Quantification of Au Nanoparticle Biouptake and Distribution to Freshwater Algae Using Single Cell - ICP-MS.

Quantifying metal and nanoparticle (NP) biouptake and distribution on an individual cellular basis has previously been impossible, given available techniques which provide qualitative data that are laborious to acquire and prone to artifacts. Quantifying metal and metal NP uptake and loss processes in environmental organisms will lead to mechanistic understanding of biouptake and improved understanding of potential hazards and risks of metals and NPs. In this work, we present a new technique, single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), which allows quantification of metal concentrations on an individual cell basis down to the attogram (ag) per cell level. We present data validating the novel method, along with the mass of metal per cell. Finally, we use SC-ICP-MS, with ancillary cell counting methods, to quantify the biouptake and strong sorption and distribution of both dissolved Au and Au NPs in a freshwater alga (Cyptomonas ovate). The data suggests differences between dissolved and NP uptake and loss. In the case of NPs, there was a dose and time dependent uptake, but individual cellular variations; at the highest realistic exposure conditions used in this study up to 40-50% of cells contained NPs, while 50-60% of cells did not.

The concentration-dependent aggregation of Ag NPs induced by cystine.

Cystine is widely used in cell culture media. Cysteine, the reduced form of cystine, is widely used to scavenge dissolved Ag in eco-toxicological studies to differentiate dissolved vs. nanoparticle uptake and toxicity. However, little is known about the impact of cysteine and cystine on the aggregation behavior of Ag NPs, in particular as a function of Ag NP concentration. Herein, we investigate how cystine (0-300µM) affects the stability of citrate-, polyvinylpyrrolidone-, and polyethylene glycol-coated silver nanoparticles (cit-Ag NPs, PVP-Ag NPs and PEG-Ag NPs, respectively) with and without Suwannee River fulvic acid (SRFA) as a function of Ag NPs concentration using UV-vis spectroscopy at environmentally and ecotoxicologically relevant Ag NP concentrations (ca. 125-1000µgL(-1)). The results demonstrate, for the first time, the concentration-dependent aggregation of cit-Ag NPs in the presence of cystine with a shift in the critical coagulation concentration (CCC) to lower cystine concentrations at lower cit-Ag NP concentrations. At the highest cit-Ag NP concentration (1000µgL(-1)), reaction limited aggregation was only observed and no CCC was measured. SRFA slowed the aggregation of cit-Ag NPs by cystine and aggregation occurred in reaction limited aggregation (RLA) regime only. No CCC value was measured in the presence of SRFA. Cystine replaces citrate, PVP and PEG coatings, resulting in aggregation of both electrostatically and sterically stabilized Ag NPs. These findings are important in understanding the factors determining the behavior of Ag NPs in cell culture media. Also due to the similarity between cystine and cysteine, these results are important in understanding the uptake and toxicity of Ag NPs vs. Ag ions, and suggest that the reduction of the toxicity of Ag NPs in the presence of cysteine could be due to a combined effect of scavenging Ag(+) ions and Ag NP aggregation in the presence of cysteine.

Subject-specific computational simulation of left ventricular flow based on magnetic resonance imaging.

A detailed investigation of left ventricle (LV) flow patterns could improve our understanding of the function of the heart and provide further insight into the mechanisms of heart failure. This study presents patient-specific modelling with magnetic resonance imaging (MRI) to investigate LV blood flow patterns in normal subjects. In the study, the prescribed LV wall movements based on the MRI measurements drove the blood flow in and out of the LV in computational fluid dynamics simulation. For the six subjects studied, the simulated LV flow swirls towards the aortic valve and is ejected into the ascending aorta with a vertical flow pattern that follows the left-hand rule. In diastole, the inflow adopts a reasonably straight route (with no significant secondary flow) towards the apex in the rapid filling phase with slight variations in the jet direction between different cases. When the jet reaches about two thirds of the distance from the inflow plane to the apex, the blood flow starts to change direction and swirls towards the apex. In the more slowly filling phase, a centrally located jet is evident with vortices located on both sides of the jet on an anterior-posterior plane that passes through the mitral and aortic valves. In the inferior-superior plane, a main vortex appears for most of the cases in which an anticlockwise vortex appears for three cases and a clockwise vortex occurs for one case. The simulated flow patterns agree well qualitatively with MRI-measured flow fields.

