1.
A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays.
Source
Department of Bioengineering and Center for Bioengineering Research, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA. jiayu.liao@ucr.edu.
Abstract
Using a fluorescent NBD amino acid, new protease substrates were developed that are attractive because of the excellent chemical stability and long wavelength of excitation (480 nm) of the NBD fluorophore. The fluorescent peptidesare synthesized by Fmoc solid-phase peptide synthesis. An example peptide was efficiently immobilized onto a microarray surface using click chemistry, and its proteolysis was monitored by fluorescence imaging. Excellent site specificity was achieved for the protease. Fluorescent peptides are also used to monitor the conjugation efficiency onto a surface using a standard microarray scanner.
Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNAiMet synthesis and systemic insulin signaling.
Source
Department of Biochemistry and Molecular Biology, Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, Calgary, AB, Canada T2N 4N1.
Abstract
The target-of-rapamycin pathway couples nutrient availability with tissue and organismal growth in metazoans. The key effectors underlying this growth are, however, unclear. Here we show that Maf1, a repressor of RNA polymerase III-dependent tRNA transcription, is an important mediator of nutrient-dependent growth in Drosophila. We find nutrients promote tRNA synthesis during larval development by inhibiting Maf1. Genetic inhibition of Maf1 accelerates development and increases body size. These phenotypes are due to a non-cell-autonomous effect of Maf1 inhibition in the fat body, the main larval endocrine organ. Inhibiting Maf1 in the fat body increases growth by promoting the expression of brain-derived insulin-like peptides and consequently enhanced systemic insulin signaling. Remarkably, the effects of Maf1 inhibition are reproduced in flies carrying one extra copy of the initiator methionine tRNA, tRNA(i)(Met). These findings suggest the stimulation of tRNA(i)(Met) synthesis via inhibition of dMaf1 is limiting for nutrition-dependent growth during development.
The Renin Angiotensin System: A Target of and Contributor to Dyslipidemias, Altered Glucose Homeostasis and Hypertension of the Metabolic Syndrome.
Source
1University of Kentucky.
Abstract
The renin-angiotensin system (RAS) is an important therapeutic target in the treatment of hypertension. Obesity has emerged as a primary contributor to essential hypertension in the US, and clusters with other metabolic disorders (hyperglycemia, hypertension, high triglycerides, low HDL cholesterol) defined within the metabolic syndrome. In addition to hypertension, RAS blockade may also serve as an effective treatment strategy to control impaired glucose and insulin tolerance and dyslipidemias in patients with the metabolic syndrome. Hyperglycemia, insulin resistance, and/or specific cholesterol metabolites have been demonstrated to activate components required for the synthesis(angiotensinogen, renin, ACE), degradation (ACE2), or responsiveness (angiotensin type 1 receptors (AT1R), mas receptors) to angiotensin peptides in cell types (e.g., pancreatic islet cells, adipocytes, macrophages) that mediate specific disorders of the metabolic syndrome. An activated local RAS in these cell types may contribute to dysregulated function by promoting oxidative stress, apoptosis, and inflammation. This review will discuss data demonstrating regulation of components of the RAS by cholesterol and its metabolites, glucose and/or insulin in cell types implicated in disorders of the metabolic syndrome. In addition, we discuss data supporting a role for an activated local RAS in dyslipidemias, glucose intolerance/insulin resistance, and the development of hypertension in the metabolic syndrome. Identification of an activated RAS as a common thread contributing to several disorders of the metabolic syndrome makes the use of ARBs and ACE inhibitors an intriguing and novel option for multi-symptom treatment.
Synthesis of Thiophenylalanine-containing Peptides via Cu(I)-mediated Cross-Coupling.
Source
Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
Abstract
Aryl thiolates have unique reactive, redox, electronic, and spectroscopic properties. A practical approach to synthesizepeptides containing thiophenylalanine has been developed via a novel Cu(I)-mediated cross-coupling reaction between thiolacetic acid and iodophenylalanine-containing peptides in the solid phase. This approach is compatible with all canonical proteinogenic functional groups, providing general access to aryl thiolates in peptides. Peptides containing thiophenylalanine (pK(a) 6.4) were readily elaborated to contain methyl, allyl, and nitrobenzyl thioethers, disulfides, sulfoxides, sulfones, or sulfonates.
