomalies of turbidity and temperature. Detecting seawater turbidity by MAPR based on deep-tow technology is established and improved during our cruises. We collected data recorded by MAPR and information from geological sampling, yielding the following results: (1)Strong hydrothermal turbidity and temperature anomalies were recorded at 1.23°N, southeast and northwest of PSMHF. According to the CTD data on the mooring system, significant temperature anomalies were observed over PSMHF at the depth of 1,470 m, with anomalies range from 0.2℃ to 0.4℃, which gave another evidence of the existence of hydrothermal plume. (2)At 1.23°N (101.4802°W/1.2305°N), the nose-shaped particle plume was concentrated at a depth interval of 1,400-1,600 m, with 200 m thickness and an east-west diffusion range of 500 m. The maximum turbidity anomaly (0.045 △NTU) was recorded at the depth of 1,500 m, while the background anomaly was about 0.01△NTU. A distinct temperature anomaly was also detected at the seafloor near 1.23°N. Deep-tow camera showed the area was piled up by hydrothermal sulfide sediments. (3) In the southeast (101.49°W/1.21°N), the thickness of hydrothermal plume was 300 m and it was spreading laterally at a depth of 1,500-1,800 m, for a distance about 800 m. The maximum turbidity anomaly of nose-shaped plume is about 0.04 △NTU at the depth of 1,600 m. Distinct temperature anomaly was also detected in the northwest (101.515°W/1.235°N). (4) Terrain and bottom current were the main factors controlling the distribution of hydrothermal plume. Different from the distribution of hydrothermal plumes on the mid-ocean ridges, which was mostly

High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity.

PubMed

Pfeilmeier, Sebastian; Saur, Isabel Marie-Luise; Rathjen, John Paul; Zipfel, Cyril; Malone, Jacob George

2016-05-01

The plant innate immune system employs plasma membrane-localized receptors that specifically perceive pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern-triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant-associated bacteria. Here, we show that cyclic-di-GMP [bis-(3'-5')-cyclic di-guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic-di-GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf-5 inhibit flagellin synthesis and help the bacteria to evade FLS2-mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic-di-GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic-di-GMP signalling on bacterial behaviour. © 2015 THE AUTHORS MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.

Three dimensional image of Isla Isabela in the western Galapagos Islands

NASA Technical Reports Server (NTRS)

1994-01-01

This is a three-dimensional image of Isla Isabela in the western Galapagos Islands off the western coast of Ecuador, South America. The view was constructed by overlaying a Spaceborne Imaging Radar-C/X-band Synthetic Aperture Radar image on a TOPSAR digital elevation map. The vertical scale in this image is exaggerated by a factor of 1.87. The SIR-C/X-SAR image was taken on the 40th orbit of the shuttle Endeavour. The image is centered at about .5 degrees south latitude and 91 degrees West longitude and covers an area of 75 km by 60 km. The radar incidence angle at the center of the image is about 20 degrees. This SIR-C/X-SAR image of Alcedo and Sierra Negra volcanoes shows the rougher lava flowas as bright features, while ash deposits and smooth Pahoehoe lava flows appear dark. A small portion of Isla Fernandina is visible in the extreme upper left corner of the image. The Jet Propulsion Laboratory alternative photo number is P-43913.

Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMP-dependent modulation of NF-κB activity.

PubMed

Siednienko, Jakub; Nowak, Joanna; Moynagh, Paul N; Gorczyca, Wojciech A

2011-06-01

Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (≤10μM) and decreased at higher (>100μM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-κB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-κB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway. Copyright © 2011 Elsevier Ltd. All rights reserved.

Nitric oxide provokes tumor necrosis factor-alpha expression in adult feline myocardium through a cGMP-dependent pathway.

PubMed

Kalra, D; Baumgarten, G; Dibbs, Z; Seta, Y; Sivasubramanian, N; Mann, D L

2000-09-12

The mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the failing heart is unknown. To determine whether NO was sufficient to provoke TNF-alpha biosynthesis, we examined the effects of an NO donor, S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff hearts. SNAP (1 micromol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF-alpha mRNA and protein biosynthesis in adult cat hearts. The effects of SNAP were completely abrogated by a NO quenching agent, 2-(4-carboxyphenyl)-4, 4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic mobility shift assays demonstrated that SNAP treatment led to the rapid induction of nuclear factor kappa-beta (NF-kappaB) but not AP-1. The importance of the cGMP pathway in terms of mediating NO-induced TNF-alpha biosynthesis was shown by studies that demonstrated that 8-bromo-cGMP mimicked the effects of SNAP and that the effects of SNAP could be completely abrogated using a cGMP antagonist, 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or protein kinase G antagonist (Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the site-specific phosphorylation (serine 32) and degradation of IkappaBalpha in isolated cardiac myocytes. Finally, protein kinase G was sufficient to directly phosphorylate IkappaBalpha on serine 32, a critical step in the activation of NF-kappaB. These studies show that NO provokes TNF-alpha biosynthesis through a cGMP-dependent pathway, which suggests that the coincident expression of TNF-alpha and NO may foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the failing heart.

The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

PubMed Central

Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

2016-01-01

Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

Convergence of Ca2+-desensitizing mechanisms activated by forskolin and phenylephrine pretreatment, but not 8-bromo-cGMP.

PubMed

Porter, Melissa; Evans, Melissa C; Miner, Amy S; Berg, Krystina M; Ward, Kevin R; Ratz, Paul H

2006-06-01

Contractile stimuli can sensitize myosin to Ca2+ by activating RhoA kinase (ROK) and PKC that inhibit myosin light chain phosphatase (MLCP) activity. Relaxant stimuli, acting through PKA and PKG (cyclic nucleotide-dependent protein kinases), and pretreatment with contractile agents such as phenylephrine (PE), can desensitize myosin to Ca2+. It is unknown precisely how these stimuli cause Ca2+ desensitization. To test the hypothesis that PKA, PKG, and PE pretreatment signaling systems converge to cause relaxation by inhibition of ROK in intact, isolated tissues, we examined the effects of forskolin (FSK; PKA activation), 8-bromo-cGMP (8br-cGMP; PKG activation), and PE pretreatment on KCl-induced force maintenance in rabbit arteries, a response nearly completely dependent on ROK activation. PE pretreatment and agents activating PKA and PKG caused Ca2+ desensitization by inhibiting KCl-induced tonic force and MLC phosphorylation without inhibiting intracellular [Ca2+]. At pCa 5 in beta-escin-permeabilized muscle, FSK and 8b-cGMP accelerated the relaxation rate when tissues were returned to pCa 9, suggesting that both agents can elevate MLCP activity. However, a component of the Ca2+ desensitization attributed to PKG activation in intact tissues appeared to involve a MLC phosphorylation-independent component. Inhibition of KCl-induced tonic force by the ROK inhibitor, Y-27632, and by PE pretreatment, were synergistically potentiated by 8b-cGMP, but not FSK. FSK and PE pretreatment, but not 8b-cGMP, inhibited the KCl-induced increase in site-specific myosin phosphatase target protein-1 phosphorylation at Thr853. These data support the hypothesis that PKA and PE pretreatment converge on a common Ca2+-desensitization pathway, but that PKG can act by a mechanism different from that activated by PKA and PE pretreatment.

Distinct patterns of genetic differentiation among annelids of eastern Pacific hydrothermal vents.

PubMed

Hurtado, L A; Lutz, R A; Vrijenhoek, R C

2004-09-01

Population genetic and phylogenetic analyses of mitochondrial COI from five deep-sea hydrothermal vent annelids provided insights into their dispersal modes and barriers to gene flow. These polychaetes inhabit vent fields located along the East Pacific Rise (EPR) and Galapagos Rift (GAR), where hundreds to thousands of kilometers can separate island-like populations. Long-distance dispersal occurs via larval stages, but larval life histories differ among these taxa. Mitochondrial gene flow between populations of Riftia pachyptila, a siboglinid worm with neutrally buoyant lecithothrophic larvae, is diminished across the Easter Microplate region, which lies at the boundary of Indo-Pacific and Antarctic deep-sea provinces. Populations of the siboglinid Tevnia jerichonana are similarly subdivided. Oasisia alvinae is not found on the southern EPR, but northern EPR populations of this siboglinid are subdivided across the Rivera Fracture Zone. Mitochondrial gene flow of Alvinella pompejana, an alvinellid with large negatively buoyant lecithotrophic eggs and arrested embryonic development, is unimpeded across the Easter Microplate region. Gene flow in the polynoid Branchipolynoe symmytilida also is unimpeded across the Easter Microplate region. However, A. pompejana populations are subdivided across the equator, whereas B. symmitilida populations are subdivided between the EPR and GAR axes. The present findings are compared with similar evidence from codistributed species of annelids, molluscs and crustaceans to identify potential dispersal filters in these eastern Pacific ridge systems.

Fumarole/plume and diffuse CO2 emission from Sierra Negra volcano, Galapagos archipelago

NASA Astrophysics Data System (ADS)

Padron, E.; Hernandez Perez, P. A.; Perez, N.; Theofilos, T.; Melian, G.; Barrancos, J.; Virgil, G.; Sumino, H.; Notsu, K.

2009-12-01

The active shield-volcano Sierra Negra is part of the Galapagos hotspot. Sierra Negra is the largest shield volcano of Isabela Island, hosting a 10 km diameter caldera. Ten historic eruptions have occurred and some involved a frequently visited east caldera rim fissure zone called Volcan Chico. The last volcanic event occurred in October 2005 and lasted for about a week, covering approximately twenty percent of the eastern caldera floor. Sierra Negra volcano has experienced some significant changes in the chemical composition of its volcanic gas discharges after the 2005 eruption. This volcanic event produced an important SO2 degassing that depleted the magmatic content of this gas. Not significant changes in the MORB and plume-type helium contribution were observed after the 2005 eruption, with a 65.5 % of MORB and 35.5 % of plume contribution. In 2006 a visible and diffuse gas emission study was performed at the summit of Sierra Negra volcano, Galapagos, to evaluate degassing rate from this volcanic system. Diffuse degassing at Sierra Negra was mainly confined in three different DDS: Volcan Chico, the southern inner margin of the caldera, and Mina Azufral. These areas showed also visible degassing, which indicates highly fractured areas where volcano-hydrothermal fluids migrate towards surface. A total fumarole/plume SO2 emission of 11 ± 2 td-1 was calculated by mini-DOAS ground-based measurements at Mina Azufral fumarolic area. Molar ratios of major volcanic gas components were also measured in-situ at Mina Azufral with a portable multisensor. The results showed H2S/SO2, CO2/SO2 and H2O/SO2 molar ratios of 0.41, 52.2 and 867.9, respectively. Multiplying the observed SO2 emission rate times the observed (gas)i/SO2 mass ratio we have estimated other volatiles emission rates. The results showed that H2O, CO2 and H2S emission rates from Sierra Negra are 562, 394, and 2.4 t d-1, respectively. The estimated total output of diffuse CO2 emission from the summit of

Isolation and characterization of 10 polymorphic microsatellite loci for the endangered Galapagos-endemic whitespotted sandbass (Paralabrax albomaculatus).

