The characterization of an epithelial chloride channel and purification of a channel component

A membrane fraction (M$\sb{\rm PS}$), enriched in Cl$\sp-$ channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The tracheal fraction shows a 37 fold enrichment of Cl$\sp-$ channels over crude tracheal homogenates by net Cl$\sp-$ measurements in membrane vesicles. Alkaline phosphatase and (Na$\sp+$ + K$\sp+$)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. The M$\sb{\rm PS}$ fraction exhibits a protein profile unlike that of other membrane fractions with major proteins of 200 kDa and 42 kDa, proteins of 30 to 35 kDa, and lesser amounts of other proteins. Reconstitution of M$\sb{\rm PS}$ fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is linear with a slope conductance of 84 pS in symmetrical 400 mM KCl, and is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (Valdivia, Dubinsky, and Coronado. Science, 1988). In addition, the voltage dependence, selectivity sequence of Cl$\sp- >$ Br$\sp- \ge$ I$\sp-$, and inhibition by low concentrations of the Cl$\sp-$ channel blocker, DIDS, correspond to those of the predominant apical membrane channel. Thus, although the M$\sb{\rm PS}$ fraction appears to be of subcellular origin, it may be functionally related to an apical membrane Cl$\sp-$ permeability. When renal M$\sb{\rm PS}$ membranes were treated with the detergent octyl-glucoside (OG, 2%) and centrifuged, the supernatant, sM$\sb{\rm PS}$, showed a 2 to 7-fold enrichment in specific Cl$\sp-$ flux activity compared with the detergent treated M$\sb{\rm PS}$. These solubilized proteins were then size fractionated on a Superose 12 HPLC gel filtration column, followed by fractionation on a Mono Q HPLC anion exchange column. Fractions that eluted in high salt consistently exhibited significant Cl$\sp-$ flux activity. These fractions had protein profiles consisting of a major band at 34 kDa, a band at 66 kDa, and variable faint bands. Fractions eluting in lower salt had protein profiles consisting of a single band at 34 kDa, and often had little or no Cl$\sp-$ flux activity. However, co-reconstitution of the low salt, solely 34 kDa protein-containing Mono Q fractions with sM$\sb{\rm PS}$ resulted in an enhancement of flux activity compared to that of sM$\sb{\rm PS}$ reconstituted alone. Flux assays of active Mono Q fractions showed that the channel retained its DIDS sensitivity. Applying sM$\sb{\rm PS}$ to a DIDS-affinity column and eluting with salt resulted in fractions with protein profiles again consisting of at least one major band at 34 kDa, a band at 66 kDa, and variable faint bands. Co-reconstitution with sM$\sb{\rm PS}$ again resulted in an enhancement of activity. Thus, the 34 kDa protein appears to be a component of the M$\sb{\rm PS}$ Cl$\sp-$ channel. ^

Method for the determination of specific molecular markers of biomass burning in lake sediments

Fire has an influence on regional to global atmospheric chemistry and climate. Molecular markers of biomass burning archived in lake sediments are becoming increasingly important in paleoenvironmental reconstruction and may help determine the interaction between climate and fire activity. Here, we present a high performance anion exchange chromatography–mass spectrometry method to allow separation and analysis of levoglucosan, mannosan and galactosan in lake sediments, with implications for reconstructing past biomass burning events. Determining mannosan and galactosan in Lake Kirkpatrick, New Zealand (45.03°S, 168.57°E) sediment cores and comparing these isomers with the more abundant biomass burning markers levoglucosan and charcoal represents a significant advancement in our ability to analyze past fire activity. Levoglucosan, mannosan and galactosan concentrations correlated significantly with macroscopic charcoal concentration. Levoglucosan/mannosan and levoglucosan/(mannosan + galactosan) ratios may help determine not only when fires occurred, but also if changes in the primary burned vegetation occurred.