The use of plants for the production of therapeutic human peptides.
Source
Laboratorio di Biotecnologie, Unità Tecnica BIORAD, ENEA C.R. Casaccia, 00123, Rome, Italy.
Abstract
Peptides have unique properties that make them useful drug candidates for diverse indications, including allergy, infectious disease and cancer. Some peptides are intrinsically bioactive, while others can be used to induce precise immune responses by defining a minimal immunogenic region. The limitations of peptides, such as metabolic instability, short half-life and low immunogenicity, can be addressed by strategies such as multimerization or fusion to carriers, to improve their pharmacological properties. The remaining major drawback is the cost of production using conventional chemical synthesis, which is also difficult to scale-up. Over the last 15 years, plants have been shown to produce bioactive and immunogenic peptides economically and with the potential for large-scale synthesis. The production ofpeptides in plants is usually achieved by the genetic fusion of the corresponding nucleotide sequence to that of a carrier protein, followed by stable nuclear or plastid transformation or transient expression using bacterial or viral vectors. Chimeric plant viruses or virus-like particles can also be used to display peptide antigens, allowing the production of polyvalent vaccine candidates. Here we review progress in the field of plant-derived peptides over the last 5 years, addressing new challenges for diverse pathologies.
Pseudomorphic synthesis of monodisperse magnetic mesoporous silica microspheres for selective enrichment of endogenous peptides.
Source
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China.
Abstract
In this work, we describe a novel synthetic strategy of magnetic mesoporous silica spheres (Fe(3)O(4)@mSiO(2)) for the selective enrichment of endogenous peptides. Fe(3)O(4) particles were coated with silica shell by a sol-gel method, followed by pseudomorphic synthesis to transform nonporous silica shell into ordered mesoporous silica shell. The core/shell structure and mesostructure were individually fabricated in two steps, which can be expedient to independently optimize the properties of monodispersion, magnetization and mesostructure. Actually, it was confirmed that the produced Fe(3)O(4)@mSiO(2) particles possess good monodispersion, high magnetization, superparamagnetism, uniform accessible mesopores, and large surface area and pore volume. With these good properties, Fe(3)O(4)@mSiO(2) spheres were applied to the rapid enrichment of peptides. Based on the size-exclusion mechanism and hydrophobic interaction with siloxane bridge group mainly on the surface of inside pores, Fe(3)O(4)@mSiO(2) can selectively capture peptides and exclude high-MW proteins and salts. Furthermore, peptides in human plasma were successfully enriched by Fe(3)O(4)@mSiO(2).
Copyright © 2011 Elsevier B.V. All rights reserved.
Structural Mass Spectrometry: Rapid Methods for Separation and Analysis of Peptide Natural Products.
Source
Department of Chemistry, Vanderbilt University , Nashville, Tennessee, United States.
Abstract
A significant challenge in natural product discovery is the initial discrimination of discrete secondary metabolites alongside functionally similar primary metabolic cellular components within complex biological samples. A property that has yet to be fully exploited for natural product identification and characterization is the gas-phase collision cross section, or, more generally, the mobility-mass correlation. Peptide natural products possess many of the properties that distinguish natural products, as they are frequently characterized by a high degree of intramolecular bonding and possess extended and compact conformations among other structural modifications. This report describes a rapid structural mass spectrometry technique based on ion mobility-mass spectrometry for the comparison of peptide natural products to their primary metabolic congeners using mobility-mass correlation. This property is empirically determined using ion mobility-mass spectrometry, applied to the analysis of linear versus modified peptides, and used to discriminate peptide natural products in a crude microbial extract. Complementary computational approaches are utilized to understand the structural basis for the separation of primary metabolism derived linear peptides from secondary metabolite cyclic and modified cyclic species. These findings provide a platform for enhancing the identification of secondary metabolic peptides with distinct mobility-mass ratios within complex biological samples.
TFAP2E-DKK4 and chemoresistance in colorectal cancer.
Source
Department of Medicine II, Universitätsmedizin Mannheim, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany. matthias.ebert@umm.de
Abstract
BACKGROUND:
Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy.
