The Role of Protein-L-isoaspartyl Methyltransferase (PIMT) in the Suppression of Toxicity of the Oligomeric Form of Aß42, in Addition to the Inhibition of Its Fibrillization.

The oligomeric form of amyloid-ß peptide (Aß42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD) and is responsible for cognitive deficits. The soluble oligomers are believed to be more toxic compared to the fibril form. Protein-L-isoaspartyl methyltransferase (PIMT) is a repair enzyme that converts aberrant isoAsp residues, formed spontaneously on isomerization of normal Asp and Asn residues, back to typical Asp. It was shown to inhibit the fibrillization of Aß42 (containing three Asp residues), and here, we investigate its effect on the size, conformation, and toxicity of Aß42 oligomers (AßO). Far-UV CD indicated a shift in the conformational feature of AßOs from the random coil to ß-sheet in the presence of PIMT. Binding of bis-ANS to different AßOs (obtained using different concentrations of Aß42 monomer) indicated the correlation of size of oligomers to hydrophobicity: the smallest AßO having the highest hydrophobicity is the most toxic. Dynamic light scattering showed an increase in size of AßO with the addition of PIMT, a contrasting role to that on Aß fibril. Assays using PC12-derived neurons showed the neuroprotective role of PIMT against AßO-induced toxicity. Furthermore, we have elaborated on the molecular mechanism of the antifibrillar action of PIMT and how this function is correlated with its enzymatic activity. PIMT has a more pronounced effect on AßO as compared to a small heat shock protein, pointing to its importance for the amelioration of the adverse effect of both Aß42 oligomers and fibrils.

The gold nanoparticle reduces Vibrio cholerae pathogenesis by inhibition of biofilm formation and disruption of the production and structure of cholera toxin.

Formation of biofilm by Vibrio cholerae plays a crucial role in pathogenesis and transmission of cholera. Lower infective dose of the biofilm form of V. cholerae compared to the planktonic counterpart, and its antibiotic resistance, make it challenging to combat cholera. Nanoparticles may serve as an effective alternative to conventional antibiotics for targeting biofilms and virulence factors. We explored the effectiveness of gold nanoparticles (AuNPs) of different size and shape (spherical: AuNS10 and AuNS100, and rod: AuNR10, the number indicating the diameter in nm) on both the inhibition of formation and eradication of biofilm of the two biotypes of V. cholerae, classical (VcO395) and El Tor (VcN16961). Inhibition of biofilm formation by spherical AuNPs was observed for both the biotypes. Considering eradication, the biofilms for both, particularly El Tor, was destroyed using both the AuNSs, AuNS100 showing higher efficacy. AuNR10 did not affect the biofilm of either biotype. Micrographs of small intestinal sections of VcO395-infected mice indicated the inhibition of biofilm formation by both AuNSs. We also studied the effect of these AuNPs on the structure of cholera toxin (CT), the major toxin produced by V. cholerae. Far-UV CD showed both AuNR10 and AuNS100 compromised the structure of CT, which was also validated from the reduction of fluid accumulation in mice ileal loop. Western blot analysis revealed the reduction of CT production upon treatment with AuNPs. AuNS100 seems to be the best suited to inhibit the formation or destruction of biofilm, as well as to disrupt CT production and function.

Virstatin-Conjugated Gold Nanoparticle with Enhanced Antimicrobial Activity against the Vibrio cholerae El Tor Biotype.

Because of the emergence of multidrug-resistant pathogenic bacteria, there is a growing interest for the development of an efficient alternative to antibiotics. Gold nanoparticles (AuNPs) are promising candidates due to their inherent non-toxicity and can be used as effective carriers of drugs. Cholera caused by Gram-negative Vibrio cholerae is still a potential threat in many developing countries. Virstatin, a small molecule, has been reported to inhibit virulence regulation in V. cholerae. Herein, we report an efficient synthesis of virstatin-conjugated gold nanoparticles (VL-AuNPs) and their antibacterial efficacy against the El Tor biotype of V. cholerae (VcN16961). The spherical-shaped NPs have an average diameter of ∼17 nm. The uniqueness of VL-AuNPs relies in the enhanced antibacterial efficacy compared to virstatin, as evidenced from the inhibitory concentration obtained from growth kinetics, and attributed to the inhibition of ATPase activity and DNA damage. More importantly, the expression of cholera toxin, the most important virulence factor of V. cholera, is reduced to a far greater extent than by any of the component molecules. The effect of VL-AuNPs on VcN16961 was monitored using various assays such as confocal microscopy, FACS, fluorescence spectroscopy, and so on. Overall, VL-AuNPs could be a potential candidate for the use as an effective agent for combating diarrheal diseases caused by V. cholera.

