Rainfall-induced release of fecal coliforms and other manure constituents: comparison and modeling

Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl-), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl-, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents.     

Effect of bovine manure on fecal coliform attachment to soil and soil particles of different sizes

Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.     

Effect of Bovine Manure on Fecal Coliform Attachment to Soil and Soil Particles of Different Sizes

Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.     

Bacteriological Quality of Runoff Water from Pastureland

Runoff from a cow-calf pasture in eastern Nebraska was monitored for total coliforms (TC), fecal coliforms (FC), and fecal streptococci (FS) during 1976, 1977, and 1978. Bacteriological counts in runoff from both grazed and ungrazed areas generally exceeded recommended water quality standards. The FC group was the best indicator group of the impact of grazing. Rainfall runoff from the grazed area contained 5 to 10 times more FC than runoff from the fenced, ungrazed area. There was little difference in TC counts between the two areas, but FS counts were higher in runoff from the ungrazed area and reflected the contributions from wildlife. Recommended bacteriological water quality standards, developed for point source inputs, may be inappropriate for characterizing nonpoint source pollution from pasture runoff. The FC/FS ratio in pasture runoff was useful in identifying the relative contributions of cattle and wildlife. Ratios below 0.05 were indicative of wildlife sources and ratios above 0.1 were characteristic of grazing cattle. Occasions when the FC/FS ratio of diluted cattle waste exceeded one resulted from differential aftergrowth and die-off between FC and FS. The FC/FS ratio and percentage of Streptococcus bovis in pasture runoff are useful indicators for evaluating the effectiveness of livestock management practices for minimizing bacterial contamination of surface water. The importance of choice of medium for the enumeration of FS in runoff derived from cattle wastes is discussed.     

Bacteriological quality of runoff water from pastureland.

Runoff from a cow-calf pasture in eastern Nebraska was monitored for total coliforms (TC), fecal coliforms (FC), and fecal streptococci (FS) during 1976, 1977, and 1978. Bacteriological counts in runoff from both grazed and ungrazed areas generally exceeded recommended water quality standards. The FC group was the best indicator group of the impact of grazing. Rainfall runoff from the grazed area contained 5 to 10 times more FC than runoff from the fenced, ungrazed area. There was little difference in TC counts between the two areas, but FS counts were higher in runoff from the ungrazed area and reflected the contributions from wildlife. Recommended bacteriological water quality standards, developed for point source inputs, may be inappropriate for characterizing nonpoint source pollution from pasture runoff. The FC/FS ratio in pasture runoff was useful in identifying the relative contributions of cattle and wildlife. Ratios below 0.05 were indicative of wildlife sources and ratios above 0.1 were characteristic of grazing cattle. Occasions when the FC/FS ratio of diluted cattle waste exceeded one resulted from differential aftergrowth and die-off between FC and FS. The FC/FS ratio and percentage of Streptococcus bovis in pasture runoff are useful indicators for evaluating the effectiveness of livestock management practices for minimizing bacterial contamination of surface water. The importance of choice of medium for the enumeration of FS in runoff derived from cattle wastes is discussed.     

Dispersion and Transport of Cryptosporidium Oocysts from Fecal Pats under Simulated Rainfall Events

The dispersion and initial transport of Cryptosporidium oocysts from fecal pats were investigated during artificial rainfall events on intact soil blocks (1,500 by 900 by 300 mm). Rainfall events of 55 mm h⁻¹ for 30 min and 25 mm h⁻¹ for 180 min were applied to soil plots with artificial fecal pats seeded with approximately 10⁷ oocysts. The soil plots were divided in two, with one side devoid of vegetation and the other left with natural vegetation cover. Each combination of event intensity and duration, vegetation status, and degree of slope (5° and 10°) was evaluated twice. Generally, a fivefold increase (P < 0.05) in runoff volume was generated on bare soil compared to vegetated soil, and significantly more infiltration, although highly variable, occurred through the vegetated soil blocks (P < 0.05). Runoff volume, event conditions (intensity and duration), vegetation status, degree of slope, and their interactions significantly affected the load of oocysts in the runoff. Surface runoff transported from 10^0.2 oocysts from vegetated loam soil (25-mm h⁻¹, 180-min event on 10° slope) to up to 10^4.5 oocysts from unvegetated soil (55-mm h⁻¹, 30-min event on 10° slope) over a 1-m distance. Surface soil samples downhill of the fecal pat contained significantly higher concentrations of oocysts on devegetated blocks than on vegetated blocks. Based on these results, there is a need to account for surface soil vegetation coverage as well as slope and rainfall runoff in future assessments of Cryptosporidium transport and when managing pathogen loads from stock grazing near streams within drinking water watersheds.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Long-term variations in abundance and distribution of sewage pollution indicator and human pathogenic bacteria along the central west coast of India

