Fine-scale spatiotemporal variations in bacterial community diversity in agricultural pond water.

Microbial communities in surface waters are affected by environmental conditions and can influence changes in water quality. To explore the hypothesis that the microbiome in agricultural waters associates with spatiotemporal variations in overall water quality and, in turn, has implications for resource monitoring and management, we characterized the relationships between the microbiota and physicochemical properties in a model irrigation pond as a factor of sampling time (i.e., 9:00, 12:00, 15:00) and location within the pond (i.e., bank vs. interior sites and cross-sectional depths at 0, 1, and 2 m). The microbial communities, which were defined by 16S rRNA gene sequencing analysis, significantly varied based on all sampling factors (PERMANOVA P < 0.05 for each). While the relative abundances of dominant phyla (e.g., Proteobacteria and Bacteroidetes) were relatively stable throughout the pond, subtle yet significant increases in α-diversity were observed as the day progressed (ANOVA P < 0.001). Key water quality properties that also increased between the morning and afternoon (i.e., pH, dissolved oxygen, and temperature) positively associated with relative abundances of Cyanobacteria, though were inversely proportional to Verrucomicrobia. These properties, among additional parameters such as bioavailable nutrients (e.g., NH3, NO3, PO4), chlorophyll, phycocyanin, conductivity, and colored dissolved organic matter, exhibited significant relationships with relative abundances of various bacterial genera as well. Further investigation of the microbiota in underlying sediments revealed significant differences between the bank and interior sites of the pond (P < 0.05 for α- and ß-diversity). Overall, our findings emphasize the importance of accounting for time of day and water sampling location and depth when surveying the microbiomes of irrigation ponds and other small freshwater sources.

Long-term safety and efficacy of anacetrapib in patients with atherosclerotic vascular disease.

AIMS: REVEAL was the first randomized controlled trial to demonstrate that adding cholesteryl ester transfer protein inhibitor therapy to intensive statin therapy reduced the risk of major coronary events. We now report results from extended follow-up beyond the scheduled study treatment period. METHODS AND RESULTS: A total of 30 449 adults with prior atherosclerotic vascular disease were randomly allocated to anacetrapib 100 mg daily or matching placebo, in addition to open-label atorvastatin therapy. After stopping the randomly allocated treatment, 26 129 survivors entered a post-trial follow-up period, blind to their original treatment allocation. The primary outcome was first post-randomization major coronary event (i.e. coronary death, myocardial infarction, or coronary revascularization) during the in-trial and post-trial treatment periods, with analysis by intention-to-treat. Allocation to anacetrapib conferred a 9% [95% confidence interval (CI) 3-15%; P = 0.004] proportional reduction in the incidence of major coronary events during the study treatment period (median 4.1 years). During extended follow-up (median 2.2 years), there was a further 20% (95% CI 10-29%; P < 0.001) reduction. Overall, there was a 12% (95% CI 7-17%, P < 0.001) proportional reduction in major coronary events during the overall follow-up period (median 6.3 years), corresponding to a 1.8% (95% CI 1.0-2.6%) absolute reduction. There were no significant effects on non-vascular mortality, site-specific cancer, or other serious adverse events. Morbidity follow-up was obtained for 25 784 (99%) participants. CONCLUSION: The beneficial effects of anacetrapib on major coronary events increased with longer follow-up, and no adverse effects emerged on non-vascular mortality or morbidity. These findings illustrate the importance of sufficiently long treatment and follow-up duration in randomized trials of lipid-modifying agents to assess their full benefits and potential harms. TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number (ISRCTN) 48678192; ClinicalTrials.gov No. NCT01252953; EudraCT No. 2010-023467-18.

Functional Evaluation of Three Manure-Borne Indicator Bacteria Release Models with Multiyear Field Experiment Data.

