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The use of copper complexes to alleviate inflammation associated with rheumatoid arthritis (RA) is well known. This study focuses on designing a new drug that could be used to increase the bioavailability of copper and hence be more effective. The ligand chosen was sarcosyl-l-lysyl-l-lysine (Sar-Lys-Lys). The thermodynamic stability of H+, CuII, NiII, and ZnII complexes of Sar-Lys-Lys was measured in an aqueous solution at 298 ± 0.01°C and an ionic strength of 0.15 M (NaCl) using glass electrode potentiometry. UV-Vis, ESR, and 1H NMR spectroscopy was used to investigate the solution structures of the different species. At physiological pH, the ligand was found to coordinate via two amide nitrogens, the terminal amine, and the terminal carboxy group. The ε-amino group of lysine did not coordinate with the metal ion. Dermal absorption is the preferred method of administration and so this study used partition coefficients and tissue permeability studies to assess the bioavailability of the different complexes. Sar-Lys-Lys was found to increase the copper lipophilicity by a factor of 10 and increased tissue permeability by 30 %.

Metal complexes involving a chelation between copper(II) and the tripeptides, sarcosyl- l -leucyl-phenylalanine (Sar-Leu-Phe) and glycyl- l -leucyl-phenylalanine (Gly-Leu-Phe), have been identified as potential therapeutic drugs for rheumatoid arthritis. However, in solution the speciation and exact coordination of these tripeptides to copper(II) is unknown and as a result this was investigated using glass electrode potentiometry, ultraviolet–visible spectrophotometry, infrared spectroscopy and 1H NMR and CW EPR spectroscopies. The same basicity was found for the terminal amine of both tripeptides, but the methyl group on Sar decreased the copper(II) complex stability constants by 0.38–1.67 log units. Four species: ML, MLH-1, ML2H-1 and MLH-2 were found for both tripeptides in the pH range 2–11. The dissociated protons are from the amide groups. Room temperature and frozen solution EPR spectra support the existence of MLH-1, MLH-2 and ML2H-1 (a large excess was required for the ML2H-1 species). The two species, MLH-2 and ML2H-1, have a CuN3O equatorial chromophore, which therefore produce similar room temperature EPR spectra, with similar magnetic parameters. Frozen solution EPR spectra revealed different anisotropic magnetic parameters in axial symmetry, thus suggesting the presence of two different geometries. The lower g|| and the higher A|| values indicate that the geometry of the MLH-2 complexes tends more towards square planar, instead of the usual expected tetragonally elongated octahedral geometry. Copper(II) could coordinate to each tripeptide via an amine-N, two amide-Ns, two carbonyl-Os and a carboxyl-O. The proposed coordination modes were validated using quantum mechanical calculations.

The quest for effective treatments of oxidative stress has concentrated over the years on new nanomaterials with improved antioxidant and antiradical activity, thereby attracting broad research interest. In that regard, research efforts in our lab were launched to pursue such hybrid materials involving a) synthesis of silica gel matrices, b) evaluation of the suitability of atoxic matrices as potential carriers for the controlled release of V(IV)(VOSO4), V(V)(NaVO3) compounds and a newly synthesized heterometallic lithium-vanadium(IV,V) tetranuclear compound containing vanadium-bound hydroxycarboxylic 1,3-diamine-2-propanol-N,N,N′,N′-tetraacetic acid (DPOT), and c) investigation of structural and textural properties of silica nanoparticles (NPs) by different and complementary characterization techniques, inquiring into the nature of the encapsulated vanadium species and their interaction with the siloxane matrix, collectively targeting novel antioxidant and antiradical nanomaterials biotechnology. The physicochemical characterization of the vanadium-loaded SiO2 NPs led to the formulation of optimized material configuration linked to the delivery of the encapsulated antioxidant-antiradical load. Entrapment and drug release studies showed a) the competence of hybrid nanoparticles with respect to encapsulation efficiency of the vanadium compound (concentration dependence), b) congruence with the physicochemical features determined, and c) a well-defined release profile of NP load. Antioxidant properties and the free radical scavenging capacity of the new hybrid materials (containing VOSO4, NaVO3, and V-DPOT) were demonstrated through a) 2-diphenyl-1-picrylhydrazyl (DPPH) free radical, and b) intracellular-extracellular reactive oxygen species (ROS) assays, through UV–Visible spectroscopy techniques, collectively showing that the hybrid silica NPs (empty-loaded) could serve as an efficient platform for nanodrug formulations counteracting oxidative stress.

We investigated the alterations of sleep regulation and promotion biomarkers as adenosine through its enzymes total adenosine deaminase (tADA)/adenosine deaminase (ADA2) in a microgravity analogue environment of head-down-tilt bed rest and their association with brain connectivity networks during non-rapid eye movement sleep stage 3 (NREM3), as well as the effectiveness of the reactive sledge (RSL) jump countermeasure to promote sleep. A total of 23 healthy male volunteers were maintained in 6° head-down-tilt position for 30 days and assigned either to a control or to a RSL group. Blood collection and polysomnographic recordings were performed on data acquisition day 1, 14, 30 and -14, 21, respectively. Immunochemical techniques and network-based statistics were employed for adenosine enzymes and cortical connectivity estimation. Our findings indicate that human blood adenosine biomarkers as well as NREM3 cortical functional connectivity are impaired in simulated microgravity. RSL physical activity intervened in sleep quality via tADA/ADA2 fluctuations lack, minor cortical connectivity increases, and limited degree of node and resting-state networks. Statistically significant decreases in adenosine biomarkers and NREM3 functional connectivity involving regions (left superior temporal gyrus, right postcentral gyrus, precuneus, left middle frontal gyrus, left postcentral gyrus, left angular gyrus and precuneus) of the auditory, sensorimotor default-mode and executive networks highlight the sleep disturbances due to simulated microgravity and the sleep-promoting role of RSL countermeasure. The head-down-tilt environment led to sleep deterioration projected through NREM3 cortical brain connectivity or/and adenosine biomarkers shift. This decline was more pronounced in the absence of the RSL countermeasure, thereby highlighting its likely exploitation during space missions.

