RSSSurveying proteolytic processes in human cancer microenvironments by microdialysis and activity-based mass spectrometry.
Source
Boston Biomedical Research Institute, Watertown, MA, USA. hardt@bbri.org.
Abstract
Purpose: We present a strategy to survey proteolytic processes that occur in human cancer microenvironments. Experimental design: In situ microdialysis during oral cancer surgery was combined with mass spectrometry-based proteomics to analyze interstitial fluid surrounding tumors and anatomically matched normal sites. Protease activity-based (18)O-profiling was utilized to reveal peptides that were processed by co-collected proteases ex vivo. Results: We demonstrated for the first time the use of microdialysis in humans to collect interstitial fluid from cancer microenvironments. Proteomic profiling identified proteases and inhibitors in the microdialysis samples. A subset ofpeptides displayed characteristic (18)O-isotope patterns that indicated processing by endogenous proteases. Conclusions and clinical relevance: The presented approach provides unprecedented views of in vivo targets of proteases without disrupting the cancer or surrounding tissue. The methodology can be broadly adapted to other physiological conditions in which proteolytic mediators are involved (e.g. arthritic joints, inflamed muscle, other types of cancer) and where a comparison of normal and pathological tissue is sought.
Copyright © 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22262628
- [PubMed - as supplied by publisher]
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-Aminononanoic acid; 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.
Published by Elsevier Inc.
- PMID:
- 22261143
- [PubMed - as supplied by publisher]
Exploiting cell surface thiols to enhance cellular uptake.
Source
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Abstract
Efficient cellular delivery is one of the key issues that has hampered the therapeutic development of novel synthetic biomolecules such as oligonucleotides, peptides and nanoparticles. The highly specialized cellular plasma membrane specifically internalizes compounds through tightly regulated mechanisms. It is possible to exploit these natural mechanisms of cellular uptake with rationally designed reagents. Here, we discuss how thiol groups (-SH) naturally present on the cell surface (exofacial thiols) can be used to enhance cellular association and internalization of various materials bearing thiol-reactive groups in their structure. We propose that such thiol modifications should be considered in future design of synthetic biomolecules for optimized cellular delivery.
Copyright © 2011 Elsevier Ltd. All rights reserved.
- PMID:
- 22260747
- [PubMed - as supplied by publisher]
Monitoring of gluten-free diet compliance in celiac patients by assessment of gliadin 33-mer equivalent epitopes in feces.
Source
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain.
Abstract
BACKGROUND:
Certain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available.
OBJECTIVE:
We assessed the capacity to determine the gluten ingestion and monitor GFD compliance in celiac patients by the detection of gluten and gliadin 33-mer equivalent peptidic epitopes (33EPs) in human feces.
DESIGN:
Fecal samples were obtained from healthy subjects, celiac patients, and subjects with other intestinal pathologies with different diet conditions. Gluten and 33EPs were analyzed by using immunochromatography and competitive ELISA with a highly sensitive antigliadin 33-mer monoclonal antibody.
RESULTS:
The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo. We were able to detect gluten peptides in feces of healthy individuals after consumption of a normal gluten-containing diet, after consumption of a GFD combined with controlled ingestion of a fixed amount of gluten, and after ingestion of <100 mg gluten/d. These methods also allowed us to detect GFD infringement in CD patients.
CONCLUSIONS:
Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake. These techniques may serve to show GFD compliance or infringement and be used in clinicalresearch in strategies to eliminate gluten immunotoxic peptides during digestion. This trial was registered at clinicaltrials.gov as NCT01478867.
Beverage consumption, appetite, and energy intake: what did you expect?
Source
Department of Nutrition Science, Purdue University, West Lafayette, IN, and the Department of Medicine, Indiana University, Indianapolis, IN.
Abstract
BACKGROUND:
Beverage consumption is implicated in the overweight/obesity epidemic through the weaker energy compensation response it elicits compared with solid food forms. However, plausible mechanisms are not documented.
OBJECTIVE:
This study assessed the cognitive and sensory contributions of differential postingestive responses to energy- and macronutrient-matched liquid (in beverage form) and solid food forms and identifies physiologic processes that may account for them.
