1.
Mass spectrometric detection of CYP450 adducts following oxidative desulfuration of methyl parathion.
Source
Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi, USA. pkyle@umc.edu.
Abstract
Cytochrome P450 (CYP)-mediated desulfuration of methyl parathion results in mechanism-based inhibition of the enzyme. Although previous data suggest that reactive sulfur is released and binds to the apoprotein, the identities of neither the adduct(s) nor the affected amino acid(s) have been clearly determined. In this study, nanospray tandem mass spectroscopy was used to analyze peptide digests of CYP resolved by SDS-PAGE from liver microsomes of male rats following incubation in the absence or presence of methyl parathion. Oxidative desulfuration was confirmed by measurement of methyl paraoxon, and inhibition of specific CYP isozymes was determined by measurement of testosterone hydroxylation. Total CYP content was quantified spectrophotometrically. Incubation of microsomes with methyl parathion decreased CYP content by 58%. This effect was not associated with a comparable increase in absorbance at 420 nm, suggesting the displacement of heme from the apoprotein. Rates of testosterone 2β- and 6β-hydroxylation, respectively, were reduced to 8 and 2%, implicating CYP3A and CYP2C11 in the oxidative desulfuration of methyl parathion. Mass spectrometric analysis identified 96 amu adducts to cysteines 64 and 378 of CYP3A1. In addition, a peptide containing cysteine 433 that coordinates with heme was possibly modified as it was detected in control, but not methyl parathion samples. A comparison of rat CYP3A1 with human CYP3A4 suggests that cysteines 64 and 378 reside along the substrate channel, remote from the active site. Alteration of these residues might modulate substrate entry to the binding pocket of the enzyme. Copyright © 2012 John Wiley & Sons, Ltd.
Copyright © 2012 John Wiley & Sons, Ltd.
The contradictory functions (activation/termination) of neutrophil proteinase 3 (PR3) in IL-33 biological activity.
Source
Konkuk University, Korea, Republic of.
Abstract
IL-1 family ligand does not possess a typical hydrophobic signal peptide and needs a processing enzyme for maturation. The maturation process of IL-33 (IL-1F11), a new member of the IL-1 family ligand, remains unclear. Precursor IL-33 ligand affinity column isolates neutrophil proteinase 3 (PR3) from human urinary proteins. PR3 is a known IL-1 family ligand processing enzyme for IL-1β (IL-1F2) and IL-18 (IL-1F4) including other inflammatory cytokines. We investigated PR3 in the maturation process of precursor IL-33 since we isolated urinary PR3 by using precursor IL-33 ligand affinity column. PR3 converted inactive human and mouse precursor IL-33 proteins to biological active forms; however, the increase of PR3 incubation time abrogated IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, PR3 cut precursor IL-33 and IL-18 at various sites and yielded multi-bands. The increased incubation period of PR3 abated mature IL-33 in a time-dependent manner. The result is consistent with the decreased bioactivity of IL-33 along with the increased PR3 incubation time. Six different human and mouse rIL-33 proteins were expressed by the predicted consensus amino acid sequence of PR3 cleavage sites and tested for bioactivities. The human IL-33/p1 was highly active but human IL-33/p2 and p3 proteins were inactive. Our results suggest the dual functions (activation/termination) of PR3 in IL-33 biological activity.
Receptor-Dependent 4D-QSAR Analysis of Peptidemimetic Inhibitors of Trypanosoma cruzi Trypanothione Reductase with Receptor Based Alignment.
Source
Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco A, Sala 609, LabMMol, CEP: 21949-900, Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Faculdade de Farmácia, ModMolQSAR, CEP 21941-590, Rio de Janeiro, RJ, Brazil. Universidade Federal Fluminense, Centro de Estudos Gerais, Instituto de Biologia, LaBioMol, CEP 24210-130, Niteroi, RJ, Brazil. University of New Mexico, College of Pharmacy, ZIP 87131-0001, Albuquerque, New Mexico, EUA.
Abstract
Receptor dependent four-dimensional quantitative structure-activity relationship (4D-QSAR) studies were applied on a series of 21 peptides reversible inhibitors of Trypanosoma cruzi Trypanothione Reductase (TR) (McKie et al. Amino Acids 2001, 20:145). The RD-4D-QSAR (Pan, Tseng, Hopfinger, J. Chem. Inform. Comp. Sci., 2003, 43:1591) approach can evaluate multiple conformations from Molecular Dynamics Simulation and several superposition structure alignments inside a box composed by unitary cubic cells. The descriptors are the occupancy frequency of the atoms types inside the grid cells. We could develop 3D-QSAR models that were highly predictive (q2 above 0.71). The 3D-QSAR models can be visualized as a spatial map of atom types that are important on the comprehension of the ligand-enzyme interaction mechanism, pointing main pharmacophoric groups and TR sub-sites described into the literature. We were able also to identify some TR sub-sites for further development in the drug discovery process against tropical diseases not yet studied. © 2012 John Wiley & Sons A/S.