CHARACTERIZATION OF CYTOCHROME P-450 MIXED FUNCTION OXIDASE OF RAT COLON MUCOSA

Non-pregnant, female adult rats pretreated with either phenobarbital (PB) or (beta)-naphthoflavone ((beta)NF) through short-course intraperitoneal injections were shown by sodium dithionite-reduced carbon monoxide difference spectroscopy and NADPH-cytochrome c in vitro assay to contain cytochrome P-450 and NADPH-dependent reductase associated with the microsomal fraction of colon mucosa. These two protein components of the mixed function oxidase system were released from the microsomal membrane, resolved from each other, and partially purified by using a combination of techniques including solubilization in nonionic detergent followed by ultracentrifugation, anion exchange and adsorption column chromatographies, native gel electrophoresis, polyethylene glycol fractionation and ultrafiltration.^ In vitro reconstitution assays demonstrated the cytochrome P-450 fraction as the site of substrate and molecular oxygen binding. By the use of immunochemical techniques including radial immunodiffusion, Ouchterlony double diffusion and protein electroblotting, the cytochrome P-450 fraction was shown to contain at least 5 forms of the protein, having molecular weights as determined by SDS gel electrophoresis identical to the corresponding hepatic cytochrome P-450. Estimation of total cytochrome P-450 content confirmed the preferential induction of particular forms in response to the appropriate drug pretreatment.^ The colonic NADPH-dependent reductase was isolated from native gel electrophoresis and second dimensional SDS gel electrophoresis was performed in parallel to that for purified reductase from liver. Comparative electrophoretic mobilities together with immunochemical analysis, as with the cytochrome P-450s, reconstitution assays, and kinetic characterization using artificial electron acceptors, gave conclusive proof of the structural and functional homology between the colon and liver sources of the enzyme.^ Drug metabolism was performed in the reconstituted mixed function oxidase system containing a particular purified liver cytochrome P-450 form or partially pure colon cytochrome P-450 fraction plus colon or liver reductase and synthetic lipid vesicles. The two drugs, benzo{(alpha)}pyrene and benzphetamine, which are most representative of the action of system in liver, lung and kidney, were tested to determine the specificity of the reconstituted system. The kinetics of benzo{(alpha)}pyrene hydroxylation were followed fluorimetrically for 3-hydroxybenzo{(alpha)}pyrene production. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Identification, purification, sequencing and characterization of human lung fibroblast motility -stimulating factor for human soft tissue sarcoma cells

Paracrine motogenic factors, including motility cytokines and extracellular matrix molecules secreted by normal cells, can stimulate metastatic cell invasion. For extracellular matrix molecules, both the intact molecules and the degradative products may exhibit these activities, which in some cases are not shared by the intact molecules. We found that human peritumoral and lung fibroblasts secrete motility-stimulating activity for several recently established human sarcoma cell strains. The motility of lung metastasis-derived human SYN-1 sarcoma cells was preferentially stimulated by human lung and peritumoral fibroblast motility-stimulating factors (FMSFs). FMSFs were nondialyzable, susceptible to trypsin, and sensitive to dithiothreitol. Cycloheximide inhibited accumulation of FMSF activity in conditioned medium; however, addition of cycloheximide to the migration assay did not significantly affect motility-stimulating activity. Purified hepatocyte growth factor/scatter factor (HGF/SF), rabbit anti-hHGF, and RT-PCR analysis of peritumoral and lung fibroblast HGF/SF mRNA expression indicated that FMSF activity was unrelated to HGF/SF. Partial purification of FMSF by gel exclusion chromatography revealed several peaks of activity, suggesting multiple FMSF molecules or complexes.^ We purified the fibroblast motility-stimulating factor from human lung fibroblast-conditioned medium to apparent homogeneity by sequential heparin affinity chromatography and DEAE anion exchange chromatography. Lysylendopeptidase C digestion of FMSF and sequencing of peptides purified by reverse phase HPLC after digestion identified it as an N-terminal fragment of human fibronectin. Purified FMSF stimulated predominantly chemotaxis but chemokinesis as well of SYN-1 sarcoma cells and was chemotactic for a variety of human sarcoma cells, including fibrosarcoma, leiomyosarcoma, liposarcoma, synovial sarcoma and neurofibrosarcoma cells. The motility-stimulating activity present in HLF-CM was completely eliminated by either neutralization or immunodepletion with a rabbit anti-human-fibronectin antibody, thus further confirming that the fibronectin fragment was the FMSF responsible for the motility stimulation of human soft tissue sarcoma cells. Since human soft tissue sarcomas have a distinctive hematogenous metastatic pattern (predominantly lung), FMSF may play a role in this process. ^

