Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo

Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pH-dependent membrane lysis and deliver genes and toxins in vitro. The aim of this study was to measure PAA cellular uptake using ISA1-OG (and as a reference ISA23-OG) in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of (125)I-labelled ISA1-tyr in liver cells after intravenous (i.v.) administration to rats. The effect of time after administration (0.5-3h) and ISA1 dose (0.04-100mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed approximately 60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density (also visible by TEM). Increasing ISA1 dose from 10mg/kg to 100mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1h. Moreover, internalised ISA1 provoked NAG release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilisation of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors.

Excellent correlation between cathepsin B inhibition and cytotoxicity for a series of palladacycles

The reaction of the five- or six-membered C,N or C,S-palladacycles [(L)PdCl](2) with PTA (1,3,5-triaza-7-phosphaadamantane) led to the monomeric complexes [(L)Pd(PTA)Cl] 6a, 6b and 7 where LH= N,N-dimethyl-1-phenylmethanamine, benzyl(methyl)sulfane or 1-methyl-5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one respectively. Dimeric complexes have also been synthesised: [Pd(2)L(2)(mu-dppe)Cl(2)], where LH = 1-methyl-5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one (1a), (R)- or (S)-3-isopropyl-1-methyl-5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one (1b, 1c), [Pd(2)L(2)(mu-dppf)Cl(2)], where L= 1-methyl-5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one (4a) or (R)-3-isopropyl-1-methyl-5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one (4b), respectively, and dppe = 1,2-bis(diphenylphosphino)ethane, dppf = 1,1'-bis(diphenylphosphino)ferrocene. The complexes were characterised in solution, by (1)H and (31)P NMR spectroscopy, and single crystals of complexes 6b and 7 were studied in the solid state by X-ray crystallography. The palladacycles were evaluated for in vitro activity as cytotoxic agents on A2780/S cells and also as cathepsin B inhibitors, an enzyme implicated in a number of cancer related events.

Achiral, selective CCK2 receptor antagonists based on a 1,3,5-benzotriazepine-2,4-dione template

Novel, achiral 1H-1,3,5-benzotriazepine-2,4(3H,5H)-diones have been prepared and structurally characterized. These compounds are potent CCK2 receptor antagonists that display a high degree of selectivity over CCK1 receptors.

Characterisation of products of tricalcium silicate hydration in the presence of heavy metals

The hydration of tricalcium silicate (C(3)S) in the presence of heavy metal is very important to cement-based solidification/stabilisation (s/s) of waste. In this work, tricalcium silicate pastes and aqueous suspensions doped with nitrate salts of Zn(2+), Pb(2+), Cu(2+) and Cr(3+) were examined at different ages by X-ray powder diffraction (XRD), thermal analysis (DTA/TG) and (29)Si solid-state magic angle spinning/nuclear magnetic resonance (MAS/NMR). It was found that heavy metal doping accelerated C(3)S hydration, even though Zn(2+) doping exhibited a severe retardation effect at an early period of time of C(3)S hydration. Heavy metals retarded the precipitation of portlandite due to the reduction of pH resulted from the hydrolysis of heavy metal ions during C(3)S hydration. The contents of portlandite in the control, Cr(3+)-doped, Cu(2+)-doped, Pb(2+)-doped and Zn(2+)-doped C(3)S pastes aged 28 days were 16.7, 5.5, 5.5, 5.5, and <0.7%, respectively. Heavy metals co-precipitated with calcium as double hydroxides such as (Ca(2)Cr(OH)(7).3H(2)O, Ca(2)(OH)(4)4Cu(OH)(2).2H(2)O and CaZn(2)(OH)(6).2H(2)O). These compounds were identified as crystalline phases in heavy metal doping C(3)S suspensions and amorphous phases in heavy metal doping C(3)S pastes. (29)Si NMR data confirmed that heavy metals promoted the polymerisation of C-S-H gel in 1-year-old of C(3)S pastes. The average numbers of Si in C-S-H gel for the Zn(2+)-doped, Cu(2+)-doped, Cr(3+)-doped, control, and Pb(2+)-doped C(3)S pastes were 5.86, 5.11, 3.66, 3.62, and 3.52. And the corresponding Ca/Si ratios were 1.36, 1.41, 1.56, 1.57 and 1.56, respectively. This study also revealed that the presence of heavy metal facilitated the formation of calcium carbonate during C(3)S hydration process in the presence of carbon dioxide.