© 2012 John Wiley & Sons A/S.
A NOVEL LECTIN FROM AGROCYBE AEGERITA SHOWS HIGH BINDING SELECTIVITY FOR TERMINAL N-ACETYLGLUCOSAMINE.
Abstract
A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with N-acetylglucosamine (GlcNAc) coupled Sepharose 6B after (NH4)2SO4 precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by mass spectrometry against the translated transcriptome of A. aegerita. The molecular weight of AAL-2 was calculated to be 43.175 kDa from mass spectrometry (MS), which was consistent with the data calculated from the amino acid sequence. To analyze the sugar binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed and the result showed that AAL-2 bound with high selectivity to terminal, nonreducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal, nonreducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as wheat germ agglutinin (WGA) and Griffonia simplicifolia lectin-II (GSL-II). Isothermal titration calorimetry (ITC) further showed that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the current study, we also evaluated the antitumor activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumor growth and prolongation of survival time of tumor bearing mice in vivo.
Effects of peptides lys-glu-asp-gly and ala-glu-asp-gly on hormonal activity and structure of the thyroid gland in hypophysectomized young chickens and old hens.
Source
Chita State Medical Academy, Russia. bi_kuznik@mail.ru.
Abstract
Hypophysectomy in 5-days chickens and old hens was followed by hormonal disturbances and structural changes in the thyroid gland. Administration of peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly synthesized on the basis the amino acid composition of extracts from the anterior and posterior lobes of the pituitary gland, respectively, to hypophysectomized birds for 40 days significantly reduced the degree of these changes. The normalizing effect of synthetic peptides on the concentration of thyrotrophic hormone and thyroid hormones in old hens was less pronounced than in chickens.
- PMID:
- 22268052
- [PubMed - in process]
Massive monoclonal expansion of CD4 T-cells specific for a M. tuberculosis ESAT-6 peptide.
Source
Saarland University, 66421 Homburg.
Abstract
T-cell responses towards tuberculin (PPD) or the M. tuberculosis-specific antigens ESAT-6 CFP-10 are indicative of prior contact with mycobacterial antigens. In this study, we investigated the exceptional case of a 75-year-old patient who devoted more than one third of his CD4 T-cells against PPD and ESAT-6.Antigen-specific T-cells were characterised using flow-cytometric intracellular cytokine-staining, ELISPOT-assay, proliferation-assays, and T-cell receptor spectratyping.T-cell frequencies were far above those found in age-matched controls (median 0.33%, 0.05-6.32%) and remained at high levels for more than two years. The patient initially presented with hemoptysis, but active tuberculosis was ruled out by repeated analysis of sputum and BAL-fluid. Skin-testing was negative and hemoptyses did not have a M. tuberculosis-related etiology. Phenotypical and functional properties of specific T-cells were consistent with a terminally differentiated effector-memory phenotype with capacity to produce IFN-γ, IL-2, and TNF-α. Epitope mapping showed that the CD4 T-cells were directed against a single peptide from ESAT-6 (amino-acid 5-20) that was presented in context of HLA-DR. T-cell receptor Vβ-spectratyping and sequencing of specific CD4 T-cells revealed a prominent peak-fraction of monoclonal origin.In conclusion, similar to monoclonal gammopathies of undetermined significance (MGUS), this may represent the first T-cell counterpart with known specificity against M. tuberculosis.
Tyrosines in the Carboxy-terminus of Syk once Phosphorylated Interact with Signaling Proteins including TULA-2, a Negative Regulator of Mast Cell Degranulation.
Source
NIDCR, United States.
Abstract
Activation of the high affinity IgE receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the carboxy terminus of the protein tyrosine kinase Syk. Synthetic peptides representing the last tenamino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2 and GADS and the proteins phosphatases SHIP-1 and UBASH3B (also known as TULA-2). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far westerns showed direct binding of the phosphorylatedpeptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2 and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-2γ. There was parallel enhancement of the receptor-induced degranulation and NFAT or NFκB activation indicating that TULA-2 like SHIP-1 functions as a negative regulator of FcεRI signaling in mast cells. Therefore the terminal tyrosines of Syk once phosphorylated bind complexes of proteins that are positive and negative regulators of signaling in mast cells.