Efficient synthesis of 1,2,4-dithiazolidine-3,5-diones [dithiasuccinoyl-amines] from bis(chlorocarbonyl)disulfane plus bis(trimethylsilyl)amines.

The 1,2,4-dithiazolidine-3,5-dione heterocycle, also referred to as a dithiasuccinoyl (Dts)-amine, serves as a readily removable amino protecting group for building blocks used in syntheses of peptides, glycopeptides, and PNA; it is also useful as a masked isocyanate and (inversely) as a sulfurization reagent for trivalent phosphorus. Bis(chlorocarbonyl)disulfane, the two-sulfur analogue of succinyl chloride, has been envisioned as a reagent for facile single-step elaboration of the heterocycle. However, reactions of bis(chlorocarbonyl)disulfane directly with primary amines fail to yield Dts-amines for reasons that are discussed. Inspired by several precedents from the organosilicon chemistry literature that a trimethylsilyl group may serve as a "large proton," a successful, high-yield preparation of Dts-amines through reactions of bis(chlorocarbonyl)disulfane with bis(trimethylsilyl)amines has been developed. Studies aimed at elucidating mechanistic reasons for these observations are also presented.

The influence of inflow boundary conditions on intra left ventricle flow predictions.

The combination of computational fluid dynamics (CFD) and magnetic resonance imaging (MRI) offers a promising tool that enables the prediction of blood flow patterns in subject-specific cardiovascular models. The influence of the model geometry on the accuracy of the simulation is well recognized. This paper addresses the impact of different boundary conditions on subject-specific simulations of left ventricular (LV) flow. A novel hybrid method for prescribing effective inflow boundary conditions in the mitral valve plane has been developed. The detailed quantitative results highlight the strengths as well as the potential pitfalls of the approach.

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling.

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.

Dual contrast TrueFISP imaging for left ventricular segmentation.

Based on varying tissue contrasts at different RF flip angles, a new TrueFISP imaging strategy for cardiac function measurement is presented. A single breath-hold dual RF flip angle cine multi-slice TrueFISP imaging sequence was implemented which provides a significant increase in signal contrast between blood and myocardium. The increase in image contrast combined with different characteristics in RF response facilitates the delineation of cardiovascular borders. Based on this imaging strategy it is demonstrated how a simple 2D histogram clustering algorithm can be used for the fully automatic segmentation of the left ventricular (LV) blood pool. The method is validated with data acquired from 10 asymptomatic subjects, and the results are shown to be comparable to that of manual delineation by experienced observers.

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase.

We prepared a stable cell line expressing the glucagon receptor to characterize the effect of G(s)-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.

Mass spectrometric evaluation of synthetic peptides as primary structure models for peptide and protein deamidation.

Solid-phase peptide synthesis and deamidation measurements using a novel mass spectrometric technique were carried out for 94 model asparaginyl peptides from 3 to 13 residues in length. Deamidation rates of these peptides in pH 7.4, 37.0 degrees C, 0.15 M Tris-HCl buffer were measured and evaluated. It was found that they validate the use of pentapeptide models as surrogates for the primary sequence dependence of peptide and protein deamidation rates and the discovery by difference of secondary, tertiary and quaternary structure effects. Deamidation of the pentapeptide models, compared with that of longer peptides of more intricate structure, is discussed, and the application of this technique to deamidation measurement of intact proteins is demonstrated.

Airblast TNT equivalence for a range of commercial blasting explosives.