PubMed

Bertolotti, Alicia C; Griffiths, Sarah M; Truelove, Nathan K; Box, Stephen J; Preziosi, Richard F; Salinas de Leon, Pelayo

2015-01-01

The white-spotted sandbass (Paralabrax albomaculatus) is a commercially important species in the Galapagos Marine Reserve, but is classified as endangered in the IUCN Red List. For this study, 10 microsatellite loci were isolated and characterized using Illumina paired-end sequencing. These loci can be used for genetic studies of population structure and connectivity to aid in the management of the white-spotted sandbass and other closely-related species. The 10 characterized loci were polymorphic, with 11-49 alleles per locus, and observed heterozygosity ranged from 0.575 to 0.964. This set of markers is the first to be developed for this species.

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

PubMed Central

Medintz, Igor; Wong, Wendy W.; Berti, Lorenzo; Shiow, Lawrence; Tom, Jennifer; Scherer, James; Sensabaugh, George; Mathies, Richard A.

2001-01-01

An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population. PMID:11230165

Comparative effects of vinpocetine and 8-Br-cyclic GMP on the contraction and /sup 45/Ca-fluxes in the rabbit aorta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiu, P.J.; Tetzloff, G.; Ahn, H.S.

1988-07-01

Vinpocetine is a highly specific inhibitor of calmodulin-dependent phosphodiesterase (CaM-PDE) with an IC50 of 19 microM and produces a significant accumulation of cyclic GMP but not cyclic AMP in rabbit aorta. In isolated rabbit aortic strips, vinpocetine (0.01 and 0.1 mM) inhibited the contraction and /sup 45/Ca uptake due to both phenylephrine (1 microM) and KCl (40 mM), whereas 8-Br-cyclic GMP (0.1-1mM) selectively impaired phenylephrine-induced responses. Furthermore, the KCl-stimulated /sup 45/Ca efflux in normal Ca2+ buffer, which reflects elevated cytosolic Ca2+, was greatly diminished by vinpocetine but not by 8-Br-cyclic GMP. However, phenylephrine-induced /sup 45/Ca efflux and contraction in Ca2+-freemore » buffer, which reflect Ca2+ release from intracellular sites, were similarly inhibited by both vinpocetine and 8-Br-cyclic GMP. The results suggest that vinpocetine may effect vasodilatation through blockade of the slow channel and selective inhibition of CaM-PDE in the vascular smooth muscle.« less

Microplate and shear zone models for oceanic spreading center reorganizations

NASA Technical Reports Server (NTRS)

Engeln, Joseph F.; Stein, Seth; Werner, John; Gordon, Richard

1988-01-01

The kinematics of rift propagation and the resulting goemetries of various tectonic elements for two plates is reviewed with no overlap zone. The formation and evolution of overlap regions using schematic models is discussed. The models are scaled in space and time to approximate the Easter plate, but are simplified to emphasize key elements. The tectonic evolution of overlap regions which act as rigid microplates and shear zones is discussed, and the use of relative motion and structural data to discriminate between the two types of models is investigated. The effect of propagation rate and rise time on the size, shape, and deformation of the overlap region is demonstrated.

Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode.

PubMed

Zhang, Jin; Tang, Ying; Teng, Liumei; Lu, Minghua; Tang, Dianping

2015-06-15

A simple and low-cost DNA sensing platform based on Pb(2+)-specific DNAzyme-modified microplate was successfully developed for highly sensitive monitoring of lead ion (Pb(2+), one kind of toxic heavy metal ion) in the environmental samples coupling with a portable personal glucometer (PGM)-based detection mode. The detection cell was first prepared simply by means of immobilizing the DNAzyme on the streptavidin-modified microplate. Gold nanoparticle labeled with single-stranded DNA and invertase (Enz-AuNP-DNA) was utilized as the signal-transduction tag to produce PGM substrate (glucose). Upon addition of lead ion into the microplate, the substrate strand of the immobilized DNAzyme was catalytically cleaved by target Pb(2+), and the newly generated single-strand DNA in the microplate could hybridize again with the single-stranded DNA on the Enz-AuNP-DNA. Accompanying with the Enz-AuNP-DNA, the carried invertase could convert sucrose into glucose. The as-produced glucose could be monitored by using a widely accessible PGM for in situ amplified digital readout. Based on Enz-AuNP-DNA amplification strategy, as low as 1.0 pM Pb(2+) could be detected under the optimal conditions. Moreover, the methodology also showed good reproducibility and high selectivity toward target Pb(2+) against other metal ions because of highly specific Pb(2+)-dependent DNAzyme, and was applicable for monitoring Pb(2+) in the naturally contaminated sewage and spiked drinking water samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Ab Initio QM/MM Study Shows a Highly Dissociated SN2 Hydrolysis Mechanism for the cGMP-Specific Phosphodiesterase-5.

PubMed

Li, Zhe; Wu, Yinuo; Feng, Ling-Jun; Wu, Ruibo; Luo, Hai-Bin

2014-12-09

Phosphodiesterases (PDEs) are the sole enzymes hydrolyzing the important second messengers cGMP and cAMP and have been identified as therapeutic targets for several diseases. The most successful examples are PDE5 inhibitors (i.e., sildenafil and tadalafil), which have been approved for the treatment of male erectile dysfunction and pulmonary hypertension. However, the side effects mostly due to nonselective inhibition toward other PDE isoforms, set back the clinical usage of PDE5 inhibitors. Until now, the exact catalytic mechanism of the substrate cGMP by PDE5 is still unclear. Herein, the first computational study on the catalytic hydrolysis mechanism of cGMP for PDE5 (catalytic domain) is performed by employing the state-of-the-art ab initio quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations. Our simulations show a SN2 type reaction procedure via a highly dissociated transition state with a reaction barrier of 8.88 kcal/mol, which is quite different from the previously suggested hydrolysis mechanism of cAMP for PDE4. Furthermore, the subsequent ligand exchange and the release of the product GMP have also been investigated by binding energy analysis and MD simulations. It is deduced that ligand exchange would be the rate-determining step of the whole reaction, which is consistent with many previous experimental results. The obtained mechanistic insights should be valuable for not only the rational design of more specific inhibitors toward PDE5 but also understanding the general hydrolysis mechanism of cGMP-specific PDEs.

Serum and supplement optimization for EU GMP-compliance in cardiospheres cell culture

PubMed Central

Chimenti, Isotta; Gaetani, Roberto; Forte, Elvira; Angelini, Francesco; De Falco, Elena; Zoccai, Giuseppe Biondi; Messina, Elisa; Frati, Giacomo; Giacomello, Alessandro

2014-01-01

Cardiac progenitor cells (CPCs) isolated as cardiospheres (CSs) and CS-derived cells (CDCs) are a promising tool for cardiac cell therapy in heart failure patients, having CDCs already been used in a phase I/II clinical trial. Culture standardization according to Good Manufacturing Practices (GMPs) is a mandatory step for clinical translation. One of the main issues raised is the use of xenogenic additives (e.g. FBS, foetal bovine serum) in cell culture media, which carries the risk of contamination with infectious viral/prion agents, and the possible induction of immunizing effects in the final recipient. In this study, B27 supplement and sera requirements to comply with European GMPs were investigated in CSs and CDCs cultures, in terms of process yield/efficiency and final cell product gene expression levels, as well as phenotype. B27− free CS cultures produced a significantly reduced yield and a 10-fold drop in c-kit expression levels versus B27+ media. Moreover, autologous human serum (aHS) and two different commercially available GMP AB HSs were compared with standard research-grade FBS. CPCs from all HSs explants had reduced growth rate, assumed a senescent-like morphology with time in culture, and/or displayed a significant shift towards the endothelial phenotype. Among three different GMP gamma-irradiated FBSs (giFBSs) tested, two provided unsatisfactory cell yields, while one performed optimally, in terms of CPCs yield/phenotype. In conclusion, the use of HSs for the isolation and expansion of CSs/CDCs has to be excluded because of altered proliferation and/or commitment, while media supplemented with B27 and the selected giFBS allows successful EU GMP-complying CPCs culture. PMID:24444305

Decreased levels of guanosine 3', 5'-monophosphate (cGMP) in cerebrospinal fluid (CSF) are associated with cognitive decline and amyloid pathology in Alzheimer's disease.

PubMed

Ugarte, Ana; Gil-Bea, Francisco; García-Barroso, Carolina; Cedazo-Minguez, Ángel; Ramírez, M Javier; Franco, Rafael; García-Osta, Ana; Oyarzabal, Julen; Cuadrado-Tejedor, Mar

2015-06-01

Levels of the cyclic nucleotides guanosine 3', 5'-monophosphate (cGMP) or adenosine 3', 5'-monophosphate (cAMP) that play important roles in memory processes are not characterized in Alzheimer's disease (AD). The aim of this study was to analyse the levels of these nucleotides in cerebrospinal fluid (CSF) samples from patients diagnosed with clinical and prodromal stages of AD and study the expression level of the enzymes that hydrolyzed them [phosphodiesterases (PDEs)] in the brain of AD patients vs. For cGMP and cAMP CSF analysis, the cohort (n = 79) included cognitively normal participants (subjective cognitive impairment), individuals with stable mild cognitive impairment or AD converters (sMCI and cMCI), and mild AD patients. A high throughput liquid chromatography-tandem mass spectrometry method was used. Interactions between CSF cGMP or cAMP with mini-mental state examination (MMSE) score, CSF Aβ(1-42) and CSF p-tau were analysed. For PDE4, 5, 9 and 10 expression analysis, brains of AD patients vs. controls (n = 7 and n = 8) were used. cGMP, and not cAMP levels, were significantly lower in the CSF of patients diagnosed with mild AD when compared with nondemented controls. CSF levels of cGMP showed a significant association with MMSE-diagnosed clinical dementia and with CSF biomarker Aβ42 in AD patients. Significant increase in PDE5 expression was detected in temporal cortex of AD patients compared with that of age-matched healthy control subjects. No changes in the expression of others PDEs were detected. These results support the potential involvement of cGMP in the pathological and clinical development of AD. The cGMP reduction in early stages of AD might participate in the aggravation of amyloid pathology and cognitive decline. © 2014 British Neuropathological Society.