Pyroglutamate A(beta) aggravates behavioral deficits in 5XFAD mice.
Source
University of Goettingen, Germany;
Abstract
Pyroglutamate-modified Abeta peptides at amino acid position three (AβpE3-42) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). AβpE3-42 is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated AβpE3-42 levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces AβpE3-42. TBA42 mice showed age-dependent behavioral deficits and AβpE3-42- accumulation. The Aβ profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-truncated and modified Aβ peptides: Aβ1-42, Aβ1-40, AβpE3-40, AβpE3-42, Aβ3-42, Aβ4-42 and Aβ5-42. 5XFAD and TBA42 mice were then crossed to generate bigenic FAD42 mice. At six months of age, FAD42 mice showed an aggravated behavioral phenotype compared to single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that AβpE3 levels were elevated in FAD42 mice. No change in Aβx-42 or other Aβ isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of AβpE-42 in FAD42 mice.
Bacillus subtilis RapA phosphatase domain interaction with its substrate Spo0F~P and inhibitor PhrA peptide.
Source
The Scripps Research Institute, Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, MEM-116, La Jolla, CA 92037.
Abstract
Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino terminal alpha-helical domain connected to a domain formed by six tetratricopeptide (TPR) repeats. In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate Spo0F∼P and its inhibitor pentapeptide PhrA was analyzed in part by generating chimeric proteins with RapC that targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression as well as in vitro biochemical assays allowed the identification of the amino terminal sixty amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3-5 repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR repeat is highly versatile to use a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.
Cu(II) Coordination Chemistry of Patellamide Derivatives: Possible Biological Functions of Cyclic Pseudopeptides.
Source
Universität Heidelberg, Anorganisch-Chemisches Institut, Im Neuenheimer Feld 270, 69120 Heidelberg (Germany). peter.comba@aci.uni-heidelberg.de.
Abstract
Two synthetic derivatives of the naturally occurring cyclic pseudooctapeptides patellamide A-F and ascidiacyclamide, that is, H(4) pat(2) , H(4) pat(3) , as well as their Cu(II) complexes are described. These cyclic peptide derivatives differ from the naturally occurring macrocycles by the variation of the incorporated heterocyclic donor groups and the configuration of the amino acids connecting the heterocycles. The exchange of the oxazoline and thiazole groups by dimethylimidazoles or methyloxazoles leads to more rigid macrocycles, and the changes in the configuration of the side chains leads to significant differences in the folding of the cyclic peptides. These variations allow a detailed study of the various possible structural changes on the chemistry of the Cu(II) complexes formed. The coordination of Cu(II) with these macrocyclic species was monitored by high-resolution electrospray mass spectrometry (ESI-MS), spectrophotometric (UV/Vis) and circular dichroic (CD) titrations, and electron paramagnetic resonance (EPR) spectroscopy. Density functional theory (DFT) calculations and molecular mechanics (MM) simulations have been used to model the structures of the Cu(II) complexes and provide a detailed understanding of their geometric preferences and conformational flexibility. This is related to the Cu(II) coordination chemistry and the reactivity of the dinuclear Cu(II) complexes towards CO(2) fixation. The variation observed between the natural and various synthetic peptide systems enables conclusions about structure-reactivity correlations, and our results also provide information on why nature might have chosen oxazolines and thiazoles as incorporated heterocycles.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
A luminescent affinity tag for proteins based on the terbium(III)-bindingpeptide.
Source
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, Japan; Research Center for Bio-Microsensing Technology, Kyushu Institute of Technology, 1-1 Sensui-cho, Tobata-ku, Kitakyushu 804-8550, Japan.
Abstract
Genetically encoded tags attached to proteins of interest are widely exploited for proteome analysis. Here, we present Tb(3+)-binding peptides (TBPs) which can be used for both luminescent measurements and affinity purification of proteins. TBPs consist of acidic amino acid residues and tryptophan residues which serve as Tb(3+)-binding sites and sensitizers for Tb(3+) luminescence, respectively. The Tb(3+) complexes of TBPs fused to a target protein exhibited luminescence characteristic of Tb(3+) by excitation of the tryptophan residue, and fusion proteins fused to one of the TPBs were successfully isolated from Escherichia coli cell lysate by affinity chromatography with a Tb(3+)-immobilized solid support.
