Computer simulations of the interaction of human immunodeficiency virus (HIV) aspartic protease with spherical gold nanoparticles: implications in acquired immunodeficiency syndrome (AIDS).

The interaction of gold nanoparticles (AuNP) with human immune-deficiency virus aspartic protease (HIVPR) is modelled using a regime of molecular dynamics simulations. The simulations of the 'docking', first as a rigid-body complex, and eventually through flexible-fit analysis, creates 36 different complexes from four initial orientations of the nanoparticle strategically positioned around the surface of the enzyme. The structural deviations of the enzymes from the initial x-ray crystal structure during each docking simulation are assessed by comparative analysis of secondary structural elements, root mean square deviations, B-factors, interactive bonding energies, dihedral angles, radius of gyration (R g), circular dichroism (CD), volume occupied by C α , electrostatic potentials, solvation energies and hydrophobicities. Normalisation of the data narrows the selection from the initial 36 to one 'final' probable structure. It is concluded that, after computer simulations on each of the 36 initial complexes incorporating the 12 different biophysical techniques, the top five complexes are the same no matter which technique is explored. The significance of the present work is an expansion of an earlier study on the molecular dynamic simulation for the interaction of HIVPR with silver nanoparticles. This work is supported by experimental evidence since the initial 'orientation' of the AgNP with the enzyme is the same as the 'final' AuNP-HIVPR complex generated in the present study. The findings will provide insight into the forces of the binding of the HIVPR to AuNP. It is anticipated that the protocol developed in this study will act as a standard process for the interaction of any nanoparticle with any biomedical target.

Multiple fold increase in activity of ferroxidase-apoferritin complex by silver and gold nanoparticles.

The effect of silver (Ag) and gold (Au) nanoparticles on the ferroxidase activity of apoferritin showed a 110-fold increase in specific activity and a 9-fold increase over the control at the respective molar ratios of Au-apoferritin and Ag-apoferritin nanoparticles (NPs) of 500:1 and 1000:1. Typical color change, from pale yellow to brown, occurred when apoferritin was mixed with AgNO(3) or AuCl(3) followed by sodium borohydride to afford respective metal-apoferritin NP complexes in a ratio of between 250:1 and 4000:1. These complexes were characterized by ultraviolet-visible inductively coupled plasma-optical emission spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and energy-dispersive x-ray spectroscopy. Transmission electron microscopy revealed that the size of NPs increased as the molar ratio of metal to apoferritin increased, with an average size of 3-6 nm generated with Au-to-apoferritin and/or Ag-to-apoferritin molar ratios of 250:1 to 4000:1. Fourier transform infrared spectrometry showed no structural changes of apoferritin when the NPs were attached to the protein. FROM THE CLINICAL EDITOR: In this paper the utility of gold and silver nanoparticles in augmenting the activity of the ferroxidase-apoferritin complex is described. Both NPs dramatically increased the ferroxidase activity.

Spectrofluorimetric analysis of the interaction of amyloid peptides with neuronal nitric oxide synthase: implications in Alzheimer's disease.

BACKGROUND: The deposition of aggregated ß-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides. METHODS: Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aß(1-40) and be catalytic towards amyloid fibril formation. RESULTS: Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aß(1-40) (Aß(17-28) and Aß(25-35)) had Stern-Volmer values (K(SV)) of 0.111µM(-1) and 0.135µM(-1) indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aß(17-28) and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aß(29-37)] and bind to the enzyme at a site remote to the active region. CONCLUSIONS: The interaction and binding of Aß(17-28) and Aß(25-35) to nNOS causes the movement of two critical tryptophan residues of 0.77nm and 0.57nm respectively towards the surface of the enzyme. GENERAL SIGNIFICANCE: The binding of Aß-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aß-peptides and fibrillogenesis in senile plaques in an AD brain.

Ferroxidase activity of apoferritin is increased in the presence of platinum nanoparticles.