METHODS:
We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation.
RESULTS:
TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism.
CONCLUSIONS:
TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).
Arginine Mimetics Using α-Guanidino Acids: Introduction of Functional Groups and Stereochemistry Adjacent to Recognition Guanidiniums in Peptides.
Source
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 (USA).
Abstract
Arginine residues are broadly employed for specific biomolecular recognition, including in protein-protein, protein-DNA, and protein-RNA interactions. Arginine recognition commonly exploits the potential for bidentate electrostatic and hydrogen-bonding interactions. However, in arginine residues, the guanidinium functional group is located at the terminus of a flexible hydrocarbon side chain, which lacks the functionality to contribute to specific arginine-mediated recognition and may entropically disfavor binding. In order to enhance the potential for specificity and affinity in arginine-mediated molecular recognition, we have developed an approach to the synthesis of peptides that incorporates an α-guanidino acid as a novel arginine mimetic. α-Guanidino acids, derived from α-amino acids, with guanidinylation of the amino group, were incorporated stereospecifically into peptides on solid phase via coupling of an Fmoc amino acid to diaminopropionic acid (Dap), Fmoc deprotection, guanidinylation of the amine on solid phase, and deprotection, generating a peptide containing an α-functionalized arginine mimetic. This approach was examined by incorporating arginine mimetics into ligands for the Src, Grb, and Crk SH3 domains at the site of the key recognition arginine. Protein binding was examined for peptides containing guanidino acids derived from Gly, L-Val, L-Phe, L-Trp, D-Val, D-Phe, and D-Trp. We demonstrate that paralogue specificity and target site affinity may be modulated with the use of α-guanidino acid-derived arginine mimetics, generating peptides that exhibit enhanced Src specificity by selection against Grb andpeptides that reverse the specificity of the native peptide ligand, with enhancements in Src target specificity of up to 15-fold (1.6 kcal mol(-1) ).
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Dietary nitrogen and fish welfare.
Source
CCMAR-CIMAR L.A., Centro de Ciências do Mar do Algarve, Universidade do Algarve, Campus de Gambelas, 8005-139, Faro, Portugal, lconcei@ualg.pt.
Abstract
Little research has been done in optimizing the nitrogenous fraction of the fish diets in order to minimize welfare problems. The purpose of this review is to give an overview on how amino acid (AA) metabolism may be affected when fish are under stress and the possible effects on fish welfare when sub-optimal dietary nitrogen formulations are used to feed fish. In addition, it intends to evaluate the current possibilities, and future prospects, of using improved dietary nitrogen formulations to help fish coping with predictable stressful periods. Both metabolomic and genomic evidence show that stressful husbandry conditions affect AA metabolism in fish and may bring an increase in the requirement of indispensable AA. Supplementation in arginine and leucine, but also eventually in lysine, methionine, threonine and glutamine, may have an important role in enhancing the innate immune system. Tryptophan, as precursor for serotonin, modulates aggressive behaviour and feed intake in fish. Bioactive peptides may bring important advances in immunocompetence, disease control and other aspects of welfare of cultured fish. Fishmeal replacement may reduce immune competence, and the full nutritional potential of plant-protein ingredients is attained only after the removal or inactivation of some antinutritional factors. This review shows that AA metabolism is affected when fish are under stress, and this together with sub-optimal dietary nitrogen formulations may affect fish welfare. Furthermore, improved dietary nitrogen formulations may help fish coping with predictable stressful events.
Comparative syntheses of peptides and peptide thioesters derived from mouse and human prion proteins.
Source
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic, jsebestik@seznam.cz.