DESIGN:
Fifty-two healthy adults [mean ± SD age: 24.7 ± 5.5 y; BMI (in kg/m(2)): 26.3 ± 6.3] completed this randomized, 4-arm crossover study. Participants consumed oral liquid and solid preloads that they perceived, through cognitive manipulation, to be liquid or solid in their stomach (ie, oral liquid/perceived gastric liquid, oral liquid/perceived gastric solid, oral solid/perceived gastric liquid, or oral solid/perceived gastric solid). However, all preloads were designed to present a liquid gastric challenge. Appetite, gastric-emptying and orocecal transit times, and selected endocrine responses were monitored for the following 4 h; total energy intake was also recorded.
RESULTS:
Oral-liquid and perceived gastric-liquid preloads elicited greater postprandial hunger and lower fullness sensations, more rapid gastric-emptying and orocecal transit times, attenuated insulin and glucagon-like peptide 1 release, and lower ghrelin suppression than did responses after oral-solid and perceived gastric-solid treatments (all P < 0.05). Faster gastric-emptying times were significantly associated with greater energy intake after consumption of perceived gastric-liquid preloads (P < 0.05). Energy intake was greater on days when perceived gastric-liquid preloads were consumed than when perceived gastric solids were consumed (2311 ± 95 compared with 1897 ± 72 kcal, P = 0.007).
CONCLUSIONS:
These data document sensory and cognitive effects of food form on ingestive behavior and identify physical and endocrine variables that may account for the low satiety value of beverages. They are consistent with findings that clear, energy-yielding beverages pose a particular risk for positive energy balance. This study is registered at clinicaltrials.gov as NCT01070199.
Spatiotemporal control of cell fate and cardiac differentiation.
Abstract
Congestive heart failure is a leading cause of morbidity and mortality in the United States and worldwide [1]. Although not the only cause of congestive heart failure, loss of myocardium due to obstructive coronary artery disease is a major contributor to this condition [2,3]. The loss of myocardium is mainly regional and, therefore, localized therapy holds the most promise. In recent years, many clinical studies have been initiated to deliver localized therapy in the form of various cell types for reconstitution of the myocardium [4-6]. However, there is much debate on the optimal cell type, whether or not stem cells can differentiate into functional myocardium and the long-term effects of these non-myocytes. In addition to exogenous cell delivery, paracrine effects arising from delivery of angiogenic factors and other biochemical agents suggest that the myocardium retains the ability to remodel and heal [7,8]. Understandably, there has been tremendous focus on both growth factor- and gene therapy-based therapeutics. While a source of great promise, direct growth factor delivery to the myocardium will most likely be inefficient as several studies have noted that many of these small proteins are carried away in the highly vascularized cardiac tissue [9,10]. Gene therapy, while providing an excellent analytical tool, has not met with enthusiasm clinically, mostly due to the inability to quantify delivery and nonspecific targeting in vivo. This body of work supports two crucial conclusions: (1) appropriate biological cues that act locally on the myocardium can improve functional outcomes, and (2) spatiotemporal control over the delivery and presentation of these cues remains challenging and inefficient. The current body of work on cardiac drug delivery and stem cell therapy support two crucial conclusions: (1) appropriate biological cues that act locally on the myocardium can improve functional outcomes, and (2) spatiotemporal control over the delivery and presentation of these cues remains challenging and inefficient. Using specialized biomaterials, we are able to address this. Our laboratory has used functional nanoparticles to deliver regenerative cues with both spatial and temporal control. By adding biomimetic signals to the surface of the nanoparticles, we are able to enhance nonphagocytic cell uptake, achieve cell-specific targeting, and deliver molecules in a time-controlled manner. Furthermore, by combining our nanoparticles with self-assembling peptide nanofibers, we can achieve some of these in 3D while retaining implanted cells. Following infarction, and during cell differentiation and survival, there are unique sequences of events that contribute to the pathophysiology of the processes. By enhanced understanding of these signals, and the spatiotemporal control needed to deliver them, it will help design better therapeutics for cardiac regeneration. Using high-throughput screening, we have identified several carbohydrates that bind with high affinity to both cardiomyocytes and cardiac progenitor cells. For example, n-acetylglucasmine (GlcNAc) and related sugars bound with high affinity to both of these important cell types. Modification of the sugars with phospholipds allowed incorporation of into hydrophobic nanoparticles with high fidelity. When delivered to cardiomyocytes or cardiac progenitor cells, these biologically-modified nanoparticles enhanced uptake by several fold. In addition to using sugars, we also used metal affinity chromatography chemistry to link Nickel-NTA to the outside of nanoparticles. This not only allowed for conjugation of Histidine-tagged proteins to the outside for temporal delivery, but also allowed for conjugation of cell-specific targeting agents as well. Indeed, when His-VE-Cadherin was used as a model targeting agent, binding and uptake by endothelial cells increased significantly. For cell therapy, incorporation of proper signals to the micoenvironment is critical. One such signal that greatly impacts differentiation is the Notch1 pathway. We were able to immobilize Jagged1, the ligand of Notch1, on to self-assembling peptide nanofibers. Inclusion of this bioactive signal significantly enhanced cardiogenic differentiation. Morever, by including time release nanoparticles, we were able to improve differentiation and survival significantly more, underscoring the need to spatiotemporal control of drug delivery for enhancing cell therapy.