Manzano : : distribución de micronutrientes en suelo

Para medir la disponibilidad de los micronutrientes en la zona de mayor densidad radical de los frutales de pepita y carozo y sugerir pautas de manejo que permitan un uso sustentable del recurso suelo, se tomaron muestras representativas de 25 montes cultivados con manzanos a lo largo del Alto Valle del río Negro (Argentina). Se extrajeron muestras a 0-25 y 25-50 cm de profundidad y se determinó la concentración disponible de Fe, Cu, Mn y Zn; granulometría; pH; materia orgánica (MO); carbonatos; P y capacidad de intercambio catiónico (CIC). Los resultados muestran que los micronutrientes se concentran mayoritariamente en la primera capa de suelo analizada, disminuyendo abruptamente en el estrato 25-50 cm. En la capa superficial, la disponibilidad de Cu y Zn está influenciada por el P mientras que el pH afecta la del Fe, Cu, y Mn en el estrato de suelo de 25-50 cm. En conclusión, es posible mejorar la nutrición mineral de los cultivos si se crean condiciones favorables para el crecimiento radical en la capa superficial del suelo.

Propiedades químicas de suelos desarrollados sobre distintos materiales originales bajo bosques de Austrocedrus chilensis

En este trabajo se caracterizaron las propiedades químicas del horizonte A de los suelos desarrollados a partir de distintos materiales originarios, sobre los cuales habitan bosques de Austrocedrus chilensis. Se seleccionaron cinco sitios, ubicados en el Valle 16 de Octubre y en el Parque Nacional Los Alerces de la Provincia del Chubut, Argentina. De cada sitio se tomaron muestras compuestas del horizonte A para la caracterización de las propiedades químicas. Todos los suelos analizados presentaron elevados contenidos de materia orgánica y nitrógeno. Los suelos originados a partir de materiales glaciarios presentaron los máximos valores de bases de intercambio, capacidad de intercambio catiónico y contenido de fósforo, diferenciándose significativamente de los suelos volcánicos. Dentro de los suelos volcánicos se detectaron diferencias en función de la granulometría del material: los suelos derivados de ceniza (< 2mm) tendieron a presentar mayores valores de capacidad de intercambio catiónico y bases de intercambio que los suelos de pumita (> 2mm).

Caracterización de tres suelos en Hidalgo y Querétaro, México: disponibilidad y daño tóxico por arsénico en garbanzo (Cicer arietinum L.)

La presencia de arsénico (As) en suelos permite su migración hacia cultivos como el garbanzo (Cicer arietinum L.). En este trabajo se comparan contenidos de As en tres suelos de dos estados de la República Mexicana, y su acumulación en C. arietinum L. Los suelos resultaron moderadamente alcalinos, no salinos, reductores intermedios y con potenciales zeta (pZ) que indican suspensiones coloidales moderadamente estables. Con moderados contenidos de humedad y texturas franco- arcillosas, densidad aparente, capacidad de campo, agregados estables y capacidad de intercambio catiónico, significativamente diferentes y semejantes en velocidad de infiltración y espesor del horizonte A. En materia orgánica, carbono y nitrógeno son significativamente diferentes y con bajos contenidos. Existen diferencias importantes entre semillas de C. arietinum L. certificadas o no en su capacidad de germinación y desarrollo de raíces. El efecto genotóxico del As en raíces de C. arietinum L. se apreció por inducción de micronúcleos, reducción de 2,8 veces la división celular de muestras tratadas con agua con As, respecto de muestras control. Se apreció incremento de As de 9,5 veces en plántulas germinadas en suelo de El Salitre, que en suelo de Bella Vista, indicativo de migración del As. La suma de efectos de concentración de As en suelos y agua se incrementa 15,3 veces. Entre suelos de Bella Vista y Querétaro la correlación fue 2,9 veces mayor. En controles positivos los IBAs resultan 3 veces mayores que en las pruebas experimentales.

