Tau protein assembles into isoform- and disulfide-dependent polymorphic fibrils with distinct structural properties

Tauopathies are neurodegenerative diseases in which insoluble fibrillar aggregates of a microtubule-binding protein, Tau, are abnormally accumulated. Pathological Tau fibrils often exhibit structural polymorphisms that differ among phenotypically distinct tauopathies; however, a molecular mechanism to generate polymorphic Tau fibrils remains obscure. Here, we note the formation of a disulfide bond in isoforms of full-length Tau and show that the thiol-disulfide status as well as the isoform composition determines structural and morphological properties of Tau fibrils in vitro. Mainly two regions in a Tau primary sequence are found to act as structural blocks for building a protease-resistant core of Tau fibrils. Interactions among those two blocks for building a core structure depend upon the thiol-disulfide status in each isoform of Tau, which results in the formation of polymorphic fibrils with distinct structural properties. Furthermore, we have found that more diverse structures of Tau fibrils emerge through a cross-seeded fibrillation between heterologous pairs of Tau isoforms. We thus propose that isoform- and disulfide-dependent combinatorial interactions among multiple regions in a Tau sequence endow Tau fibrils with various structures, i.e. polymorphism.     

Study of structural and rheological properties of starch isolated from genetically modified potato

It has been found in this work that the starchiness and starch content in tubers of GM potato and tubers of initial Lugovskoi potato variety are identical. No differences and difficulties were observed upon starch preparation from GM and common potato. Starch samples isolated from GM potato conform to the current standard for potato starch. The starch isolated from GM potato has a higher melting point, i.e., it is more thermally stable as compared with the starch isolated from a control sample. The bulk modulus of gels obtained from starch isolated from GM potato is higher in comparison with that of gels prepared from starch isolated from the control sample. Thermodynamic and rheological properties of starch from GM potato provide a possibility to predict its application in the manufacture of thermostable and strong polymeric materials.     

Relation between the rheological properties and the swallowing characteristics of vegetable juices fortified with carrot puree

The relation between the rheological properties and the swallowing characteristics of vegetable juices fortified with 0-30.0% carrot puree (CP) was evaluated. The apparent viscosity of vegetable juices increased with increasing CP concentrations, and a increases in yield stress were observed at and above 17.5% CP. In a sensory evaluation, texture perceived in the oral cavity varied as between vegetable juices with >17.5% CP and those with <12.5% CP. The maximum velocity in the pharyngeal region was classified into three same-quality subgroups: vegetable juices with 0-12.5% CP, with 10.0-25.0% CP, and with 17.5-30.0% CP. It significantly decreased with increasing CP concentrations.     

Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms

Changes in the viscoelastic material properties of bacterial biofilms resulting from chemical and antimicrobial treatments were measured by rheometry. Colony biofilms of Staphylococcus epidermidis or a mucoid Pseudomonas aeruginosa were subjected to a classical creep test performed using a parallel plate rheometer. Data were fit to the 4-parameter Burger model to quantify the material properties. Biofilms were exposed to the chloride salts of several common mono-, di-, and tri- valent cations, and to urea, industrial biocides, and antibiotics. Many of these treatments resulted in statistically significant alterations in the material properties of the biofilm. Multivalent cations stiffened the P. aeruginosa biofilm, while ciprofloxacin and glutaraldehyde weakened it. Urea, rifampin, and a quaternary ammonium biocide weakened the S. epidermidis biofilm. In general, there was no correspondence between the responses of the two different types of biofilms to a particular treatment. These results underscore the distinction between the killing power of an antimicrobial agent and its ability to alter biofilm mechanical properties and thereby influence biofilm removal. Understanding biofilm rheology and how it is affected by chemical treatment could lead to improvements in biofilm control.     

Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms

Changes in the viscoelastic material properties of bacterial biofilms resulting from chemical and antimicrobial treatments were measured by rheometry. Colony biofilms of Staphylococcus epidermidis or a mucoid Pseudomonas aeruginosa were subjected to a classical creep test performed using a parallel plate rheometer. Data were fit to the 4-parameter Burger model to quantify the material properties. Biofilms were exposed to the chloride salts of several common mono-, di-, and tri- valent cations, and to urea, industrial biocides, and antibiotics. Many of these treatments resulted in statistically significant alterations in the material properties of the biofilm. Multivalent cations stiffened the P. aeruginosa biofilm, while ciprofloxacin and glutaraldehyde weakened it. Urea, rifampin, and a quaternary ammonium biocide weakened the S. epidermidis biofilm. In general, there was no correspondence between the responses of the two different types of biofilms to a particular treatment. These results underscore the distinction between the killing power of an antimicrobial agent and its ability to alter biofilm mechanical properties and thereby influence biofilm removal. Understanding biofilm rheology and how it is affected by chemical treatment could lead to improvements in biofilm control.     