The ferroxidase activity of horse spleen apoferritin (HSAF) is increased by nine-fold in the presence of platinum nanoparticles. HSAF was mixed with varying concentrations of K2PtCl4 followed by a 20-fold concentration of sodium borohydride to afford Pt:HSAF nanoparticle complexes in a ratio of between 1:250 and 1:4000. Typical colour changes, from colourless or pale yellow to brown, occurred that were dependent on the amount of platinum present. These complexes were characterized by UV/vis, inductively coupled plasma optical emission spectroscopy, Fourier transform infrared, transmission electron microscopy and energy dispersive x-ray spectroscopy. Transmission electron microscopy analysis revealed that the size of nanoparticles increased as the molar ratio of platinum to HSAF increased with an average size diameter of 2-6 nm generated with HSAF:platinum molar ratios of 1:250-1:4000. Fourier transform infrared spectroscopy (FTIR) spectra showed no distinct changes in the structure of HSAF but confirmed that the nanoparticles were attached to the protein. The effect of platinum nanoparticles on the ferroxidase activity of HSAF showed a specific activity of 360 ρmol min(-1) mg(-1), (nine-fold increase over the control) at the molar ratio of HSAF:platinum nanoparticles of 1:1000.

Association of ß-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease.

Neuronal nitric oxide synthase (nNOS) was purified on DEAE-Sepharose anion-exchange in a 38% yield, with 3-fold recovery and specific activity of 5 µmol.min(-1).mg(-1). The enzyme was a heterogeneous dimer of molecular mass 225 kDa having a temperature and pH optima of 40°C and 6.5, K(m) and V(max) of 2.6 µM and 996 nmol.min(-1).ml(-1), respectively and was relatively stable at the optimum conditions (t(½) = 3 h). ß-Amyloid peptide fragments Aß(17-28) was the better inhibitor for nNOS (K(i) = 0.81 µM). After extended incubation of nNOS (96 h) with each of the peptide fragments, Congo Red, turbidity and thioflavin-T assays detected the presence of soluble and insoluble fibrils that had formed at a rate of 5 nM.min(-1). A hydrophobic fragment Aß(17-21) [Leu(17) - Val(18) - Phe(19) - Phe(20) - Ala(21)] and glycine zipper motifs within the peptide fragment Aß(17-35) were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.

Bioreduction of platinum salts into nanoparticles: a mechanistic perspective.

A mechanism for the bioreduction of H2PtCl6 and PtCl2 into platinum nanoparticles by a hydrogenase enzyme from Fusarium oxysporum is proposed. Octahedral H2PtCl6 is too large to fit into the active region of the enzyme and, under conditions optimum for nanoparticle formation (pH 9, 65 degrees C), undergoes a two-electron reduction to PtCl2 on the molecular surface of the enzyme. This smaller molecule is transported through hydrophobic channels within the enzyme to the active region where, under conditions optimal for hydrogenase activity (pH 7.5, 38 degrees C) it undergoes a second two-electron reduction to Pt(0). H2PtCl6 was unreactive at pH 7.5, 38 degrees C; PtCl2 was unreactive at pH 9, 65 degrees C.

Recovery of rhodium(III) from solutions and industrial wastewaters by a sulfate-reducing bacteria consortium.

A quantitative analysis of the rate of removal of rhodium(III) by a resting sulfate-reducing bacteria (SRB) consortium under different initial rhodium and biomass concentrations, pH, temperature, and electron donor was studied. Rhodium speciation was found to be the main factor controlling the rate of its removal from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependent rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg of rhodium per gram of resting SRB biomass. Electron microscopy studies revealed a time-dependent localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. When a purified hydrogenase enzyme was incubated with rhodium chloride solution under hydrogen, 88% of the rhodium was removed within 1 h, whereas with a soluble extract from SRB 77% was removed within 10 min. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly limited, and it was apparent that an industrial effluent pretreatment was necessary before the application of an enzymatic treatment. In the present study, however, it was established that SRB are good candidates for the enzymatic recovery of rhodium from both aqueous solution and industrial effluent.

Bacterial diguanylate cyclases: structure, function and mechanism in exopolysaccharide biofilm development.

The ubiquitous bacterial cyclic di-guanosine monophosphate (c-di-GMP) emerges as an important messenger for the control of many bacterial cellular functions including virulence, motility, bioluminescence, cellulose biosynthesis, adhesion, secretion, community behaviour, biofilm formation and cell differentiation. The synthesis of this cyclic nucleotide arises from external stimuli on various signalling domains within the N-terminal region of a dimeric diguanylate cyclase. This initiates the condensation of two molecules of guanosine triphosphate juxtaposed to each other within the C-terminal region of the enzyme. The biofilm from pathogenic microbes is highly resistant to antimicrobial agents suggesting that diguanylate cyclase and its product - c-di-GMP - are key biomedical targets for the inhibition of biofilm development. Furthermore the formation and long-term stability of the aerobic granule, a superior biofilm for biological wastewater treatment, can be controlled by stimulation of c-di-GMP. Any modulation of the synthetic pathways for c-di-GMP is clearly advantageous in terms of medical, industrial and/or environmental bioremediation implications. This review discusses the structure and reaction of individual diguanylate cyclase enzymes with a focus on new directions in c-di-GMP research. Specific attention is made on the molecular mechanisms that control bacterial exopolysaccharide biofilm formation and aerobic granules.