Modeling the fate and transport of Escherichia coli is of substantial interest because of how this organism serves as an indicator of fecal contamination in microbial water quality assessment. The efficacy of models used to assess the export of E. coli from agricultural fields is dependent, in part, on submodels they utilize to simulate E. coli release from land-applied manure and animal waste. Although several release submodels have been proposed, they have only been evaluated and compared with data from laboratory or small plot E. coli release experiments. Our objective was to evaluate and compare performances of three manure-borne bacteria release submodels at field-scale: exponential release (EM), two-parametric Bradford and Schijven (B-S), and two-parametric Vadas-Kleinman-Sharpley (VKS); each was independently incorporated and tested as a submodel within the export model KINEROS2/STWIR, using E. coli. Dairy manure was uniformly applied via surface broadcasting once a year for six consecutive years on a 0.28 ha experimental field site. Two irrigation events followed each application: the first immediately followed the initial application and the second occurred one week later. Manure and soil samples were collected before and after irrigation, respectively, and manure, soil, and edge-of-field runoff samples were analyzed for E. coli. Model performance was evaluated with the Akaike criterion, coefficients of determination (R2), and root mean squared errors (RMSE) values. The percentage of exported manure-borne E. coli varied from 0.1% to 10% in most cases, generally reflecting the lag time between initiation of irrigation and initiation ofedge-of-field runoff. The export model performed better when using the VKS submodel which was preferred in 55% of cases. The B-S and EM submodels were preferred in 27% and 18% of cases, respectively. Two-parametric submodels were ultimately preferred over the single parameter submodel.

Chronic Administration of Anacetrapib Is Associated With Accumulation in Adipose and Slow Elimination.

Anacetrapib is a novel cholesteryl-ester transfer protein (CETP) inhibitor in late-stage clinical development, shown in preceding clinical trials to have residual pharmacological activity after prolonged washout after chronic dosing. Preclinical findings suggest that white adipose tissue is a potential depot and that accumulation into adipose tissue governs the long-term kinetics of anacetrapib in mice. A phase I study performed to test this hypothesis in humans revealed that plasma exposure was correlated with fat content in food administered with the drug. Plasma concentrations of anacetrapib seemed to reach plateau faster than adipose concentrations. Anacetrapib continued to accumulate in adipose during the treatment period despite apparent plateau in plasma with only minimal decline in adipose levels up to 1 year postdose. Because of its high lipophilicity, anacetrapib partitions into adipose tissue, this likely forms a drug reservoir that, in turn, contributes to the long residence time of the drug in plasma.

Differential release of manure-borne bioactive phosphorus forms to runoff and leachate under simulated rain.

Limited information exists on the unhindered release of bioactive phosphorus (P) from a manure layer to model the partitioning and transport of component P forms before they reach an underlying soil. Rain simulations were conducted to quantify effects of intensity (30, 60, and 90 mm h-1) on P release from an application of 60 Mg ha-1 of dairy manure. Runoff contained water-extractable- (WEP), exchangeable and enzyme-labile bioactive P (TBIOP), in contrast to the operationally defined "dissolved-reactive P" form. The released P concentrations and flow-weighed mass loads were described by the log-normal probability density function. At a reference condition of 30 mm h-1 and maintaining the surface at a 5% incline, runoff was minimal, and WEP accounted for 20.9% of leached total P (TP) concentrations, with an additional 25-30% as exchangeable and enzyme-labile bioactive P over the 1-h simulation. On a 20% incline, increased intensity accelerated occurrence of concentrationmax and shifted the skewed P concentration distribution more to the left. Differences in trends of WEP, TBIOP, or net enzyme-labile P (PHPo) cumulative mass released per unit mass of manure between intensities were attributable to the higher frequency of raindrops striking the manure layer, thus increasing detachment and load of colloidal PHPo of the water phases. Thus, detailed knowledge of manure physical characteristics, bioactive P distribution in relation to rain intensity, and attainment of steady-state of water fluxes were critical factors in improved prediction of partitioning and movement of manure-borne P under rainfall.

Response of coliform populations in streambed sediment and water column to changes in nutrient concentrations in water.