Abstract
Prions are suspected as causative agents of several neuropathogenic diseases, even though the mode of their action is still not clear. A combination of chemical and recombinant syntheses can provide suitable probes for explanation of prions role in pathogenesis of neurodegenerative diseases. However, the prions contain several difficult sequences forsynthesis by Fmoc/tBu approach. For that reason, the peptide thioesters as the key building blocks for chemical syntheses of proteins by native chemical ligation were employed. A scan of the mouse prion domain 93-231 was carried out in order to discover availability of derived thioesters as the suitable building blocks for a total chemical synthesis of the prion protein based probes. The synthesis on 2-chlorotritylchloride resin was utilized and after a deprotection of the samples for analysis, the peptide segments were purified and characterized. If the problems were detected during thesynthesis, the segment was re-synthesized either using the special pseudoproline dipeptides or by splitting its molecule to two or three smaller segments, which were prepared easier. The protected segments, prepared correctly without any deletion and in sufficient amounts, were coupled either with EtSH after DIC/DMAP activation or with p-Ac-NH-Ph-SH using PyBOP activation to yield corresponding thioesters. In some special cases, the other techniques of thioester formation, like sulfonamide-safety catch and/or trimethylaluminium approach were utilized.
PET Imaging of Integrin Positive Tumors Using F Labeled Knottin Peptides.
Source
1. Molecular Imaging Program at Stanford (MIPS), Bio-X Program, Department of Radiology, Stanford University, California, 94305-5344, USA.
Abstract
Purpose: Cystine knot (knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of (18)F-labeled knottins in a U87MG glioblastoma model.Procedures: Engineered knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP). The resulting probes, (18)F-FP-2.5D and (18)F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies.Results: MicroPET imaging studies with (18)F-FP-2.5D and (18)F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that (18)F-FP-2.5D and (18)F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent.Conclusions: (18)F-FP-2.5D and (18)F-FP-2.5F showed reduced gallbladder uptake compared with previously published (18)F-FB-2.5D. (18)F-FP-2.5D and (18)F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. (18)F-FP-2.5D demonstrated more desirable pharmacokinetics compared to (18)F-FP-2.5F, and thus has greater potential for clinical translation.
N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression.
Source
Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin Medical University, Harbin, 150001, China.
Abstract
RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90 min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15 min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.
The sclerostin-bone protein interactome.
Source
Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Abstract
The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptidesderived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.
Copyright © 2011 Elsevier Inc. All rights reserved.
[Vitamin D and autoimmunity. First part: Fundamental aspects.]
Source
Service de médecine interne, université Versailles Saint-Quentin-en-Yvelines, hôpital Foch, 40, rue Worth, 92151 Suresnes cedex, France.
Abstract
The effects of vitamin D on calcium homeostasis and bone metabolism are well known. In recent years, suboptimal vitamin D status has been recognized as a pandemic. Meanwhile, extra-skeletal effects of vitamin D are becoming better documented, particularly its effects on immunity. The authors present their actions on myeloid dendritic cells, T cells, B cells, as well as on the synthesis of antimicrobial peptides and autophagy, and the potential beneficial effects in autoimmune and inflammatory diseases.
Copyright © 2011. Published by Elsevier SAS.
Plant defensins and defensin-like peptides - biological activities and biotechnological applications.
Source
Laboratorio de Fisiologia e Bioquimica de Microrganismos, Centro de Biociencias e Biotecnologia,Universidade Estadual do Norte Fluminense; Av. Alberto Lamego, 2000;Campos dos Goytacazes-RJ RJ; CEP: 28013-600; Brazil. andre@uenf.br.
Abstract
Plant defensins are cationic peptides that are ubiquitous within the plant kingdom and belong to a large superfamily of antimicrobial peptides found in several organisms collectively called defensins. The primary structure of these peptidesincludes 45 to 54 amino acid residues with considerable sequence variation. At the level of three-dimensional structure, they are small and globular, composed of three anti-parallel β-sheets and one α-helix, which is highly conserved among these peptides. The three-dimensional structure is stabilized by four disulfide bridges formed by eight strictly conserved Cys residues. Two of these bridges compose the Cys-stabilized α-helix β-strand motif, which is found in other peptideswith biological activities. Plant defensins present numerous biological activities, such as inhibiting protein synthesis, ion channel function and α-amylase and trypsin activity; impairing microbial, root hair and parasitic plant growth; mediating abiotic stress and Zn tolerance; altering ascorbic acid redox state; stimulating sweet taste sensation; serving as epigenetic factors; affecting self-incompatibility; and promoting male reproductive development. Some of these biological activities, such as microbial growth inhibition and sweet taste induction, coupled with a scaffold that provides thesepeptides with incredible physicochemical resistance to harsh environments and the potential for simple amino acid substitution, raise the opportunity to improve the function of defensins or introduce new activities, endowing thesepeptides with great biotechnological and medical significance. This review will cover the biological activities and roles of plant defensins and will focus on their application in the field of biotechnology.