Novel quaternary ammonium surfactants (QUATs) derived from phenylalaninyl-proline dipeptide with chain length C12 and C14 were synthesised as potential active ingredients to be used in body cleansing formulations. The physicochemical properties and biological activities of the QUATs were determined in both single and in mixed surfactant system with either the conventional anionic surfactant sodium dodecyl sulphate (SDS) or sodium N-dodecyl prolinate. The C12 QUAT derivative showed antagonistic behaviour in both SDS and sodium N-dodecyl prolinate mixed surfactant system. Comparing the mixed system of the C12 QUAT with SDS and sodium N-dodecyl prolinate, it was found that the latter displayed better antibacterial activity together with the lower ocular irritation. The C12 QUAT-sodium N-dodecyl prolinate mixture were non cytotoxic at a concentration corresponding to its MIC value, showing that the mixture was selective towards bacterial cells rather than mammalian cell lines. Diffusion measurements showed that the sodium N-dodecyl prolinate surfactant consisted of 26 molecules per micelle in water but only 3 molecules per micelle in DMSO/water (1:1). On the other hand, C12 QUAT did not form a micelle in DMSO/Water. Membrane permeability studies of the C12 QUAT and sodium N-dodecyl prolinate showed that these surfactants are capable to penetrate into deeper skin layers to exert their antibacterial and cleansing action and hence can be used as a promising candidate as active ingredients in body wash formulations.

Neuropeptides belonging to the adipokinetic hormone (AKH) family elicit metabolic effects as their main function in insects, by mobilizing trehalose, diacylgycerol, or proline, which are released from the fat body into the hemolymph as energy sources for muscle contraction required for energy-intensive processes, such as locomotion. One of the AKHs produced in locusts is a decapeptide, Locmi-AKH-I (pELNFTPNWGT-NH2). A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure. In vitro, cycloAKH selectively retains full efficacy on a pest insect (desert locust) AKH receptor, while showing little or no activation of the AKH receptor of a beneficial insect (honeybee). Molecular dynamic analysis incorporating NMR data indicate that cycloAKH preferentially adopts a type II β-turn under micelle conditions, whereas its linear counterpart and natural AKH adopts a type VI β-turn under similar conditions. CycloAKH, linear LNFTPNWG-NH2, and Locmi-AKH-I feature the same binding site during docking simulations with the desert locust AKH receptor (Schgr-AKHR), but differ in the details of the ligand/receptor interactions. However, cycloAKH failed to enter the binding pocket of the honeybee receptor 3D model during docking simulations. Since the locust AKH receptor has a greater tolerance than the honeybee receptor for the cyclic conformational constraint in vitro receptor assays, it could suggest a greater tolerance for a shift in the direction of the type II β turn exhibited by cycloAKH from the type VI β turn of the linear octapeptide and the native locust decapeptide AKH. Selectivity in biostable mimetic analogs could potentially be enhanced by incorporating conformational constraints that emphasize this shift. Biostable mimetic analogs of AKH offer the potential of selectively disrupting AKH-regulated processes, leading to novel, environmentally benign control strategies for pest insect populations. ispartof: Insect Biochemistry And Molecular Biology vol:125 ispartof: location:England status: published

The advent of biodegradable nanomaterials with enhanced antibacterial activity stands as a challenge to the global research community. In an attempt to pursue the development of novel antibacterial medicinal nanotechnology, we herein a) synthesized ionic-gelated chitosan nanoparticles, b) compared and evaluated the antibacterial activity of essential oils extracted from nine different herbs (Greek origin) and their combinations with a well-defined antibacterial Zn(II)-Schiff base compound, and c) encapsulated the most effective hybrid combination of Zn(II)-essential oils inside the chitosan matrix, thereby targeting well-formulated nanoparticles of distinct biological impact. The empty and loaded chitosan nanoparticles were physicochemically characterized by FT-IR, Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), with the entrapment and drug release studies being conducted through UV-Visible and atomic absorption techniques. The antimicrobial properties of the novel hybrid materials were demonstrated against Gram positive (S. aureus, B. subtilis, and B. cereus) and Gram negative (E. coli and X. campestris) bacteria using modified agar diffusion methods. The collective physicochemical profile of the hybrid Zn(II)-essential oil cocktails, formulated so as to achieve optimal activity when loaded to chitosan nanoparticles, signifies the importance of design in the development of efficient nanomedicinal pharmaceuticals a) based on both natural products and biogenic metal ionic cofactors, and b) targeting bacterial infections and drug resistance.

The data presented in this article are related to the publication âInteraction of the red pigment-concentrating hormone of the crustacean Daphnia pulex, with its cognate receptor, Dappu-RPCHR: A nuclear magnetic resonance and modeling studyâ (Jackson et al., 2017) [1]. This article contains the data for homology modeling of the red pigment-concentrating hormone (RPCH) receptor of the water flea, Daphnia pulex (Dappu-RPCHR), which was constructed from its primary sequence. This is the first 3D model of a crustacean G-protein coupled receptor. Docking of the agonist, pGlu-Val-Asn-Phe-Ser-Thr-Ser-Trp amide (Dappu-RPCH), was used to find a binding pocket on the receptor and compared to the binding pocket of the adipokinetic hormone (AKH) receptor from the malaria mosquito. Data for the receptor, with and without loop refinement, together with the docked agonist, are presented. Keywords: Daphnia pulex, Red pigment-concentrating hormone, Homology modeling, Molecular docking