The role of isoaspartate in fibrillation and its prevention by Protein-L-isoaspartyl methyltransferase.

BACKGROUND: Isomerization of aspartate to isoaspartate (isoAsp) on aging causes protein damage and malfunction. Protein-L-isoaspartyl methyltransferase (PIMT) performs a neuroprotective role by repairing such residues. A hexapeptide, Val-Tyr-Pro-(isoAsp)-His-Ala (VA6), a substrate of PIMT, is shown to form fibrils, while the normal Asp-containing peptide does not. Considering the role of PIMT against epileptic seizure, the combined effect of PIMT and two antiepileptic drugs (AEDs) (valproic acid and stiripentol) was investigated for anti-fibrillation activity. METHODS: Structural/functional modulations due to the binding of AEDs to PIMT were investigated using biophysical techniques. Thioflavin T (ThT) fluorescence assay and microscopic methods were employed to study fibril formation by VA6. In vitro experiments with PC12 cells were carried out with PIMT/AEDs. RESULTS: ThT assay indicated reduction of fibrillation of VA6 by PIMT. AEDs stabilize PIMT, bind close to the cofactor binding site, possibly exerting allosteric effect, increase the enzymatic activity, and anti-fibrillation efficacy. Furthermore, Aß42, implicated in Alzheimer's disease, undergoes ß-sheet to α-helix transition in presence of PIMT. Studies with PC12 derived neurons showed that PIMT and PIMT/AEDs exerted neuroprotective effect against anti-NGF induced neurotoxicity. This was further validated against neurotoxicity induced by Aß42 in primary rat cortical neurons. CONCLUSIONS: The study provides a new perspective to the role isoAsp in protein fibrillation, PIMT in its prevention and AEDs in enhancing the activity of the enzyme. GENERAL SIGNIFICANCE: IsoAsp, with an additional C atom in the main-chain of polypeptide chain, may make it more susceptible to fibrillation. PIMT alone, or in association with AEDs prevents this.

The Sialic Acid-Dependent Nematocyst Discharge Process in Relation to Its Physical-Chemical Properties Is A Role Model for Nanomedical Diagnostic and Therapeutic Tools.

Formulas derived from theoretical physics provide important insights about the nematocyst discharge process of Cnidaria (Hydra, jellyfishes, box-jellyfishes and sea-anemones). Our model description of the fastest process in living nature raises and answers questions related to the material properties of the cell- and tubule-walls of nematocysts including their polysialic acid (polySia) dependent target function. Since a number of tumor-cells, especially brain-tumor cells such as neuroblastoma tissues carry the polysaccharide chain polySia in similar concentration as fish eggs or fish skin, it makes sense to use these findings for new diagnostic and therapeutic approaches in the field of nanomedicine. Therefore, the nematocyst discharge process can be considered as a bionic blue-print for future nanomedical devices in cancer diagnostics and therapies. This approach is promising because the physical background of this process can be described in a sufficient way with formulas presented here. Additionally, we discuss biophysical and biochemical experiments which will allow us to define proper boundary conditions in order to support our theoretical model approach. PolySia glycans occur in a similar density on malignant tumor cells than on the cell surfaces of Cnidarian predators and preys. The knowledge of the polySia-dependent initiation of the nematocyst discharge process in an intact nematocyte is an essential prerequisite regarding the further development of target-directed nanomedical devices for diagnostic and therapeutic purposes. The theoretical description as well as the computationally and experimentally derived results about the biophysical and biochemical parameters can contribute to a proper design of anti-tumor drug ejecting vessels which use a stylet-tubule system. Especially, the role of nematogalectins is of interest because these bridging proteins contribute as well as special collagen fibers to the elastic band properties. The basic concepts of the nematocyst discharge process inside the tubule cell walls of nematocysts were studied in jellyfishes and in Hydra which are ideal model organisms. Hydra has already been chosen by Alan Turing in order to figure out how the chemical basis of morphogenesis can be described in a fundamental way. This encouraged us to discuss the action of nematocysts in relation to morphological aspects and material requirements. Using these insights, it is now possible to discuss natural and artificial nematocyst-like vessels with optimized properties for a diagnostic and therapeutic use, e.g., in neurooncology. We show here that crucial physical parameters such as pressure thresholds and elasticity properties during the nematocyst discharge process can be described in a consistent and satisfactory way with an impact on the construction of new nanomedical devices.

The antibacterial and anticancer properties of zinc oxide coated iron oxide nanotextured composites.

Core-shell α-Fe2O3-ZnO structures of different nanotextured morphology were synthesized through wet chemical routes using different solvents like ethanol, ethanolamine, water and acetaldehyde. Morphological tuning using different solvents resulted in the formation of different shapes, such as disc, spindle, rod and sphere (abbreviated as FZ-ND, FZ-NSP, FZ-NR and FZ-NS, respectively). Structural, morphological and compositional characterization of these nanoparticles (NPs) has been carried out. Antibacterial efficacy of the synthesized NPs was checked against Gram negative V. cholerae N16961 (VcN16961) and Gram positive S. aureus bacteria by recording optical density (OD) at different time points. Among the NPs tested, FZ-NSP was found to be the most effective against VcN16961, while FZ-NR showed maximum efficacy against S. aureus, implying the importance of nanotextured surface as well as the morphology in the manifestation of antibacterial activity. The kinetics of growth for both the bacteria has been modelled using logistic approach. Cytotoxicity was evaluated through MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay against human breast adenocarcinoma cell line (MCF-7), human hepatocarcinoma cell line (HepG2) and against normal human embryonic kidney cell line (HEK-293). The lesser toxicity of α-Fe2O3-ZnO towards HEK-293 and the potent anticancer activity against MCF-7 and HepG2 cells underline its applicability as anticancer agent. With continued improvement of nanotechnology, this study may pave the way for designing and construction of various morphologically diverse, nanotextured materials with desired functional attributes.

Modelling of growth kinetics of Vibrio cholerae in presence of gold nanoparticles: effect of size and morphology.

Emergence of multiple drug resistant strains of pathogenic bacteria calls for new initiatives to combat infectious diseases. Gold nanoparticles (AuNPs), because of their non-toxic nature and size/shape dependent optical properties, offer interesting possibility. Here we report the antibacterial efficacy of AuNPs of different size and shape (AuNS10, AuNS100 and AuNR10; the number indicating the diameter in nm; S stands for sphere and R for rod) against the classical (O395) and El Tor (N16961) biotypes of Vibrio cholerae, the etiological agent responsible for cholera. Growth kinetics was monitored by measuring optical density at different time intervals and fitted by non-linear regression of modified Buchanan model. Sigmoidal growth curve for VcO395 indicated the existence of single phenotype population and was affected by AuNR10 only, implying the importance of morphology of AuNP. Growth of VcN16961 was affected by all three AuNPs indicating the vulnerability of El Tor biotype. Interestingly, VcN16961 exhibited the occurrence of two phenotypic subpopulations - one with shorter (vulnerable Type 1) and the other with extended (tolerant Type 2) lag phase. Various assays were conducted to probe the impact of AuNPs on bacterial cells. Apart from AuNR10, antimicrobial efficacy of AuNS10 was better compared to AuNS100.

Structure and function of Vibrio cholerae accessory cholera enterotoxin in presence of gold nanoparticles: Dependence on morphology.