Results are reported from a programme of work undertaken by the UK Health and Safety Executive to investigate the airblast produced by commercial sector explosives having velocities of detonation (VoD) in the range 2000-8200 m s(-1). The data produced will be useful in evaluating the blast hazards of such explosives in industrial circumstances and also as a means of assessing post-accident damage. All of the solid explosive materials studied produced blast waves which ramped up into shock-wave form close to the point of initiation. The dependence of peak overpressure and positive phase impulse on scaled distance is presented and compared to that of TNT. The TNT equivalence (TNT(e)) technique is shown to be applicable to solid phase explosives with a wide range of VoD, although the precise values of TNT(e) vary with distance.

Fire and explosion hazards to flora and fauna from explosives.

Deliberate or accidental initiation of explosives can produce a range of potentially damaging fire and explosion effects. Quantification of the consequences of such effects upon the surroundings, particularly on people and structures, has always been of paramount importance. Information on the effects on flora and fauna, however, is limited, with probably the weakest area lying with fragmentation of buildings and their effects on different small mammals. Information has been used here to gain an appreciation of the likely magnitude of the potential fire and explosion effects on flora and fauna. This is based on a number of broad assumptions and a variety of data sources including World War II bomb damage, experiments performed with animals 30-40 years ago, and more recent field trials on building break-up under explosive loading.

Selective stabilization of the high affinity binding conformation of glucagon receptor by the long splice variant of Galpha(s).

To analyze functional differences in the interactions of the glucagon receptor (GR) with the two predominant splice variants of Galpha(s), GR was covalently linked to the short and the long forms Galpha(s)-S and Galpha(s)-L to produce the fusion proteins GR-Galpha(s)-S and GR-Galpha(s)-L. GR-Galpha(s)-S bound glucagon with an affinity similar to that of GR, while GR-Galpha(s)-L showed a 10-fold higher affinity for glucagon. In the presence of GTPgammaS, GR-Galpha(s)-L reverted to the low affinity glucagon binding conformation. Both GR-Galpha(s)-L and GR-Galpha(s)-S were constitutively active, causing elevated basal levels of cAMP even in the absence of glucagon. A mutant GR that failed to activate G(s) (G23D1R) was fused to Galpha(s)-L. G23D1R-Galpha(s)-L bound glucagon with high affinity, but failed to elevate cAMP levels, suggesting that the mechanisms of GR-mediated Galpha(s)-L activation and Galpha(s)-L-induced high affinity glucagon binding are independent. Both GR-Galpha(s)-S and GR-Galpha(s)-L bound the antagonist desHis(1)[Nle(9),Ala(11),Ala(16)]glucagon amide with affinities similar to GR. The antagonist displayed partial agonist activity with GR-Galpha(s)-L, but not with GR-Galpha(s)-S. Therefore, the partial agonist activity of the antagonist observed in intact cells appears to be due to GRs coupled to Galpha(s)-L. We conclude that Galpha(s)-S and Galpha(s)-L interact differently with GR and that specific coupling of GR to Galpha(s)-L may account for GTP-sensitive high affinity glucagon binding.

Orientation of cecropin A helices in phospholipid bilayers determined by solid-state NMR spectroscopy.

The orientation of the insect antibiotic peptide cecropin A (CecA) in the phospholipid bilayer membrane was determined using (15)N solid-state NMR spectroscopy. Two peptide samples, each specifically labeled with (15)N at Val(11) or Ala(27), were synthesized by solid phase techniques. The peptides were incorporated into phospholipid bilayers, prepared from a mixture of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, and oriented on glass slides. The (15)N chemical shift solid-state NMR spectra from these uniaxially oriented samples display a single (15)N chemical shift frequency for each labeled residue. Both frequencies are near the upfield end of the (15)N chemical shift powder pattern, as expected for an alpha-helix with its long axis in the plane of the membrane and the NH bonds perpendicular to the direction of the magnetic field. These results support a mechanism of action in which CecA binds to and covers the membrane surface, thereby causing a general destabilization and leakiness of the lipid bilayer membrane. The data are discussed in relation to a proposed mechanism of membrane lysis and bacterial killing via an ion channel activity of CecA.