Due to its physical and chemical properties, bismuth (Bi(III)) is widely used in the treatment of several gastrointestinal and skin diseases, and infections caused by bacteria. Herein, its known antimicrobial potential was taken into consideration in the synthesis of two new hybrid ternary materials of Bi(III) with the physiological α-hydroxycarboxylic glycolic acid and 1,10-phenanthroline (phen), [Bi2(C2H2O3)2(C2H3O3)(NO3)]n. nH2O (1) and [Bi(C12H8N2)(NO3)4](C10H8N4) (2), aiming at improving its antibacterial properties. Their physicochemical characterization was carried out through elemental analysis, FT-IR, atomic absorption spectroscopy, single crystal X-ray diffraction, thermogravimetric analysis (TGA), photoluminescence, and 13C MAS-NMR techniques. The antimicrobial activity of the title complexes was directly linked to Bi(III) coordination environment and the incipient aqueous chemistry. For their antibacterial assessment, minimum inhibitory concentration (MIC), zone of inhibition (ZOI), and bacteriostatic-bacteriocidal activity were determined in various Gram positive (Staphylococcus aureus, Bacillus subtilis and Bacillus cereus) and Gram negative (Escherichia coli and Xanthomonas campestris) bacterial cultures, in reference to a positive control (ampicillin), encompassing further comparisons with literature data. The findings reveal that the new hybrid bismuth materials have significant antimicrobial effects against the employed bacteria. Specifically, 2 exhibits better antimicrobial properties than free Bi(NO3)3 and phen. On the other hand, 1 is bacteriostatic toward four microorganisms except X. campestris, with 2 being bacteriocidal toward four microorganisms except B. cereus. Collectively, the new hybrid, well-defined, and two of the rarely crystallographically characterized Bi(III) materials a) exhibit properties reflecting their physicochemical nature and reactivity, and b) are expected to contribute to the development of efficient metallodrugs against drug-resistant bacterial infections.

Chondroitin sulfate (CS) occurs in human urine. It has several potential binding sites for calcium and as such may play an inhibitory role in calcium oxalate and calcium phosphate (kidney stone disease by reducing the supersaturation (SS) and crystallization of these salts. Urinary magnesium is also a role player in determining speciation in stone forming processes. This study was undertaken to determine the thermodynamic parameters for binding of the disaccharide unit of two different CS isomers with calcium and magnesium. These included the binding constant K. Experiments were performed using an isothermal titration calorimeter (ITC) at 3 different pH levels in the physiological range in human urine. Data showed that interactions between the CS isomers and calcium and magnesium occur via one binding site, thought to be sulfate, and that log K values are 1.17–1.93 and 1.77–1.80 for these two metals respectively. Binding was significantly stronger in Mg-CS than in Ca-CS complexes and was found to be dependent on pH in the latter but not in the former. Furthermore, binding in Ca-CS complexes was dependent on the location of the sulfate binding site. This was not the case in the Mg-CS complexes. Interactions were shown to be entropy driven and enthalpy unfavourable. These findings can be used in computational modeling studies to predict the effects of the calcium and magnesium CS complexes on the speciation of calcium and the SS of calcium salts in real urine samples.

Stone formation in the urinary tract is a multifactorial world-wide disease afflicting between 8 and 20% of population groups in different geographical locations. Discrimination between stone formers and healthy persons on the basis of urine composition remains a crucial goal among researchers. Since 1 H NMR is able to monitor the metabolic function of the kidney we applied it to the urine of 60 stone formers (34 females, 26 males) and 38 healthy persons (14 females, 24 males). Spectra were normalized relative to an internal standard and integrated over 37 consecutive regions. The resulting data were subjected to principal component and canonical discriminant analysis. Excellent discrimination between patient and controls for both genders was achieved, with all the data falling within the 95% confidence interval. The most important variables allowing for this inter-group separation correspond to those associated with protein signals. We therefore speculate that the discrimination between patients and controls may be due to the presence or absence of macromolecular stone promoters and/or inhibitors. This supports numerous in vitro studies demonstrating that urinary macromolecules play significant roles in stone formation and prevention. Our finding that 1 H NMR analysis of urine differentiates between stone formers and healthy persons represents an important breakthrough for rapid screening of individuals who are at risk for this disease.

Stone formation in the urinary tract is a multifactorial world-wide disease afflicting between 8 and 20% of population groups in different geographical locations. Discrimination between stone formers and healthy persons on the basis of urine composition remains a crucial goal among researchers. Since

Dietary hydroxyproline may be involved in the endogenous synthesis of oxalate. Glycolate, produced during the metabolism of hydroxyproline, may exert physicochemical effects on urinary calcium by virtue of its dihydroxycarboxylic acid structure. The aim of this study was to investigate these possible stone-risk scenarios. We modelled the effect of different glycolic acid concentrations on ionized calcium (iCa2+) and relative supersaturation (RSS) of calcium oxalate (CaOx) using the program JESS. Thereafter, three healthy white males and two healthy black males ingested 30 g gelatin for 3 days. 24-h urines were collected at baseline and after completion of the protocol. Urines were analysed for physicochemical risk factors and for iCa2+ and glycolic acid. Speciation concentrations and RSS values were calculated. Theoretical modelling showed that binding between calcium and glycolate does not occur and that iCa2+ and RSS CaOx are unaffected. However, after ingestion of hydroxyproline, iCa2+ decreased significantly. Urinary pH and glycolate increased significantly. Oxalate excretion and RSS CaOx did not change We attribute the decrease in iCa2+ to increases in the concentrations of several Ca–phosphate species, the formation of which is due to the increase in pH. We speculate that the absence of an increase in oxalate excretion despite an increase in glycolate excretion may be due to the mixed racial composition of our test group in which some pathways may be preferred to others. Our findings alert stone researchers to the importance of measuring urinary pH in their workup of subjects and to select racially homogenous groups for investigation.

Hydroxycitrate (HCA), has been mooted as a potentially equivalent, if not superior therapy to citrate for calcium urolithiasis. However, past studies were mainly mechanistic while thermodynamic experiments were limited. We determined stability constants for HCA protonation and metal ion complexation, and measured HCA speciation by potentiometric titration. We modelled the effect of HCA, citrate and pH on calcium oxalate (CaOx) and calcium phosphate (CaP) supersaturation (SS) using the program JESS to which we had added our determined thermodynamic constants. Finally, we measured the effects of HCA on CaOx metastable limit (MSL) in artificial urine using turbidimetry. Our experiments showed that fully dissociated HCA is the dominant species at urinary pH 6. Modelling predicted that HCA decreases SS CaOx and CaP to a greater extent than citrate. In vitro experiments showed increases in the MSLs of CaOx and CaP thereby confirming favorable thermodynamic effects of HCA on SS. Clinical trials are warranted to investigate the promise of HCA for reducing this important risk factor for calcium nephrolithiasis.