Mouse and human indoleamine 2,3-dioxygenase display some distinct biochemical and structural properties

The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the most significant pathway for mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its dual role in immunity and the pathogenesis of many diseases. Reported here are differences and similarities between biochemical behaviour and structural features of recombinant human IDO and recombinant mouse IDO. Significant differences were observed in the conversion of substrates and pH stability. Differences in inhibitor potency and thermal stability were also noted. Secondary structural features were broadly similar but variation between species was apparent, particularly in the α-helix portion of the enzymes. With mouse models substituting for human diseases, the differences between mouse and human IDO must be recognised before applying experimental findings from one system to the next.     

Relation between rheological properties and structural changes in monolayers of model lung surfactant under compression

rSP-C surfactant monolayers spread on a native physiological model substrate show two plateau regions in the pi/A-isotherm. The first corresponds to the main phase transition in the monolayer from a LE to a LC phase. Its course is non-horizontal because of the complex composition of the lung surfactant. The second plateau, which is much more pronounced, cannot be attributed to a change of the phase state. Brewster angle microscopy images taken in this region show a sharp apparent decrease of the aggregation degree from the LE to the LC state. This process can be considered as a change in the monolayer orientation relative to the direction of the propagated light. Such a change can be the result of monolayer folding and formation of a thicker layer, which is supported by results of rheological measurements. The dilatation elasticity obtained from oscillating barrier and longitudinal wave measurements reveals a pure elastic behaviour with a steep increase in the second plateau region. Because of the insolubility of the pure lipid components, a possible explanation is squeezing protein components of rSP-C or its complexes with lipids out of the monolayer into the bulk.     

Isomers of human alpha-synuclein stabilized by disulfide bonds exhibit distinct structural and aggregative properties

The discovery of three mutants in the -synuclein (alphaSyn) gene and the identification of alphaSyn as the major component of Lewy body have opened a new field for understanding the pathogenesis of Parkinson's disease (PD). AlphaSyn is a natively unfolded protein with unknown function and unspecified conformational heterogeneity. In this study, we introduce four Ser/Ala --> Cys mutations at positions 9, 42, 69, and 89 in human wild-type alphaSyn (wt-alphaSyn) and two PD-associated alphaSyn mutants, A30P-alphaSyn and A53T-alphaSyn. This allows expression of three alphaSyn mutants, wt-alphaSyn(4C), A30P-alphaSyn(4C), and A53T-Syn(4C). Subsequent oxidative folding enables each alphaSyn(4C) mutant to form three partially stabilized two-disulfide isomers, designated as alphaSyn(2SS), that are amenable to further isolation and characterization. These alphaSyn mutants exhibit the following properties. (a) A30P-alphaSyn(4C) exhibits a lower folding flexibility than wt-alphaSyn(4C) and A53T-alphaSyn(4C). (b) All three alphaSyn(4C) mutants, like wt-alphaSyn, exhibit a predominant structure of random coil. However, wt-alphaSyn(2SS) adopts an alpha-helical conformation, whereas A30P-alphaSyn(2SS) and A53T-alphaSyn(2SS) take on significant beta-sheet structure. (c) A30P-alphaSyn(2SS) shows a stronger tendency to aggregate than A53T-alphaSyn(2SS) and wt-alphaSyn(2SS). (d) Three isolated isomers of wt-alphaSyn(2SS) exhibit a propensity for forming oligomers different yet enhanced versus that for wt-alphaSyn. These data together substantiate the notion that under physiological conditions, human alphaSyn exists as diverse conformational isomers which exhibit distinct propensities for aggregation and fibril formation.     

Detailed structural characterization of succinoglycan, the major exopolysaccharide of Rhizobium meliloti Rm1021.

The detailed structure of the symbiotically important exopolysaccharide succinoglycan from Rhizobium meliloti Rm1021 was determined by mass spectrometry with electrospray ionization and collision-induced dissociation of the octameric oligosaccharide repeating unit. Previously undetermined locations of the succinyl and acetyl modifications were determined, in respect to both residue locations within the octamer and the carbon positions within the pyranose ring. Glycosidic linkages determined previously by methylation analysis were also verified.     

