Acid Position Research Articles

Effect of zeolite structure on light aromatics formation during upgrading of cellulose fast pyrolysis vapor

Promising technology for the conversion of cellulose to aromatics by catalytic fast pyrolysis (CFP) was investigated using five zeolite catalysts, i.e., 5A, SAPO-34, HY, BETA and HZSM-5. The relationship between the porosity and acidity of different zeolites with product selectivity was studied. The results showed that both the acidity and pore size of the zeolite significantly affected the production of aromatics and coke, especially the bio-oil composition. The bio-oils obtained over 5A or SAPO-34 (small pore<5.5 nm) have relatively high oxygen content. The BTEXN (benzene, toluene, ethylbenzene, xylenes and naphthalene) carbon yields over weak acidic zeolites of HY and BETA are only 6.5% and 9.0%, respectively. Due to the appropriate pore size distribution and acid position, HZSM-5 gave the highest BTEXN carbon yield of 21.1%. Moreover, the coke deposited on the spent zeolites was analyzed by temperature programmed oxidation. Furthermore, three possible mechanisms that the acid sites catalyze vapor towards non-condensable gases, aromatics and coke were also studied. HZSM-5 achieved satisfactory deoxygenation and aromatic production simultaneously, made it a potential catalyst for producing light aromatics from reforming the biomass pyrolytic vapors.

Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is highly amidated. Amidation of α-D-isoglutamic acid in position 2 of the stem peptide plays a decisive role in the polymerization of cell wall building blocks. S. aureus mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin, indicating that targeting the amidation reaction could be a useful strategy to combat this pathogen. The enzyme complex that catalyzes the formation of α-D-isoglutamine in the Lipid II stem peptide was identified recently and shown to consist of two subunits, the glutamine amidotransferase-like protein GatD and the Mur ligase homolog MurT. We have solved the crystal structure of the GatD/MurT complex at high resolution, revealing an open, boomerang-shaped conformation in which GatD is docked onto one end of MurT. Putative active site residues cluster at the interface between GatD and MurT and are contributed by both proteins, thus explaining the requirement for the assembled complex to carry out the reaction. Site-directed mutagenesis experiments confirm the validity of the observed interactions. Small-angle X-ray scattering data show that the complex has a similar conformation in solution, although some movement at domain interfaces can occur, allowing the two proteins to approach each other during catalysis. Several other Gram-positive pathogens, including Streptococcus pneumoniae, Clostridium perfringens and Mycobacterium tuberculosis have homologous enzyme complexes. Combined with established biochemical assays, the structure of the GatD/MurT complex provides a solid basis for inhibitor screening in S. aureus and other pathogens.

Structural Optimization and Characterization of Potent Analgesic Macrocyclic Analogues of Neurotensin (8–13)

The neurotensin receptors are attractive targets for the development of new analgesic compounds. They represent potential alternatives or adjuvants to opioids. Herein, we report the structural optimization of our recently reported macrocyclic peptide analogues of NT(8-13). The macrocycle was formed via ring-closing metathesis (RCM) between an ortho allylated tyrosine residue in position 11 and the side chain of alkene-functionalized amino acid in position 8 of NT(8-13). Minute modifications led to significant binding affinity improvement ( Ki improved from 5600 to 15 nM) with greatly improved plasma stability compared to NT(8-13). This study also delineates the structural features influencing these parameters. The signaling profiles of the new macrocycles were determined on the NTS1 receptor, and the physiological effects of the two most potent and stable analogues were assessed in vivo using rodent models. Both compounds displayed strong analgesic effects.

Effect of ferric citrate on amyloid-beta peptides behavior.