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean

35S-Methionine and 3H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.

The effect of pressure on leucine and thymidine incorporation by free-living bacteria and by bacteria attached to sinking oceanic particles

The effect of pressure on upper ocean free-living bacteria and bacteria attached to rapidly sinking particles was investigated through studying their ability to synthesize DNA and protein by measuring their rate of 3H-thymidine and 3H-leucine incorporation. Studies were carried out on samples from the NE Atlantic under the range of pressures (1–430 atm) encountered by sinking aggregates during their journey to the deep-sea bed. Thymidine and leucine incorporation rates per bacterium attached to sinking particles from 200 m were about six and ten times higher, respectively, than the free-living bacterial assemblage. The ratio of leucine incorporation rate per cell to thymidine incorporation rate per cell was significantly different between the larger attached (18.9:1) and smaller free-living (10.4:1) assemblages. The rates of leucine and thymidine incorporation decreased exponentially with increasing pressure for the free-living and linearly for attached bacteria, while there was no significant influence of pressure on cell numbers. At 100 atm leucine and thymidine incorporation rate per free-living bacterium was reduced to 73 and 20%, respectively, relative to that measured at 1 atm. Pressure of 100 atm reduced leucine and thymidine incorporation per attached bacterium to 94 and 70%, and at 200 atm these rates were reduced to 34 and 51%, respectively, relative to those measured at 1 atm. There was no significant uncoupling of thymidine and leucine incorporation for either the free-living or attached bacterial assemblages with increasing pressure, indicating that the processess of DNA and protein synthesis may be equally affected by increasing pressure. It is therefore unlikely that bacteria, originating from surface waters, attached to rapidly sinking particles play a role in particle remineralization below approximately 1000–2000 m. These results may help to explain the occurrence of relatively fresh aggregates on the deep-sea bed that still contain sufficient organic carbon to fuel the rapid growth of benthic micro-organisms; they also indicate that the effect of pressure on microbial processes may be important in oceanic biogeochemical cycles.

Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.

Hydrochemical and isotopic tracing of mixing dynamics and water quality evolution under pumping conditions in the mine shaft of the abandoned Frances Colliery, Scotland.

Since 1995, when pumps were withdrawn from deep mines in East Fife (Scotland), mine waters have been rebounding throughout the coalfield. Recently, it has become necessary to pump and treat these waters to prevent their uncontrolled emergence at the surface. However, even relatively shallow pumping to surface treatment lagoons of the initially chemically-stratified mine water from a shaft in the coastal Frances Colliery during two dynamic step-drawdown tests to establish the hydraulic characteristics of the system resulted in rapid breakdown of the stratification within 24 h and a poor pumped water quality with high dissolved Fe loading. Further, data are presented here of hydrochemical and isotopic sampling of the extended pump testing lasting up to several weeks. The use in particular of the environmental isotopes d18O, d2H, d34S, 3H, 13C and 14C alongside hydrochemical and hydraulic pump test data allowed characterisation of the Frances system dynamics, mixing patterns and water quality sources feeding into this mineshaft under continuously pumped conditions. The pumped water quality reflects three significant components of mixing: shallow freshwater, seawater, and leakage from the surface treatment lagoons. In spite of the early impact of recirculating lagoon waters on the hydrochemistries, the highest Fe loadings in the longer-term pumped waters are identified with a mixed freshwater–seawater component affected by pyrite oxidation/melanterite dissolution in the subsurface system.

