Formation of β-sheets in glutamine and alanine tripeptides

The misfolding and aggregation of proteins is associated with many different diseases including the trinucleotide repeat disorders and Prion diseases. We have studied three residue peptides comprising alanine and glutamine in order to understand the short range interactions affecting the formation of β-rich aggregates. Using infrared spectroscopy, we have found that trialanine and triglutamine form significant amounts of β-sheet, but that tripeptides containing alanine and glutamine are only able to form β-sheet if the glutamine side-chains extend outward on both faces of the sheet. From our data, we conclude that different stabilizing interactions are responsible for β-sheet formation in trialanine and triglutamine.     

Peculiarities of Formation of the Wakefulness—Sleep Cycle in Ontogenesis of Rats

Electrophysiological changes in the wakefulness—sleep cycle were studied in early postnatal ontogenesis of rat pups. EEG was recorded and its spectral power was determined, as well as spatial-temporal synchronization between the brain cortex zones (visual and sensomotor) and hippocampus in the process of sleep at various periods of ontogenesis. These data were compared with the literature data on studying of cytoarchitectonics and ultrastructure of rat neocortex as well as on formation of neuronal activity and maturation of transmitter systems at the same periods. Based on time of formation of interneuronal and interstructural connections and of maturation of transmitter systems, 3 stages of functional development of sleep in ontogenesis were identified: the first stage—undifferentiated sleep, the interneuronal connections are absent; the second stage—partially differentiated sleep, interneuronal connections function and control from subcortical structures appears; the third stage—differentiated sleep, clear division into sleep phase, additional control from all three transmitter systems: noradrenergic, cholinergic, and serotoninergic, action of the latter providing inhibitory mechanisms in CNS.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 2, 2005, pp. 154–159.Original Russian Text Copyright © 2005 by Titkov, Aristakesyan, Oganesyan.     

Formation of β-Fructosyl Compounds of Pyridoxine in Growing Culture of Aspergillus niger

Two pyridoxine compounds were found to be formed in a culture filtrate of Aspergillus niger and A. sydowi, when grown in a medium containing sucrose and pyridoxine. Each of the two compounds I and II was obtained as a white powdered preparation by preparative paper chromatography, gel filtration on Toyopearl HW-40S and Sephadex G-10 columns, DEAE-cellulose column chromatography, and lyophilization. Compounds I and II were identified as 5?-O-(β-D-fructofuranosyl)-pyridoxine and 5?-O-(β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl]-pyridoxine, on the basis of the various experimental results, viz., elementary analyses, UV, ¹H-, and ¹³C-NMR spectra, products by hydrolysis with acid and yeast β-D-fructofuranosidase, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid. Levansucrase from Microbacterium laevaniformans and yeast β-D-fructofuranosidase did not catalyze the β-D-fructofuranosyl transfer from sucrose to pyridoxine to give rise to β-D-fructofuranosyl-pyridoxine.     

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.     

Formation of Toxic Oligomeric α-Synuclein Species in Living Cells

Background

Misfolding, oligomerization, and fibrillization of α-synuclein are thought to be central events in the onset and progression of Parkinson''s disease (PD) and related disorders. Although fibrillar α-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells.

Methodology/Principal Findings

Here we used bimolecular fluorescence complementation (BiFC) to directly visualize α-synuclein oligomerization in living cells, allowing us to study the initial events leading to α-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting α-synuclein aggregation affect the process over time. Stabilization of α-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of α-synuclein oligomers. Introduction of PD-associated mutations in α-synuclein did not affect oligomer formation but the biochemical properties of the mutant α-synuclein oligomers differ from those of wild type α-synuclein.

Conclusions/Significance

This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of α-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies.     

Thermodynamics of β-Sheet Formation in Polyglutamine

The role of β-sheets in the early stages of protein aggregation, specifically amyloid formation, remains unclear. Interpretations of kinetic data have led to a specific model for the role of β-sheets in polyglutamine aggregation. According to this model, monomeric polyglutamine, which is intrinsically disordered, goes through a rare conversion into an ordered, metastable, β-sheeted state that nucleates aggregation. It has also been proposed that the probability of forming the critical nucleus, a specific β-sheet conformation for the monomer, increases with increasing chain length. Here, we test this model using molecular simulations. We quantified free energy profiles in terms of β-content for monomeric polyglutamine as a function of chain length. In accord with estimates from experimental data, the free energy penalties for forming β-rich states are in the 10-20 kcal/mol range. However, the length dependence of these free energy penalties does not mirror interpretations of kinetic data. In addition, although homodimerization of disordered molecules is spontaneous, the imposition of conformational restraints on polyglutamine molecules does not enhance the spontaneity of intermolecular associations. Our data lead to the proposal that β-sheet formation is an attribute of peptide-rich phases such as high molecular weight aggregates rather than monomers or oligomers.     