Copyright © 2012 Elsevier Inc. All rights reserved.
Yellowtail insulin-like growth factor 1: molecular cloning and response to various nutritional conditions.
Source
Faculty of Agriculture, Kochi University, 200 Monobe, Nankoku, Kochi 783-8,502, Japan.
Abstract
Insulin-like growth factor 1 (IGF1) plays an important role in fish growth. This study investigated the IGF1 response to various nutritional conditions in yellowtail. First, we cloned 1,075 bp of yellowtail IGF1 cDNA, which codes for a protein of 185 amino acids (aa). This is composed of 44 aa for the signal peptide; 68 aa for the mature peptide comprising the B, C, A, and D domains; and 73 aa for the E domain. The mature yellowtail IGF1 showed high identity to IGF1 of other teleosts. Insulin-like growth factor 1 mRNA expression in the liver and white muscle was measured to observe the IGF1 response to various nutritional conditions, because the liver has the highest IGF1 expression and white muscle comprises the largest fraction of the fish body. Only white muscle IGF1 mRNA expression decreased significantly by 3 wk of fasting and recovered by refeeding. In subsequent feeding ratio (1%, 2%, and 3%/BW/d) experiments, significant correlations to growth were observed in white muscle IGF1 mRNA expression at 2- and 6-wk points and in hepatic IGF1 mRNA expression at 4 wk point. These data suggest that IGF1 expression both in hepatic and white muscle is important for somatic growth in yellowtail. Furthermore, white muscle IGF1 mRNA expression showed better responses to somatic growth and nutrition status in our two experiments than hepatic IGF1 mRNA expression.
Copyright © 2012 Elsevier Inc. All rights reserved.
Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization.
Abstract
ABSTRACT:
BACKGROUND:
To characterize the human humoral immune response against enterovirus 71 (EV71) infection and map human epitopes on the viral capsid proteins.
METHODS:
A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3) of BJ08 strain (genomic C4) were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA) was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 mug/mul). Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel.
RESULTS:
A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3) were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15) was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71) were identified and mapped to VP1.
CONCLUSION:
These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.
JBIR-78 and JBIR-95: Phenylacetylated Peptides Isolated from Kibdelosporangium sp. AK-AA56.
Source
Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
Abstract
The search for metabolites of Kibdelosporangium sp. AK-AA56 resulted in the discovery of novel N-phenylacetylatedpeptides, JBIR-78 (1) and JBIR-95 (2). Compounds 1 and 2 were established to be N-phenylacetylated heptapeptides by extensive NMR and HRESIMS analyses. The absolute configuration of the standard amino acids including a cysteicacid moiety was determined using Marfey's method on the acid hydrolysates of 1 and 2. The relative and absolute configurations of a nonstandard amino acid, β-hydroxyleucine, were elucidated using the J-based and modified Mosher's methods, respectively. In an antimicrobial test, 1 showed antibacterial activity against Micrococcus luteus.
Atrial natriuretic peptide-Fc, ANP-Fc, fusion proteins: semi-synthesis, in vitro activity and pharmacokinetics in rats.
Abstract
Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short in vivo half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semi-synthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly-expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied between 2, 11 or 16 amino acids. In addition, either one ("monomeric") or two ("dimeric") ANP peptides were linked to Fc to study whether this modification had an effect on in vitro activity and/or in vivo half-life. The various constructs were studied for in vitro activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16 and ~375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc's were found to be approximately two orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8-5.5 h), but a trend was observed where the Cmax of the monomeric constructs was approximately 3-fold higher than the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases.
QSAR Modeling and Design of Cationic Antimicrobial Peptides Based on Structural Properties of Amino Acids.
Source
College of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 400050, P.R. China. zhlin@cqut.edu.cn.
Abstract
Drug resistance to existing antibiotics poses alarming threats to global public health, which inspires heightened interests in searching for new antibiotics, including antimicrobial peptides (AMPs). Accurate prediction of antibacterial activities of AMPs may expedite novel AMP design and reduce the costs and efforts involved in laboratory screening. In the present study, a novel quantitative prediction method of AMP was established by quantitative structure-activity relationship (QSAR) modeling based on the physicochemical properties of amino acids. The indices of these physicochemical properties were used to define AMP. The structural variables were optimized by stepwise regression (STR). Three series of AMPs from the QSAR model were constructed by multiple linear regressions (MLR). These QSAR models showed good performance in reliability and predictability. The normalized regression coefficients of the QSAR model and the contribution of amino acids at each position of AMP may determine the suitableness of a particular residue at any given position. QSAR models constructed by STR-MLR should prove to be useful tools inpeptide design with respect to the calculation, explanation, good and reliable performance, and definition of physiochemical properties.