Structure-activity studies on alpha-conotoxins.
Source
Institute for Molecular Bioscience, The University of Queensland, St Lucia Queensland 4072, Australia. p.alewood@imb.uq.edu.au.
Abstract
Conotoxins are small bioactive highly structured peptides from the venom of marine cone snails (genus Conus). Over the past 50 million years these molluscs have developed a complex venom cocktail for each species that is comprised of 100-2000 distinct cysteine- rich peptides for prey capture and defence. This review focuses on an important and well-studied class of conotoxins, the α- conotoxins. These α-conotoxins are potent and selective antagonists of various subtypes of the nicotinic acetylcholine receptors (nAChRs). Key structure-activity relationship studies are presented to illustrate the common motifs, structural features and pharmacophores that define this interesting peptide class. Additionally, their synthesis, chemical modifications, the development of more selective and stable analogues and their therapeutic potential are discussed.
Angiotensinase and Vasopressinase Activities in Hypothalamus, Plasma, and Kidney after Inhibition of Angiotensin-converting Enzyme: Basis for a New Working Hypothesis.
Source
Unit of Physiology, Department of Health Sciences, University of Jaén, Jaen, Spain.
Abstract
Reducing angiotensin II (Ang II) production via angiotensin-converting enzyme (ACE) inhibitors is a key approach for the treatment of hypertension. However, these inhibitors may also affect other enzymes, such as angiotensinases and vasopressinase, responsible for the metabolism of other peptides also involved in blood pressure control, such as Ang 2-10, Ang III, Ang IV, and vasopressin. We analyzed the activity of these enzymes in the hypothalamus, plasma, and kidney of normotensive adult male rats after inhibition of ACE with captopril. Aspartyl- (AspAP), glutamyl- (GluAP), alanyl- (AlaAP) and cystinyl-aminopeptidase (CysAP) activities were measured fluorimetrically using arylamides as substrates. Systolic blood pressure (SBP), water intake, and urine flow were also measured. Captopril reduced SBP and increased urine flow. In the hypothalamus, GluAP and AspAP increased, without significant changes in either AlaAP or CysAP. In contrast with effects in plasma, GluAP was unaffected, AspAP decreased, while AlaAP and CysAP increased. In the kidney, enzymatic activities did not change in the cortex, but decreased in the medulla. These data suggest that after ACE inhibition, the metabolism of Ang I in hypothalamus may lead mainly to Ang 2-10 formation. In plasma, the results suggest an increased formation of Ang IV together with increased vasopressinase activity. In the kidney, there is a reduction of vasopressinase activity in the medulla, suggesting a functional reduction of vasopressin in this location. The present data suggest the existence of alternative pathways in addition to ACE inhibition that might be involved in reducing BP after captopril treatment.
© Georg Thieme Verlag KG Stuttgart · New York.
Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei.
Source
Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Straße 1, D- 85354 Freising, Germany.
Abstract
The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively. Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles. It is suggested, that after transport into the cell peptidesare only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Studying protein-peptide interactions using benzophenone units: A case study of protein kinase B/Akt and its inhibitor PTR6154.
Source
Institute of Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Abstract
Protein-protein interactions (PPIs) govern nearly all processes in living cells. Peptides play an important role in studying PPIs. Peptides carrying photoaffinity labels that covalently bind the interacting protein can be used to obtain more accurate information regarding PPIs. Benzophenone (BP) is a useful photoaffinity label that is widely used to study PPIs. We developed a one-pot two-step synthesis for the preparation of novel BP units. To map the binding site more thoroughly, linkers of various lengths were attached to the BP moiety. These units can be incorporated into peptide sequences using well-established solid phase peptide synthesis (SPPS) protocols. As a proof of concept, we studied the interaction between protein kinase B (PKB/Akt) and its synthetic peptide inhibitor, PTR6154. The methodology is general and can be implemented to study PPIs in a variety of biological systems.
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