Formation of Phage-Induced γ-Polyglutamic Acid Depolymerase in Lysogenic Strain of Bacillus natto

The influence of tea catechins on the absorption of starch or sucrose was investigated in vivo. Tea catechins were administered orally to rats before soluble starch or sucrose administration. Saccharide-dosed rats were killed and the blood and the contents of the intestine were collected at intervals over two hours. Catechins of certain concentrations suppressed the increase of plasma glucose levels, thus concurrently suppressing insulin activity. Increased activity of intestinal α-amylase by starch dosing was inhibited markedly in the catechin-administered rats. Sucrase on the brush border membrane was also inhibited by prior catechin administration. From these results it was assumed that orally administered catechins will inhibit intestinal α-amylase or sucrase, thereby deterring the digestion of certain amounts of starch or sucrose and eventually reducing the plasma glucose levels.     

Formation of Hydroxyl Radicals in Biological Systems. Does Myoglobin Stimulate Hydroxyl Radical Formation from Hydrogen Peroxide?

Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H₂O₂ causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H₂O₂ to form OH detectable by deoxyribose, but that H₂O₂ eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H₂O₂ is discussed in relation to myocardial reoxygenation injury.     

Chain Formation in Œcophylla longinoda

The aggregation phenomenon is very common in numerous activities of social insects, however, it is often their functional aspects that are studied, leaving their mechanisms not so well understood. With the example of chain formation in cophylla longinoda, we present the mechanisms responsible for these collective structures. Our experimental results show that a change in the probability that a worker will decide to join or leave a chain is (1) strongly dependent on the number of ants present in the chain and (2) slightly dependent on the presence of a visual stimulus. The determining role of these probabilities is validated with the use of a mathematical model that reproduces the formation and breakup of the chain. Moreover, it predicts other properties of aggregation such as the influence of nest population size.     

Formation and Development of Different Types of Embryo Sacs in Polyembryonic Rice Strain APⅣ

The process of different types of embryo sac formation was studied in AP IV by using the technic of seni-thin sectioning. Different types of embryo sacs were formed by different ways of development. The embryo sac of 5-2-1 type, 6-2-0 type, and 5-3-0 type were formed through three respectively new Polygonum-variant ways of development. The factors causing the three different ways of development were positional change of functional megaspore nucleus, change of orientation of the dividing-spindle of the embryo sac nucleus, nonsynchronous division of embryo sac nucleus and orientation of nucleus after division. Double set of embryo sac resulted possibly from mutual change in position of the primordial low polar nucleus and the primordial egg cell, i. e. the polar nucleus did not move toward the center of the embryo sac, but remained in the micropyle part, whereas the egg cell moved to the center and replaced the low polar nucleus.     

Roles of β-Tubulin Residues Ala428 and Thr429 in Microtubule Formation in Vivo

The C termini of β-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human βI and βIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from βIII and 16 from βI can assemble normally. Interestingly, removal of the next residue (Ala⁴²⁸) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human βI and βIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1–429 of human β-tubulins make no contribution to microtubule assembly. Ala⁴²⁸, in the C-terminal sequence motif N-QQYQDA⁴²⁸, lies at the end of helix H12 of β-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala⁴²⁸ may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr⁴²⁹ plays a more complex role. In the βI isotype of tubulin, Thr⁴²⁹ is not at all necessary for assembly; however, in the βIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of βIII as well as with the observation that evolutionary conservation is total for Ala⁴²⁸ and frequent for Thr⁴²⁹.Microtubules are involved in a great variety of cellular functions. Their constituent protein tubulin is an αβ heterodimer, both α- and β-tubulin existing as multiple isotypes, encoded by different genes and differing in amino acid sequence (1). The differences among the isotypes are highly conserved in evolution. In mammals, the β isotypes are βIa, βIb, βII, βIII, βIVa, βIVb, βV, and βVI. There is evidence that the isotype differences have functional significance. For instance, the βIV isotype is found in all axonemes (2).Structurally, both α- and β-tubulin consist of a globular region of 427 amino acids followed by a C-terminal region of 17–24 amino acids (3–5). The C-terminal region is highly negatively charged, being especially rich in glutamate residues and lacking in basic residues, and is likely to project outward from the rest of the molecule, because of its high negative charge and the electrostatic repulsion among the glutamate residues (3). The three-dimensional structure of the globular domain has been determined by electron and x-ray crystallography (4, 5). However, the C-terminal region has never been localized in the three-dimensional reconstructions except by computer modeling. The probable reasons for this are 1) that, if the C-terminal region projects out from the rest of molecule, it is likely to be very flexible with respect to the rest of the molecule and 2) the C-terminal region undergoes post-translational modification. Both of these can lead to structural heterogeneity and cause the C terminus to be invisible to crystallographic techniques.In this work, we examine the role of the C termini of human β-tubulins to determine the minimal sequence requirement for microtubule incorporation through structure/function analyses. The human βI and βIII tubulin isotypes were utilized based on their high degree of sequence variability clustered at the C terminus (Fig. 1) and the fact that βI is broadly distributed among normal tissues, whereas βIII has a very narrow tissue distribution. These two isotypes share 92% sequence identity, with differences among these isotypes occurring in both the globular domain and the C-terminal region (1).Open in a separate windowFIGURE 1.Sequence alignment of human βIa and βIII tubulin isotypes. Human βIa and βIII tubulin isotypes were aligned with ClustalX 1.83 and processed with BioEdit. Hyphens denote identical residues between sequences.