As sediments increasingly become recognized as reservoirs of indicator and pathogen microorganisms, an understanding of the persistence of indicator organisms becomes important for assessment and predictions of microbial water quality. The objective of this work was to observe the response of water column and sediment coliform populations to the change in nutrient concentrations in the water column. Survival experiments were conducted in flow-through chambers containing sandy sediments. Bovine feces were collected fresh and introduced into sediment. Sixteen days later, the same fecal material was autoclaved and diluted to provide three levels - 1×, 0.5×, and 0.1× of nutrient concentrations - spike in water column. Total coliforms, Escherichia coli, and total aerobic heterotrophic bacterial concentrations were monitored in water and sediment. Bacteria responded to the nutrient spike with initial growth both in the water column and in sediment. The response of bacterial concentrations in water column was nonlinear, with no significant changes at 0.1 and .5× spikes, but a substantial change at 1× spike. Bacteria in sediment responded to the spikes at all added nutrient levels. Coliform inactivation rates both in sediment and in water after the initial growth occurred, were not significantly different from the inactivation rates before spike. These results indicate that introduction of nutrients into the water column results in nonlinear response of E. coli concentrations both in water and in sediments, followed by the inactivation with the same rate as before introduction of nutrients.

Comparing temperature effects on Escherichia coli, Salmonella, and Enterococcus survival in surface waters.

UNLABELLED: The objective of this study was to compare dependency of survival rates on temperature for indicator organisms Escherichia coli and Enterococcus and the pathogen Salmonella in surface waters. A database of 86 survival datasets from peer-reviewed papers on inactivation of E. coli, Salmonella and Enterococcus in marine waters and of E. coli and Salmonella in lake waters was assembled. The Q10 model was used to express temperature effect on survival rates obtained from linear sections of semi-logarithmic survival graphs. Available data were insufficient to establish differences in survival rates and temperature dependencies for marine waters where values of Q10  = 3 and a survival rate of 0·7 day(-1) could be applied. The Q10 values in lake waters were substantially lower in marine waters, and Salmonella inactivation in lake water was, on average, twice as fast as E. coli; data on E. coli substantially outnumber data on Enterococcus and Salmonella. The relative increase in inactivation with increase in temperature is higher in marine waters than lake water, and differences in inactivation between Salmonella and E. coli at a given temperature were significant in lake water but not in marine waters. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiological quality of surface waters is of paramount importance for public health. The novelty of this work is using a large compendium of published data to develop the first comparison of temperature effects on survival of the pathogen Salmonella and water quality indicator micro-organisms Escherichia coli and Enterococcus in natural waters. The existing relatively large body of knowledge on E. coli survival appears to be useful to assess the effect of temperature on survival of Salmonella. Moreover, results of this work constitute an essential input in models to support environmental management decisions on the use of surface water sources in agriculture, aquaculture and recreation.

Escherichia coli survival in waters: temperature dependence.

Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 model. This suggestion was made 34 years ago based on 20 survival curves taken from published literature, but has not been revisited since then. The objective of this study was to re-evaluate the accuracy of the Q10 equation, utilizing data accumulated since 1978. We assembled a database of 450 E. coli survival datasets from 70 peer-reviewed papers. We then focused on the 170 curves taken from experiments that were performed in the laboratory under dark conditions to exclude the effects of sunlight and other field factors that could cause additional variability in results. All datasets were tabulated dependencies "log concentration vs. time." There were three major patterns of inactivation: about half of the datasets had a section of fast log-linear inactivation followed by a section of slow log-linear inactivation; about a quarter of the datasets had a lag period followed by log-linear inactivation; and the remaining quarter were approximately linear throughout. First-order inactivation rate constants were calculated from the linear sections of all survival curves and the data grouped by water sources, including waters of agricultural origin, pristine water sources, groundwater and wells, lakes and reservoirs, rivers and streams, estuaries and seawater, and wastewater. Dependency of E. coli inactivation rates on temperature varied among the water sources. There was a significant difference in inactivation rate values at the reference temperature between rivers and agricultural waters, wastewaters and agricultural waters, rivers and lakes, and wastewater and lakes. At specific sites, the Q10 equation was more accurate in rivers and coastal waters than in lakes making the value of the Q10 coefficient appear to be site-specific. Results of this work indicate possible sources of uncertainty to be accounted for in watershed-scale microbial water quality modeling.