A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

PubMed

Vijayakumar, Paul Priyesh; Muriana, Peter M

2015-06-11

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action

PubMed Central

Vijayakumar, Paul Priyesh; Muriana, Peter M.

2015-01-01

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes. PMID:26111195

The effects of nitric oxide-cGMP pathway stimulation on dopamine in the medial preoptic area and copulation in DHT-treated castrated male rats

PubMed Central

Sato, Satoru M.; Wersinger, Scott R.; Hull, Elaine M.

2007-01-01

Dopamine (DA) in the medial preoptic area (MPOA) provides important facilitative influence on male rat copulation. We have shown that the nitric oxide-cGMP (NO-cGMP) pathway modulates MPOA DA levels and copulation. We have also shown that systemic estradiol (E2) maintains neuronal NO synthase (nNOS) immunoreactivity in the MPOA of castrates, as well as relatively normal DA levels. This effect of E2 on nNOS probably accounts for at least some of the previously demonstrated behavioral facilitation by intra-MPOA E2 administration in castrates. Therefore, we hypothesized that stimulation of the MPOA NO-cGMP pathway in dihydrotestosterone (DHT)-treated castrates should restore DA levels and copulatory behaviors. Reverse-dialysis of a NO donor, sodium nitroprusside (SNP), increased extracellular DA in the MPOA of DHT-treated castrates and restored the ability to copulate to ejaculation in half of the animals. A cGMP analog, 8-Br-cGMP, also increased extracellular DA, though not as robustly, but did not restore copulatory ability. The effectiveness of the NO donor in restoring copulation and MPOA DA levels is consistent with our hypothesis. However, the lack of behavioral effects of 8-Br-cGMP, despite its increase in MPOA DA, suggests that NO may have additional mediators in the MPOA in the regulation of copulation. Furthermore, the suboptimal copulation seen in the NO donor-treated animals suggests the importance of extra-MPOA systems in the regulation of copulation. PMID:17467707

Population subdivision of hydrothermal vent polychaete Alvinella pompejana across equatorial and Easter Microplate boundaries.

PubMed

Jang, Sook-Jin; Park, Eunji; Lee, Won-Kyung; Johnson, Shannon B; Vrijenhoek, Robert C; Won, Yong-Jin

2016-10-28

The Equator and Easter Microplate regions of the eastern Pacific Ocean exhibit geomorphological and hydrological features that create barriers to dispersal for a number of animals associated with deep-sea hydrothermal vent habitats. This study examined effects of these boundaries on geographical subdivision of the vent polychaete Alvinella pompejana. DNA sequences from one mitochondrial and eleven nuclear genes were examined in samples collected from ten vent localities that comprise the species' known range from 23°N latitude on the East Pacific Rise to 38°S latitude on the Pacific Antarctic Ridge. Multi-locus genotypes inferred from these sequences clustered the individual worms into three metapopulation segments - the northern East Pacific Rise (NEPR), southern East Pacific Rise (SEPR), and northeastern Pacific Antarctic Ridge (PAR) - separated by the Equator and Easter Microplate boundaries. Genetic diversity estimators were negatively correlated with tectonic spreading rates. Application of the isolation-with-migration (IMa2) model provided information about divergence times and demographic parameters. The PAR and NEPR metapopulation segments were estimated to have split roughly 4.20 million years ago (Mya) (2.42-33.42 Mya, 95 % highest posterior density, (HPD)), followed by splitting of the SEPR and NEPR segments about 0.79 Mya (0.07-6.67 Mya, 95 % HPD). Estimates of gene flow between the neighboring regions were mostly low (2 Nm < 1). Estimates of effective population size decreased with southern latitudes: NEPR > SEPR > PAR. Highly effective dispersal capabilities allow A. pompejana to overcome the temporal instability and intermittent distribution of active hydrothermal vents in the eastern Pacific Ocean. Consequently, the species exhibits very high levels of genetic diversity compared with many co-distributed vent annelids and mollusks. Nonetheless, its levels of genetic diversity in partially isolated populations are inversely

Characterization of growth inhibition of oral bacteria by sophorolipid using a microplate-format assay.

PubMed

Solaiman, Daniel K Y; Ashby, Richard D; Uknalis, Joseph

2017-05-01

Sophorolipid (SL) is a class of glycolipid biosurfactant produced by yeast and has potent antimicrobial activity against many microorganisms. In this paper, a microplate-based method was developed to characterize the growth inhibition by SL on five representative species of caries-causing oral bacteria. Bacterial growth on microplate in the absence and presence of varying concentrations of SL was continuously monitored by recording the absorbance at 600nm of the cultures using a microplate reader. The results showed that SL completely inhibited the growth of the Lactobacilli at ≥1mg/ml and the Streptococci at much lower concentrations of ≥50μg/ml. More importantly, we further defined the mechanism of antimicrobial activity of SL by analyzing the pattern of the cell growth curves. SL at sublethal concentrations (<1mg/ml) is bactericidal towards the Lactobacilli; it lengthens the apparent cell-doubling time (T d ) and decreases the final cell density (as indicated by A 600nm ) in a concentration-dependent manner. Against the oral Streptococci, on the other hand, SL at sublethal concentrations (<50μg/ml) is bacteriostatic; it delays the onset of cell growth in a concentration-dependent fashion, but once the cell growth is commenced there is no noticeable adverse effect on T d and the final A 600nm . Scanning electron microscopic (SEM) study of L. acidophilus grown in sublethal concentration of SL reveals extensive structural damage to the cells. S. mutans grown in sublethal level of SL did not show morphological damage to the cells, but numerous protruding structures could be seen on the cell surface. At the respective lethal levels of SL, L. acidophilus cells were lysed (at 1mg/ml SL) and the cell surface structure of S. mutans (at 130μg/ml SL) was extensively deformed. In summary, this paper presents the first report on a detailed analysis of the effects of SL on Lactobacilli and Streptococci important to oral health and hygiene. Published by Elsevier B.V.

An optical microplate biosensor for the detection of methyl parathion pesticide using a biohybrid of Sphingomonas sp. cells-silica nanoparticles.

PubMed

Mishra, Archana; Kumar, Jitendra; Melo, Jose Savio

2017-01-15

The previously developed Sphingomonas sp. based optical microplate biosensor for methyl parathion (MP) was good as it detected multiple samples but had poor stability and low sensitivity. The present study aims to overcome these limitations. Silica nanoparticles (Si NP) were thus functionalized with polyethyleneimine (PEI) and the functionalized silica nanoparticles ( f Si NP) were then integrated with Sphingomonas sp. cells. The process was optimized for hydrolysis of MP into p-nitrophenol (PNP). Integration of f Si NP with cells was confirmed by FT-IR analysis. Biohybrid of Sphingomonas sp.- f Si NP was immobilized on the wells of microplate and associated directly with the optical transducer of microplate reader. Immobilized biohybrid of Sphingomonas sp.- f Si NP was characterized using SEM. A detection range of 0.1-1ppm MP was achieved from the linear range of calibration plot. After integration with f Si NP the storage stability of biohybrid was enhanced ten times from 18 to 180 days. This study proves that after interaction of cells with f Si NP, improved the sensitivity and stability of the biosensor. Spiked samples were also analyzed and correlated using this biohybrid based biosensor. Copyright © 2016 Elsevier B.V. All rights reserved.

The Oxidation State of Fe in Glasses from the Galapagos Archipelago: Variable Oxygen Fugacity as a Function of Mantle Source

NASA Astrophysics Data System (ADS)

Peterson, M. E.; Kelley, K. A.; Cottrell, E.; Saal, A. E.; Kurz, M. D.

2015-12-01

The oxidation state of the mantle plays an intrinsic role in the magmatic evolution of the Earth. Here we present new μ-XANES measurements of Fe3+/ΣFe ratios (a proxy for ƒO2) in a suite of submarine glasses from the Galapagos Archipelago. Using previously presented major, trace, and volatile elements and isotopic data for 4 groups of glass that come from distinct mantle sources (depleted upper mantle, 2 recycled, and a primitive mantle source) we show that Fe3+/ΣFe ratios vary both with the influence of shallow level processes and with variations in mantle source. Fe3+/ΣFe ratios increase with differentiation (i.e. decreasing MgO), but show a large variation at a given MgO. Progressive degassing of sulfur accompanies decreasing Fe3+/ΣFe ratios, while assimilation of hydrothermally altered crust (as indicated by increasing Sr/Sr*) is shown to increase Fe3+/ΣFe ratios. After taking these processes into account, there is still variability in the Fe3+/ΣFe ratios of the isotopically distinct sample suites studied, yielding a magmatic ƒO2 that ranges from ΔQFM = +0.16 to +0.74 (error < 0.5 log units) and showing that oxidation state varies as a function of mantle source composition in the Galapagos hotspot system. After correcting back to a common MgO content = 8.0 wt%, the trace element depleted group similar to MORB (ITD), and the group similar to Pinta (WD = high Th/La, Δ7/4, Δ8/4 ratios) show Fe3+/ΣFe ratios within the range of MORB (average ITD = 0.162 ± 0.003 and WD = 0.164 ± 0.006). Another trace element enriched group similar to Sierra Negra and Cerro Azul (ITE = enriched Sr and Pb isotopes) shows evidence of mixing between oxidized and reduced sources (ITE oxidized end-member = 0.177). This suggests that mantle sources in the Galapagos that are thought to contain recycled components (i.e., WD and ITE groups) have distinct oxidation states. The high 3He/4He Fernandina samples (HHe group) are shown to be the most oxidized (ave. 0.175 ± 0

GMP-compliant automated synthesis of [(18)F]AV-45 (Florbetapir F 18) for imaging beta-amyloid plaques in human brain.