Safe water quality criteria on the load and types of microbial populations are important for human use from fishery, tourism and navigational viewpoints. To understand the variations in sewage pollution indicator and certain human pathogenic bacteria, data collected from various locations along central west coast of India during 2002–2007 were analyzed. Water and sediment samples were examined for total viable counts (TVC), pollution indicator bacteria (total coliforms – TC, fecal coliforms – FC and Escherichia coli – EC) and potential pathogens (Vibrio cholerae – VC, Shigella – SH, and Salmonella spp. – SA). In both Mandovi and Zuari estuaries, where fishing and tourist-related activities are sizable and long-term data collection was regular, we observed high counts of TC, FC, VC, SH and SA in particular during monsoon due to increased land runoff. Further, the abundance of TC and FC has increased significantly over the years in the water column to much above either USEPA or India permissible limits. The concentrations of Vibrio cholerae, and Shigella correlated with those of coliforms. Pathogenic bacteria were detected even 20 km and/or 25 km offshore mainly due to dumping of raw or improperly treated sewage effluents either from land, fishing trawlers and/or ships in the anchorage. Higher concentrations of fecal coliforms and pathogenic bacteria in neretic waters signify threats to environmental and human health.     

Method for Detection and Enumeration of Cryptosporidium parvum Oocysts in Feces, Manures, and Soils

Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 10², 10³, 10⁴, and 10⁵ oocysts g of bovine feces⁻¹. The percentages of recovery ranged from 10.8% (25 oocysts g⁻¹) to 17.0% (10⁴ oocysts g⁻¹) (r² = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces⁻¹. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (10⁴ oocysts g⁻¹). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO₄), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil⁻¹ depending on the soil type. The percentages of recovery without dispersant (distilled H₂O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.     

Cryo‐EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity

The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na⁺‐dependent Cl⁻‐ and K⁺‐ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo‐electron microscopy structure of human NKCC1 in the substrate‐loaded (Na⁺, K⁺, and 2 Cl⁻) and occluded, inward‐facing state that has also been observed for the SLC6‐type transporters MhsT and LeuT. Cl⁻ binding at the Cl1 site together with the nearby K⁺ ion provides a crucial bridge between the LeuT‐fold scaffold and bundle domains. Cl⁻‐ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl⁻‐sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na⁺ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members.     

Multi-Ion Mechanism for Ion Permeation and Block in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

The mechanism of Cl⁻ ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl⁻ channels by binding to a site ∼40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl⁻ concentration. Increasing extracellular Cl⁻ concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl⁻ and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl⁻ is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl⁻ concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl⁻ and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl⁻ permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl⁻ (but not gluconate) ions and the dependence of channel conductance on Cl⁻ concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN⁻) ions and anomalous mole fraction behavior seen in Cl⁻/SCN⁻ mixtures.     

Impacts of Agricultural Management and Climate Change on Future Soil Organic Carbon Dynamics in North China Plain