TABLE 1

The C-terminal amino acid sequences of the human β-tubulin isotypes

Formation of Microspherules of Lunar Regolith in Plasma–Dust Processes Initiated by Meteoroid Impacts

Plasma Physics Reports - The possibility of the formation of microspherules in plasma-dust processes initiated by meteoroids impacting the lunar surface is discussed. It is demonstrated that...     

Involvement of Nuclear Receptor REV-ERBβ in Formation of Neurites and Proliferation of Cultured Adult Neural Stem Cells

Neural stem cells (NSCs) serve as the source of both neurons and support cells, and neurogenesis is reportedly linked to the circadian clock. This study aimed to clarify the functional role of the circadian rhythm-related nuclear receptor, REV-ERBβ, in neurogenesis of NSCs from adult brain. Accordingly, Rev-erbβ expression and the effect of Rev-erbβ gene-specific knockdown on neurogenesis in vitro was examined in adult rodent NSCs. Initial experiments confirmed REV-ERBβ expression in cultured adult NSCs, while subsequent gene expression and gene ontogeny analyses identified functional genes upregulated or downregulated by REV-ERBβ. In particular, expression levels of factors associated with proliferation, stemness, and neural differentiation were affected. Knockdown of Rev-erbβ showed involvement of REV-ERBβ in regulation of cellular proliferation and self-renewal of cultured adult NSCs. Moreover, Rev-erbβ-knockdown cells formed neurons with a slightly shrunken morphology, fewer new primary neurites, and reduced length and branch formation of neurites. Altogether, this suggests that REV-ERBβ is involved in neurite formation during neuronal differentiation of cultured adult NSCs. In summary, REV-ERBβ is a known circadian regulatory protein that appears to be involved in neurogenesis via regulation of networks for cell proliferation and neural differentiation/maturation in adult NSCs.     

Formation and participation of nano-amyloids in pathogenesis of Alzheimer’s disease and other amyloidogenic diseases

Studies of neurodegenerative disorders attract much attention of the world scientific community due to increasing dissemination of Alzheimer’s disease. The reason for such pathologies consists in transition of a “healthy” molecule or peptide from its native conformation into a very stable “pathological” form. During this process, molecules existing in the “pathological” conformation aggregate and form amyloid fibrils that can undergo an uncontrolled increase. Novel knowledge is required on sporadic forms of Alzheimer’s disease, on the nature of triggering mechanisms of the conformational transitions of beta-amyloid fragments from normally functioning proteins into new structure, nano-beta-amyloids, that escape of neuronal and whole-body control resulted in the loss of neurons. This review summarized results of studies on the formation of amyloid fibrils and their role in pathogenesis of amyloid diseases.     

Formation of γ-BTC in Flooded Rice Field Soils Treated with γ-BHC

The amino acids of the B-chains of two abrins (designated as abrin-a and abrin-b) from the seeds of Abrus precatorius have been sequenced. The sequence of the B-chain of abrin-a was solved by analysis of peptides derived by enzymatic digestions with trypsin, Iysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The sequence of the B-chain of abrin-b was analyzed by sequence analysis of tryptic peptides and comparing these sequences with those of corresponding peptides of the B-chain of abrin-a. The B-chains of abrin-a and abrin-b consist of 268 amino acid residues and share 256 identical residues. Comparison of their sequences with that of the ricin B-chain shows that 60% of the residues of both abrin B-chains are identical to those of the ricin B-chain and that two saccharide-binding sites in ricin B-chain identified by a crystallographic study are highly conserved in both abrin B-chains.     

UV Circular Polarisation in Star Formation Regions: The Origin of Homochirality?

Ultraviolet circularly polarised light has been suggested as the initial cause of the homochirality of organic molecules in terrestrial organisms, via enantiomeric selection of prebiotic molecules by asymmetric photolysis. We present a theoretical investigation of mechanisms by which ultraviolet circular polarisation may be produced in star formation regions. In the scenarios considered here, light scattering produces only a small percentage of net circular polarisation at any point in space, due to the forward throwing nature of the phase function in the ultraviolet. By contrast, dichroic extinction can produce a fairly high percentage of net circular polarisation (10%) and may therefore play a key role in producing an enantiomeric excess.     

Azithromycin Reduces the Production of α-hemolysin and Biofilm Formation in Staphylococcus aureus

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases which are controlled by quorum sensing. In this study, we show that azithromycin was active against methicillin-resistant Staphylococcus aureus (MRSA) strains with MICs ranged from 32 to 64 μg/mL. Azithromycin at subinhibitory concentration, markedly reduced the production of α-hemolysin at (1/16MIC, 1/8MIC) and biofilm formation at (1/16MIC, 1/8MIC), respectively. The results indicated that sub-inhibitory concentrations of azithromycin decreased the production of α-hemolysin and biofilm formation in MRSA in a dose-dependent manner. Therefore, azithromycin may be useful in the treatment of α-hemolysin producing and biofilm formation MRSA infections.