The aim of this study was to develop complexes that could be used to eleviate the inflammation associated with arthritis by facilitating the dermal absorption of copper. To this end, copper(II) complexes of two tripeptides sarcosyl- l -histidyl- l -lysine (L1) and sarcosyl- l -lysyl- l -histidine (L2) were studied using potentiometric and spectroscopic (UV–vis, ESR, 1H NMR) techniques as well as molecular mechanics (MM) calculations. Since Zn(II) and Ni(II) are in vivo competitors of Cu(II), equilibrium constants of H+, Cu(II), Ni(II) and Zn(II) with the peptides were measured in aqueous solution at 25 ± 0.01 °C and an ionic strength of 0.15 M (NaCl) using glass electrode potentiometry. In the pH range 2.00–11.00, four species; MLH, ML, MLH−1, and MLH−2 were observed. 1H NMR identified the binding sites to be the imidazole nitrogen, the amide nitrogen and the terminal amino group. The e-amino group of lysine did not coordinate to the Cu(II). The ML2H−2 species involved coordination of two deprotonated amide groups to give a neutral complex. UV/vis and EPR spectroscopy confirmed the mode of coordination, which was different in L1 and L2. Molecular mechanics calculations were used to support the structures postulated from potentiometric and spectroscopic data. Speciation calculations of Cu(II) using a computer model of blood plasma indicated that Sar-Lys-His (L2) was the best at mobilising copper in silico.

The well-known host molecule C-methylcalix[4]resorcinarene is shown to exist in two unique hexameric arrangements within the same crystal. These two hexameric assemblies are linked via a water molecule into a supra-heterodimer.

Fruit flies are a widely distributed pest insect that pose a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, has the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extra-cellular binding site of a G protein-coupled receptor causes its activation. In this paper, the structure of Phote-HrTH in sodium dodecyl sulfate (SDS) micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be fairly rigid, with an S2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, D. melanogaster, Drome-AKHR, was used to construct two models of the receptor. It is proposed that these two models represent the active and inactive state of the receptor. The model based on the crystal structure of the β-2 adrenergic receptor was found to bind Phote-HrTH with a binding constant of −102kJmol−1, while the other model, based on the crystal structure of rhodopsin, did not bind the peptide. Under molecular dynamic simulation, in a palmitoyloleoylphosphatidylcholine (POPC) membrane, the receptor complex changed from an inactive to an active state. The identification and characterisation of the ligand binding site of Drome-AKHR provide novel information of ligand–receptor interaction, which could lead to the development of species-specific control substances to use discriminately against the fruit fly.

Kidney stone formation is governed by thermodynamic (supersaturation) and kinetic (crystal nucleation, growth, aggregation) mechanisms. We adopted a dual theoretical and experimental approach to investigate the potential role of urinary phytate in this regard. Thermodynamic constants for eight protonated phytate species and seven calcium-phytate complexes were determined by potentiometry and incorporated into the speciation program JESS. Urine was collected from 16 heathy males and their urine compositions were used as input for JESS. Phytate concentration was varied during modelling. No statistically significant decreases in Ca2+ concentrations or in supersaturation values were predicted by JESS. Crystallization experiments were then performed in pooled urine. Endogenous phytate concentration was determined using a metal-dye assay. The pool was dosed with various concentrations of phytate to achieve final concentrations equivalent to those used for modelling. Experiments showed that phytate had no effects on Ca2+ concentrations (as predicted by our theoretical modelling), metastable limits or crystal nucleation and growth kinetics. However, crystal aggregation kinetics was inhibited. We speculate that HPhy-11, small amounts of which were revealed by modelling, may bind to crystal surfaces and inhibit aggregation. We conclude that phytate exerts a kinetic, but not a thermodynamic inhibitory effect on crystallization in urine.

Neurodegenerative diseases entail deeply complex processes, intimately associated with progressive brain damage reflecting cellular demise. Biochemical reactivity linked to such processes in Alzheimer's disease involves, among others, metal-induced oxidative stress contributing to neuronal cell death. Prominent among redox active metals inducing oxidative stress is Cu(II). Poised to develop molecular technology counteracting oxidative stress, efforts were launched to prepare bioactive hybrid nanoparticles, capable of working as host-carriers of potent antioxidants, such as the natural flavonoid quercetin. Employing synthetic protocols consistent with the assembly of silica nanoparticles, PEGylated and CTAB-modified materials were synthesized. Subsequent concentration-dependent loading of quercetin led to well-defined molecular carriers, the antioxidant efficiency of which was determined through drug release studies using UV-visible spectroscopy. The physicochemical characterization (elemental analysis, particle size, z-potential, FT-IR, thermogravimetric analysis, scanning electron microscopy) of the empty and loaded silica nanoparticles led to the formulation of optimized material linked to the delivery of the encapsulated antioxidant to primary rat hippocampal cultures under oxidative stress. Entrapment and drug release studies showed a) the competence of hybrid nanoparticles as far as the loading capacity in quercetin (concentration dependence), b) congruence with the physicochemical features determined, and c) the release profile of the nanoparticle load under oxidative stress in neuronal cultures. The bio-activity profile of quercetin nanoparticles in a neurodegenerative environment brought on by Cu(II) a) denotes the improved specificity of antioxidant reactivity counteracting oxidative stress, and b) sets the stage for the development of molecular protection and preventive medical nanotechnology of relevance to neurodegenerative Alzheimer's disease.

Alzheimer's disease (AD) belongs to one of the most multifactorial, complex and heterogeneous morbidity-leading disorders. Despite the extensive research in the field, AD pathogenesis is still at some extend obscure. Mechanisms linking AD with certain comorbidities, namely diabetes mellitus, obesity and dyslipidemia, are increasingly gaining importance, mainly because of their potential role in promoting AD development and exacerbation. Their exact cognitive impairment trajectories, however, remain to be fully elucidated. The current review aims to offer a clear and comprehensive description of the state-of-the-art approaches focused on generating in-depth knowledge regarding the overlapping pathology of AD and its concomitant ailments. Thorough understanding of associated alterations on a number of molecular, metabolic and hormonal pathways, will contribute to the further development of novel and integrated theranostics, as well as targeted interventions that may be beneficial for individuals with age-related cognitive decline.