Dynamics of cropland soil organic carbon (SOC) in response to different management practices and environmental conditions across North China Plain (NCP) were studied using a modeling approach. We identified the key variables driving SOC changes at a high spatial resolution (10 km×10 km) and long time scale (90 years). The model used future climatic data from the FGOALS model based on four future greenhouse gas (GHG) concentration scenarios. Agricultural practices included different rates of nitrogen (N) fertilization, manure application, and stubble retention. We found that SOC change was significantly influenced by the management practices of stubble retention (linearly positive), manure application (linearly positive) and nitrogen fertilization (nonlinearly positive) – and the edaphic variable of initial SOC content (linearly negative). Temperature had weakly positive effects, while precipitation had negligible impacts on SOC dynamics under current irrigation management. The effects of increased N fertilization on SOC changes were most significant between the rates of 0 and 300 kg ha⁻¹ yr⁻¹. With a moderate rate of manure application (i.e., 2000 kg ha⁻¹ yr⁻¹), stubble retention (i.e., 50%), and an optimal rate of nitrogen fertilization (i.e., 300 kg ha⁻¹ yr⁻¹), more than 60% of the study area showed an increase in SOC, and the average SOC density across NCP was relatively steady during the study period. If the rates of manure application and stubble retention doubled (i.e., manure application rate of 4000 kg ha⁻¹ yr⁻¹ and stubble retention rate of 100%), soils across more than 90% of the study area would act as a net C sink, and the average SOC density kept increasing from 40 Mg ha⁻¹ during 2010s to the current worldwide average of ∼55 Mg ha⁻¹ during 2060s. The results can help target agricultural management practices for effectively mitigating climate change through soil C sequestration.     

Trigeminal Ganglion Neurons of Mice Show Intracellular Chloride Accumulation and Chloride-Dependent Amplification of Capsaicin-Induced Responses

Intracellular Cl⁻ concentrations ([Cl⁻]_i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl⁻ is accumulated by the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1), resulting in a [Cl⁻]_i above electrochemical equilibrium and a depolarizing Cl⁻ efflux upon Cl⁻ channel opening. Here, we investigate the [Cl⁻]_i and function of Cl⁻ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1^−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl⁻]_i of WT TG neurons indicated active NKCC1-dependent Cl⁻ accumulation. Gamma-aminobutyric acid (GABA)_A receptor activation induced a reduction of [Cl⁻]_i as well as Ca²⁺ transients in a corresponding fraction of TG neurons. Ca²⁺ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca²⁺ channels (VGCCs). Ca²⁺ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1^−/− TG neurons, but elevated under conditions of a lowered [Cl⁻]_o suggesting a Cl⁻-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca²⁺-activated Cl⁻ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca²⁺ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1^−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca²⁺-activated Cl⁻-dependent signal amplification mechanism in TG neurons that requires intracellular Cl⁻ accumulation by NKCC1 and the activation of CaCCs.     

Quantitative and Physiological Analyses of Chloride Dependence of Growth of Halobacillus halophilus

A quantitative analysis of the Cl⁻ dependence of growth of Halobacillus halophilus was performed. Optimal growth rates were obtained at Cl⁻ concentrations of between 0.5 and 2.0 M, and the final yield was also strictly dependent on the Cl⁻ concentration. Br⁻ but not I⁻, SO₄²⁻, NO₂⁻, SO₂⁻, OCN⁻, SCN⁻, BO₂⁻, or BrO₃⁻ could substitute for Cl⁻. To analyze the function of chloride, chloride concentration was determined. At low external Cl⁻ (Cl_e⁻) concentrations, the growth rate was low and Cl⁻ was excluded from the cytoplasm; increasing the Cl_e⁻ concentration led to an increase in the growth rate and an energy-dependent uptake of Cl⁻, thus decreasing the Cl_e⁻/internal Cl_i⁻ gradient from ≥10 at 0.1 M Cl_e⁻ to a nearly constant value of 2 at Cl_e⁻ concentrations which allowed optimal growth. Two membrane proteins with apparent molecular masses of 31 and 16 kDa which were identified to be specific for Cl⁻-grown cultures are possible candidates for a chloride uptake system.     

Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl? Secretion by Human Airway Epithelia

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl⁻ and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl⁻ secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca²⁺ from the endoplasmic reticulum (ER), lowering [Ca²⁺] in the ER and thereby activating the Ca²⁺-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca²⁺] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl⁻ current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca²⁺ buffer that lowers [Ca²⁺] in the ER similar to the effect of 3O-C12 also increased cAMP and I_Cl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl⁻ and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca²⁺] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl⁻ and fluid secretion.     