Persistence of Escherichia coli introduced into streambed sediments with goose, deer and bovine animal waste.

AIMS: The focus of this work was to compare the survival of Escherichia coli introduced into streambed sediments from goose, deer and bovine faeces vs indigenous E. coli. METHODS AND RESULTS: The survival experiments were conducted in flow-through chambers for 32days using two sediments (mineral and organic) obtained from a first-order creek in Maryland. Bovine, goose and deer faeces were collected fresh and diluted or enriched so that added E. coli and indigenous populations were equivalent. Escherichia coli and total coliforms were enumerated using the Colilert-18 Quanti-Tray system. Patterns of E. coli survival and inactivation rates were virtually identical for indigenous strains in both mineral and organic sediments. The addition of E. coli strains from bovine, goose or deer faeces had relatively little impact on final E. coli concentrations, with the exception of deer-borne E. coli populations in the organic sediment. CONCLUSION: These results indicate that indigenous sediment-borne E. coli strains are generally, or more, persistent than those deposited into sediments, including wildlife. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the survival of E. coli originating from wildlife faeces, in sediments, as opposed to bovine faeces or laboratory-cultured strains. As wildlife are likely to be the primary source of E. coli in most non agricultural watersheds, an understanding of the persistence of these strains is important to understanding microbial water quality.

The diphtheria toxin channel-forming T-domain translocates its own NH2-terminal region and the catalytic domain across planar phospholipid bilayers.

The T-domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy-terminal 57-amino acid segment (residues 322-378) in the T-domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T-domain upstream from this. Here we show that in association with channel formation by the T-domain, its hydrophilic 63-amino acid NH2-terminal region (residues 202-264) as well as the entire catalytic A fragment (residues 1-201) cross the lipid bilayer. The phenomenon that enabled us to demonstrate this was the rapid closure of channels at cis negative voltages when a histidine tag was placed at various positions in the NH2-terminal region of the T-domain or in the A fragment; the inhibition of this effect by trans nickel established that the histidine tag was present on the trans side of the membrane. Thus, all of the machinery necessary to translocate the A fragment across membranes is built into the 114 residues at the carboxy-terminal end of the T-domain (residues 265-378), without the requirement of any proteins in the plasma membrane (e.g., toxin receptor) or of any other cellular components.

Topography of diphtheria Toxin's T domain in the open channel state.

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a "double dagger" model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed "daggers," TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of approximately 70 residues, is translocated across the membrane to the trans side.

Tethered blockers as molecular 'tape measures' for a voltage-gated K+ channel.

The propagation of electrical signals in excitable cells is orchestrated by a molecular family of voltage-dependent ion channel proteins. These K+, Na+, and Ca++ channels are all composed of four identical or similar units, each containing six transmembrane segments (S1-S6) in a roughly four-fold symmetric structure. The S5-S6 sequences fold into a central pore unit, which is surrounded by a voltage-gating module composed of S1-S4. The recent structure of KcsA, a two-transmembrane bacterial K+ channel, illuminates the physical character of the pore unit, but little is known about the arrangement of the surrounding S1-S4 sequences. To locate regions of this gating module in space, we synthesized a series of compounds of varying length that function as molecular 'tape measures': quaternary ammonium (QA) pore blockers that can be tethered to specific test residues. We show that in a Shaker K+ channel, the extracellular ends of S1 and S3 are approximately 30 ¿ from the tetraethylammonium (TEA) blocking site at the external opening of the pore. A portion of the S3-S4 loop is, at 17-18 ¿, considerably closer.

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance.

