Chemotactic assay for biological effects of silver nanoparticles

A method is proposed for assessing the biocidal efficacy of water-dispersed nanoparticles of silver. It is based on negative chemotaxis of the plasmodia of the slime mold Physarum polycephalum. Biocidal and repellent effects were compared for silver nanoparticles, Ag⁺ ions, and AOT in solution and in the agar gel. In such characteristics as increasing the period of auto-oscillations of contractile activity, decreasing the area of spreading on substrate, and substrate preference in spatial tests, silver nanoparticles proved to be substantially more effective than Ag⁺ and AOT. The lethal concentrations of the nanoparticles were close to those found earlier for bacteria and viruses. The chemotactic tests allow quantitative assessment of the biological reaction and monitoring its dynamics; in resolution, they are superior to the tests based on the lethal action of biocidal agents.     

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the structural transformation of native protein to the highly ordered cross‐β structure, the key feature of amyloid fibrils, is under active investigation. Conventional biophysical methods have limited application in addressing the problem because of the heterogeneous nature of the system. In this study, we demonstrated that deep‐UV resonance Raman (DUVRR) spectroscopy in combination with circular dichroism (CD) and intrinsic tryptophan fluorescence allowed for quantitative characterization of protein structural evolution at all stages of hen egg white lysozyme fibrillation in vitro. DUVRR spectroscopy was found to be complimentary to the far‐UV CD because it is (i) more sensitive to β ‐sheet than to α ‐helix, and (ii) capable of characterizing quantitatively inhomogeneous and highly light‐scattering samples. In addition, phenylalanine, a natural DUVRR spectroscopic biomarker of protein structural rearrangements, exhibited substantial changes in the Raman cross section of the 1000‐cm^–1 band at various stages of fibrillation. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

146 Amyloid fibril polymorphism probed by advanced vibrational spectroscopy

Amyloid fibrils are associated with many neurodegenerative diseases. All known amyloids including pathogenic and nonpathogenic forms display functional and structural heterogeneity (polymorphism) which determines the level of their toxicity. Despite a significant biological and biomedical importance, the nature of the amyloid fibril polymorphism remains elusive. We utilized for the first time three most advanced vibrational techniques to probe the core, the surface, and supramolecular chirality of fibril polymorphs. A new type of folding, aggregation phenomenon, spontaneous refolding from one polymorph to another, was discovered (Kurouski, Lauro et al., 2010). Hydrogen–deuterium exchange deep UV resonance Raman spectroscopy (Oladepo, Xiong et al., 2012) combined with advanced statistical analysis (Shashilov & Lednev, 2010) allowed for structural characterization of the highly ordered cross-β core of amyloid fibrils. We reported several examples showing significant variations in the core structure for fibril polymorphs. Amyloid fibrils are generally composed of several protofibrils and may adopt variable morphologies, such as twisted ribbons or flat-like sheets. We discovered the existence of another level of amyloid polymorphism, namely, that associated with fibril supramolecular chirality. Two chiral polymorphs of insulin, which can be controllably grown by means of small pH variations, exhibit opposite signs of vibrational circular dichroism (VCD) spectra (Kurouski, Dukor et al. 2012). VCD supramolecular chirality is correlated not only by the apparent fibril handedness but also by the sense of supramolecular chirality from a deeper level of chiral organization at the protofilament level of fibril structure. A small pH change initiates spontaneous transformation of insulin fibrils from one polymorph to another. As a result, fibril supramolecular chirality overturns both accompanying morphological and structural changes (Kurouski, Dukor et al. 2012). No conventional methods could probe the fibril surface despite its significant role in the biological activity. We utilized tip-enhanced Raman spectroscopy (TERS) to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range (Kurouski, Deckert-Gaudig et al. 2012). It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations and amino acid composition.     

Effects of extremely weak alternating magnetic fields on the plant gravitropism

It was shown that the rate of gravitropic response in apical segments excised from the 4-day-old seedlings of flax (Linum bienne) may be substantially influenced by combined magnetic fields (CMF) of two different types: 1) CMF tuned to the parametric resonance for Ca2+; 2) CMF containing extremely weak alternating component with the values of magnetic density ranging from 10(-6) to 10(-10) T. Our experimental data indicate that CMF affect the gravitropic response via at least two different mechanisms. The first one corresponds to the ion parametric resonance well established earlier in studies with test-systems prepared from animals. The origin of the bioeffects induced by CMF containing extremely weak alternating component remains to be established.     

Comparison between UV Raman and circular dichroism detection of short alpha helices in bombolitin III

We have used UV resonance Raman (UVRR) and circular dichroism (CD) spectroscopies to examine the dilute solution-phase secondary structure of the 17 amino acid peptide Bombolitin III (BIII). Both UVRR and CD clearly observe the alpha-helix structure induced by the addition of trifluoroethanol (TFE) to BIII. In contrast, only UVRR is able to detect the single alpha-helical turn induced by increasing the pH of BIII from pH 1.8 to 6.4. This alpha-helical turn is formed because of a stabilizing salt bridge formed between Lys(2) and Asp(5). Further increases in the alpha-helix content occur as the pH is raised further. We compare the relative sensitivity of UVRR and CD to short alpha helices and find, as expected, that the CD cannot detect short alpha helices. This study demonstrates that UV Raman measurements can detect the formation of single alpha-helical turns which cannot be detected by CD measurements.     

Dependence of gravitotropic reaction in segments of flax stems on frequency and amplitude of variable components of a weak combined magnetic field

We determined the dependence of the effect of weak combined magnetic field (CMF) tuned to Ca2+ resonance (Ca(2+)-CMF) on the rate of gravitropic response in apical segments (25 mm long) excised from the 4-day-old seedlings of flax (Linum bienne) on the amplitude and frequency of the alternating component of Ca(2+)-CMF. The results indicate that test-systems derived from plant and animal sources are affected by Ca(2+)-CMF via an identical mechanism.     

Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide

A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)₃GY(GA)₃GE(GA)₃GH(GA)₃GK, forms large β-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of β-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel β-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125°C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state, as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of ~1 and ~60 min, respectively, indicating no detectable formation of hairpin-type nuclei in the millisecond timescale that could be attributed to nonlocal “nonnative” interactions. The polypeptide folding dynamics agree with a general property of β-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less frustrated free-energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.     

Conjugates of Tacrine and Its Cyclic Homologues with <Emphasis Type="Italic">p</Emphasis>-Toluenesulfonamide as Novel Acetylcholinesterase and Butyrylcholinesterase Inhibitors

Using the acylation reaction with tosyl chloride of N-aminopropyl analogues of tacrine and its cyclic homologues with different size of the aliphatic cycle (5–8), we synthesized a number of new derivatives of p-toluenesulfonamide. It is shown that the synthesized hybrid compounds of tacrine and p-toluenesulfonamide are effective inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with the preferential inhibition of BChE. They also displace propidium from the peripheral anionic site of the electric eel AChE (Electrophorus electricus). The characteristics of the efficiency and selectivity of cholinesterase inhibition by the test compounds were confirmed by the results of molecular docking.