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1e) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M-r of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M-r 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to -9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 mug (.) kg(-1) (.) min(-1), followed by 2 h at 83.0 mug (.) kg(-1) (.) min(-1); corresponding to 0.4 and 2.0 mU (.) kg(-1) (.) min(-1)). At the lower dose, the exogenons glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P

First-principles study of oxygenates on Co surfaces in Fischer-Tropsch synthesis

Extensive density function theory calculations are performed to study the mechanism of the formation of aldehyde and alcohol on Co surfaces in Fischer-Tropsch synthesis, a challenging issue in heterogeneous catalysis. Three possible pathways for the production of formaldehyde and methanol on flat and stepped Co(0001) surfaces are investigated: (i) CO + 4H -> CHO + 3H -> CH2O + 2H -> CH3O + H -> CH3OH; (ii) CO + 4H -> COH + 3H -> CHOH + 2H -> CH2OH + H -> CH3OH; and (iii) the coupling reactions of CH2 + O -> CH2O and CH3 + OH -> CH3OH. It is found that these pathways are generally favored at step sites, and the preferred mechanism is pathway (i) via CHO. Furthermore, the three traditional chain growth mechanisms in Fischer-Tropsch synthesis are semi quantitatively compared and discussed. Our results suggest that the two mechanisms involving oxygenate intermediates (the CO-insertion and hydroxycarbene mechanisms) are less important than the carbene mechanism in the production of long chain hydrocarbons. However, the CO-insertion mechanism may be responsible for the production of long-chain oxygenates.

Insight into association reactions on metal surfaces: Density-functional theory studies of hydrogenation reactions on Rh(111)

Hydrogenation reaction, as one of the simplest association reactions on surfaces, is of great importance both scientifically and technologically. They are essential steps in many industrial processes in heterogeneous catalysis, such as ammonia synthesis (N-2+3H(2)-->2NH(3)). Many issues in hydrogenation reactions remain largely elusive. In this work, the NHx (x=0,1,2) hydrogenation reactions (N+H-->NH, NH+H-->NH2 and NH2+H-->NH3) on Rh(111) are used as a model system to study the hydrogenation reactions on metal surfaces in general using density-functional theory. In addition, C and O hydrogenation (C+H-->CH and O+H-->OH) and several oxygenation reactions, i.e., C+O, N+O, O+O reactions, are also calculated in order to provide a further understanding of the barrier of association reactions. The reaction pathways and the barriers of all these reactions are determined and reported. For the C, N, NH, and O hydrogenation reactions, it is found that there is a linear relationship between the barrier and the valency of R (R=C, N, NH, and O). Detailed analyses are carried out to rationalize the barriers of the reactions, which shows that: (i) The interaction energy between two reactants in the transition state plays an important role in determining the trend in the barriers; (ii) there are two major components in the interaction energy: The bonding competition and the direct Pauli repulsion; and (iii) the Pauli repulsion effect is responsible for the linear valency-barrier trend in the C, N, NH, and O hydrogenation reactions. For the NH2+H reaction, which is different from other hydrogenation reactions studied, the energy cost of the NH2 activation from the IS to the TS is the main part of the barrier. The potential energy surface of the NH2 on metal surfaces is thus crucial to the barrier of NH2+H reaction. Three important factors that can affect the barrier of association reactions are generalized: (i) The bonding competition effect; (ii) the local charge densities of the reactants along the reaction direction; and (iii) the potential energy surface of the reactants on the surface. The lowest energy pathway for a surface association reaction should correspond to the one with the best compromise of these three factors. (C) 2003 American Institute of Physics.

Detection of Mixing Dynamics During Pumping of a Flooded Coal Mine

In complex hydrogeological environments the effective management of groundwater quality problems by pump-and-treat operations can be most confidently achieved if the mixing dynamics induced within the aquifer by pumping are well understood. The utility of isotopic environmental tracers (C-, H-, O-, S-stable isotopic analyses and age indicators—14C, 3H) for this purpose is illustrated by the analysis of a pumping test in an abstraction borehole drilled into flooded, abandoned coal mineworkings at Deerplay (Lancashire, UK). Interpretation of the isotope data was undertaken conjunctively with that of major ion hydrochemistry, and interpreted in the context of the particular hydraulic setting of flooded mineworkings to identify the sources and mixing of water qualities in the groundwater system. Initial pumping showed breakdown of initial water quality stratification in the borehole, and gave evidence for distinctive isotopic signatures (d34S(SO4) ~= -1.6‰, d18O(SO4) ~= +15‰) associated with primary oxidation of pyrite in the zone of water table fluctuation—the first time this phenomenon has been successfully characterized by these isotopes in a flooded mine system. The overall aim of the test pumping—to replace an uncontrolled outflow from a mine entrance in an inconvenient location with a pumped discharge on a site where treatment could be provided—was swiftly achieved. Environmental tracing data illustrated the benefits of pumping as little as possible to attain this aim, as higher rates of pumping induced in-mixing of poorer quality waters from more distant old workings, and/or renewed pyrite oxidation in the shallow subsurface.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Structural effects on the pH-dependent fluorescence of naphthalenic derivatives and consequences for sensing/switching