The primary sequence of the red pigment-concentrating hormone (RPCH) receptor of the water flea, Daphnia pulex, was used in homology modeling to construct the first 3D model of a crustacean G-protein coupled receptor, Dappu-RPCHR. This receptor was found to belong to the class A subfamily of GPCRs with a disulfide bridge between Cys72 and Cys150 and an ionic lock between Arg97 and Thr224 and Thr220. NMR restrained molecular dynamics was used to determine the structure of an agonist, Dappu-RPCH, in a membrane-mimicking environment. The agonist was found to be flexible but has two main conformations in solution, both having β-turns. Docking of the predominant structure was used to find a binding pocket on the receptor. The pocket's spatial location was similar to that of the AKH receptor of Anopheles gambiae. The binding affinity was -69kcalmol-1 with the N-terminus of Dappu-RPCH inserted between helices 4 and 6, and the C-terminus interacting with extra-cellular loop, ECL2. Upon binding, H-bonding to the peptide may activate the receptor. This development of the first Dappu-RPCH/Dappu-RPCHR model could be useful for understanding ligand-receptor interactions in crustaceans.

Copper complexes have been reported to alleviation the inflammation associated with rheumatoid arthritis (RA). The aim of this research was to design and test new ligands that are able to promote the percutaneous absorption of copper and/or mobilise endogenous copper reserves, with the ultimate aim of testing these as anti-inflammatory agents. Formation constants of H + and Cu(II) with the tripeptides sarcosyl- l -histidyl- l -histidine (Sar-HH) and sarcosyl- l -glycyl- l -histidine (Sar-GH) were measured at 25 ± 0.01 °C and at an ionic strength of 0.15 M (NaCl) using glass electrode potentiometry. In the pH range 2–11, four species; CuLH, CuL, CuLH -1 , and CuLH -2 were observed. The two tripeptides showed significantly different coordination behaviour at physiological pH, Sar-HH forming a CuLH -1 species while Sar-GH formed a CuLH -2 species. The solution structures of the different Cu(II) species formed with these ligands were investigated using visible spectroscopy (UV–Vis), electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy. The CuLH -2 species involved coordination of two deprotonated amide groups to give a neutral complex. 1H NMR identified the binding sites to be the imidazole nitrogen, the two-amide nitrogens and the terminal amino group. UV–Vis and EPR spectroscopy confirmed this mode of coordination. Speciation calculations using a blood plasma model were used to estimate the complexing ability of the ligands in silico . Octanol/water partition coefficients and Franz cell permeation studies were used to evaluate percutaneous skin absorption. The results showed that the Cu(II) complexes are hydrophilic but that Sar-GH caused a 2 fold increase in membrane permeability of Cu(II).

We used computer modeling to investigate the influence of physicochemical stone risk factors on urinary supersaturation (SS) of calcium oxalate (CaOx) in patients with severe hyperoxaluria, relative hypocalciuria, hypocitraturia, and CaOx nephrolithiasis after extensive small bowel resection, usually performed for Crohn's disease. We also simulated different treatment strategies, including oral calcium supplements and citrate, in such patients.A baseline urine model was derived by consolidating data acquired by ourselves with those from another patient cohort. Calcium and oxalate excretions in this model were altered to obtain an extreme case. For comparison, additional models were based on published urine data from normal subjects (N) and idiopathic CaOx stone formers (SF). The Joint Expert Speciation System was used to simulate different urine situations based on reported compositional values.[Ca(2+)][Ox(2-)] ionic concentration products and SS(CaOx) are substantially higher in enteric hyperoxaluric patients than in N and SF, despite their relatively lower calcium excretions. Molar Ca:Ox ratios are substantially lower in enteric hyperoxalurics than in N and SF. Oral calcium supplements can reduce SS(CaOx), but monitoring is required to avoid exceeding a safe dosing threshold. A simple calculation can alert the clinician that this threshold is being approached or even exceeded. Increasing urinary pH and citrate decreases SS(CaOx) but not to the same extent as decreasing Ox excretion.Calcium supplements can help reduce stone risk in patients with severe enteric hyperoxaluria, but initial efforts should be directed toward reducing urinary oxalate by reducing dietary oxalate. Citrate therapy that increases both urine pH and urinary citrate provides an additional therapeutic benefit.

Potential application of Kenyan bentonite for adsorption of iron, cobalt, copper, nickel and zinc and for analysis of electroactive species in water from a polluted water course using a 3-electrode potentiostat and cyclic voltammetry was studied. Polished carbon graphite electrodes were used either bare or modified with Kenyan bentonite using an electrochemically inert adhesive to a thickness of about 0.8 mm. These were used to prepare calibration curves of iron, cobalt, copper, nickel and zinc by plotting cyclic voltammograms of the ions at different concentrations and using 0.1 M sulphuric acid as supporting electrolyte. The slopes of the curves from bentonite-modified electrodes were observed to be higher than those obtained from bare carbon electrodes by a factor of between 1.7 and 24, implying that bentonite enhanced electron transfer kinetics of the metal ions. It was also observed that the magnitude of the ratio depended on the proximity of the element to either filled or half-filled 3d orbitals, which implied that a chemical reaction may have taken place between the bentonite and the ions (chemisorption). Carbon graphite electrodes were modified with bentonite that had been soaked in water samples from a polluted water course at a ratio of 1:1 w/w. The cyclic voltammograms showed clear oxidative and reductive peaks indicating that electroactive species that previously could not be detected on the potentiostat were pre-concentrated on the bentonite and could thus be detected. Thus Kenyan bentonite is observed to chemically adsorb zinc, cobalt, copper, nickel and iron species in aqueous solution and can be used to monitor electroactive pollutants in aqueous systems using electroanalytical techniques. Key words: Bentonite, heavy metals, surface modified electrodes, cyclic voltammetry, preconcentration, adsorption.

It has been shown that the inflammation associated with rheumatoid arthritis can be reduced using copper complexes. In order to improve the bioavailability of copper and hence efficacy, 3-(2-aminoacetamido)-N-(pyridin-2-ylmethyl)propanamide, H2(5 6 5)NH2, was designed as a potential chelator of copper. Solution equilibrium measurements show that the [Cu(LH-2)] species predominates at physiological pH and blood plasma speciation calculations predict that this ligand is able to mobilise Cu(II) in vivo. A structural study of the Cu(II)/H2(5 6 5)NH2 system was conducted in the solid and solution state using Uv–Vis, CD, 1H NMR and EPR spectroscopy and single crystal X-ray crystallography. The result indicate that the structure of [Cu(H2(5 6 5)NH2)H-2] in the solid and solution state are similar and confirm that, the metal binds to the pyridine nitrogen, the two amide nitrogens and the terminal amino group in a distorted square planar geometry.