Structure-activity studies of normal and retro pig cecropin-melittin hybrids.

Chimeric analogs of cecropin P1 and melittin with normal and retro sequences were synthesized to explore the effect of sequence, amide bond direction (helical dipole), charge, amphipathicity and hydrophobicity on their antibacterial activity and channel-forming ability. When viewed from the opposite end by rotation in the plane 180 degrees retro analogs have the same sequence as the parent with reversed amide bond and helical dipole directions. The expected activities were related to the important structural features and a series of assumptions were made. Retro analogs are expected to be inactive if both sequence and amide bond direction make critical contributions to the activity. CP1(1-10)M(2-9) amide, (SWLSKTAKKLIGAVLKVL), showed a broad antibacterial spectrum with high activity against the two Gram-negative and three Gram-positive bacteria tested. Retro-CP1(1-10)M(2-9) was less active compared to its normal peptide. CP1(1-9)M(1-8) and CP1(1-9)M(2-8) amides were found to be active against Gram-negative Escherichia coli and also Gram-positive Streptococcus pyogenes, but inactive against the other test organisms. The corresponding retro analogs were inactive against all the five bacteria tested. These results suggest that both sequence and amide bond direction (helix dipole) are important structural requirements for the activity of CP1-M hybrids. Acetylation of the N-terminal amine in both normal and retro analogs lowered their activity, indicating the contribution of free amine to the activity. These analogs form ion-conducting channels in lipid bilayers. The action of the peptides may be explained by self-aggregation and formation of ion-conducting pores across bacterial membranes. Conformational analysis obtained from CD measurements showed that all analogs form amphipathic alpha-helices in presence of 12-20% hexafluoro isopropanol. The retro CP1(1-10)M(2-9) amide showed higher helicity and is more potent compared to other retro analogs synthesized. These studies show the effect of small sequence modifications on the biological activity of the peptides and on their alpha-helical conformation in HFIP, the structure-inducing organic solvent.

Positively charged residues at positions 12, 17, and 18 of glucagon ensure maximum biological potency.

Glucagon is a peptide hormone that plays a central role in the maintenance of normal circulating glucose levels. Structure-activity studies have previously demonstrated the importance of histidine at position 1 and the absolute requirement for aspartic acid at position 9 for transduction of the hormonal signal. Site-directed mutagenesis of the receptor protein identified Asp64 on the extracellular N-terminal tail to be crucial for the recognition function of the receptor. In addition, antibodies generated against aspartic acid-rich epitopes from the extracellular region competed effectively with glucagon for receptor sites, which suggested that negative charges may line the putative glucagon binding pocket in the receptor. These observations led to the idea that positively charged residues on the hormone may act as counterions to these sites. Based on these initial findings, we synthesized glucagon analogs in which basic residues at positions 12, 17, and 18 were replaced with neutral or acidic residues to examine the effect of altering the positive charge on those sites on binding and adenylyl cyclase activity. The results indicate that unlike N-terminal histidine, Lys12, Arg17, and Arg18 of glucagon have very large effects on receptor binding and transduction of the hormonal signal, although they are not absolutely critical. They contribute strongly to the stabilization of the binding interaction with the glucagon receptor that leads to maximum biological potency.

Synthesis and study of normal, enantio, retro, and retroenantio isomers of cecropin A-melittin hybrids, their end group effects and selective enzyme inactivation.

