Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V j-sensitive gating of I j (V j, gap junction voltage; I j, gap junction current), (2) contribution and (3) kinetics of I j deactivation and (4) single-channel conductance. The first three reflect alterations of fast V j gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.

Effects of drilling and stress release on hydraulic properties and porewater chemistry of crystalline rocks

The experimental verification of matrix diffusion in crystalline rocks largely relies on indirect methods performed in the laboratory. Such methods are prone to perturbations of the rock samples by collection and preparation and therefore the laboratory-derived transport properties and fluid composition might not represent in situ conditions. We investigated the effects induced by the drilling process and natural rock stress release by mass balance considerations and sensitivity analysis of analytical out-diffusion data obtained from originally saturated, large-sized drillcore material from two locations drilled using traced drilling fluid. For in situ stress-released drillcores of quartz-monzodiorite composition from the Aspo HRL, Sweden, tracer mass balance considerations and 1D and 2D diffusion modelling consistently indicated a contamination of <1% of the original pore water. This chemically disturbed zone extends to a maximum of 0.1 mm into the drillcore (61.8 mm x 180.1 mm) corresponding to about 0.66% of the total pore volume (0.77 vol.%). In contrast, the combined effects of stress release and the drilling process, which have influenced granodioritic drillcore material from 560 m below surface at Forsmark. Sweden, resulted in a maximum contamination of the derived porewater Cl(-) concentration of about 8%. The mechanically disturbed zone with modified diffusion properties covers the outermost similar to 6 mm of the drillcore (50 mm x 189 mm), whereas the chemically disturbed zone extends to a maximum of 0.3 mm based on mass balance considerations, and to 0.15 mm to 0.2 mm into the drillcore based on fitting the observed tracer data. This corresponds to a maximum of 2.4% of the total pore volume (0.62 vol.%) being affected by the drilling-fluid contamination. The proportion of rock volume affected initially by drilling fluid or subsequently with experiment water during the laboratory diffusion and re-saturation experiments depends on the size of the drillcore material and will become larger the smaller the sample used for the experiment. The results are further in support of matrix diffusion taking place in the undisturbed matrix of crystalline rocks at least in the cm range.

Development And Use Of A Model To Predict Properties Of Infiltrated Solid Oxide Fuel Cell Anodes

Solid oxide fuel cells (SOFCs) provide a potentially clean way of using energy sources. One important aspect of a functioning fuel cell is the anode and its characteristics (e.g. conductivity). Using infiltration of conductor particles has been shown to be a method for production at lower cost with comparable functionality. While these methods have been demonstrated experimentally, there is a vast range of variables to consider. Because of the long time for manufacture, a model is desired to aid in the development of the desired anode formulation. This thesis aims to (1) use an idealized system to determine the appropriate size and aspect ratio to determine the percolation threshold and effective conductivity as well as to (2) simulate the infiltrated fabrication method to determine the effective conductivity and percolation threshold as a function of ceramic and pore former particle size, particle fraction and the cell¿s final porosity. The idealized system found that the aspect ratio of the cell does not affect the cells functionality and that an aspect ratio of 1 is the most efficient computationally to use. Additionally, at cell sizes greater than 50x50, the conductivity asymptotes to a constant value. Through the infiltrated model simulations, it was found that by increasing the size of the ceramic (YSZ) and pore former particles, the percolation threshold can be decreased and the effective conductivity at low loadings can be increased. Furthermore, by decreasing the porosity of the cell, the percolation threshold and effective conductivity at low loadings can also be increased

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

Characterisation of Gas Transport Properties of the Opalinus Clay, a Potential Host Rock Formation for Radioactive Waste Disposal