Naphthalenic compounds are a rich resource for designers of fluorescent sensing/switching/logic systems. The degree of internal charge transfer (ICT) character in the fluorophore excited states can vary from negligible to substantial. Naphthalene-1,8;4,5-diimides (11–13), 1,8-naphthalimides (16) and 4-chloro-1,8-naphthalimides (15) are of the former type. The latter type is represented by the 4-alkylamino-1,8-naphthalimides (1). Whether ICT-based or not, these serve as the fluorophore in ‘fluorophore-spacer-receptor’ switching systems where PET holds sway until the receptor is bound to H+. On the other hand, 4-dialkylamino-1,8-naphthalimides (3–4) show modest H+-induced fluorescence switching unless the 4-dialkylamino group is a part of a small ring (5). Electrostatic destabilization of a non-emissive twisted internal charge transfer (ICT) excited state is the origin of this behaviour. An evolution to the non-emissive twisted ICT excited state is responsible for the weak emission of the model compound 6 (and related structures 7 and 8) across the pH range. Twisted ICT excited states are also implicated in the switch 9 and its model compound 10, which are based on the 6-dialkylamino-3H-benzimidazo[2,1-a]benz[d,e]isoquinolin-3-one fluorophore.

Optimisation of Kinetic Parameters for an After-treatment Catalyst

Mathematical modelling has become an essential tool in the design of modern catalytic systems. Emissions legislation is becoming increasingly stringent, and so mathematical models of aftertreatment systems must become more accurate in order to provide confidence that a catalyst will convert pollutants over the required range of conditions. 
Automotive catalytic converter models contain several sub-models that represent processes such as mass and heat transfer, and the rates at which the reactions proceed on the surface of the precious metal. Of these sub-models, the prediction of the surface reaction rates is by far the most challenging due to the complexity of the reaction system and the large number of gas species involved. The reaction rate sub-model uses global reaction kinetics to describe the surface reaction rate of the gas species and is based on the Langmuir Hinshelwood equation further developed by Voltz et al. [1] The reactions can be modelled using the pre-exponential and activation energies of the Arrhenius equations and the inhibition terms. 
The reaction kinetic parameters of aftertreatment models are found from experimental data, where a measured light-off curve is compared against a predicted curve produced by a mathematical model. The kinetic parameters are usually manually tuned to minimize the error between the measured and predicted data. This process is most commonly long, laborious and prone to misinterpretation due to the large number of parameters and the risk of multiple sets of parameters giving acceptable fits. Moreover, the number of coefficients increases greatly with the number of reactions. Therefore, with the growing number of reactions, the task of manually tuning the coefficients is becoming increasingly challenging. 
In the presented work, the authors have developed and implemented a multi-objective genetic algorithm to automatically optimize reaction parameters in AxiSuite®, [2] a commercial aftertreatment model. The genetic algorithm was developed and expanded from the code presented by Michalewicz et al. [3] and was linked to AxiSuite using the Simulink add-on for Matlab. 
The default kinetic values stored within the AxiSuite model were used to generate a series of light-off curves under rich conditions for a number of gas species, including CO, NO, C_{3H8 and C3H6. These light-off curves were used to generate an objective function.} 
This objective function was used to generate a measure of fit for the kinetic parameters. The multi-objective genetic algorithm was subsequently used to search between specified limits to attempt to match the objective function. In total the pre-exponential factors and activation energies of ten reactions were simultaneously optimized. 
The results reported here demonstrate that, given accurate experimental data, the optimization algorithm is successful and robust in defining the correct kinetic parameters of a global kinetic model describing aftertreatment processes.