In our effort to understand the structural requirements for the antimicrobial activity of cecropin A (CA) and melittin (M), we synthesized the normal, enantio, retro and retroenantio hybrid analogs; we related activity to their sequence, chirality, amide bond direction (helix dipole) and end group charges. To compare the effect of the end groups, each of these analogs was synthesized both with an acid and an amide C-terminus and also with and without an N alpha-acetyl N-terminus. The all-L- and all-D-enantiomers of several cecropin-melittin hybrids were previously found to be equally potent against several bacterial species, and no chiral effect was observed. This general rule has now been confirmed and extended. However, two exceptions have been found. All-L-CA(1-13)M(1-13) acid was 5 times and 9 times less potent than the all-D-analog, respectively, toward gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa. All-L-CA(1-7)M(2-9) acid was 5 times and 14 times less active against S. aureus and P. aeruginosa, respectively, than its all-D acid isomer. The corresponding D- and L-retro analogs differed only marginally. A role for proteolytic enzymes has been implicated as a cause for these differences in the activities of L- and D-enantiomers. In all cases, blocking the alpha-amine by acetylation had no significant effect on potency. The retro and retroenantio analogs of CA(1-13)M(1-13) acid were as potent as their normal and enantio analogs against all the test bacteria. The C-terminal amides also showed similar potency against four test bacteria. It should be noted that the negative end of the helix dipole of a normal peptide points toward the C-terminus, whereas it points away in the case of a retro derivative when viewed in the direction of the normal sequence.

Prevention of insect-borne disease: an approach using transgenic symbiotic bacteria.

Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting insects may serve as a powerful approach to control certain arthropod-borne diseases. The endosymbiont of the Chagas disease vector, Rhodnius prolixus, has been transformed to express cecropin A, a peptide lethal to the parasite, Trypanosoma cruzi. In insects carrying the transformed bacteria, cecropin A expression results in elimination or reduction in number of T. cruzi. A method has been devised to spread the transgenic bacteria to populations of R. prolixus, in a manner that mimics their natural coprophagous route of symbiont acquisition.

Synthesis and antibacterial action of cecropin and proline-arginine-rich peptides from pig intestine.

Two antimicrobial peptides, cecropin P1 (CP1), with a C-terminal carboxyl group, and PR-39, with an amidated, C-terminus, are found in the small intestine of the pig. Each is active against both Gram-positive and Gram-negative bacteria. We have synthesized these peptides and several analogs, including the D-enantiomers and the retro sequences, each with a free or acetylated amino terminus. The CP1 amide was also prepared. The retro CP1 peptides were much less active than the parent CP1 peptide, confirming the importance of sequence or the amide bond and helix dipole direction, and the N alpha-acetyl peptides were also less active, indicating that a free amino terminus is essential for high activity. The ratio of the lethal concentration of L/D isomers of CP1 is less than 1 for Gram-negative, but greater than 1 for Gram-positive bacteria. PR-39 showed no significant chiral selectivity toward Escherichia coli, Bacillus subtilis and Streptococcus pyogenes, but the L/D ratio was high for Pseudomonas aeruginosa (66), and very high for Staphylococcus aureus (> 1000). In the latter case the lethal concentration for the D-isomer was 0.57 microM, whereas this organism was quite resistant to the L-isomer (> 600 microM). Thus the enantiomers of CP1 and PR-39 are not equally active for all species. In a plate assay with a very small log-phase inoculum of Staph aureus, D-PR-39 produced a clear zone of killing surrounded by a zone of stimulated growth. After prolonged incubation the two zones became one clear zone. Addition of D-PR-39 to the wells of a dense turbid plate of growing cells showed a cleared zone for each of the test organisms, indicating that PR-39 lyses the bacteria rather than simply inhibiting their multiplication.

Hydrophobic effects on antibacterial and channel-forming properties of cecropin A-melittin hybrids.

The design of cecropin-melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile8 in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH2, [CA(1-7)M(2-9)NH2] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile8 reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile8 by a hydrophobic leucine maintained good activity and Ala8 was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser8 strongly reduced potency against all five organisms. Deletion of Leu15 decreased activity, but addition of Thr16 maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic alpha-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in alpha-helicity with an increase in hexafluoro 2-propanol concentration.