Amyloid beta (Aβ) aggregation and oxidative stress are two of the central events in Alzheimer's Disease (AD). Both these phenomena can be caused by the interaction of Aβ with metal ions. In the last years the interaction between ZnII , CuII , and Aβ was much studied, but between iron and Aβ it is still little known. In this work we determine how three Aβ peptides, present in AD, interact with FeIII -citrate. The three Aβ peptides are: full length Aβ1-42, an isoform truncated at Glutamic acid in position three, Aβ3-42, and its pyroglutamated form AβpE3-42. Conformation and morphology of the three peptides, aggregated with and without FeIII -citrate were studied. Besides, we have determined the strength of the interactions Aβ/FeIII -citrate studying the effect of ethylenediaminetetraacetic acid as chelator. Results reported here demonstrate that FeIII -citrate promotes the aggregation in all the three peptides. Moreover, Aspartic acid 1, Glutamic acid 3, and Tyrosine 10 have an important role in the coordination with iron, generating a more stable complex for Aβ1-42 compared to that for the truncated peptides.

Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.

Collision-induced dissociation of doubly-charged barium-cationized lipids generated from liquid samples by atmospheric pressure matrix-assisted laser desorption/ionization provides structurally diagnostic product ions

Obtaining structural information for lipids such as phosphatidylcholines, in particular the location of double bonds in their fatty acid constituents, is an ongoing challenge for mass spectrometry (MS) analysis. Here, we present a novel method utilizing the doping of liquid matrix-assisted laser desorption/ionization (MALDI) samples with divalent metal chloride salts, producing ions with the formula [L+M]2+ (L = lipid, M = divalent metal cation). Multiply charged lipid ions were not detected with the investigated trivalent metal cations. Collision-induced dissociation (CID) product ions from doubly charged metal-cationized lipids include the singly charged intact fatty acids [snx+M–H]+, where ‘x’ represents the position of the fatty acid on the glycerol backbone. The preference of the divalent metal cation to locate on the sn2 fatty acid during CID was found, enabling stereochemical assignment. Pseudo-MS3 experiments such as in-source decay (ISD)-CID and ion mobility-enabled time-aligned parallel (TAP) MS of [snx+M–H]+ provided diagnostic product ion spectra for determining the location of double bonds on the acyl chain and were applied to identify and characterize lipids extracted from soya milk. This novel method is applicable to lipid profiling in the positive ion mode, where structural information of lipids is often difficult to obtain.Graphical abstractMALDI of liquid lipid samples doped with divalent metal salt (e.g. BaCl2) produces doubly charged lipid-barium ions and enables structural elucidation via MS/MS and MS3 analysis

Characterisation and pharmacological analysis of a crustacean G protein-coupled receptor: the red pigment-concentrating hormone receptor of Daphnia pulex

This is the first pharmacological characterisation of a neuropeptide G protein-coupled receptor (GPCR) in a crustacean. We cloned the ORF of the red pigment-concentrating hormone from a German strain of Daphnia pulex (Dappu-RPCH), as well as that of the cognate receptor (Dappu-RPCHR). Dappu-RPCHR has the hallmarks of the rhodopsin superfamily of GPCRs, and is more similar to insect adipokinetic hormone (AKH) receptor sequences than to receptor sequences for AKH/corazonin-like peptide or corazonin. We provide experimental evidence that Dappu-RPCH specifically activates the receptor (EC50 value of 65 pM) in a mammalian cell-based bioluminescence assay. We further characterised the properties of the ligands for the Dappu-RPCHR by investigating the activities of a variety of naturally-occurring peptides (insect AKH and crustacean RPCH peptides). The insect AKHs had lower EC50 values than the crustacean RPCHs. In addition, we tested a series of Dappu-RPCH analogues, where one residue at a time is systematically replaced by an alanine to learn about the relative importance of the termini and side chains for activation. Mainly amino acids in positions 1 to 4 and 8 of Dappu-RPCH appear responsible for effective activation of Dappu-RPCHR. The substitution of Phe4 in Dappu-RPCH had the most damaging effect on its agonistic activity.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10–800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.