Arginine metabolising enzymes as targets against Alzheimers' disease.

Even though the accumulation of arginine and the deposit of aggregated Aß-peptides (senile plaques) in the brain of an Alzheimer patient are classic points of evidence in the neuropathology of the disease considerable dispute remains on their method of formation. One acceptable mechanism to initiate events is a 'seed' aggregation of free monomeric peptides into toxic soluble amyloid oligomers and subsequently into deposits of insoluble fibrils. Since all of these events take place in the brain astrocytes it suggests an interference between arginine-metabolising enzymes and the Aß-peptides. Through kinetic, fluorimetric and thermodynamic analyses two such enzymes - neuronal nitric oxide synthase and peptidyl arginine deiminase - are, not only inhibited by structural fragments of Aß1-42 but are catalytic towards fibrillogenesis. The interaction of the peptide fragments with each enzyme is endothermic, non-spontaneous and involves hydrophobic-hydrophobic associations with a single binding site. The trigger for this series of events focusses in particularly on Aß17-21 with two phenylalanines [Phe19; Phe20], the three glycine zipper motifs [Aß25-29; Aß29-33; Aß33-37] and the triple sequence [Aß25-37] that includes two isoleucine residues [Ile31; Ile32]. FRET studies show the Aß-peptide fragments bind to the enzymes <3.0nm from a single surface tryptophan. Free Aß monomers bind to an enzyme, formulate a nucleus, initiate their aggregation and subsequently become entrapped and couple to the existing aggregated monomers, leading to an elongated fibril. Silver and gold nanoparticles reverse fibrillogenesis! They are surrounded by the amyloid peptide molecules in vivo to effectively deplete their concentration. This does not allow any 'lag' time, nor prevent the formation of critical nuclei for the initial association phase and, inevitably, prevent fibril initiation and elongation. This review focusses on the function and action of arginine metabolising enzymes with respect to the formation of senile plaques and amyloid peptide aggregation to facilitate more of an understanding of neurodegeneration in Alzheimer disease.

Arginine metabolising enzymes as therapeutic tools for Alzheimer's disease: peptidyl arginine deiminase catalyses fibrillogenesis of beta-amyloid peptides.

The accumulation of arginine in the cerebrospinal fluid and brains of patients suffering from acute neurodegenerative diseases like Alzheimer's disease, point to defects in the metabolic pathways involving this amino acids. The deposits of neurofibrillary tangles and senile plaques perhaps as a consequence of fibrillogenesis of beta-amyloid peptides has also been shown to be a hallmark in the aetiology of certain neurodegenerative diseases. Peptidylarginine deiminase (PAD II) is an enzyme that uses arginine as a substrate and we now show that PAD II not only binds with the peptides Abeta(1-40), Abeta(22-35), Abeta(17-28), Abeta(25-35) and Abeta(32-35) but assists in the proteolytic degradation of these peptides with the concomitant formation of insoluble fibrils. PAD was purified in 12.5% yield and 137 fold with a specific activity of 59 micromol min(-1) mg(-1) from bovine brain by chromatography on diethylaminoethyl (DEAE)-Sephacel. Characterisation of the enzyme gave a pH and temperature optima of 7.5 degrees C and 68 degrees C, respectively, and the enzyme lost 50% activity within 38 min at this temperature. Michaelis-Menten kinetics established a V(max) and K(m) of 1.57 micromol min(-1) ml(-1) and 1.35 mM, respectively, with N-benzoyl arginine ethyl ester as substrate. Kinetic analysis was used to measure the affinity (K(i)) of the amyloid peptides to PAD with values between 1.4 and 4.6 microM. The formation of Abeta fibrils was rate limiting involving an initial lag time of about 24 h that was dependent on the concentration of the amyloid peptides. Turbidity measurements at 400 nm, Congo Red assay and Thioflavin-T staining fluorescence were used to establish the aggregation kinetics of PAD-induced fibril formation.