PubMed

Yao, Cheng-Hsiang; Lin, Kun-Ju; Weng, Chi-Chang; Hsiao, Ing-Tsung; Ting, Yi-Shu; Yen, Tzu-Chen; Jan, Tong-Rong; Skovronsky, Daniel; Kung, Mei-Ping; Wey, Shiaw-Pyng

2010-12-01

We report herein the Good Manufacturing Practice (GMP)-compliant automated synthesis of (18)F-labeled styrylpyridine, AV-45 (Florbetapir), a novel tracer for positron emission tomography (PET) imaging of beta-amyloid (Abeta) plaques in the brain of Alzheimer's disease patients. [(18)F]AV-45 was prepared in 105 min using a tosylate precursor with Sumitomo modules for radiosynthesis under GMP-compliant conditions. The overall yield was 25.4+/-7.7% with a final radiochemical purity of 95.3+/-2.2% (n=19). The specific activity of [(18)F]AV-45 reached as high as 470+/-135 TBq/mmol (n=19). The present studies show that [(18)F]AV-45 can be manufactured under GMP-compliant conditions and could be widely available for routine clinical use. Copyright 2010 Elsevier Ltd. All rights reserved.

Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

1988-07-01

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

Role of selective cyclic GMP phosphodiesterase inhibition in the myorelaxant actions of M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine.

PubMed Central

Souness, J. E.; Brazdil, R.; Diocee, B. K.; Jordan, R.

1989-01-01

1. The mechanism by which M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine (MIMAX), which have been described as selective cyclic GMP phosphodiesterase (PDE) inhibitors, relax rat aorta was investigated. 2. Three cyclic nucleotide PDEs were identified in the soluble fraction of rat aorta; a Ca2+-insensitive form exhibiting substrate selectivity for cyclic GMP (cGMP PDE), a Ca2+/calmodulin-stimulated form which also preferentially hydrolyzed cyclic GMP (Ca2+ PDE), and a form demonstrating substrate selectivity for cyclic AMP (cAMP PDE). 3. M&B 22,948 and MIMAX inhibited cGMP PDE (Ki = 0.16 microM and 0.43 microM, respectively) and Ca2+ PDE (Ki = 9.9 microM and 0.55 microM, respectively), but exhibited weak activity against cAMP PDE (Ki = 249 microM and 42 microM, respectively). MY-5445 selectivity inhibited cGMP PDE (Ki = 1.3 microM) and vinpocetine selectively inhibited Ca2+ PDE (Ki = 14 microM). 4. M&B 22,948 and MIMAX induced dose-dependent increases in the accumulation of cyclic GMP, but not cyclic AMP, in rat aorta pieces. These effects were greatly reduced by endothelial denudation and by methylene blue (5 microM) which blocks the actions of endothelium-derived relaxant factor. MY-5445 and vinpocetine had no effect on rat aorta cyclic GMP or cyclic AMP accumulation. 5. All four compounds caused dose-related relaxation of 5-hydroxytryptamine (10 microM) contracted, endothelium-intact rat aorta, the effects of M&B 22,948 and MIMAX being greatly reduced by methylene blue (5 microM). Methylene blue also caused 10 fold and 100 fold rightward shifts in the dose-response curves of MY-5445 and vinpocetine, respectively. 6. The results are consistent with the smooth muscle relaxant actions of M&B 22,948 and MIMAX, but not vinpocetine and MY-5445, being mediated through a mechanism involving inhibition of cyclic GMP hydrolysis. PMID:2480168

Phosphodiesterase 9A regulates central cGMP and modulates responses to cholinergic and monoaminergic perturbation in vivo.

PubMed

Kleiman, Robin J; Chapin, Douglas S; Christoffersen, Curt; Freeman, Jody; Fonseca, Kari R; Geoghegan, Kieran F; Grimwood, Sarah; Guanowsky, Victor; Hajós, Mihály; Harms, John F; Helal, Christopher J; Hoffmann, William E; Kocan, Geralyn P; Majchrzak, Mark J; McGinnis, Dina; McLean, Stafford; Menniti, Frank S; Nelson, Fredrick; Roof, Robin; Schmidt, Anne W; Seymour, Patricia A; Stephenson, Diane T; Tingley, Francis David; Vanase-Frawley, Michelle; Verhoest, Patrick R; Schmidt, Christopher J

2012-05-01

Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.

Atrial natriuretic peptide provokes a dramatic increase in cyclic GMP formation and markedly inhibits muscarinic-stimulated Ca2+ mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells.

PubMed

Ding, K H; Ali, N; Abdel-Latif, A A

1999-02-01

We investigated the effects of cGMP-elevating agents, including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP), on cGMP accumulation and on carbachol (CCh)-stimulated intracellular calcium ([Ca2+]i) mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and in primary cultured cat iris sphincter smooth muscle (CISM) cells. The stimulatory effects of the natriuretic peptides on cGMP production correlated well with their inhibitory effects on CCh-induced [Ca+1]i mobilisation, and these effects were significantly more pronounced in the SV-CISM-2 cells than in the CISM cells. Thus, ANP (1 microM) increased cGMP production in the SV-CISM-2 cells and CISM cells by 487- and 1.7-fold, respectively, and inhibited CCh-induced [Ca2+]i mobilisation by 95 and 3%, respectively. In the SV-CISM-2 cells, ANP and CNP dose dependently inhibited CCh-induced [Ca2+]i mobilisation with IC50 values of 156 and 412 nM, respectively, and dose dependently stimulated cGMP formation with EC50 values of 24 and 88 nM, respectively, suggesting that the inhibitory actions of the peptides are mediated through cGMP. Both ANP and CNP stimulated cGMP accumulation in a time-dependent manner. The potency of the cGMP-elevating agents were in the following order: ANP>CNP>SNP; these agents had no effect on cAMP accumulation. The inhibitory effects of the natriuretic peptides were mimicked by 8-Br-cGMP, a selective activator of cGMP-dependent protein kinase. LY83583, a soluble guanylyl cyclase inhibitor, significantly inhibited SNP-induced cGMP formation but had no effect on those of ANP and CNP. The basal activities of the guanylyl cyclase and the dissociation constant (Kd) and total receptor density (Bmax) values of the natriuretic peptide receptor for [125I]ANP binding were not significantly different between the two cell types. The cGMP system, as with the cAMP system, has a major inhibitory influence on the muscarinic

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

PubMed

Wuchter, Patrick; Vetter, Marcel; Saffrich, Rainer; Diehlmann, Anke; Bieback, Karen; Ho, Anthony D; Horn, Patrick

2016-06-01

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

GMP-Compliant Expansion of Clinical-Grade Human Mesenchymal Stromal/Stem Cells Using a Closed Hollow Fiber Bioreactor.

PubMed

Barckhausen, Christina; Rice, Brent; Baila, Stefano; Sensebé, Luc; Schrezenmeier, Hubert; Nold, Philipp; Hackstein, Holger; Rojewski, Markus Thomas

2016-01-01

This chapter describes a method for GMP-compliant expansion of human mesenchymal stromal/stem cells (hMSC) from bone marrow aspirates, using the Quantum(®) Cell Expansion System from Terumo BCT. The Quantum system is a functionally closed, automated hollow fiber bioreactor system designed to reproducibly grow cells in either GMP or research laboratory environments. The chapter includes protocols for preparation of media, setup of the Quantum system, coating of the hollow fiber bioreactor, as well as loading, feeding, and harvesting of cells. We suggest a panel of quality controls for the starting material, the interim product, as well as the final product.

A Method for Identifying Small-Molecule Aggregators Using Photonic Crystal Biosensor Microplates

PubMed Central

Chan, Leo L.; Lidstone, Erich A.; Finch, Kristin E.; Heeres, James T.; Hergenrother, Paul J.; Cunningham, Brian T.

2010-01-01

Small molecules identified through high-throughput screens are an essential element in pharmaceutical discovery programs. It is now recognized that a substantial fraction of small molecules exhibit aggregating behavior leading to false positive results in many screening assays, typically due to nonspecific attachment to target proteins. Therefore, the ability to efficiently identify compounds within a screening library that aggregate can streamline the screening process by eliminating unsuitable molecules from further consideration. In this work, we show that photonic crystal (PC) optical biosensor microplate technology can be used to identify and quantify small-molecule aggregation. A group of aggregators and nonaggregators were tested using the PC technology, and measurements were compared with those gathered by three alternative methods: dynamic light scattering (DLS), an α-chymotrypsin colorimetric assay, and scanning electron microscopy (SEM). The PC biosensor measurements of aggregation were confirmed by visual observation using SEM, and were in general agreement with the α-chymotrypsin assay. DLS measurements, in contrast, demonstrated inconsistent readings for many compounds that are found to form aggregates in shapes, very different from the classical spherical particles assumed in DLS modeling. As a label-free detection method, the PC biosensor aggregation assay is simple to implement and provides a quantitative direct measurement of the mass density of material adsorbed to the transducer surface, whereas the microplate-based sensor format enables compatibility with high-throughput automated liquid-handling methods used in pharmaceutical screening. PMID:20930952

Cyclic GMP signaling in cardiomyocytes modulates fatty acid trafficking and prevents triglyceride accumulation

USDA-ARS?s Scientific Manuscript database

While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the im...

Analysis of proton wires in the enzyme active site suggests a mechanism of c-di-GMP hydrolysis by the EAL domain phosphodiesterases.