In a recent paper (Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein, 1989. Proc. Natl. Acad. Sci. USA. 86:2209-2213) we described the general channel-forming properties of the PA65 fragment of anthrax toxin in planar phospholipid bilayer membranes. In the present paper we extend our previous studies of the permeability properties of this channel, using a series of symmetric tetraalkylammonium (TAA) ions. Our main finding is that at micromolar concentrations on either the cis (toxin-containing) or trans side of a membrane containing many (greater than 1,000) channels, these ions, ranging in size from tetramethylammonium to tetrahexylammonium, induce a voltage-dependent reduction of membrane conductance. (We attribute a similar voltage-dependent reduction of membrane conductance by millimolar concentrations of HEPES to a cationic form of this buffer present at micromolar concentrations.) In going from large negative to large positive voltages (on the TAA side) one sees that the conductance first decreases from its value in the absence of TAA, reaches a minimum, and then rises back at larger positive voltages toward the level in the absence of TAA. Our interpretation of this behavior is that these symmetric TAA ions block the cation-selective PA65 channel in a voltage-dependent manner. We postulate that there is a single site within the channel to which TAA ions can bind and thereby block the passage of the major current-carrying ion (potassium). A blocking ion is driven into the site by modest positive voltages, but is driven off the site and through the channel by larger positive voltages, thus explaining the relief of block. (In the accompanying paper [Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942] we confirm this interpretation of the data by analysis at the single-channel level.) This means that these blocking ions can pass through the channel; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A.

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Single-channel analysis.

Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PA65 fragment of anthrax toxin. In this paper we analyze this phenomenon at the single-channel level for tetrabutylammonium ion (Bu4N+). We find that Bu4N+ induces a flickery block of the PA65 channel when present on either side of the membrane, and this block is relieved by large positive voltages on the blocking-ion side. At high frequencies (greater than 2 kHz) we have resolved individual blocking events and measured the dwell times in the blocked and unblocked states. These dwell times have single-exponential distributions, with time constants tau b and tau u that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction of time the channel spends unblocked [tau u/(tau u + tau b)] as a function of voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements of blocking, thus demonstrating that these single channels mirror the behavior seen with many (greater than 10,000) channels in the membrane. In going from large negative to large positive voltages (-100 to +160 mV) on the cis (PA65-containing) side of the membrane, one sees the mean blocked time (tau b) increase to a maximum at +60 mV and then steadily decline for voltages greater than +60 mV, thereby clearly demonstrating that Bu4N+ is driven through the channel by positive voltages on the blocking-ion side. In other words, the channel is permeable to Bu4N+. An interesting finding that emerges from analysis of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with Bu4N+ present on the cis side of the membrane, plateaus at large positive cis voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited.

Diffusion limitation in the block by symmetric tetraalkylammonium ions of anthrax toxin channels in planar phospholipid bilayer membranes.

Current flow through the channel formed in planar phospholipid bilayer membranes by the PA65 fragment of anthrax toxin is blocked, in a voltage-dependent manner, by tetraalkylammonium ions (at micromolar concentrations), which bind to a blocking site within the channel lumen. We have presented evidence that diffusion plays a significant role in the kinetics of blocking by tetrabutylammonium ion (Bu4N+) from the cis (toxin-containing) side of the membrane (Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942); in this paper we examine the implications and consequences of diffusion control for binding kinetics. As expected for a diffusion-affected reaction, both the entry rate constant (kcis1) of Bu4N+ from the cis solution to the blocking site and the exit rate constant (kcis-1) of Bu4N+ from the blocking site to the cis solution are reduced if the viscosity of that medium is increased by the addition of dextran. In conformity with both thermodynamics and kinetic arguments, however, the voltage-dependent equilibrium binding constant, Keq (= kcis-1/kcis1), is not altered by the dextran-induced viscosity increase of the cis solution. The entry rate constants (kcis1) for tetrapentylammonium (Pe4N+), tetrahexylammonium (Hx4N+), and tetraheptylammonium (Hp4N+) are also diffusion controlled, and all of them, including that for Bu4N+, attain a voltage-independent plateau value at large positive cis voltages consistent with diffusion limitation. Although the plateau value of kcis1 for Hx4N+ is only a factor of 3 less than that for Bu4N+, the plateau value for Hp4N+ is a factor of 35 less. This precipitous fall in value indicates, from diffusion-limitation theory, that the diameter of the channel entrance facing the cis solution is not much larger than the diameter of Hp4N+, i.e., approximately 12 A.