Advances in aerobic granule formation and granule stability in the course of storage and reactor operation.

Aerobic granulation is drawing increasing global interest in a quest for an efficient and innovative technology in wastewater treatment. Developed less than two decades ago, extensive research work on aerobic granulation has been reported. The instability of the granule, which is one of the main problems that hinder practical application of aerobic granulation technology, is still to be resolved. This paper presents a review of the literature in aerobic granulation focusing on factors that influence granule formation, granule development and their stability in the context of sludge granulation. The review attempts to shed light on the potential of developing granules with adequate structural stability for practical applications. The possibilities and perspective of using stored granule as inoculums for rapid startup, and as microbial supplement to enhance treatment of bioreactor systems are also discussed.

Stereological assessment of extracellular polymeric substances, exo-enzymes, and specific bacterial strains in bioaggregates using fluorescence experiments.

This review addresses the introduction of fluorescent molecular tags into exo-enzymes and extra polymeric substances of bioaggregates and the use of confocal laser scanning microscopy (CLSM) to map their role, purpose and quantitative description of the biological processes they undertake. Multiple color staining coupled with CLSM and fluorescent in situ hybridisation (FISH) and flow cytometry have identified the individual polymeric substances, whether they are proteins, lipids, polysaccharides, nucleic acids or antibodies, as well as the microorganisms in the bioaggregate. Procedures are presented for simultaneous multicolor staining with seven different fluorochromes - SYTOX Blue for nucleic acids; Nile red for lipids; Calcofluor white [CW] for beta-polysaccharides; concanavalin A [Con A] for alpha-poly-saccharides; fluorescein-isothiocyanate [FITC] for proteins; SYTO 63 for live microbial cells and Calcium Green for monitoring calcium levels in the microbial cells. For the distribution of certain microbial strains, metabolic enzymes and extrapolymeric substances to be quantitatively described the generated colored images are converted into digital forms under specific predefined criteria. Procedures and computer software programs (Amira; MATLAB) are presented in order to quantitatively establish grid patterns from the CLSM images. The image is digitized using a threshholding algorithm followed by a reconstruction of the image as a volumetric grid for finite element simulation. The original color image is first converted to a grey followed by resizing, detection and modification of bilevel images and finally a total reversal of the image colors. The grid file is then used by specific computer software (Gambit, Fluent) for further numerical studies incorporating chemical reactions, transport processes and computational fluid dynamics including intra-bioaggregate fluid flow, and heat and mass transfer within the bioaggregate matrix.

Arginine deiminases: therapeutic tools in the etiology and pathogenesis of Alzheimer's disease.

There is, at present, no definitive pre-mortem diagnostic tool for Alzheimer's disease, (AD) which relates to a poor understanding of its etiology. Brains of AD patients contain large amounts of the toxic plaque-forming beta-amyloid1-42 fragment in addition to elevated concentrations of the amino acid L-arginine. This work proposes that lowering levels of arginine in the astrocytes surrounding amyloid plaques may serve as a therapeutic tool in this neurodegenerative disorder. Arginine deiminase (ADI), from Pseudomonas aeruginosa, and peptidylarginine deiminase [PAD II], from bovine brain, are inhibited by amyloid peptides that contain arginine (amyloid1-42) and those that have no arginine (amyloid12-28/22-35). Enhanced activity of PAD II is noted with free L-arginine.

Acetamidoquinone and acetamidohydroxy derivatives as inhibitors for both dihydroxyacetamido epoxidase and dehydrogenase.

A series of monohydroxy and dihydroxyacetanilides, acetamidoquinones and bromoacetamidoquinones have been synthesised and tested as substrates and/or inhibitors of highly purified dihydroxyacetamido epoxidase (DHAE) and dihydroxy acetamido dehydrogenase (DHADH) from Streptomyces LL-C10337. None was found to act as substrates but many selectively inhibit the enzymes. Kinetic analysis has shown that all the compounds act as reversible competitive inhibitors with respect to the substrates 2,5-dihydroxyacetanilide and 2,3-epoxy-1,4-benzoquinone-5-acetanilide. Monohydroxy acetanilides showed weak inhibition to these enzymes compared to the dihydroxy derivatives while the more powerful inhibitors were the benzoquinoneacetanilide and its 5-bromo equivalent.