Evaluación del proceso de compostaje de dos mezclas de residuos avícolas

Los residuos del sector avícola, principalmente guano (aves ponedoras) y cama de parrilleros (aves de engorde), pueden generar un impacto negativo en el ambiente contribuyendo a la contaminación de suelo, agua y aire. La estabilización aeróbica a través del compostaje es una alternativa de tratamiento para reducir la contaminación. El objetivo de este trabajo fue evaluar el proceso de compostaje en dos mezclas con diferentes porcentajes de residuos avícolas (guano de aves ponedoras y cama de pollos parrilleros). Se compostaron dos mezclas que contenían 81% y 70% de residuos avícolas durante 16 semanas. Las variables analizadas fueron: temperatura (T°), pH, conductividad eléctrica (CE), humedad (H), capacidad de intercambio catiónico (CIC), carbono orgánico total (COT), amonio (NH4+), nitrato (NO3 - ), nitrógeno total (NT ) y carbono soluble (CS). Las características finales de los compost A y B fueron: pH 7,1 - 6,8, CE 3,3 - 2,9 (mS. cm- 1), COT 14,8 - 17,9 %, NT 0,97 - 0,88 %, NH4 + 501 - 144,9 mg kg-1, NO3-552,3 - 543,0 mg kg-1 respectivamente. El proceso de compostaje podría ser una herramienta para estabilizar los residuos avícolas minimizando su impacto en el ambiente.

Chemical composition of clay minerals from ODP Leg 168 sites

The exchangeable cation compositions of organic-poor terrigenous sediments containing smectite as primary ion exchanger from a series of holes along ODP Leg 168 transect on the eastern flank of the Juan de Fuca Ridge have been examined as a function of distance from the ridge axis and burial depth. The total cation exchange capacity (CEC) values of the sediments ranged from 2 to 59 meq/100 g, increasing with increases in the wt.% smectite. At the seafloor, the exchangeable cation compositions involving Na, K, Mg, and Ca, expressed in terms of equivalent fraction, are nearly constant regardless of the different transect sites: XNa = 0.21 ± 0.04, XK = 0.08 ± 0.01, XMg = 0.33 ± 0.09, and XCa = 0.38 ± 0.09. The calculated selectivity coefficients of the corresponding quaternary exchange reactions, calculated using porewater data, are in log units -5.45 ± 0.39 for Na, 1.97 ± 0.49 for K, 0.42 ± 0.41 for Mg, and 3.06 ± 0.69 for Ca. The exchangeable cation compositions below the seafloor change systematically with distance from the ridge crest and burial depth, conforming to the trends of the same cations in the porewaters. The selectivities for Na and Mg are roughly constant at temperatures from 2 to 66°C, indicating that the equivalent fractions of these two cations are independent of sediment alteration taking place on the ridge flank. Unlike Na and Mg, the temperature influence is significant for K and Ca, with Ca-selectivity decreases being coupled with increases in K-selectivity. Although potentially related to diagenetic and/or hydrothermal mineral precipitation or recrystallization, no evidence of such alteration was detected by XRD and TEM. In sites where upwelling of hydrothermal fluids from basement is occurring, the K-selectivity of the sediment is appreciably higher than at the other sites and corresponds to the formation of (Fe, Mg) rich smectite and zeolites. Our study indicates that local increases in K-selectivity at hydrothermal sites are caused by the formation of these authigenic minerals.

(Supplement) Regeneration of Pinus sylvestris, disturbance residuals and soil properties 5 years after a mixed-severity wildfire in the inner Italian Alps

We analyzed the abundance of Scots pine regeneration in a 257 ha wildfire in an inner-alpine forest. We sampled regeneration, percent soil cover by classes, physical and chemical properties of topsoils (A horizon, 0-5 cm) under four fire severity levels (unburned, moderate, moderate/high, high severity). 5 plots per severity level, circular (R= 3m). Analysis methods for soil properties as described in the paper.

Species and composition of tundra meadows

Observations of hummock and string-like microrelief features were made in High Arctic hydric meadows. Thermal shearing of thick bryophyte mats, and subsequent roll back during spring flooding appears to be one way in which this topography is formed. Hummocky and non-hummocky (flat) meadows show distinct floristic differences which may in part be due to observed differences in temperature, nutrient concentrations and moisture relations.

Electrical properties of basalts of ODP Sites 124-768 and 124-770

Conductivity of 54 basalt samples from ODP Sites 768 and 770 was measured as a function of temperature and fluid salinity. Porosity was also measured for all samples, and cation exchange capacity was measured for 46 of the samples. Porosity measurements indicated that porosity is underestimated for basalts like these, unless one uses extensive drying at high vacuum. At salinities greater than 29 ppt, and throughout the range of salinity and temperatures likely in situ, sample conductivity (Co) is controlled by porosity (phi) according to the Archie relation Co = 0.22*Cw phi*1-3 (orFF = 4.5/f1.3), where Cw is conductivity of the pore fluids and FF = Cw/CO is the formation factor. At lower salinity, clay-surface conduction or microcrack conduction may dominate. We are unable to distinguish reliably between the two mechanisms, but we do detect their effects subtly at high salinity and strongly at low salinity.