Fine-needle aspiration of a metastatic breast carcinoma in the lung with melanin pigmentation: a case report.

The presence of melanin pigment in the cytoplasm of breast carcinoma cells has been reported. Fine-needle aspiration of a solitary lung lesion in a woman who had undergone mastectomy for ductal carcinoma revealed malignant cells consistent with the primary mammary tumor; many of these tumor cells contained melanin pigment.

Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers.

The three separate proteins that make up anthrax toxin--protective antigen (PA), edema factor (EF), and lethal factor (LF)--act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF). PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. PA can be separated, after mild trypsinolysis, into two fragments, PA65 (65 kDa) and PA20 (20 kDa). We demonstrate that trypsin-cleaved PA is capable of forming cation-selective channels in planar phospholipid bilayer membranes and that this activity is confined to the PA65 fragment; PA20, LF, and EF are devoid of channel-forming activity. These PA65 channels exhibit pH-dependent and voltage-dependent activity--a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins.

A hydroxide ion carrier in planar phospholipid bilayer membranes: (C6F5)2Hg (dipentafluorophenylmercury).

Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C6F5)2Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na2SO4 solutions, membranes treated with (C6F5)2Hg are almost ideally selective for H+/OH- between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH-] over this pH range and with the square of the (C6F5)2Hg concentration. The presumed current-carrying species is the dimer [(C6F5)2Hg]2OH-, which, along with the neutral molecule (C6F5)2Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH- and Cl- conductances are approximately equal. Thus, the carrier displays an approximately 10(4)-fold preference for OH- over Cl-.

The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers.

The heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayer membranes. Channel activity is confined to the N-terminal half of this chain; the C-terminal half is inactive. Channel activity is stimulated by low pH (4.5-5.5) on the cis side (the side to which protein is added), neutral pH on the opposite (trans) side, and cis positive voltages. These findings are strikingly similar to those previously reported for analogous fragments of diphtheria and tetanus toxins.

[3H]mazindol binding associated with neuronal dopamine and norepinephrine uptake sites.

[3H]Mazindol labels neuronal dopamine uptake sites in corpus striatum membranes (KD = 18 nM) and neuronal norepinephrine uptake sites in cerebral cortex and submaxillary/sublingual gland membranes (KD = 4 nM). The potencies of various inhibitors of biogenic amine uptake in reducing [3H]mazindol binding in striatal membranes correlate with their potencies for inhibition of neuronal [3H]dopamine accumulation, whereas their potencies in reducing [3H]mazindol binding to cortical and salivary gland membranes correlate with their potencies for inhibition of neuronal [3H]norepinephrine accumulation. Similar to the dopamine and norepinephrine uptake systems, [3H]mazindol binding in all three tissues is dependent upon sodium (with potassium, lithium, rubidium, and Tris being ineffective substitutes) and chloride (with sulfate and phosphate being ineffective substitutes). In membranes of the cerebral cortex and salivary gland, half-maximal stimulation is observed at 50-80 mM NaCl, whereas in membranes of the corpus striatum half-maximal stimulation occurs at 240 mM NaCl. In striatal membranes NaCl increases the affinity of [3H]mazindol binding with no effect on the maximal number of sites. The enhancement of affinity is due to a selective slowing of the dissociation of the ligand from its binding site. The association of [3H]mazindol binding sites with neuronal dopamine uptake sites in the corpus striatum is further supported by the reduction of [3H]mazindol binding sites in striatal membranes following destruction of dopaminergic neurons by 6-hydroxydopamine. Similarly, the association of [3H]mazindol binding sites with neuronal norepinephrine uptake sites in cerebral cortex is supported by the reduction of [3H]mazindol binding to cortical membranes following destruction of noradrenergic neurons by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine.