Complex formation and stability of westiellamide derivatives with copper(II)

The Cu^II coordination chemistry of three synthetic analogues of westiellamide (H₃L^wa) with an [18]azacrown-6 macrocyclic structure and imidazole (H₃L¹), oxazole (H₃L²), or thiazole (H₃L³) heterocyclic donors in addition to the peptide groups, is reported. The N_heterocycle–N_peptide–N_heterocycle binding sites are highly preorganized for the coordination to Cu^II ions. The stability constants of mono- and dinuclear Cu^II complexes of H₃L¹, H₃L², and H₃L³, obtained by isothermal titration microcalorimetry, are reported. EPR and NMR spectroscopy as well as electrospray ionization mass spectrometry (ESI-MS) were used to characterize the complexes formed in solution. The stabilities of the mononuclear and dinuclear Cu^II complexes of the three ligands are in the range of 10⁵ M⁻¹, but there are subtle differences; specifically the oxazole-derived ligand has, in contrast to the other two macrocycles, a negative formation entropy for coordination to the first Cu^II ion and a higher stability for complexation to a second Cu^II center in comparison with the first Cu^II center (cooperativity). Differences between the three ligands are also apparent in terms of the formation mechanism. With the oxazole-based ligand H₃L², NMR spectroscopy, EPR spectroscopy, and ESI-MS indicate the formation of a ligand–Cu^II 2:1 intermediate, and this may explain the differences in the formation entropy as well as the cooperativity.     

A new approach to the isolation of RNA-DNA hybrids and its application to the quantitative determination of labeled tumor virus RNA

A procedure has been developed by which the hybrid formed between a labeled RNA and complementary DNA can be selectively separated from all other single and double-stranded nucleic acids. We describe the application of this procedure to the quantitative determination of labeled avian tumor virus RNA. Purified DNA complementary to avian myeloblastosis virus RNA is extended at its 3′ terminus with 40 to 60 dCMP residues, using terminal deoxynucleotidyl-transferase. The elongated DNA is annealed with the labeled nucleic acid preparation and the mixture is passed through a column of Sephadex to which poly(I) has been covalently bound. The poly(I) retains the specific RNA-DNA hybrids by virtue of their poly(C) extension. The column is washed with RNAase to degrade nonhybridized RNA, the RNA retained on the column is eluted with formamide and its radioactivity is determined. The background hybridization was reduced to 0.003 to 0.008% by addition of oligo(C)_5.20 to the hybridization mixture and by carrying out the adsorption to the poly(I)-Sephadex column in the presence of poly(U). The hybridization efficiency was about 50%. The content of radioactive Rous sarcoma virus-specific RNA was determined in infected and uninfected cells after labeling with [³H]uridine for two hours. The content of labeled virus-specific RNA in infected cells was 0.6 to 0.9% and 0.05% in uninfected cells. The value found for monkey cell RNA was 0.009%. This method can be used for the detection of hybrids between labeled RNA and complementary DNAs too short to allow quantitation by conventional methods. If the RNAase step is omitted the procedure can be used for the isolation of any RNA for which a complementary DNA is available, as well as for its precursor.     

Complex formation of rutin and quercetin with copper alters the mode of inhibition of ribonuclease A

Rutin and quercetin, both minor components of green tea and their Cu(II) complexes interact with Ribonuclease A (RNase A) in a novel way. The effects of rutin, quercetin and their copper complexes on the catalytic activity of the protein were investigated. Rutin shows an enhancement in the ribonucleolytic activity whereas the copper complexes and quercetin behave as non-competitive type inhibitors with K(i) values in the μM range. The secondary structural changes of RNase A in presence of the ligands were measured by circular dichroism and Fourier transform infrared spectroscopy. The binding parameters were obtained using a fluorescence quenching analysis.     

A model for one-dimensional morphoelasticity and its application to fibroblast-populated collagen lattices

The mechanical behaviour of solid biological tissues has long been described using models based on classical continuum mechanics. However, the classical continuum theories of elasticity and viscoelasticity cannot easily capture the continual remodelling and associated structural changes in biological tissues. Furthermore, models drawn from plasticity theory are difficult to apply and interpret in this context, where there is no equivalent of a yield stress or flow rule. In this work, we describe a novel one-dimensional mathematical model of tissue remodelling based on the multiplicative decomposition of the deformation gradient. We express the mechanical effects of remodelling as an evolution equation for the effective strain, a measure of the difference between the current state and a hypothetical mechanically relaxed state of the tissue. This morphoelastic model combines the simplicity and interpretability of classical viscoelastic models with the versatility of plasticity theory. A novel feature of our model is that while most models describe growth as a continuous quantity, here we begin with discrete cells and develop a continuum representation of lattice remodelling based on an appropriate limit of the behaviour of discrete cells. To demonstrate the utility of our approach, we use this framework to capture qualitative aspects of the continual remodelling observed in fibroblast-populated collagen lattices, in particular its contraction and its subsequent sudden re-expansion when remodelling is interrupted.     

Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation

BackgroundCopper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson’s Disease. Hence, residue specific interaction of Cu²⁺ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail.MethodsWe investigated the residue specific mapping of Cu²⁺ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu²⁺ were studied using fluorescence, CD, and AFM respectively.ResultsCopper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu²⁺ showed an enhanced rate of fibril formation with a pronounced effect for G51D.ConclusionCu²⁺ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu²⁺ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q.General SignificanceThese findings will help in the better understanding of Cu²⁺ catalyzed aggregation of synucleins.     