Microgravity (MG) is one of the main problems that astronauts have to cope with during space missions. Long-duration space travel can have detrimental effects on human neurophysiology. Despite scientific efforts, these effects are still insufficiently investigated. Animal earth-based analogs are used to investigate potential nervous system associated perturbations that might occur during prolonged space missions. Hindlimb unloading, Tail suspension and Pelvic suspension models are currently used in MG studies. Loss of homeostasis of certain biological pathways in the nervous system can lead to the functioning and expression of receptors/genes, and the release and functioning of neurotransmitters and neuronal membrane ion channels into specific brain regions. The potential impact of MG on molecular mechanisms linked to neurophysiology through animal earth-based analogs is reviewed. The effect of molecular signalling pathways on the decline of neuronal connectivity and cognitive and neuroplasticity function under MG simulated conditions will be studied. The role of biomarkers including neurotransmitters, genes or receptors will be highlighted in the healthy and MG-affected brain. MG-mediated neurodegenerative mechanisms linked to learning and memory impairment will be highlighted. This review depicts the current rodent models applied to simulate MG ground based approaches and investigates the MG induced changes in the nervous system. The neuropathological profile of the above animal MG ground-based models can be comparable to the effects of ageing, anxiety and other neurological disorders. The advantages and limitations of the existing approaches are discussed. MG induced neurophysiology outcomes can be extrapolated to study other clinical applications.

Background Supersaturation (SS) of urinary salts has been extensively invoked for assessing the risk of renal stone formation, but precursors have often been ignored. Our objectives were to establish by computer modeling, which urinary components are essential for calculating reliable SS values, to investigate whether unique equilibrium processes occur in the urine of stone formers (SF) which might account for their higher SS levels relative to healthy controls (N), to determine the relative efficacies of three different, widely-used protocols for lowering urinary SS of calcium salts and to examine the influence of precursors. Methods Urinary data from published studies were consolidated into three N–SF cohort pairs and were selected for modeling using the speciation program JESS. SS values of calcium oxalate monohydrate (COM) and its precursors calcium oxalate di- and tri-hydrate (COD, COT), as well as those of hydroxyapatite (HAP) and its precursors octacalcium phosphate (OCP) and brushite (Bru) were calculated. A pragmatic approach for the statistical analysis of the modeling outputs was developed and applied. Results Thirteen urinary parameters were identified as being crucial for the accurate calculation of SS of calcium oxalate (CaOx) and calcium phosphate (CaP) salts. Chloride, sulfate and ammonium, or combinations thereof, have frequently been omitted from SS calculations, thereby giving erroneously high values for these parameters. Subtle differences occur in the urinary equilibrium processes of SF and N. SS (OCP) and SS (HAP) are more sensitive to dilution effects than the other urinary salts. Decreasing urinary Ca and decreasing Ox are equally efficacious in reducing SS of CaOx salts, and are marginally superior to increasing urinary volume. Decreasing urinary calcium and increasing urinary volume are equally efficacious in reducing SS of CaP salts. SS of precursors follow the Ostwald Lussac empirical rule and undergo the same changing trends in different urinary scenarios. Conclusions Researchers are advised to include all of the 13 identified urinary parameters in their calculations of SS of Ca salts, in order to attain better accuracy. Differences in achieving speciation equilibria in N and SF contribute towards higher values of SS in SF, but in a minor way only. Increasing urinary volume is confirmed as the most realistically efficacious strategy for decreasing SS of urinary calcium salts. SS of HAP should be assigned equal importance to that of Bru in considering the stone-forming potential of urine. Precursors of CaOx and CaP salts should be taken into account when investigating stone forming mechanisms.

Copper complexes have anti-inflammatory activity in the treatment of inflammation associated with rheumatoid arthritis (RA). The preferred route of administration is through the skin, so the rate of dermal absorption and bioavailability of copper is important. Based on previous studies, 3-amino-N-(pyridin-2-ylmethyl)-propanamide, [H(56)NH2], was designed as a potential chelator of copper. The stability constant measurements revealed that MLH−1 is the most stable species at the physiological pH of 7.4. The X-ray crystal structure of this species was solved and copper was found in a rectangular pyramidal geometry. The ligand occupied three coordination sites while bridging chloride linked copper ions together in a chain. The ligand bound to the metal ion through the pyridyl nitrogen, the amide nitrogen and the terminal amino group. Spectroscopic studies confirmed that this structure persisted in aqueous solution. Octanol/water partition coefficients and Franz cell permeation studies showed that [H(56)NH2] is able to promote the dermal absorption of Cu(II).

Insect flight requires rapid mobilization of energy reserves during flight, which is mediated and regulated by hormonal control via adipokinetic hormones. The structure of the G-protein receptors to which these hormones bind, are crucial in understanding many of the physiological processes in which they play a central role. To date no 3D structure of an insect G-protein coupled receptor (GPCR) is available. Here, the first models of the 3D structures of a GPCR from the malaria mosquito are presented. Homology modeling of the receptor identified from the genome of Anopheles gambiae was used to construct two models of the receptor. The 7 transmembrane helical bundles of these two models are based on the crystal structures of beta2-adrenergic receptor and rhodopsin. The flexible loop regions were modeled using high temperature simulated annealing and constrained molecular dynamic simulations. The two receptor models differ in a number of critical features, the most important of which is that the rhodopsin-based model has a ‘closed’ structure while the beta2-based structure is ‘open’. The ‘open’ conformation provides easy access of the hormone to the binding pocket. Docking calculations with the insect adipokinetic hormones, AKH-1 (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH2) from the malaria mosquito and Del-CC (pGlu-Lys-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH2) from the blister beetle showed that while the binding motif of the two is similar, AKH-1 has more than 30 times higher affinity than Del-CC, which strongly suggests that the binding is specific, and that the correct binding site was identified. Using these models it is possible to design antagonists, which block the binding site and are thus species-specific insecticides.