BACKGROUND: Accessory cholera enterotoxin (Ace) is a classical enterotoxin produced by Vibrio cholerae, the causative agent for cholera. Considering the crucial role of Ace in pathogenesis of cholera, we explored the modulation of structure/function of Ace using gold nanoparticles (AuNPs) of different size and shape - spherical (AuNS10 and AuNS100, the number indicating the diameter in nm) and rod (AuNR10). METHODS: Biophysical techniques have been used to find out structural modulation of Ace by AuNPs. Effect of AuNP on Ace conformation was monitored by far-UV CD; urea-induced unfolding and binding of Ace to various AuNPs were studied by tryptophan fluorescence. In vivo experiments using mouse ileal loop and Ussing chamber were carried out to corroborate biophysical data. RESULTS: Biophysical data revealed degradation of Ace by AuNR10 and AuNS100, not by AuNS10. The feature of AuNR10 having high aspect ratio, but with the same transverse diameter as that of AuNS10 enabled us to explore the importance of morphology on modulation of protein structure/function. The equilibration time for adsorption shows dependence on the radius of curvature, being largest for AuNR10. In vivo experiments revealed the efficacy of AuNR10 and AuNS100 for reduced fluid accumulation, indicative of the loss of activity of Ace. CONCLUSIONS: We show how biophysical studies and in vivo experiments go hand-in-hand in establishing the efficacy and role of size/shape of AuNPs on a toxin structure. GENERAL SIGNIFICANCE: The effect of AuNP on toxin depends on its morphology. The targeted modulation of Ace could be of therapeutic benefit for gastrointestinal disorders.

Crystal structure and activity of protein L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of AdoHcy binding.

The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39-58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 10(6) M(-1), quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.

Antibacterial effect of silver nanoparticles and the modeling of bacterial growth kinetics using a modified Gompertz model.

BACKGROUND: An alternative to conventional antibiotics is needed to fight against emerging multiple drug resistant pathogenic bacteria. In this endeavor, the effect of silver nanoparticle (Ag-NP) has been studied quantitatively on two common pathogenic bacteria Escherichia coli and Staphylococcus aureus, and the growth curves were modeled. METHODS: The effect of Ag-NP on bacterial growth kinetics was studied by measuring the optical density, and was fitted by non-linear regression using the Logistic and modified Gompertz models. Scanning Electron Microscopy and fluorescence microscopy were used to study the morphological changes of the bacterial cells. Generation of reactive oxygen species for Ag-NP treated cells were measured by fluorescence emission spectra. RESULTS: The modified Gompertz model, incorporating cell death, fits the observed data better than the Logistic model. With increasing concentration of Ag-NP, the growth kinetics of both bacteria shows a decline in growth rate with simultaneous enhancement of death rate constants. The duration of the lag phase was found to increase with Ag-NP concentration. SEM showed morphological changes, while fluorescence microscopy using DAPI showed compaction of DNA for Ag-NP-treated bacterial cells. CONCLUSIONS: E. coli was found to be more susceptible to Ag-NP as compared to S. aureus. The modified Gompertz model, using a death term, was found to be useful in explaining the non-monotonic nature of the growth curve. GENERAL SIGNIFICANCE: The modified Gompertz model derived here is of general nature and can be used to study any microbial growth kinetics under the influence of antimicrobial agents.

Interaction of virstatin with human serum albumin: spectroscopic analysis and molecular modeling.

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ∼pH 7.2, B form ∼pH 9.0 and F form ∼pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K(a) for N and B isomers were found to be 6.09×10(5 )M(-1) and 4.47×10(5) M(-1), respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1:1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.

Ion mobility studies of electronically excited States of atomic transition metal cations: development of an ion mobility source for guided ion beam experiments.

The design of an ion mobility source developed to couple to a guided ion beam tandem mass spectrometer is presented. In these exploratory studies, metal ions are created continuously by electron ionization of the volatile hexacarbonyls of the three group 6 transition metals. These ions are focused into a linear hexapole ion trap, which collects the ions and then creates high intensity pulses of ions, avoiding excessive ion losses resulting from the low duty cycle of pulsed operation. The ion pulses are injected into a six-ring drift cell filled with helium where ions having different electronic configurations can separate because they have different ion mobilities. Such separation is observed for chromium ions and compares favorably with the pioneering work of Kemper and Bowers (J. Phys. Chem.1991, 95, 5134). The results are then extended to Mo(+) and W(+), which also show efficient configuration separation. The source conditions needed for high intensities and good configuration separation are discussed in detail and suggestions for further improvements are also provided.