Association of Japanese cedar pollinosis and sensitization with HLA-DPB1 in the Japanese adolescent

Allergic rhinitis (AR) is a heterogeneous disorder that significantly affects daily activity, work productivity, sleep, learning, and quality of life in all generations. Japanese cedar (JC) pollen is the most common allergen responsible for the development of AR in Japan. AR caused by JC pollen is considered to be a multifactorial inheritance disease that is caused by both environmental and genetic factors. The aim of this study was to investigate whether Human Leukocyte Antigen-DPB1 (HLA-DPB1) is associated with JC sensitization/pollinosis. Subjects in the present study were 544 students at the University of Tsukuba from 2013 to 2015. PCR-SSOP was performed to determine each individual's HLA-DPB1 alleles. Logistic regression analysis was performed to examine relationships between JC-related phenotypes and alleles/amino acid polymorphisms of HLA-DPB1. HLA-DPB1*02 allele were significantly associated with both JC sensitization/pollinosis (q<0.05). Furthermore, HLA-DPB1*02:01 and HLA-DPB1*02:02 had a protective tendency for JC sensitization/pollinosis, and HLA-DPB1*05:01 had a susceptible tendency for sensitization (P<0.05). In amino acid polymorphism analyses, Glutamic acid in position 69, Glycine-Glycine-Proline-Methionine in positions 84-87, Threonine in position 170 and Methionine in position 205 were also observed to have a protective tendency for JC sensitization (P<0.05). Amino acid positions 69 and 84-87 were located in binding pocket 5 and 1 of HLA-DPβ1, respectively. Amino acid changes in the allergen-binding pocket of HLA-DPβ1 are likely to influence pollinosis/sensitization to the allergenic peptide of JC pollen and determine the pollinosis risk for each individual exposed to JC pollen.

Theoretical investigation on the binding specificity of fluorinated sialyldisaccharides Neu5Acα(2–3)Gal and Neu5Acα(2–6)Gal with influenza hemagglutinin H1 – A Molecular Dynamics Study

ABSTRACTIn the present study, the hydroxyl groups at the C4 and C7 positions of sialic acid and C6 position of galactose in Neu5Acα(2–3)Gal (N23G) and the hydroxyl groups at the C8 position of sialic acid and C3 and C4 positions of galactose in Neu5Acα(2–6)Gal (N26G) were substituted with fluorine atoms, respectively. Molecular dynamics simulations of 100 ns duration were carried out to investigate the structural and dynamical behavior of H1 bound with the tri-fluorinated N23G and N26G (FN23G and FN26G). Based on energy analysis, it was concluded that FN26G should be a better binder for hemagglutinin (H1) than FN23G and it might act as an inhibitor for influenza.

Physical Characteristics of a Citrullinated Pro-Filaggrin Epitope Recognized by Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Sera.

Rheumatoid arthritis (RA) is an autoimmune disease of complex etiology. A characteristic feature of a subset of RA is the presence of anti-citrullinated protein antibodies (ACPA), which correlate with a progressive disease course. In this study, we employed streptavidin capture enzyme-linked immunosorbent assay to analyze ACPA reactivity. Using the pro-filaggrin peptide HQCHQEST-Cit-GRSRGRCGRSGS, as template, we analyzed the reactivity of RA sera and healthy donor sera to various peptides in order to determine the physical characteristics of the citrullinated pro-filaggrin epitope and to examine whether biotin labelling influence antibody recognition. The full-length cyclic pro-filaggrin peptide and a linear form with a N-terminal biotin, was recognized to the same level, whereas, a notable difference in ACPA reactivity to the linear peptides with a C-terminal biotin was found, probably due to steric hindrance. Screening of linear and cyclic truncated peptides, revealed that small cyclic peptides containing 10–12 amino acids are favored over the linear. Moreover, the charged amino acids C-terminal to citrulline were found to be essential for antibody reactivity, most important was the charged amino acid in position 4 C-terminal to citrulline. Collectively, peptide structure, length, the presence of charged amino acids and biotin labelling markedly influence antibody reactivity. In relation to the clinical diagnostics of ACPA, these findings may reflect the differences in diagnostic assays used for detection of ACPA, which relates to differences in sensitivity and specificity dependent on the assay applied.

The impact of β-azido(or 1-piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues.