Theoretical modeling of urinary crystallization processes affords opportunities to create and investigate scenarios which would be extremely difficult or impossible to achieve in in vivo experiments. Researchers have previously hypothesized that calcium renal stone formation commences in the nephron. In the present study, concentrations of urinary components and pH ranges in different regions of the nephron were estimated from concentrations in blood combined with a knowledge of the renal handling of individual ions. These were used in the chemical speciation program JESS to determine the nature of the solution complexes in the different regions of the nephron and the saturation index (SI) of the stone-forming salts calcium oxalate (CaOx), brushite (Bru), hydroxyapatite (HAP) and octacalcium phosphate (OCP). The effect of independent precipitation of each of the latter on the SI values of other salts was also investigated. HAP was the only salt which was supersaturated throughout the nephron. All of the other salts were supersaturated only in the middle and distal regions of the collecting duct. Supersaturations were pH sensitive. When precipitation of CaOx, Bru and OCP was simulated in the distal part of the collecting duct, little or no effect on the SI values of the other stone forming salts was observed. However, simulation of HAP precipitation caused all other salts to become unsaturated. This suggests that if HAP precipitates, a pure stone comprising this component will ensue while if any of the other salts precipitates, a mixed CaOx/CaP stone will be formed. Application of Ostwald's Rule of Stages predicts that the mixed stone is likely to be CaOx and Bru. Our modelling demonstrates that precipitation of stone-forming salts in the nephron is highly dependent on the delicate nature of the chemical equilibria which prevail and which are themselves highly dependent on pH and component concentrations.

Copper complexes of N , N ′-di(aminoethylene)-2,6-pyridinedicarbonylamine and bis-( N , N -dimethylethyl)-2,6-pyridinedicarboxamide have been studied by glass electrode potentiometry, NMR, UV and IR spectroscopy as potential anti-inflammatory agents for the alleviation of inflammation associated with rheumatoid arthritis. The protonation and formation constants with Cu(II), Zn(II) and Ca(II), determined at 25 °C and an ionic strength of 0.15 mol dm −3 were used to calculate the copper plasma mobilizing index of the ligands. Spectroscopic studies suggested that metal ion complexation promotes deprotonation and coordination of the amide nitrogens resulting in overall tetragonal distorted copper complexes. Bio-distribution and dermal absorption studies showed the complexes to have relatively long biological half-lives with 50% of the injected dose remaining in the body 24 h after administration.

A simple liquid scintillation counting technique to measure the activity composition of a mixture containing two known pure beta-emitting radionuclides was recently developed at the NMISA. The method has been applied to various two-component mixtures of (32)P, (33)P and (35)S, primarily to gauge the effect of spectral energy differences on the method's ability to extract the individual activities. Excellent results were obtained for mixtures of (33)P and (35)S, radionuclides with similar, low beta energies. Mixtures containing the high-energy beta-emitter (32)P were more difficult to resolve, although quenching of the counting sources with CHCl(3) improved mixture resolution.

The thermodynamic equilibria of copper(II), zinc(II) and calcium(II) with N,N′-bis[2(2-pyridyl)-methyl]pyridine-2,6-dicarboxamide (L1) have been studied at 25 °C and an ionic strength of 0.15 mol dm−3. Spectroscopic studies suggest metal ion complexation promotes deprotonation and coordination of the amide nitrogens resulting in overall tetragonal coordination of Cu2+. Blood–plasma modelling predicts that Cu(II) competes effectively against Zn(II) and Ca(II) for L1 in vivo. Octanol–water partition coefficient studies show that Cu(II)–L1 complexes are reasonably lipophilic. However, the CuL1H−2 species which predominates at the physiological pH of 7.4 has poor superoxide dismutase activity. Bio-distribution experiments showed activity accumulation and retention in the body of about 50% of the injected dose for the [64Cu]Cu(II)–L1 complex after 24 h.

A comparison of the conformational characteristics of the related hormones [Nle(15)] gastrin-17 and [Tyr(9)-SO(3)] cholecystokinin-15, in membrane-mimetic solutions of dodecylphosphocholine micelles and water, was undertaken using NMR spectroscopy to investigate the possibility of a structural motif responsible for the two hormones common ability to stimulate the CCK(2) receptor. Distance geometry calculations and NOE-restrained molecular dynamics simulations in biphasic solvent boxes of decane and water pointed to the two peptides adopting near identical helical C-terminal configurations, which extended one residue further than their shared pentapeptide sequence of Gly-Trp-Met-Asp-Phe-NH(2). The C-terminal conformation of [Nle(15)] gastrin-17 contained a short alpha-helix spanning the Ala(11)-Trp(14) sequence and an inverse gamma-turn centered on Nle(15) while that of [Tyr(9)-SO(3)] cholecystokinin-15 contained a short 3(10) helix spanning its Met(10) to Met(13) sequence and an inverse gamma-turn centered on Asp(14). Significantly, both the C-terminal helices were found to terminate in type I beta-turns spanning the homologous Gly-Trp-Met-Asp sequences. This finding supports the hypothesis that this structural motif is a necessary condition for CCK(2) receptor activation given that both gastrin and cholecystokinin have been established to follow a membrane-associated pathway to receptor recognition and activation. Comparison of the conformations for the non-homologous C-terminal tyrosyl residues of [Nle(15)] gastrin-17 and [Tyr(9)-SO(3)] cholecystokinin-15 found that they lie on opposite faces of the conserved C-terminal helices. The positioning of this tyrosyl residue is known to be essential for CCK(1) activity and non-essential for CCK(2) activity, pointing to it as a possible differentiator in CCK(1)/CCK(2) receptor selection. The different tyrosyl orientations were retained in molecular models for the [Nle(15)] gastrin-17/CCK(2) receptor and [Tyr(9)-SO(3)] cholecystokinin-15/CCK(1) receptor complexes, highlighting the role of this residue as a likely CCK(1)/CCK(2) receptor differentiator.