The Opalinus Clay in Northern Switzerland has been identified as a potential host rock formation for the disposal of radioactive waste. Comprehensive understanding of gas transport processes through this low-permeability formation forms a key issue in the assessment of repository performance. Field investigations and laboratory experiments suggest an intrinsic permeability of the Opalinus Clay in the order of 10(-20) to 10(-21) m(2) and a moderate anisotropy ratio < 10. Porosity depends on clay content and burial depth; values of similar to 0.12 are reported for the region of interest. Porosimetry indicates that about 10-30 of voids can be classed as macropores, corresponding to an equivalent pore radius > 25 nm. The determined entry pressures are in the range of 0.4-10 MPa and exhibit a marked dependence on intrinsic permeability. Both in situ gas tests and gas permeameter tests on drillcores demonstrate that gas transport through the rock is accompanied by porewater displacement, suggesting that classical flow concepts of immiscible displacement in porous media can be applied when the gas entry pressure (i.e. capillary threshold pressure) is less than the minimum principal stress acting within the rock. Essentially, the pore space accessible to gas flow is restricted to the network of connected macropores, which implies a very low degree of desaturation of the rock during the gas imbibition process. At elevated gas pressures (i.e. when gas pressure approaches the level of total stress that acts on the rock body), evidence was seen for dilatancy controlled gas transport mechanisms. Further field experiments were aimed at creating extended tensile fractures with high fracture transmissivity (hydro- or gasfracs). The test results lead to the conclusion that gas fracturing can be largely ruled out as a risk for post-closure repository performance.

Impact of normal and shear stresses due to wheel slip on hydrological properties of an agricultural clay loam: Experimental and new computerized approach

The main purpose of this study was to evaluate the effect that mechanical stresses acting under the slipping driving wheels of agricultural equipment have on the soil’s pore system and water flow process (surface runoff generation during extreme event). The field experiment simulated low slip (1%) and high slip (27%) on a clay loam. The stress on the soil surface and changes in the amounts of water flowing from macropores were simulated using the Tires/tracks And Soil Compaction (TASC) tool and the MACRO model, respectively. Taking a 65 kW tractor on a clay loam as a reference, results showed that an increase in slip of the rear wheels from 1% to 27% caused normal stress to increase from 90.6 kPa to 104.4 kPa at the topsoil level, and the maximum shear contact stress to rise drastically from 6.0 kPa to 61.6 kPa. At 27% slip, topsoil was sheared and displaced over a distance of 0.35 m. Excessive normal and shear stress values with high slip caused severe reductions of the soil’s macroporosity, saturated hydraulic conductivity, and water quantities flowing from topsoil macropores. Assuming that, under conditions of intense rainfall on sloping land, a loss in vertical water flow would mean an increase in surface runoff, we calculated that a rainfall intensity of 100 mm h-1 and a rainfall duration of 1 h would increase the runoff coefficient to 0.79 at low slip and to 1.00 at high slip, indicating that 100% of rainwater would be transformed into surface runoff at high slip. We expect that these effects have a significant impact on soil erosion and floods in steeper terrain (slope > 15°) and across larger surface areas (> 16 m2) than those included in our study.

The Effects Of Reinforcement Inclusions On Wear Tolerance, Playing Quality And Physical Properties In A Silt Loam And Sand Rootzone Matrix

Reinforcement inclusions have been advocated to alleviate wear, compaction, and unstable surfaces in sports fields, but little research on the effects of these materials has been conducted in the USA. Experiments were established on a native silt loam and a sand rootzone matrix, seeded with a Kentucky bluegrass (Poa pratensis L.) blend, at the Joseph Troll Turf Research Center, University of Massachusetts, Amherst, USA to determine the effects of reinforcement inclusions on wear, surface hardness, traction, ball roll, ball bounce resilience, water infiltration rate, soil bulk density, air porosity, total porosity, and root weights. Three types of reinforcement inclusions (Sportgrass, Netlon, Turfgrids) were tested along with a non-reinforced control in a three year study. The treatments were set out in a randomized complete block design with four replications in both soils. No inclusion provided less wear or greater infiltration or air-filled porosity relative to the control. Reinforcement inclusions showed significant differences, however, in surface hardness, traction, and ball roll relative to the control, although this varied with the time of year. Infiltration rates, airfilled porosity, total pore space, bulk density, hardness, traction, ball roll, and ball rebound were greater on the sand rootzone than on the silt loam. Significant correlations were present between soil bulk density, surface hardness, traction, and ball roll. Based on our study, the use of reinforcement inclusions to provide better wear tolerance for sand or native soil athletic fields is not warranted. Certain playing surface characteristics, however, may be slightly improved with the use of reinforcement inclusions. The use of sands for sports surfaces is justified based upon the improvement in playing quality characteristics and soil physical properties important to a good playing surface.

Anelastic strain recovery and elastic properties of ODP Leg 123 oceanic basalt samples

A knowledge of rock stress is fundamental for improving our understanding of oceanic crustal mechanisms and lithospheric dynamic processes. However, direct measurements of stress in the deep oceans, and in particular stress magnitudes, have proved to be technically difficult. Anelastic strain recovery measurements were conducted on 15 basalt core samples from Sites 765 and 766 during Leg 123. Three sets of experiments were performed: anelastic strain recovery monitoring, dynamic elastic property measurements, and thermal azimuthal anisotropy observations. In addition, a range of other tests and observations were recorded to characterize each of the samples. One common feature of the experimental results and observations is that apparently no consistent orientation trend exists, either between the different measurements on each core sample or between the same sets of measurements on the various core samples. However, some evidence of correspondence between velocity anisotropy and anelastic strain recovery exists, but this is not consistent for all the core samples investigated. Thermal azimuthal anisotropy observations, although showing no conclusive correlations with the other results, were of significant interest in that they clearly exhibited anisotropic behavior. The apparent reproducibility of this behavior may point toward the possibility of rocks that retain a "memory" of their stress history, which could be exploited to derive stress orientations from archived core. Anelastic strain recovery is a relatively new technique. Because use of the method has extended to a wider range of rock types, the literature has begun to include examples of rocks that contracted with time. Strong circumstantial evidence exists to suggest that core-sample contractions result from the slow diffusion of pore fluids from a preexisting microcrack structure that permits the rock to deflate at a greater rate than the expansion caused by anelastic strain recovery. Both expansions and contractions of the Leg 123 cores were observed. The basalt cores have clearly been intersected by an abundance of preexisting fractures, some of which pass right through the samples, but many are intercepted or terminate within the rock matrix. Thus, the behavior of the core samples will be influenced not only by the properties of the rock matrix between the fractures, but also by how these macro- and micro-scale fractures mutually interact. The strain-recovery curves recorded during Leg 123 for each of the 15 basalt core samples may reflect the result of two competing time dependent processes: anelastic strain recovery and pore pressure recovery. Were these the only two processes to influence the gauge responses, then one might expect that given the additional information required, established theoretical models might be used to determine consistent stress orientations and reliable stress magnitudes. However, superimposed upon these competing processes is their respective interaction with the preexisting fractures that intersect each core. Evidence from our experiments and observations suggests that these fractures have a dominating influence on the characteristics of the recovery curves and that their effects are complex.

Molecular properties of gases of ODP Leg 204 sites

The recognition of finely disseminated gas hydrate in deep marine sediments heavily depends on various indirect techniques because this mineral quickly decomposes upon recovery from in situ pressure and temperature conditions. Here, we discuss molecular properties of closely spaced gas voids (formed as a result of core recovery) and gas hydrates from an area of relatively low gas flux at the flanks of the southern Hydrate Ridge offshore Oregon (ODP Sites 1244, 1245 and 1247). Within the gas hydrate occurrence zone (GHOZ), the concentration of ethane (C2) and propane (C3) in adjacent gas voids shows large variability. Sampled gas hydrates are enriched in C2 relative to void gases but do not contain C3. We suggest that the observed variations in the composition of void gases is a result of molecular fractionation during crystallization of structure I gas hydrate that contains C2 but excludes C3 from its crystal lattice. This hypothesis is used to identify discrete intervals of finely disseminated gas hydrate in cored sediments. Variations in gas composition help better constrain gas hydrate distribution near the top of the GHOZ along with variations in pore water chemistry and core temperature. Sediments near the base of the gas hydrate stability zone are relatively enriched in C2+ hydrocarbon gases. Complex and poorly understood geological and geochemical processes in these deeper sediments make the identification of gas hydrate based on molecular properties of void gases more ambiguous. The proposed technique appears to be a useful tool to better understand the distribution of gas hydrate in marine sediments and ultimately the role of gas hydrate in the global carbon cycle.

Consolidation properties and strength characteristics of ODP Site 204-1244 sediments

Eight whole-core samples from Ocean Drilling Program Site 1244, Hydrate Ridge, Cascadia continental margin, were provided to Massachusetts Institute of Technology (Cambridge, Massachusetts, USA) for geotechnical characterization. The samples were collected from depths ranging from 5 to 136 meters below seafloor (mbsf). Seven of the eight whole-core samples were located within the gas hydrate stability zone, whereas the eighth sample was located in the free gas zone. Atterberg limits testing showed that the average liquid limit of the soil is 81% and the average plastic limit is 38%, giving an average plasticity index of 43%. The liquid limit is sensitive to oven drying, shown by a drop in liquid limit to 64% when tests were performed on an oven-dried sample. Loss on ignition averages 5.45 wt%. Constant rate of strain consolidation (CRSC) tests were performed to obtain the compression characteristics of the soil, as well as to determine the stress history of the site. CRSC tests also provided hydraulic conductivity and coefficient of consolidation characteristics for these sediments. The compression ratio (Cc) ranges from 0.340 to 0.704 (average = 0.568). Cc is fairly constant to a depth of 79 mbsf, after which Cc decreases downhole. The recompression ratio (Cr) ranges from 0.035 to 0.064 (average = 0.052). Cr is constant throughout the depth range. In situ hydraulic conductivity varies between 1.5 x 10**-7 and 3 x 10**-8 cm/s and shows no trend with depth. Ko-consolidated undrained compression/extension (CKoUC/E) tests were also performed to determine the peak undrained shear strength, stress-strain curve, and friction angle. The normalized undrained strength ranges from 0.29 to 0.35. The friction angle ranges from 27 to 37. Because of the limited amount of soil, CRSC and CKoUC/E tests were also conducted on resedimented specimens.

Physical properties of sediments at DSDP Holes 78-541 and 78-543

Sediment cored within the Barbados subduction complex at Sites 541 and 542 are underconsolidated. Underconsolidation and changes in physical properties of the cored section can be related to excess pore water pressure that equals the lithostatic load at Site 542 and to major thrust faulting observed at Site 541. Apparently, the pore fluids within the subduction complex are absorbing the tectonic shock of underthrusting. Sediment sampled from the reference Site 543 on the adjacent Atlantic Plate are also underconsolidated. However, underconsolidation in Hole 543 is apparently due to the movement of excess nitrogen gas observed deeper in the hole. Excess gas was not observed at Sites 541 and 542.

Electromagnetic, rock magnetic, and geochemical properties of surficial sediments in Eckernförde Bay

Submarine groundwater discharge in coastal settings can massively modify the hydraulic and geochemical conditions of the seafloor. Resulting local anomalies in the morphology and physical properties of surface sediments are usually explored with seismo-acoustic imaging techniques. Controlled source electromagnetic imaging offers an innovative dual approach to seep characterization by its ability to detect pore-water electrical conductivity, hence salinity, as well as sediment magnetic susceptibility, hence preservation or diagenetic alteration of iron oxides. The newly developed electromagnetic (EM) profiler Neridis II successfully realized this concept for a first time with a high-resolution survey of freshwater seeps in Eckernförde Bay (SW Baltic Sea). We demonstrate that EM profiling, complemented and validated by acoustic as well as sample-based rock magnetic and geochemical methods, can create a crisp and revealing fingerprint image of freshwater seepage and related reductive alteration of near-surface sediments. Our findings imply that (1) freshwater penetrates the pore space of Holocene mud sediments by both diffuse and focused advection, (2) pockmarks are marked by focused freshwater seepage, underlying sand highs, reduced mud thickness, higher porosity, fining of grain size, and anoxic conditions, (3) depletion of Fe oxides, especially magnetite, is more pervasive within pockmarks due to higher concentrations of organic and sulfidic reaction partners, and (4) freshwater advection reduces sediment magnetic susceptibility by a combination of pore-water injection (dilution) and magnetite reduction (depletion). The conductivity vs. susceptibility biplot resolves subtle lateral litho- and hydrofacies variations.