The synthesis of new dermorphin analogues is described. The (R)-alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α-methyl-β-azidoalanine or α-benzyl-β-azido(1-piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [(3) H]DAMGO (a μ ligand) and [(3) H]DELT (a δ ligand). The most active analogue in this series, Tyr-(R)-Ala-(R)-α-benzyl-β-azidoAla-Gly-Tyr-Pro-Ser-NH2 and its epimer were analysed by (1) H and (13) C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C(α) in the α-benzyl-β-azidoAla residue in position 3. The (R) configuration led to the formation of a type I β-turn, whilst switching to the (S) configuration gave rise to an inverse β-turn of type I', followed by the formation of a very short β-sheet. The selectivity of Tyr-(R)-Ala-(R) and (S)-α-benzyl-β-azidoAla-Gly-Tyr-Pro-Ser-NH2 was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation.

The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic C-terminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the four cationic amino acids in positions 21-24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner than a specific lipid-peptide affinity. The present work on the lipid extraction by melittin and citrullinated melittin with model membranes emphasizes the complex relation between the affinity, the lipid extraction/membrane fragmentation, and the lipid specificity.

Разработка кандидатной субстанции рекомбинантного белка CRM197

CRM197 is a non-toxic mutant of diphtheria toxin having a single amino acid substitution of a glycine for a glutamic acid in position 52. Being naturally nontoxic, CRM197 is a promising adjuvant and ideal carrier protein for conjugate vaccines. Typically, production of diphtheria toxic and non-toxic mutants are carried out in Corynebacterium diphtheria. Production of recombinant CRM197 in Escherichia coli is advantageous. It is simple, cheap and permits production of the target protein in a short time using a non-pathogenic microorganism. In this study the pharmacology grade substance of recombinant protein CRM197 was developed based on chemical properties of the protein. The substance of recombinant protein CRM197 is characterized by presence of 0.054 ng/mcg of CRM197 E. coli host cell protein, 1.26 pg/mcg of CRM197 E.coli Host Cell DNA and less than 0,078 EU endotoxins per 1 mg of recombinant protein CRM197.

Palladium‐Catalyzed Divergent Arylation with Triazolopyridines: One‐Pot Synthesis of 6‐Aryl‐2‐α‐styrylpyridines

AbstractWe have developed a new strategy for palladium‐catalyzed arylation reactions with triazolopyridines, wherein two different chemical transformations (C‐3 vs. C‐7) are observed by differentiating the substrates using different bases. The reactive palladium carbenoids were directly generated from triazolopyridines and underwent denitrogenative arylations with aryl bromides. Intriguingly, when potassium carbonate was replaced with potassium tert‐butoxide, direct CH arylation occurred at the most acidic position (C‐7). Moreover, two different catalytic arylation events were successfully performed in a one‐pot sequence, providing a convenient access to 6‐aryl‐2‐α‐styrylpyridines.magnified image

Virulence of viral haemorrhagic septicaemia virus (VHSV) genotype III in rainbow trout.

In general, viral haemorrhagic septicaemia virus (VHSV) isolates from marine fish species in European waters (genotypes GIb, GII and GIII) are non- to low virulent in rainbow trout. However, a VHSV isolation was made in 2007 from a disease outbreak in sea farmed rainbow trout in Norway. The isolate, named NO-2007-50-385, was demonstrated to belong to GIII. This isolate has attracted attention to assess which of the viral genome/proteins might be associated with the virulence in rainbow trout. In this study, we describe the difference of virulence in rainbow trout between the NO-2007-50-385 and 4p168 isolates as representatives of virulent and non-virulent GIII isolates, respectively. Rainbow trout were bath challenged with VHSV NO-2007-50-385 for 1 and 6 h, resulting in cumulative mortalities of 5 and 35%, respectively. No mortality was observed in the rainbow trout groups immersed with the genotype III VHSV isolate 4p168 for 1 and 6 h. The viral titre in organs from fish challenged with NO-2007-50-385 for 6 h increased more rapidly than those exposed for 1 h. By in vitro studies it was demonstrated that the final titres of VHSV DK-3592B (GI), NO-2007-50-385 and 4p168 inoculated on EPC cells were very similar, whereas when inoculated on the rainbow trout cell line RTG-2 the titre of the non-virulent 4p168 isolate was 3–4 logs below the two other VHSV isolates. Based on a comparative analysis of the entire genome of the genotype III isolates, we suggest that substitutions of amino acids in positions 118–123 of the nucleo-protein are candidates for being related to virulence of VHSV GIII in rainbow trout.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0303-z) contains supplementary material, which is available to authorized users.