The protonation equilibria of a pentadentate ligand, N,N'-(2,2'-azanediylbis(ethane-2,1-diyl))dipicolinamide ([H(2)(5555)-N]) and the complexation of this ligand with Cu(II) Ca(II), Zn(II) and Ni(II) have been studied by pH-potentiometry, (1)H NMR spectroscopy and UV-vis spectrophotometry. (1)H NMR detected the protonation of the pyridyl groups and formation of Cu[H(2)(5555)-N]H species at low pH, while amide group deprotonation at higher pH resulted in the formation of Cu[H(2)(5555)-N]H(-1) and Cu[H(2)(5555)-N]H(-2) species in solution. Potentiometric detection of protonated species was limited by the acidic nature of the pyridyl nitrogen donors. From UV-vis spectroscopy it is suggested that the amide nitrogens are coordinated. This conclusion is supported by Molecular Mechanics calculations. Water-octanol partition coefficients for the Cu(II)-[H(2)(5555)-N] system indicated that although the Cu[H(2)(5555)-N]H(-1) species is largely hydrophilic, approximately 54% of the complex goes into the organic phase. This percentage is able to promote dermal absorption of copper with a calculated penetration rate of 1.92x10(-1)cmh(-1). This was confirmed by dermal absorption studies which illustrate the role of hydrophobicity in promoting percutaneous drug administration.

The conformational preferences of human little gastrin, [Nle(15)] gastrin-17, and its short analogues, gastrin-4 and [beta-Ala(1)] gastrin-5, which include the C-terminal tetrapeptide sequence Trp-Met-Asp-Phe-NH(2) crucial for gastrin bioactivity, were determined by NMR spectroscopy in aqueous solutions of zwitterionic dodecylphosphocholine micelles. Backbone HN chemical shift temperature variance, Halpha chemical shift deviations and complex non-sequential NOE patterns pointed to the C-terminal of [Nle(15)] gastrin-17 adopting an ordered conformation. Distance geometry calculations and NOE-restrained molecular dynamics simulations in membrane mimetic solvent boxes of decane and water indicated the C-terminal tetrapeptide sequence of all three peptides adopted a similar, well defined structure, with a general type IV beta-turn observed for all three peptides. The conformation of [Nle(15)] gastrin-17 consisted of two short helices between Leu(5)-Glu(9) and Ala(11)-Trp(14), with the one helix terminating in a type I beta-turn spanning Gly(13)-Asp(16). The experimental evidence and conformational characteristics of the three peptides in micellar media support a membrane-associated mechanism of receptor recognition and activation for the gastrin hormone family and furthermore point to a possible biologically relevant structural motif for gastrin activity.

Formation constants of copper(ii), zinc(ii) and calcium(ii) with 3,5-diaminodiamido-4-oxahexacyclododecane (cageL) has been studied by glass electrode potentiometry at 25 degrees C and an ionic strength of 0.15 mol dm(-3). Copper(ii) forms more stable complexes with cageL than zinc(ii) and calcium(ii). Metal ion complexation promotes deprotonation and coordination of the amide nitrogens resulting in overall tetragonal coordination of Cu(2+) suggested by the UV-visible electronic spectra. Speciation calculations using a blood plasma model suggest that zinc(ii) and calcium(ii) are good competitors of copper(ii) in vivo. Bio-distribution experiments using (64)Cu-labelled Cu(ii)-cageL show that about 50% dose of the complex is retained in the body after 24 h.

Background Garlic has been used for centuries in folk medicine for its health promoting and cancer preventative properties. The bioactive principles in crushed garlic are allyl sulphur compounds which are proposed to chemically react through (i) protein S-thiolation and (ii) production of ROS. Methods A collection of R-propyl disulphide and R-thiosulfonate compounds were synthesised to probe the importance of thiolysis and ROS generation in the cytotoxicity of garlic-related compounds in WHCO1 oesophageal cancer cells. Results A significant correlation (R2 = 0.78, Fcrit (7,1) α = 0.005) was found between the cytotoxicity IC50 and the leaving group pKa of the R-propyl disulphides and thiosulfonates, supporting a mechanism that relies on the thermodynamics of a mixed disulphide exchange reaction. Disulphide (1) and thiosulfonate (11) were further evaluated mechanistically and found to induce G2/M cell-cycle arrest and apoptosis, inhibit cell proliferation, and generate ROS. When the ROS produced by 1 and 11 were quenched with Trolox, ascorbic acid or N-acetyl cysteine (NAC), only NAC was found to counter the cytotoxicity of both compounds. However, NAC was found to chemically react with 11 through mixed disulphide formation, providing an explanation for this apparent inhibitory result. Conclusion Cellular S-thiolation by garlic related disulphides appears to be the cause of cytotoxicity in WHCO1 cells. Generation of ROS appears to only play a secondary role. General significance Our findings do not support ROS production causing the cytotoxicity of garlic-related disulphides in WHCO1 cells. Importantly, it was found that the popular ROS inhibitor NAC interferes with the assay.

In the emerging issue of enhanced multi-resistant properties in infectious pathogens, new nanomaterials with optimally efficient antibacterial activity and lower toxicity than other species attract considerable research interest. In an effort to develop such efficient antibacterials, we a) synthesized acid-catalyzed silica-gel matrices, b) evaluated the suitability of these matrices as potential carrier materials for controlled release of ZnSO4 and a new Zn(II) binary complex with a suitably designed well-defined Schiff base, and c) investigated structural and textural properties of the nanomaterials. Physicochemical characterization of the (empty-loaded) silica-nanoparticles led to an optimized material configuration linked to the delivery of the encapsulated antibacterial zinc load. Entrapment and drug release studies showed the competence of hybrid nanoparticles with respect to the a) zinc loading capacity, b) congruence with zinc physicochemical attributes, and c) release profile of their zinc load. The material antimicrobial properties were demonstrated against Gram-positive (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus) and negative (Escherichia coli, Pseudomonas aeruginosa, Xanthomonas campestris) bacteria using modified agar diffusion methods. ZnSO4 showed less extensive antimicrobial behavior compared to Zn(II)-Schiff, implying that the Zn(II)-bound ligand enhances zinc antimicrobial properties. All zinc-loaded nanoparticles were less antimicrobially active than zinc compounds alone, as encapsulation controls their release, thereby attenuating their antimicrobial activity. To this end, as the amount of loaded zinc increases, the antimicrobial behavior of the nano-agent improves. Collectively, for the first time, sol-gel zinc-loaded silica-nanoparticles were shown to exhibit well-defined antimicrobial activity, justifying due attention to further development of antibacterial nanotechnology.