PubMed

Grigorenko, Bella L; Knyazeva, Marina A; Nemukhin, Alexander V

2016-11-01

We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A genetic signature of the evolution of loss of flight in the Galapagos cormorant

USGS Publications Warehouse

Burga, Alejandro; Wang, Weiguang; Ben-David, Eyal; Wolf, Paul C.; Ramey, Andy M.; Verdugo, Claudio; Lyons, Karen; Parker, Patricia G.; Kruglyak, Leonid

2017-01-01

INTRODUCTIONChanges in the size and proportion of limbs and other structures have played a key role in the evolution of species. One common class of limb modification is recurrent wing reduction and loss of flight in birds. Indeed, Darwin used the occurrence of flightless birds as an argument in favor of his theory of natural selection. Loss of flight has evolved repeatedly and is found among 26 families of birds in 17 different orders. Despite the frequency of these modifications, we have a limited understanding of their underpinnings at the genetic and molecular levels.RATIONALETo better understand the evolution of changes in limb size, we studied a classic case of recent loss of flight in the Galapagos cormorant (Phalacrocorax harrisi). Cormorants are large water birds that live in coastal areas or near lakes, and P. harrisi is the only flightless cormorant among approximately 40 extant species. The entire population is distributed along the coastlines of Isabela and Fernandina islands in the Galapagos archipelago. P. harrisi has a pair of short wings, which are smaller than those of any other cormorant. The extreme reduction of the wings and pectoral skeleton observed in P. harrisi is an attractive model for studying the evolution of loss of flight because it occurred very recently; phylogenetic evidence suggests that P. harrisi diverged from its flighted relatives within the past 2 million years. We developed a comparative and predictive genomics approach that uses the genome sequences of P. harrisi and its flighted relatives to find candidate genetic variants that likely contributed to the evolution of loss of flight.RESULTSWe sequenced and de novo assembled the whole genomes of P. harrisi and three closely related flighted cormorant species. We identified thousands of coding variants exclusive to P. harrisi and classified them according to their probability of altering protein function based on conservation. Variants most likely to alter

The RNA binding protein CsrA controls c-di-GMP metabolism by directly regulating the expression of GGDEF proteins

PubMed Central

Jonas, Kristina; Edwards, Adrianne N.; Simm, Roger; Romeo, Tony; Römling, Ute; Melefors, Öjar

2009-01-01

Summary The carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobility shift assays confirmed the direct and specific binding of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH resulted in a more than 20-fold increase in the cellular concentration of the second messenger c-di-GMP, implying that both proteins possess diguanylate cyclase activity. Phenotypic characterization revealed that both proteins are involved in the regulation of motility in a c-di-GMP dependent manner. CsrA was also found to regulate the expression of five additional GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels of c-di-GMP. All together, these data demonstrate a global role for CsrA in the regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins at the post-transcriptional level. PMID:18713317

Bis-(3'-5')-cyclic dimeric GMP-linked quorum sensing controls swarming in Vibrio parahaemolyticus.

PubMed

Trimble, Michael J; McCarter, Linda L

2011-11-01

Movement over and colonization of surfaces are important survival strategies for bacteria, and many find it advantageous to perform these activities as a group, using quorum sensing to sample population size and synchronize behavior. It is puzzling however, that swarming-proficient and virulent strains of Vibrio parahaemolyticus are silenced for the vibrio archetypal pathway of quorum sensing. Here we describe the S-signal, a pheromone that can be communicated between cells in coculture to regulate surface colonization. This signal was harvested in cell-free supernatants and demonstrated to stimulate swarming gene expression at low cell density. The S-signal was generated by the pyridoxal phosphate-dependent aminotransferase ScrA; signal reception required the periplasmic binding protein ScrB and the membrane-bound GGDEF-EAL domain-containing protein ScrC. ScrC is a bifunctional enzyme that has the ability to form and degrade the second messenger bis-(3'-5') cyclic dimeric GMP (c-di-GMP). ScrA in neighboring cells was able to alter the activity of ScrC in a ScrB-dependent manner, transforming ScrC's repressing ability to inducing activity with respect to swarming. Conversely, cell-cell signaling repressed capsule gene expression. In summary, we report that quorum sensing can stimulate swarming in V. parahaemolyticus; it does so via an alternative pathway capable of generating an autoinducing signal that influences c-di-GMP, thereby expanding the lexicon and language of cell-cell communication.

Hematology, plasma chemistry, serology, and Chlamydophila status of the waved albatross (Phoebastria irrorata) on the Galapagos Islands.

PubMed

Padilla, Luis R; Huyvaert, Kathryn P; Merkel, Jane; Miller, R Eric; Parker, Patricia G

2003-09-01

Venipuncture was performed on 50 adult, free-ranging waved albatrosses (Phoebastria irrorata) on Española, Galapagos Islands, Ecuador, to establish hematologic and plasma biochemistry reference ranges and to determine the prevalence of exposure to important domestic avian pathogens. Weights and plasma creatine phosphokinase activities differed significantly between males and females. Serum was tested for evidence of exposure to avian influenza, avian paramyxoviruses 1, 2, and 3, avian cholera, adenovirus groups 1 and 2, avian encephalomyelitis, Marek's disease, infectious bursal disease, and infectious bronchitis virus (Connecticut and Massachusetts strains). Of 44 birds, 29 (66%) seroreacted to adenovirus group 1, and four seroreacted to avian encephalomyelitis. Cloacal swabs were negative for Chlamydophila psittaci DNA.

Rapid solid-phase immunoassay for 6-keto prostaglandin F1 alpha on microplates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schramm, W.; Smith, R.H.; Jackson, T.M.

1990-03-01

We describe, for the measurement of 6-keto prostaglandin F1 alpha in biological media, a solid-phase immunoassay with immobilized antibodies that requires a total processing time of less than 2 h with hands-on time less than 30 min for 40 samples. The method combines the convenience of the microplate format with the sensitivity of radiolabeled prostaglandin derivatives as tracers in a competitive immunoassay. The intra- and interassay variations at 50% displacement of the radiolabeled prostaglandin derivative as tracer were 9.0% and 11.8%, respectively. At 50% displacement of the radiolabeled tracer, the sensitivity is about 20 pg per well. Optimal incubation timemore » is between 60 and 90 min. Nonspecific binding was less than 1% if about 8 pg of tracer (approximately 25,000 counts/min per well) was used. Inhibition curves of samples in different dilutions were parallel to standard curves. The variation of bound radiolabeled prostaglandin derivative within the wells of one microplate (n = 96) was less than 3%. Human plasma samples and medium from tissue culture assayed for 6-keto prostaglandin F1 alpha correlated well with results obtained with a solid-phase assay based on use of magnetic particles (r = 0.99, n = 24) for culture-medium samples; r = 0.99; n = 26 for plasma samples.« less

Brunn: an open source laboratory information system for microplates with a graphical plate layout design process.

PubMed

Alvarsson, Jonathan; Andersson, Claes; Spjuth, Ola; Larsson, Rolf; Wikberg, Jarl E S

2011-05-20

Compound profiling and drug screening generates large amounts of data and is generally based on microplate assays. Current information systems used for handling this are mainly commercial, closed source, expensive, and heavyweight and there is a need for a flexible lightweight open system for handling plate design, and validation and preparation of data. A Bioclipse plugin consisting of a client part and a relational database was constructed. A multiple-step plate layout point-and-click interface was implemented inside Bioclipse. The system contains a data validation step, where outliers can be removed, and finally a plate report with all relevant calculated data, including dose-response curves. Brunn is capable of handling the data from microplate assays. It can create dose-response curves and calculate IC50 values. Using a system of this sort facilitates work in the laboratory. Being able to reuse already constructed plates and plate layouts by starting out from an earlier step in the plate layout design process saves time and cuts down on error sources.

Biochemical activity and multiple locations of particulate guanylate cyclase in Rhyacophila dorsalis acutidens (Insecta: Trichoptera) provide insights into the cGMP signalling pathway in Malpighian tubules.

PubMed

Secca, T; Sciaccaluga, M; Marra, A; Barberini, L; Bicchierai, M C

2011-04-01

In insect renal physiology, cGMP and cAMP have important regulatory roles. In Drosophila melanogaster, considered a good model for molecular physiology studies, and in other insects, cGMP and cAMP act as signalling molecules in the Malpighian tubules (MTs). However, many questions related to cyclic nucleotide functions are unsolved in principal cells (PC) and stellate cells (SC), the two cell types that compose the MT. In PC, despite the large body of information available on soluble guanylate cyclase (sGC) in the cGMP pathway, the functional circuit of particulate guanylate cyclase (pGC) remains obscure. In SC, on the other side, the synthesis and physiological role of the cGMP are still unknown. Our biochemical data regarding the presence of cyclic nucleotides in the MTs of Rhyacophila dorsalis acutidens revealed a cGMP level above the 50%, in comparison with the cAMP. The specific activity values for the membrane-bound guanylate cyclase were also recorded, implying that, besides the sGC, pGC is a physiologically relevant source of cGMP in MTs. Cytochemical studies showed ultrastructurally that there was a great deal of pGC on the basolateral membranes of both the principal and stellate cells. In addition, pGC was also detected in the contact zone between the two cell types and in the apical microvillar region of the stellate cells bordering the tubule lumen. The pGC signal is so well represented in PC and, unexpectedly in SC of MTs, that it is possible to hypothesize the existence of still uncharacterized physiological processes regulated by the pGC-cGMP system. Copyright © 2011 Elsevier Ltd. All rights reserved.

Photoaffinity labelling of cyclic GMP-inhibited phosphodiesterase (PDE III) in human and rat platelets and rat tissues: effects of phosphodiesterase inhibitors.

PubMed

Tang, K M; Jang, E K; Haslam, R J

1994-06-15

Ultraviolet irradiation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: trequinsin (IC50 = 13 +/- 2 nM), lixazinone (IC50 = 22 +/- 4 nM), milrinone (IC50 = 56 +/- 12 nM), cilostamide (IC50 = 70 +/- 9 nM), siguazodan (IC50 = 117 +/- 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC50 = 3950 +/- 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [32P]cGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated accumulation of cAMP in intact

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

PubMed

Banasik, Michał; Sachadyn, Paweł

2016-09-01

A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

[Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

PubMed

Wu, Jian-Hui; Sun, Zu-Yue

2013-06-01

To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.

Specificity of Good Manufacturing Practice (GMP) for Biomedical Cell Products.

PubMed

Tulina, M A; Pyatigorskaya, N V

2018-03-01

The article describes special aspects of Good Manufacturing Practice (GMP) for biomedical cell products (BMCP) that imply high standards of aseptics throughout the entire productio process, strict requirements to donors and to the procedure of biomaterial isolation, guaranty of tracing BMCP products, defining processing procedures which allow to identify BMCP as minimally manipulated; continuous quality control and automation of the control process at all stages of manufacturing, which will ensure product release simultaneously with completion of technological operations.

Working draft of the FDA GMP final rule (Part I).