- PMID:
- 22263858
- [PubMed - as supplied by publisher]
Structure and Vibrational Motion of Insulin from Raman Optical Activity Spectra.
Abstract
The Raman optical activity (ROA) spectroscopic technique has been applied in the past to many biologically relevant systems including peptides, proteins, sugars, and even viruses. However, theoretical interpretation of the spectra relies on lengthy quantum-chemical computations, which are difficult to extend to larger molecules. In the present study, ROA and Raman spectra of insulin under a range of various conditions were measured and interpreted with the aid of the Cartesian-coordinate tensor transfer (CCT) method. The CCT methodology yielded spectra of insulin monomer and dimer of nearly ab initio quality, while at the same time reproducing the experimental data very well. The link between the spectra and the protein structure could thus be studied in detail. Spectral contributions from the peptide backbone and the amino acid side chains were calculated. Likewise, specific intensity features originating from the -helical, coil, -sheet and 310-helical parts of the protein could be deciphered. The assignment of the Raman and ROA bands to intrinsic molecular coordinates as based on the harmonic force field calculation revealed their origin and degree of locality. Alternatively, the relation of the structural flexibility of insulin to the inhomogeneous broadening of spectral bands was studied by a combination of CCT and molecular dynamics (MD). The present study confirms the sensitivity of the ROA technique to some subtle static and dynamic changes in molecular geometry, and many previous ad hoc or semi-empirical spectral-structure assignments could be verified. On the other hand, a limitation in longer-range tertiary structure sensitivity was revealed. Unlike for smaller molecules with approximately equal contributions of the electric dipole (), quadrupole (A), and magnetic dipole (G') polarizabilities, only the electric dipolar polarization () interactions seem to dominate in the protein ROA signal. The simulations concern the largest molecule for which such spectra were interpreted by a priori procedures, and significantly enhance protein folding studies undertaken by this technique.
Roles of tRNA in cell wall biosynthesis.
Source
Department of Microbiology, Ohio State University, Columbus, OH, USA.
Abstract
Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorterpeptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. WIREs RNA 2012. doi: 10.1002/wrna.1108 For further resources related to this article, please visit the WIREs website.
Copyright © 2012 John Wiley & Sons, Ltd.
In vitro and in vivo evaluation of a (64)Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer.
Source
Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA.
Abstract
INTRODUCTION:
Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with (64)Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate (64)Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with (64)Cu and to evaluate the resulting peptide.
METHODS:
p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and (125)I-Tyr(4)-BN to determine the IC(50) value. The peptide was radiolabeled with (64)Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies.
RESULTS:
The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC(50) of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, thepeptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported (64)Cu-labeled BN analogues.
CONCLUSIONS:
These studies demonstrate that (64)Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer.
Copyright © 2011 Elsevier Inc. All rights reserved.
Bombesin analogues for gastrin-releasing peptide receptor imaging.
Source
Department of Radiology, University of Missouri School of Medicine, Columbia, Missouri 65211, USA.
Abstract
OBJECTIVES:
The present study describes the design and development of a series of new bombesin (BBN) antagonistpeptide ligands of the form [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)], where Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; X=6-amino hexanoic acid, 8-amino octanoic acid or 9-Aminononanoicacid; and BBN(6-13)NHEt=Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, an antagonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPR).
METHODS:
[NO2A-X-D-Phe(6)-BBN(6-13)NHEt] conjugates were manually conjugated with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugates were labeled with (64)Cu to yield [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)]. The metallated and nonmetallated conjugates were purified via reversed-phase high-performance liquid chromatography and characterized by electrospray ionization-mass spectrometry.
RESULTS:
Competitive displacement binding assays displayed nanomolar binding affinities toward human GRPR for all of the newly formed peptide analogues. Biodistribution studies showed very high uptake and retention of tumor-associated radioactivity in PC-3 (a prostate tumor model known to express the GRPR) tumor-bearing rodent models. The radiolabeled conjugates also exhibited rapid urinary excretion and very high tumor to background ratios. Micro-positron emission tomography (PET) molecular imaging investigations showed clear visualization of tumors in female PC-3 tumor-bearing mice 15 h postinjection.
CONCLUSION:
The biodistribution and molecular imaging study suggests that these conjugates can be considered as potential PET tracer candidates for the diagnosis of GRPR-positive tumors in human patients.
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