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as in-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ;C,D' complex and a presumed ;AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the ;C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 in-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [(125)I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ;C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B-A-D-C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.     

Bauhinia Kunitz-type proteinase inhibitors: structural characteristics and biological properties

Abstract Plant proteinase inhibitors are involved in the regulation of the activity of many proteinases and, in consequence, in biological processes driven by proteolysis. In this review, we summarize recent results on the activity of native Bauhinia inhibitors and synthetic derivatives. Structural and functional characteristics and the potential therapeutic use of these inhibitors are also discussed.     

Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca

An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm(-1) and form 2 with ν(FeC) and ν(CO) at 518 and 1920 cm(-1), respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.     

Chemical and rheological properties of an extracellular polysaccharide produced by the cyanobacterium Anabaena sp. ATCC 33047

The cyanobacterium (blue-green alga) Anabaena sp. ATCC 33047 produces an exopolysaccharide (EPS) during the stationary growth phase in batch culture. Chemical analysis of EPS revealed a heteropolysaccharidic nature, with xylose, glucose, galactose, and mannose the main neutral sugars found. The infrared (IR) spectrum of EPS showed absorption bands of carboxylate groups. The average molecular mass of the polymer was 1.35 MDa. Aqueous dispersions at EPS concentrations ranging from 0.2% to 0.6% (w/w) showed marked shear-thinning properties (power-law behavior). Linear dynamic viscoelastic properties showed that the elastic component was always higher than the viscous component. Viscous and viscoelastic properties demonstrated the absence of conformational changes within the concentration range studied. Stress-growth experiments revealed that 0.4% and 0.6% (w/w) EPS dispersions showed thixotropic properties. A detailed comparison of the linear dynamic viscoelasticity, transient flow, and decreasing shear rate flow curve properties was made for 0.4% (w/w) dispersions of xanthan gum (XG), Alkemir 110 (AG), and EPS. Viscoelastic spectra demonstrated that the EPS dispersion turned out to be more "fluidlike" than the AG and XG dispersions. The flow indexes indicated that the EPS dispersion was less shear-sensitive than that of XG, showing essentially the same viscosity, that is, >50 s(-1). The fact that viscosities of EPS and AG dispersions were not substantially different within the shear-rate range covered must be emphasized, in relation to EPS potential applications. The rheological behavior of EPS dispersions indicates the formation of an intermediate structure between a random-coil polysaccharide and a weak gel.     

Chemical characteristics and immuno-stimulating properties of biopolymers extracted from Acanthopanax sessiliflorus

During our search for macrophage stimulating compounds from medicinal plants, we isolated biopolymers from Acanthopanax sessiliflorus. Isolated fraction AS-5 showed maximum potential, and stimulated lysosonal enzymatic activity by 230% at 300 microg/ml. The nitric oxide (NO) producing ability of AS-5 100 microg/ml was 58 microM when treated with interferon-gamma and lipopolysaccharide 20 micro/ml.The lymphocyte proliferating effects of isolated biopolymer fractions were also investigated. Highest lymphoproliferative activity (a 2.8-fold enhancement compared to salines treated group was exhibited by AS-3 at 200 micro/ml followed by AS-5 and AS-6. The AS-3 fraction stimulated only T-lymphocytes and had little or no effect on B-lymphocyte proliferation.Partially methylated alditol acetates were prepared to elucidate the glycosyl linkage-compositions of the AS-3 and AS-5 biopolymers, and were analyzed by GC-MS. The AS-3 and AS-5 biopolymer fractions were found to contain 2,3,4-tri-O-methyl-D-glucitol, 2,3,4-tri-O-methyl-D-galacitol 3,4,6-tri-O-methyl-galacitol, 2-O-methyl-arabinitol and 2,4,6-tri-O-methyl-D-glucitol, 2,3,6-tri-O-methyl-D-galacitol linkages, respectively.     

Liposomal membranes XI. A suggestion to structural characteristics of acido-thermophilic bacterial membranes

To understand the role of ω-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1,2-di-11-cyclohexylundecanoyl-l-α-phosphatidylcholine (11_CYPC), 1,2-di-13-cyclohexyltridecanoyl-l-α-phosphatidylcholine (13_CYPC), and 1–13-cyclohexyltridecanoyl-2–11-cyclohexylundecanoyl-l-α-phosphatidylcholine (1–13_CY-2–11_CYPC) were prepared and the steady-state fluorescence anisotropy of 1,6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined. Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the ω-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures. The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations. Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.