Incidence of Infectious Drug Resistance Among Fecal Coliforms Isolated from Raw Sewage

Raw sewage was examined for the incidence of antibiotic-resistant coliforms present among both total and fecal coliforms. In both groups, it was found that approximately 3% of the coliform bacteria were resistant to two or more antibiotics. Of these organisms, 48% were capable of transferring all or part of their antibiotic resistance to an antibiotic-sensitive, F⁻, derivative of Escherichia coli K-12. Among the R factors identified, those conferring resistance to streptomycin-tetracycline, ampicillin-streptomycin-tetracycline, and ampicillin or ampicillin-streptomycin accounted for 23, 20, and 15%, respectively, of the total R factors detected. The data indicate a significant level of infectious drug resistance among the fecal coliforms of the urban population. The data indicate further that because of the high incidence of coliform bacteria found to be doubly resistant to streptomycin and tetracyline, the inclusion of these antibiotics in selective media used for routine total or fecal coliform counts may serve to identify domestic sources of pollution.     

Chloride channels are necessary for full platelet phosphatidylserine exposure and procoagulant activity

Platelets enhance thrombin generation at sites of vascular injury by exposing phosphatidylserine during necrosis-like cell death. Anoctamin 6 (Ano6) is required for Ca²⁺-dependent phosphatidylserine exposure and is defective in patients with Scott syndrome, a rare bleeding disorder. Ano6 may also form Cl⁻ channels, though the role of Cl⁻ fluxes in platelet procoagulant activity has not been explored. We found that Cl⁻ channel blockers or removal of extracellular Cl⁻ inhibited agonist-induced phosphatidylserine exposure. However, this was not due to direct inhibition of Ca²⁺-dependent scrambling since Ca²⁺ ionophore-induced phosphatidylserine exposure was normal. This implies that the role of Ano6 in Ca²⁺⁻dependent PS exposure is likely to differ from any putative function of Ano6 as a Cl⁻ channel. Instead, Cl⁻ channel blockade inhibited agonist-induced Ca²⁺ entry. Importantly, Cl⁻ channel blockers also prevented agonist-induced membrane hyperpolarization, resulting in depolarization. We propose that Cl⁻ entry through Cl⁻ channels is required for this hyperpolarization, maintaining the driving force for Ca²⁺ entry and triggering full phosphatidylserine exposure. This demonstrates a novel role for Cl⁻ channels in controlling platelet death and procoagulant activity.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Modeling the Impact of Soil and Water Conservation on Surface and Ground Water Based on the SCS and Visual Modflow

Soil and water conservation measures can impact hydrological cycle, but quantitative analysis of this impact is still difficult in a watershed scale. To assess the effect quantitatively, a three-dimensional finite-difference groundwater flow model (MODFLOW) with a surface runoff model–the Soil Conservation Service (SCS) were calibrated and applied based on the artificial rainfall experiments. Then, three soil and water conservation scenarios were simulated on the sand-box model to assess the effect of bare slope changing to grass land and straw mulching on water volume, hydraulic head, runoff process of groundwater and surface water. Under the 120 mm rainfall, 60 mm/h rainfall intensity, 5 m² area, 3° slope conditions, the comparative results indicated that the trend was decrease in surface runoff and increase in subsurface runoff coincided with the land-use converted from bare slope to grass land and straw mulching. The simulated mean surface runoff modulus was 3.64×10⁻² m³/m²/h in the bare slope scenario, while the observed values were 1.54×10⁻² m³/m²/h and 0.12×10⁻² m³/m²/h in the lawn and straw mulching scenarios respectively. Compared to the bare slope, the benefits of surface water reduction were 57.8% and 92.4% correspondingly. At the end of simulation period (T = 396 min), the simulated mean groundwater runoff modulus was 2.82×10⁻² m³/m²/h in the bare slope scenario, while the observed volumes were 3.46×10⁻² m³/m²/h and 4.91×10⁻² m³/m²/h in the lawn and straw mulching scenarios respectively. So the benefits of groundwater increase were 22.7% and 60.4% correspondingly. It was concluded that the soil and water conservation played an important role in weakening the surface runoff and strengthening the underground runoff. Meanwhile the quantitative analysis using a modeling approach could provide a thought for the study in a watershed scale to help decision-makers manage water resources.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.