Abundance and sizes of European oak species (Quercus petraea (Matt.) Liebl., Quercus robur L.) in dependence of neighborhood and environmental factors

Quercus robur L. (pedunculate oak) and Quercus petraea (Matt.) Liebl. (sessile oak) are two European oak species of great economic and ecological importance. Even though both oaks have wide ecological amplitudes of suitable growing conditions, forests dominated by oaks often fail to regenerate naturally. The regeneration performance of both oak species is assumed to be subject to a variety of variables that interact with one another in complex ways. The novel approach of this research was to study the effect of many ecological variables on the regeneration performance of both oak species together and identify key variables and interactions for different development stages of the oak regeneration on a large scale in the field. For this purpose, overstory and regeneration inventories were conducted in oak dominated forests throughout southern Germany and paired with data on browsing, soil, and light availability. The study was able to verify the assumption that the occurrence of oak regeneration depends on a set of variables and their interactions. Specifically, combinations of site and stand specific variables such as light availability, soil pH and iron content on the one hand, and basal area and species composition of the overstory on the other hand. Also browsing pressure was related to oak abundance. The results also show that the importance of variables and their combinations differs among the development stages of the regeneration. Light availability becomes more important during later development stages, whereas the number of oaks in the overstory is important during early development stages. We conclude that successful natural oak regeneration is more likely to be achieved on sites with lower fertility and requires constantly controlling overstory density. Initially sufficient mature oaks in the overstory should be ensured. In later stages, overstory density should be reduced continuously to meet the increasing light demand of oak seedlings and saplings.

Biogeochemical measurements of cold seep sediments inhabit by vesicomyid clams in the Japan Deep Sea Trench

Vesicomyidae clams harbor sulfide-oxidizing endosymbionts and are typical members of cold seep communities associated with tectonic faults where active venting of fluids and gases takes place. We investigated the central biogeochemical processes that supported a vesicomyid clam colony as part of a locally restricted seep community in the Japan Trench at 5346 m water depth, one of the deepest seep settings studied to date. An integrated approach of biogeochemical and molecular ecological techniques was used combining in situ and ex situ measurements. During the cruise YK06-05 in 2006 with the RV Yokosuka to the Japan Trench, we investigated a clam colony inhabited by Abyssogena phaseoliformis (former known as Calyptogena phaseoliformis) and Isorropodon fossajaponicum (former known as Calyptogena fossajaponica). The targeted sampling and precise positioning of the in situ instruments were achieved with the manned research submersible Shinkai 6500 (JAMSTEC, Nankoku, Kochi, Japan). Sampling was first performed close to the rim of the JTC colony and then at the center. Immediately after sample recovery onboard, the sediment core was sub-sampled for ex situ rate measurements or preserved for later analyses. In sediment of the clam colony, low sulfate reduction (SR) rates (max. 128 nmol ml**-1 d**-1) were coupled to the anaerobic oxidation of methane (AOM). They were observed over a depth range of 15 cm, caused by active transport of sulfate due to bioturbation of the vesicomyid clams. A distinct separation between the seep and the surrounding seafloor was shown by steep horizontal geochemical gradients and pronounced microbial community shifts. The sediment below the clam colony was dominated by anaerobic methanotrophic archaea (ANME-2c) and sulfate-reducing Desulfobulbaceae (SEEP-SRB-3, SEEP-SRB-4). Aerobic methanotrophic bacteria were not detected in the sediment and the oxidation of sulfide seemed to be carried out chemolithoautotrophically by Sulfurovum species. Thus, major redox processes were mediated by distinct subgroups of seep-related microorganisms that might have been selected by this specific abyssal seep environment. Fluid flow and microbial activity was low but sufficient to support the clam community over decades and to build up high biomasses. Hence, the clams and their microbial communities adapted successfully to a low-energy regime and may represent widespread chemosynthetic communities in the Japan Trench.