Importance of Intermolecular Hydrogen Bonding for the Stereochemical Control of Allene-Enone (3+2) Annulations Catalyzed by a Bifunctional, Amino Acid Derived Phosphine Catalyst.

The origin of stereoselectivity in the (3+2) annulation of allenes and enones catalyzed by an amino acid derived phosphine catalyst has been investigated by the use of dispersion-corrected density functional theory. An intermolecular hydrogen bond between the intermediate zwitterion and the enone was found to be the key interaction in the two enantiomeric transition states. Additional stabilization is provided by intermolecular hydrogen-bonding interactions between acidic positions on the catalyst backbone and the substrate. Enantioselectivity occurs because the intermolecular hydrogen bond in the transition state leading to the minor enantiomer is only possible at the expense of reactant distortion.

Importance of Intermolecular Hydrogen Bonding for the Stereochemical Control of Allene–Enone (3+2) Annulations Catalyzed by a Bifunctional, Amino Acid Derived Phosphine Catalyst

AbstractThe origin of stereoselectivity in the (3+2) annulation of allenes and enones catalyzed by an amino acid derived phosphine catalyst has been investigated by the use of dispersion‐corrected density functional theory. An intermolecular hydrogen bond between the intermediate zwitterion and the enone was found to be the key interaction in the two enantiomeric transition states. Additional stabilization is provided by intermolecular hydrogen‐bonding interactions between acidic positions on the catalyst backbone and the substrate. Enantioselectivity occurs because the intermolecular hydrogen bond in the transition state leading to the minor enantiomer is only possible at the expense of reactant distortion.

Development of a tactical screening method to investigate the characteristics of functional peptides

Using spot-synthesized peptide arrays, a functional peptide can be screened as a high-binding peptide for a target molecule. We have developed a rational screening method for functional peptides by analyzing the physicochemical rules of high-binding peptide sequences. To screen the peptides simply and strategically, we prepared an exhaustive 4-mer peptide library consisting of 256 peptides (44 = 256) characterized by four physicochemical groups of 20 amino acids: Group 1, non-charged hydrophobic amino acids; Group 2, non-charged hydrophilic amino acids; Group 3, positive-charged hydrophilic amino acids; Group 4, negative-charged hydrophilic amino acids. First, our previous screening data from cell adhesion, bile acid-binding, and nanoparticle-binding peptides were applied to the four-category analysis, and target-specific physicochemical characteristics were obtained. We then prepared an exhaustive 4-mer peptide library using these four physicochemical groups, and screened for high-binding peptides that bind model proteins interleukin-2 and IgG. We obtained individual physicochemical rules for high-binding peptides: group 1 or 4 amino acids in position (P) 1, group 1 in P2 and P4 for IL-2, and group 2 and 3 amino acids at all position for IgG. Therefore, this system, which employs the use of a simple and strategic peptide library, will be useful in the development of functional peptides.

ChemInform Abstract: Catalyst‐Switchable Regiocontrol in the Direct Arylation of Remote C—H Groups in Pyrazolo[1,5‐a]pyrimidines.

The regiodivergent palladium-catalyzed C–H arylation of pyrazolo[1,5-a]pyrimidine has been achieved, wherein the switch in regioselectivity between positions C3 and C7 is under complete catalyst control. A phosphine-containing palladium catalyst promotes the direct arylation at the most acidic position (C7), whereas a phosphine-free catalyst targets the most electron-rich position (C3).