A quantitative collagen fibers orientation assessment using birefringence measurements: calibration and application to human osteons

Even though mechanical properties depend strongly on the arrangement of collagen fibers in mineralized tissues, it is not yet well resolved. Only a few semi-quantitative evaluations of the fiber arrangement in bone, like spectroscopic techniques or circularly polarized light microscopy methods are available. In this study the out-of-plane collagen arrangement angle was calibrated to the linear birefringence of a longitudinally fibered mineralized turkey leg tendon cut at variety of angles to the main axis. The calibration curve was applied to human cortical bone osteons to quantify the out-of-plane collagen fibers arrangement. The proposed calibration curve is normalized to sample thickness and wavelength of the probing light to enable a universally applicable quantitative assessment. This approach may improve our understanding of the fibrillar structure of bone and its implications on mechanical properties.     

Horseradish peroxidase. XLI. Complex formation with nitrate and its effect upon compound I formation

Ferric horseradish peroxidase reacts with nitrate and acetate in acidic solution to form weakly bound complexes. Competitive binding experiments with cyanide show that the nitrate binding site is not at the sixth coordination position of the heme iron. The nitrate inhibits compound I formation apparently by binding inside the heme pocket. One physical manifestation of this binding is to increase the apparent pK_a value of the conjugate acid of a catalytic distal group.     

Mechanism of post-translational quinone formation in copper amine oxidases and its relationship to the catalytic turnover

Copper amine oxidases (CAOs) post-translationally construct a redox-active quinone from an amino acid side chain in their polypeptide chain. As such, these enzymes illustrate how nature is able to expand upon naturally-occurring side chains to create new, catalytically powerful functionalities. The active sites of the CAOs are highly unusual in their ability to catalyze two very different reactions: single-turnover, oxygen-dependent quinone formation, followed by catalytic oxidation (formally dehydrogenation) of amines. This review summarizes our current understanding of the pathway whereby the 2,4,5-trihydroxyphenylalanyl quinone (TPQ) cofactor is generated from the phenolic side chain of tyrosine. This reaction occurs spontaneously intermediates in the presence of O(2) and active site bound Cu(II), without the assistance of other proteins or cofactors. Ongoing work has focused on uncovering the details of the TPQ formation mechanism. A larger goal is to understand how a single active site is capable of supporting both quinone formation and subsequent catalytic turnover.     

The role of hydrophobic bonding in collagen fibril formation: a quantitative model

A model has been developed for approximating the free energy of collagen fibril formation (ΔF_f) and the equilibrium solubility of collagen under physiological conditions. The model utilizes an expression of Flory for rodlike polymers, with the modification that the “pure” anisotropic phase is defined as a collagen fibril containing about 0.3 g water/g collagen. The model also assumes that χ₁, the polymer–solvent interaction term, is entirely due to hydrophobic effects. χ₁ is estimated from hydrophobic bond energies of amino acid side chains, using the results of Némethy and Scheraga. The temperature dependence of χ₁ is utilized to calculate equilibrium solubilities and ΔF_f as a function of temperature.     

Fluorimetric determination of sphingosine and its application to natural mixtures of glycosphingolipids

A sensitive estimation of sphingosine, by measurement of the fluorescence of a complex formed with 1-naphthylamino-4-sulfonic acid, is described. The practical range is 5-35 nmoles sphingosine. The method is used to estimate, in terms of sphingosine, amounts of ceramide and glycosphingolipids. The isolation of microamounts (5-30 micro g) of individual glycosphingolipids from a mixture, and their quantitative estimation is described. The percentage composition of a glycosphingolipid mixture from the kidneys of adult C57/BL male mice is given.     

Chiral doping of nematic phases and its application to the determination of absolute configuration

Doping nematic liquid crystals with nonracemic chiral compounds induces a twisted nematic (cholesteric) phase. The ability of solutes to twist the nematic phase may be related to the overall shape of the chiral dopant and consequently to its absolute configuration. The cholesteric induction is therefore a powerful tool complementary to chiroptical techniques to obtain stereochemical information on chiral molecules.     

Comparative study of the quantitative determination of kanamycin sulfate in ophthalmic films with a collagen base by microbiological and chemical methods

The results of the comparative study on microbiological and chemical quantitative determination of kanamycin sulfate in the ophthalmic films with the collagen base are presented. The intraocular films prepared with the use of 1 per cent collagen solution contain dexamethasone and kanamycin. The agar diffusion method with Bacillus pumilus NCTC 8241 as the test microbe and the photocolorimetric method based on estimation of the optical density of the colored compound formed after acid hydrolysis of kanamycin with orcinol and ferric chloride were used for the quantitative determination of kanamycin. The results of the quantitative determinations of kanamycin in the films with the two methods did not differ significantly. However, the error of the microbiological method was +/- 3,75 per cent, whereas that of the chemical method was +/- 1.23 per cent or approximately 3 times lower. The time of the analysis decreased from 24 to 1.5-2 h. Moreover, the chemical method is simple and readily reproducible.