PubMed

Donawa, M E

1995-10-01

On 24 July 1995, the US Food and Drug Administration (FDA) published a notice of availability of a working draft of a final rule for new good manufacturing practice (GMP) regulations for medical devices. The new regulations could be in force by late 1996. This is the first of a two-part series of articles discussing key provisions of the working draft and their importance to companies marketing or planning to market devices in the US.

Did the Kyrenia Range of northern Cyprus rotate with the Troodos-Hatay microplate during the tectonic evolution of the eastern Mediterranean?

NASA Astrophysics Data System (ADS)

Morris, Antony; Robertson, Alastair H. F.; Anderson, Mark W.; Hodgson, Emma

2016-01-01

Previous palaeomagnetic studies have allowed the recognition of a distinctive area of Neotethyan oceanic rocks, including the Troodos ophiolite in Cyprus and the Hatay ophiolite to the east in southern Turkey, that underwent 90° of anticlockwise rotation between Late Cretaceous (Campanian) and Early Eocene time. The southern and western boundaries of this rotated Troodos-Hatay microplate have been inferred to lie within, or adjacent to, zones of deformed oceanic and continental margin rocks that are now exposed in southern and western Cyprus; however, the northern boundary of the microplate remains undefined. Relevant to this problem, palaeomagnetic data are presented here from basaltic lavas exposed along the Kyrenia Range, mostly from Late Cretaceous (Maastrichtian) sites and one Eocene site. A positive inclination-only fold test demonstrates that remanences are pre-deformational in age, and positive conglomerate tests show that magnetic remanences were acquired before Late Eocene-Early Oligocene time, together suggesting that primary magnetizations are preserved. Data from the eastern Kyrenia Range and the Karpas Peninsula (the easternmost extension of the Kyrenia Range) document significant relative tectonic rotation between these localities, with no rotation in the eastern range versus 30° of anticlockwise rotation of the Karpas Peninsula. Unfortunately, palaeomagnetic sites from the western Kyrenia Range did not yield tectonically interpretable magnetization directions, probably due to complex poly-phase thrusting and folding, and the central range also yielded no interpretable data. However, the available palaeomagnetic data are sufficient to demonstrate that the Kyrenia terrane underwent a separate rotation history to the Troodos-Hatay microplate and also implies that the northern boundary of the Troodos-Hatay microplate was located between the Troodos ophiolite and the Kyrenia Range. The former microplate margin has since been overridden and concealed by

Measurement of the distribution of non-structural carbohydrate composition in onion populations by a high-throughput microplate enzymatic assay.

PubMed

Revanna, Roopashree; Turnbull, Matthew H; Shaw, Martin L; Wright, Kathryn M; Butler, Ruth C; Jameson, Paula E; McCallum, John A

2013-08-15

Non-structural carbohydrate (NSC; glucose, fructose, sucrose and fructan) composition of onions (Allium cepa L.) varies widely and is a key determinant of market usage. To analyse the physiology and genetics of onion carbohydrate metabolism and to enable selective breeding, an inexpensive, reliable and practicable sugar assay is required to phenotype large numbers of samples. A rapid, reliable and cost-effective microplate-based assay was developed for NSC analysis in onions and used to characterise variation in tissue hexose, sucrose and fructan content in open-pollinated breeding populations and in mapping populations developed from a wide onion cross. Sucrose measured in microplates employing maltase as a hydrolytic enzyme was in agreement with HPLC-PAD results. The method revealed significant variation in bulb fructan content within open-pollinated 'Pukekohe Longkeeper' breeding populations over a threefold range. Very wide segregation from 80 to 600 g kg(-1) in fructan content was observed in bulbs of F2 genetic mapping populations from the wide onion cross 'Nasik Red × CUDH2150'. The microplate enzymatic assay is a reliable and practicable method for onion sugar analysis for genetics, breeding and food technology. Open-pollinated onion populations may harbour extensive within-population variability in carbohydrate content, which may be quantified and exploited using this method. The phenotypic data obtained from genetic mapping populations show that the method is well suited to detailed genetic and physiological analysis. © 2013 Society of Chemical Industry.

Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains

PubMed Central

Povolotsky, Tatyana L.

2015-01-01

ABSTRACT The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive “degenerate” proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. IMPORTANCE Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be

Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains.

PubMed

Povolotsky, Tatyana L; Hengge, Regine

2016-01-01

The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive "degenerate" proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c-di-GMP

Three cyanobacteriochromes work together to form a light color-sensitive input system for c-di-GMP signaling of cell aggregation.

PubMed

Enomoto, Gen; Ni-Ni-Win; Narikawa, Rei; Ikeuchi, Masahiko

2015-06-30

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that have diverse spectral properties and domain compositions. Although large numbers of CBCR genes exist in cyanobacterial genomes, no studies have assessed whether multiple CBCRs work together. We recently showed that the diguanylate cyclase (DGC) activity of the CBCR SesA from Thermosynechococcus elongatus is activated by blue-light irradiation and that, when irradiated, SesA, via its product cyclic dimeric GMP (c-di-GMP), induces aggregation of Thermosynechococcus vulcanus cells at a temperature that is suboptimum for single-cell viability. For this report, we first characterize the photobiochemical properties of two additional CBCRs, SesB and SesC. Blue/teal light-responsive SesB has only c-di-GMP phosphodiesterase (PDE) activity, which is up-regulated by teal light and GTP. Blue/green light-responsive SesC has DGC and PDE activities. Its DGC activity is enhanced by blue light, whereas its PDE activity is enhanced by green light. A ΔsesB mutant cannot suppress cell aggregation under teal-green light. A ΔsesC mutant shows a less sensitive cell-aggregation response to ambient light. ΔsesA/ΔsesB/ΔsesC shows partial cell aggregation, which is accompanied by the loss of color dependency, implying that a nonphotoresponsive DGC(s) producing c-di-GMP can also induce the aggregation. The results suggest that SesB enhances the light color dependency of cell aggregation by degrading c-di-GMP, is particularly effective under teal light, and, therefore, seems to counteract the induction of cell aggregation by SesA. In addition, SesC seems to improve signaling specificity as an auxiliary backup to SesA/SesB activities. The coordinated action of these three CBCRs highlights why so many different CBCRs exist.

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

PubMed

Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

2018-04-09

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Detection of rabbit IgG by using functional magnetic particles and an enzyme-conjugated antibody with a homemade magnetic microplate.

PubMed

Tsai, Hweiyan; Lu, Yi-Hsuan; Liao, Huan-Xuan; Wu, Shih-Wei; Yu, Feng-Yih; Fuh, Chwan Bor

2015-01-01

The enzyme-linked immunosorbent assay (ELISA) has been used for diagnosing medical and plant pathologies. In addition, it is used for quality-control evaluations in various industries. The ELISA is the simplest method for obtaining excellent results; however, it is time consuming because the immunoreagents interact only on the contact surfaces. Antibody-labeled magnetic particles can be dispersed in a solution to yield a pseudohomogeneous reaction with antigens which improved the efficiency of immunoreaction, and can be easily separated from the unreactive substances by applying a magnetic force. We used a homemade magnetic microplate, functional magnetic particles (MPs) and enzyme-labeled secondary antibody to perform the sandwich ELISA successfully. Using antibody-labeled MPs enabled reducing the analysis time to one-third of that required in using a conventional ELISA. The secondary antibody conjugated with horseradish peroxidase (HRP) was affinity-bound to the analyte (IgG in this study). The calibration curve was established according to the measured absorbance of the 3, 3', 5, 5'-tetramethybezidine-HRP reaction products versus the concentrations of standard IgG. The linear range of IgG detection was 114 ng/mL-3.5 ng/mL. The limit of detection (LOD) of IgG was 3.4 ng/mL. The recovery and coefficient of variation were 100% (±7%) and 116% (±4%) for the spiked concentrations of 56.8 ng/mL and 14.2 ng/mL, respectively. Pseudohomogeneous reactions can be performed using functional MPs and a magnetic microplate. Using antibody-labeled MPs, the analysis time can be reduced to one-third of that required in using a conventional ELISA. The substrate-enzyme reaction products can be easily transferred to another microplate, and their absorbance can be measured without interference by light scattering caused by magnetic microbeads. This method demonstrates great potential for detecting other biomarkers and in biochemical applications. Graphical AbstractA magnetic

Ethanol extract of seeds of Oenothera odorata induces vasorelaxation via endothelium-dependent NO-cGMP signaling through activation of Akt-eNOS-sGC pathway.

PubMed

Kim, Hye Yoom; Oh, Hyuncheol; Li, Xiang; Cho, Kyung Woo; Kang, Dae Gill; Lee, Ho Sub

2011-01-27

The vasorelaxant effect of ethanol extract of seeds of Oenothera odorata (Onagraceae) (one species of evening primroses) (ESOO) and its mechanisms involved were defined. Changes in vascular tension, guanosine 3',5'-cyclic monophosphate (cGMP) levels, and Akt expression were measured in carotid arterial rings from rats. Seeds of Oenothera odorata were extracted with ethanol (94%) and the extract was filtered, concentrated and stored at -70°C. ESOO relaxed endothelium-intact, but not endothelium-denuded, carotid arterial rings in a concentration-dependent manner. Similarly, ESOO increased cGMP levels of the carotid arterial rings. Pretreatment of endothelium-intact arterial rings with L-NAME, an inhibitor of nitric oxide synthase (NOS), or ODQ, an inhibitor of soluble guanylyl cyclase (sGC), blocked the ESOO-induced vasorelaxation and increase in cGMP levels. Nominally Ca(2+)-free but not L-typed Ca(2+) channel inhibition attenuated the ESOO-induced vasorelaxation. Thapsigargin, Gd(3+), and 2-aminoethyl diphenylborinate, modulators of store-operated Ca(2+) entry (SOCE), significantly attenuated the ESOO-induced vasorelaxation and increase in cGMP levels. Further, wortmannin, an inhibitor of Akt, attenuated the ESOO-induced vasorelaxation and increases in cGMP levels and phosphorylated Akt2 expression. K(+) channel blockade with TEA, 4-aminopyridine, and glibenclamide attenuated the ESOO-induced vascular relaxation. Taken together, the present study demonstrates that ESOO relaxes vascular smooth muscle via endothelium-dependent NO-cGMP signaling through activation of the Akt-eNOS-sGC pathway. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

PubMed Central

Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

2014-01-01

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

Nitric oxide augments single Ca(2+) channel currents via cGMP-dependent protein kinase in Kenyon cells isolated from the mushroom body of the cricket brain.