Summary of physical properties on cores used for petrofabric analyses triaxial experiments at DSDP Leg 67 Holes

Deformation features within the cores are studied with a view towards elucidating the structure of the Middle America Trench along the transect drilled during Leg 67. Where possible, inferences are made as to the physical environment of deformation. Extensional tectonics prevails in the area of the seaward slope and trench. Fracturing and one well-preserved normal fault are found mostly within the lower Miocene chalks, at the base of the sedimentary section. These chalks have high porosities (40%-60%) and water content (30%-190%, based on % dry wt.). Experimental triaxial compression tests conducted on both dry and water-saturated samples of chalk from Holes 495 and 499B show that only in the saturated samples is more brittle behavior observed. Brittle failure of the chalks is greatly facilitated by pore fluid pressures that lead to low effective pressures. Additional embrittlement (weakening) can take place as a result of the imposed extensional stress resulting from bending of a subducting elastic oceanic plate. The chalks exhibit, in a landward direction, an increase in density and mechanical strength and a decrease in water content. These changes are attributed to mechanical compaction that may have resulted from tectonic horizontal compression. The structure of the landward slope is not well understood because the slope sites had to be abandoned due to the presence of gas hydrate. The relationship of the chaotic, brittle deformation (observed in the cores from Hole 494A) at the base of the landward slope to tectonic processes remains unclear. The deformation observed on the slope sites (Holes 496 and 497) is mostly fracturing and near-vertical sigmoidal veinlets. These are interpreted as being the result of gas/fluid overpressurization due to the decomposition of the gas hydrate, and not due to tectonic loading of accreted sediments. Aside from four small displacement (less than 1cm) reverse faults observed in the lower Miocene chalks (which may be the product of soft-sediment deformation), there is a noticeable absence of structures reflecting a dominance of horizontal (tectonic) compression along the transect drilled. The absence of such features, the lack of continuity of sediment types across the trench-landward slope, and the normal stratigraphic sequence in Hole 494A do not support any known accretionary model.

Geotechnical properties of sediments at DSDP Leg 84 Holes

The geotechnical characteristics of 22 sediment samples from Leg 84 sites were studied in an effort to associate these with processes active along the Middle America slope and with sedimentation mechanisms. Geotechnical properties measured include water content, porosity, bulk density, Atterberg limits, consolidation characteristics, permeability, and vane shear strength. A majority of samples obtained from Sites 565, 568, and 570 show significant disturbance resulting from degassing. This disturbance apparently results in underconsolidation, although other mechanisms such as excess pore pressures generated from the subduction process can also contribute to this state. Overconsolidated sediments were found at Sites 565, 566, and 569. The overconsolidated sediments at Sites 565 and 569 may result from downslope transport mechanisms rearranging and stressing the sediment mass under consideration. The sediment condition at Site 566 is probably a result of eroded overburden: an estimated 87 m of overlying sediments may have been removed. Geotechnical and permeability relationships with depth are consistent with those found for other hemipelagic sediments of silty clay to clayey silt textures.

The physical properties of basalt at DSDP Hole 45-395A

A detailed study has been made of the physical properties of core samples from Deep Sea Drilling Project Hole 395A. The properties include: density, porosity, compressional and shear wave velocity, thermal conductivity, thermal diffusivity, and electrical resistivity. Of particular importance are the relations among the parameters. Most of the variations in the basalt properties follow the porosity, with smaller inferred dependence on pore structure, original mineralogy differences, and alteration. The sample measurements give very similar results to (and extend previous data from) Mid-Atlantic Ridge drillholes, the sample data from this site and previous data are used to estimate relations between porosity and other large-scale physical properties of the upper oceanic crust applicable to this area. These relations are important for the analysis and interpretation of downhole logging measurements and marine geophysical data.