PubMed

Kosakai, Kumiko; Tsujiuchi, Yuuki; Yoshino, Masami

2015-07-01

Behavioral and pharmacological studies in insects have suggested that the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is involved in the formation of long-term memory (LTM) associated with olfactory learning. However, the target molecules of NO and the downstream signaling pathway are still not known. In this study, we investigated the action of NO on single voltage-dependent Ca(2+) channels in the intrinsic neurons known as Kenyon cells within the mushroom body of the cricket brain, using the cell-attached configuration of the patch-clamp technique. Application of the NO donor S-nitrosoglutathione (GSNO) increased the open probability (NPO) of single Ca(2+) channel currents. This GSNO-induced increase was blocked by ODQ, a soluble guanylate cyclase (sGC) inhibitor, suggesting that the NO generated by GSNO acts via sGC to raise cGMP levels. The membrane-permeable cGMP analog 8-Bro-cGMP also increased the NPO of single Ca(2+) channel currents. Pretreatment of cells with KT5823, a protein kinase G blocker, abolished the excitatory effect of GSNO. These results suggest that NO augments the activity of single Ca(2+) channels via the cGMP/PKG signaling pathway. To gain insight into the physiological role of NO, we examined the effect of GSNO on action potentials of Kenyon cells under current-clamp conditions. Application of GSNO increased the frequency of action potentials elicited by depolarizing current injections, indicating that NO acts as a modulator resulting in a stimulatory signal in Kenyon cells. We discuss the increased Ca(2+) influx through these Ca(2+) channels via the NO/cGMP signaling cascade in relation to the formation of olfactory LTM. Copyright © 2015 Elsevier Ltd. All rights reserved.

ChIP-Seq and RNA-Seq Reveal an AmrZ-Mediated Mechanism for Cyclic di-GMP Synthesis and Biofilm Development by Pseudomonas aeruginosa

PubMed Central

Jones, Christopher J.; Newsom, David; Kelly, Benjamin; Irie, Yasuhiko; Jennings, Laura K.; Xu, Binjie; Limoli, Dominique H.; Harrison, Joe J.; Parsek, Matthew R.; White, Peter; Wozniak, Daniel J.

2014-01-01

The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for Amr

cGMP signaling as a target for the prevention and treatment of breast cancer.

PubMed

Windham, Perrin F; Tinsley, Heather N

2015-04-01

One in eight women in the United States will be diagnosed with invasive breast cancer in her lifetime. Advances in therapeutic strategies, diagnosis, and improved awareness have resulted in a significant reduction in breast cancer related mortality. However, there is a continued need for more effective and less toxic drugs for both the prevention and the treatment of breast cancer in order to see a continued decline in the morbidity and mortality associated with this disease. Recent studies suggest that the cGMP signaling pathway may be aberrantly regulated in breast cancer. As such, this pathway may serve as a source of novel targets for future breast cancer drug discovery efforts. This review provides an overview of cGMP signaling in normal physiology and in breast cancer as well as current strategies being investigated for targeting this pathway in breast cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Galapagos hotspot-spreading center system: 1. Spatial petrological and geochemical variations (83/sup 0/W-101/sup 0/W)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schilling, J.; Kingsley, R.H.; Devine, J.D.

We report on the petrology and geochemistry of basalts dredged at 40--50 km intervals along the Galapagos Spreading Center, between 83/sup 0/W and 101/sup 0/W (40 stations). Emphasis is on spatial variations of 'whole rock' major elements, rare earths, trace metals of the first transition series, and the nature of phenocryst assemblages and their abundances. These results provide new constraints on the nature and scale of mantle source heterogeneities, melting conditions, thermal field, and dynamics of crustal formation of the region. We suggest that ridge segments outside the high magnetic amplitude zone are at a steady state as a resultmore » of passive seafloor spreading. Basalts from these segments are apparently derived from an asthenosphere relatively uniformally depleted in incompatible elements, which appears of worldwide extent. We reject Vogt and DeBoer's (1976) model invoking damming at fracture zones of subaxial asthenosphere flow of crystal slushes and increasing fractional crystallization down the flow line, because this model would not explain the gradients in REE observed about the Galapagos Platform. Our preferred model combines the mantle-plume binary mixing model of Schilling (1973) with the concept of recurring rift propagation proposed by Hey (1977a). We further propose that pulsating mantle plume flux, perhaps in the form of a chain of blobs, may initiate the development of new rifts and their propagation. The present position of the tips of such new propagating rifts locate the wave fronts of such pulsating mantle plume flow. A two million year period is suggested for the last 4 m.y. from Wilson and Hey's (1979) information Rigorous testing of our preferred model is possible.« less

Microplate technique for determining accumulation of metals by algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hassett, J.M.; Jennett, J.C.; Smith, J.E.

1981-05-01

A microplate technique was developed to determine the conditions under which pure cultures of algae removed heavy metals from aqueous solutions. Variables investigated included algal species and strain, culture age (11 and 44 days), metal (mercury, lead, cadmium, and zinc), pH, effects of different buffer solutions, and time of exposure. Plastic, U-bottomed microtiter plates were used in conjunction with heavy metal radionuclides to determine concentration factors for metal-alga combinations. The technique developed was rapid, statistically reliable, and economical of materials and cells. All species of algae studied removed mercury from solution. Green algae proved better at accumulating cadmium than didmore » blue-green algae. No alga studied removed zinc, perhaps because cells were maintained in the dark during the labeling period. Chlamydomonas sp. proved superior in ability to remove lead from solution.« less

Mapping lava morphology of the Galapagos Spreading Center at 92°W: fuzzy logic provides a classification of high-resolution bathymetry and backscatter

NASA Astrophysics Data System (ADS)

McClinton, J. T.; White, S. M.; Sinton, J. M.; Rubin, K. H.; Bowles, J. A.

2010-12-01

Differences in axial lava morphology along the Galapagos Spreading Center (GSC) can indicate variations in magma supply and emplacement dynamics due to the influence of the adjacent Galapagos hot spot. Unfortunately, the ability to discriminate fine-scale lava morphology has historically been limited to observations of the small coverage areas of towed camera surveys and submersible operations. This research presents a neuro-fuzzy approach to automated seafloor classification using spatially coincident, high-resolution bathymetry and backscatter data. The classification method implements a Sugeno-type fuzzy inference system trained by a multi-layered adaptive neural network and is capable of rapidly classifying seafloor morphology based on attributes of surface geometry and texture. The system has been applied to the 92°W segment of the western GSC in order to quantify coverage areas and distributions of pillow, lobate, and sheet lava morphology. An accuracy assessment has been performed on the classification results. The resulting classified maps provide a high-resolution view of GSC axial morphology and indicate the study area terrain is approximately 40% pillow flows, 40% lobate and sheet flows, and 10% fissured or faulted area, with about 10% of the study area unclassifiable. Fine-scale features such as eruptive fissures, tumuli, and individual pillowed lava flow fronts are also visible. Although this system has been applied to lava morphology, its design and implementation are applicable to other undersea mapping applications.

Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP

PubMed Central

Okegbe, Chinweike; Fields, Blanche L.; Cole, Stephanie J.; Beierschmitt, Christopher; Morgan, Chase J.; Price-Whelan, Alexa; Stewart, Richard C.; Lee, Vincent T.; Dietrich, Lars E. P.

2017-01-01

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttle-mediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth. In the pathogenic bacterium Pseudomonas aeruginosa PA14, antibiotics called phenazines act as oxidants to balance the intracellular redox state of cells in anoxic biofilm subzones. PA14 colony biofilms show a profound morphogenic response to phenazines resulting from electron acceptor-dependent inhibition of ECM production. This effect is reminiscent of the developmental responses of some eukaryotic systems to redox control, but for bacterial systems its mechanistic basis has not been well defined. Here, we identify the regulatory protein RmcA and show that it links redox conditions to PA14 colony morphogenesis by modulating levels of bis-(3′,5′)-cyclic-dimeric-guanosine (c-di-GMP), a second messenger that stimulates matrix production, in response to phenazine availability. RmcA contains four Per-Arnt-Sim (PAS) domains and domains with the potential to catalyze the synthesis and degradation of c-di-GMP. Our results suggest that phenazine production modulates RmcA activity such that the protein degrades c-di-GMP and thereby inhibits matrix production during oxidizing conditions. RmcA thus forms a mechanistic link between cellular redox sensing and community morphogenesis analogous to the functions performed by PAS-domain–containing regulatory proteins found in complex eukaryotes. PMID:28607054

Large-scale manufacturing of GMP-compliant anti-EGFR targeted nanocarriers: production of doxorubicin-loaded anti-EGFR-immunoliposomes for a first-in-man clinical trial.

PubMed

Wicki, Andreas; Ritschard, Reto; Loesch, Uli; Deuster, Stefanie; Rochlitz, Christoph; Mamot, Christoph

2015-04-30

We describe the large-scale, GMP-compliant production process of doxorubicin-loaded and anti-EGFR-coated immunoliposomes (anti-EGFR-ILs-dox) used in a first-in-man, dose escalation clinical trial. 10 batches of this nanoparticle have been produced in clean room facilities. Stability data from the pre-GMP and the GMP batch indicate that the anti-EGFR-ILs-dox nanoparticle was stable for at least 18 months after release. Release criteria included visual inspection, sterility testing, as well as measurements of pH (pH 5.0-7.0), doxorubicin HCl concentration (0.45-0.55 mg/ml), endotoxin concentration (<1.21 IU/ml), leakage (<10%), particle size (Z-average of Caelyx ± 20 nm), and particle uptake (uptake absolute: >0.50 ng doxorubicin/μg protein; uptake relatively to PLD: >5 fold). All batches fulfilled the defined release criteria, indicating a high reproducibility as well as batch-to-batch uniformity of the main physico-chemical features of the nanoparticles in the setting of the large-scale GMP process. In the clinical trial, 29 patients were treated with this nanoparticle between 2007 and 2010. Pharmacokinetic data of anti-EGFR-ILs-dox collected during the clinical study revealed stability of the nanocarrier in vivo. Thus, reliable and GMP-compliant production of anti-EGFR-targeted nanoparticles for clinical application is feasible. Copyright © 2015 Elsevier B.V. All rights reserved.

Chlorine in mid-ocean ridge magmas: Evidence for assimilation of seawater-influenced components

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michael, P.J.; Schilling, J.G.

1989-12-01

Suites of depleted MORB glasses from the fast-spreading Pacific-Nazca Ridge at 28{degree}S and 32{degree}S and the slow-spreading eastern boundary of the Juan Fernandez microplate were analyzed for chlorine by electron microprobe. The Cl concentrations in FeTi basalts exceed by a factor of 5 to 10 the amounts that can be generated by fractional crystallization of the primitive magmas. Selective melting or breakdown of amphibole and incorporation of Cl-rich brine contained in the wall rocks may be important processes. A magmatic source for the additional Cl and H{sub 2}O cannot be ruled out on geochemical grounds but is physically unrealistic becausemore » it requires that large volumes of magma have crystallized and exsolved a Cl-rich vapor phase that has somehow migrated to a small magma chamber. Excess Cl in evolved magmas is best developed in evolved MORB from propagating or overlapping spreading centers such as the Galapagos Spreading Center at 85{degree}W and 95{degree}W and the west ridge of the Juan Fernandez microplate. Cl overenrichment has not been observed on slow-spreading ridges including the eastern ridge of the Juan Fernandez microplate, the Southwest Indian Ridge, and the mid-Atlantic Ridge. The assimilation of hydrothermally altered material could influence the concentration and isotopic ratios of other elements which have low abundances in MORB relative to seawater.« less

Cardioprotective cGMP favors exogenous fatty acid incorporation into tyiglycerides over direct beta-oxidation

USDA-ARS?s Scientific Manuscript database

While cardiac hypertrophy has been associated with a shift in substrate selection for energy production from fatty acids (FA) to carbohydrates (CHO), it remains controversial whether this shift is adaptive or maladaptive. Since enhanced cGMP signalling can prevent hypertrophy, we hypothesized that t...

Noradrenaline activates the NO/cGMP/ATP-sensitive K(+) channels pathway to induce peripheral antinociception in rats.

PubMed

Romero, Thiago R L; Guzzo, Luciana S; Perez, Andrea C; Klein, André; Duarte, Igor D G

2012-03-31

Despite the classical peripheral pronociceptive effect of noradrenaline (NA), recently studies showed the involvement of NA in antinociceptive effect under immune system interaction. In addition, the participation of the NO/cGMP/KATP pathway in the peripheral antinociception has been established by our group as the molecular mechanism of another adrenoceptor agonist xylazine. Thus the aim of this study was to obtain pharmacological evidences for the involvement of the NO/cGMP/KATP pathway in the peripheral antinociceptive effect induced by exogenous noradrenaline. The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E(2) (2μg/paw). All drugs were locally administered into the right hind paw of male Wistar rats. NA (5, 20 and 80ng/paw) elicited a local inhibition of hyperalgesia. The non-selective NO synthase inhibitor l-NOarg (12, 18 and 24μg/paw) antagonized the antinociception effect induced by the highest dose of NA. The soluble guanylyl cyclase inhibitor ODQ (25, 50 and 100μg/paw) antagonized the NA-induced effect; and cGMP-phosphodiesterase inhibitor zaprinast (50μg/paw) potentiated the antinociceptive effect of NA low dose (5ng/paw). In addition, the local effect of NA was antagonized by a selective blocker of an ATP-sensitive K(+) channel, glibenclamide (20, 40 and 80μg/paw). On the other hand, the specifically voltage-dependent K(+) channel blocker, tetraethylammonium (30μg/paw), Ca(2+)-activated K(+) channel blockers of small and large conductance types dequalinium (50μg/paw) and paxilline (20μg/paw), respectively, were not able to block local antinociceptive effect of NA. The results provide evidences that NA probably induces peripheral antinociceptive effects by activation of the NO/cGMP/KATP pathway. Copyright © 2012 Elsevier Inc. All rights reserved.

Eruptive patterns and structure of Isla Fernandina, Galapagos Islands, from SPOT-1 HRV and large format camera images

NASA Technical Reports Server (NTRS)

Munro, Duncan C.; Mouginis-Mark, Peter J.

1990-01-01

SPOT-1 HRV, and large format-camera images were used to investigate the distribution and structure of erupted materials on Isla Fernandina, Galapagos Islands. Maps of lava flows, fissures, cones and topography derived from these data allow the first study of the entire subaerial segment of this geographically remote and ecologically sensitive volcano. No significant departure from a uniform distribution of erupted lava with azimuth can be detected. Short (less than 4 km) lava flows commonly have their source in the summit region and longer (greater than 8 km) lava flows originate from vents at lower elevations. Catastrophic landslides are proposed as a possible explanation for the asymmetry of the coastline with respect to the caldera.

Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium.

PubMed

Jonas, Kristina; Edwards, Adrianne N; Ahmad, Irfan; Romeo, Tony; Römling, Ute; Melefors, Ojar

2010-02-01

Bacterial survival depends on the ability to switch between sessile and motile lifestyles in response to changing environmental conditions. In many species, this switch is governed by (3'-5')-cyclic-diguanosine monophosphate (c-di-GMP), a signalling molecule, which is metabolized by proteins containing GGDEF and/or EAL domains. Salmonella Typhimurium contains 20 such proteins. Here, we show that the RNA-binding protein CsrA regulates the expression of eight genes encoding GGDEF, GGDEF-EAL and EAL domain proteins. CsrA bound directly to the mRNA leaders of five of these genes, suggesting that it may regulate these genes post-transcriptionally. The c-di-GMP-specific phosphodiesterase STM3611, which reciprocally controls flagella function and production of biofilm matrix components, was regulated by CsrA binding to the mRNA, but was also indirectly regulated by CsrA through the FlhDC/FliA flagella cascade and STM1344. STM1344 is an unconventional (c-di-GMP-inactive) EAL domain protein, recently identified as a negative regulator of flagella gene expression. Here, we demonstrate that CsrA directly downregulates expression of STM1344, which in turn regulates STM3611 through fliA and thus reciprocally controls motility and biofilm factors. Altogether, our data reveal that the concerted and complex regulation of several genes encoding GGDEF/EAL domain proteins allows CsrA to control the motility-sessility switch in S. Typhimurium at multiple levels.

[Effects of nitric oxide on peritoneal lymphatic stomata and lymph drainage via NO-cGMP-Ca2+ pathway].

PubMed

Li, Yan-Yuan; Li, Ji-Cheng

2005-02-25

To study the cell signal transduction mechanism of nitric oxide (NO) on the peritoneal lymphatic stomata and lymph drainage in the rat, cGMP content were measured by a commercially available radioimmunoassay kit, and the [Ca(2+)](i) were observed by a confocal laser scanning microscope in the cultured peritoneal mesothelial cell. Animal experiment was practiced to study the effect of NO-cGMP-Ca(2+) pathway on the lymphatic stomata and lymph absorption. The results showed that: (1) Sper/NO increased cGMP of the rat peritoneal mesothelial cell (RPMC) in a dose-dependent manner (P<0.01) compared to the control group. This effect was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) (P<0.05), a specific inhibitor of soluble guanylyl cyclase (sGC). The level of [Ca(2+)](i) in single RPMC decreased by adding Sper/NO (P<0.05). Pretreatment with ODQ for 10 min blocked the Sper/NO-induced decrease in [Ca(2+)](i). L-typed calcium channel blocker nifedipine induced an immediate and marked decrease in [Ca(2+)](i) (P<0.05).. After [Ca(2+)](i) reached a balance again, adding Sper/NO could not change [Ca(2+)](i) (P>0.05). (2) Sper/NO increased the area of the stomata (P<0.01) and the quantity of the tracer in a dose-dependent manner (P<0.05) compared to the control group. Pretreatment with ODQ significantly inhibited Sper/NO-induced change of lymphatic stomata and lymph drainage (P<0.01). Nifedipine increased the opening area of the lymphatic stomata (P< 0.01) and the concentration of absorbed trypan blue of the diaphragm (P<0.05). Sper/NO could not make a further change in the samples pretreated by nifedipine (P> 0.05). The results indicate that NO can decrease [Ca(2+)](i) in the RPMC through the NO-cGMP pathway. This procession is related with the L- type voltage-gated Ca(2+) channel. NO enlarges the opening area of the lymphatic stomata and enhances the lymph drainage of tracer by NO-cGMP-[Ca(2+)](i) pathway.

Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer.

PubMed

Chang, Yanli; Xu, Jianjun; Zhang, Qingyun

2017-10-01

Survivin is a tumor marker for bladder cancer; however the role of urinary survivin levels has not been fully elucidated due to the limitations of current detection methods. Based on two survivin-specific monoclonal antibodies (McAbs) already confirmed through enzyme linked immunosorbent assays, the present study aimed to establish a microplate magnetic chemiluminescence immunoassay (CLIA) for the detection of urinary survivin levels and evaluate its application for the diagnosis of patients with bladder cancer. Horseradish peroxidase and biotin conjugates were used to label two different anti-survivin McAbs, respectively. The labeled antibodies combined with survivin to form a sandwiched immune complex. The streptavidin magnetic particles (MPs) served as the solid phase and the separator. The relevant parameters involved in the immunoassay, including the immunoassay reagents used and the physicochemical parameters were optimized. Then, urine samples from 130 patients with bladder cancer and 113 healthy controls were detected, and analyzed using the established method. The method was linear to 1,000 ng/ml survivin with a detection limit of 0.83 ng/ml. The intra- and inter-assay coefficients of variation were <8, and <11%, respectively. The concentration of diluted survivin and the dilution ratios gave a linear correlation of 0.9989. The results demonstrated that the urinary survivin levels in patients with bladder cancer were significantly higher (P<0.001) compared with that in healthy controls. At a survivin concentration of 2.0884 ng/ml, the sensitivity and specificity were 86.9 and 61.9%, respectively. Furthermore, the urinary survivin levels were positively correlated with metastatic stage, histological stage and recurrence (P<0.01). In conclusion, the present study preliminarily proposed a microplate magnetic CLIA for survivin detection and further evaluated the value of urinary survivin as a diagnostic marker for bladder cancer.