Immunological Evaluation of Lipopeptide Group A Streptococcus (GAS) Vaccine: Structure-Activity Relationship.
Source
The University of Queensland, School of Chemistry and Molecular Biosciences (SCMB), St. Lucia, Queensland, Australia.
Abstract
Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29 amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.
Novel Nonphosphorylated Peptides with Conserved Sequences Selectively Bind to Grb7 SH2 Domain with Affinity Comparable to Its Phosphorylated Ligand.
Source
National Key Laboratory of Medical Molecular Biology, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/School of Basic Medicine, Peking Union Medical College, Beijing, China.
Abstract
The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. Thesepeptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.
Targeting RNA Polymerase Primary σ as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid.
Source
Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, Shaanxi, China.
Abstract
BACKGROUND:
Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to "druggable" target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ(70) (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition.
METHODOLOGY/PRINCIPAL FINDINGS:
By synthesizing a serial of cell penetrating peptide conjugated peptidenucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)(3)K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ(70) in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ(70) were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM.
CONCLUSIONS:
The present result suggested that RNAP primary σ(70) is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.
Synthesis, evaluation and molecular modeling of cyclic tetrapeptide histone deacetylase inhibitors as anticancer agents.
Source
School of Life Science and Biotechnology, Dalian University of Technology, Dalian, 116024, China.
Abstract
Histone deacetylase inhibitors (HDACIs) are a promising class of anticancer agents. To examine whether a slight change in the recognition domain could alter their inhibitory activity, we synthesized a series of cyclo(-l-Am7(S2Py)-Aib-l-Phe(n-Me)-d-Pro)derivatives and evaluated their HDAC inhibitory and anticancer activities. The peptides exhibited potent HDAC inhibitory activity and inhibited three human cancer cell lines with IC(50) in the micromolar range. Docking and molecular dynamics simulation were conducted to explore the interaction mechanisms of class I and II HDACs with these inhibitors. It revealed that the zinc ion in the active site coordinated five atoms of HDACs and the sulfur atom of the inhibitor. The metal binding domains of these compounds interacted with HDAC2, and the surface recognition domains of these compounds interacted with HDAC4 through hydrogen bonding. The hydrophobic interactions also provided favorable contributions to stabilize the complexes. The results obtained from this study would be helpful for us to design some novel cyclic tetrapeptides that may act as potent HDACIs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Enhanced cellular uptake and metabolic stability of lipo-oligoarginine peptides.
Source
Department of Radiology, The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX 77030.
Abstract
Developing efficient cellular delivery vectors is crucial for designing novel therapeutic agents to enhance their plasma membrane permeability and metabolic stability in cells. Previously, we engineered cell penetrating peptide vectors named as "lipo-oligoarginine peptides" (LOAPs) by conjugating a proper combination of fatty acid and oligoarginine that translocated into cell easily without adverse effect on cell viability. In the present study, we report a systemic evaluation of cellular uptake and metabolic stability of LOAPs in Jurkat cells by introducing different combination of D-Arg residues in the peptide backbone. The cellular uptake and intracellular fate, cell viability, and metabolic stability and proteolytic degradation patterns of various LOAPs consisted of different combination of L- and D-Arg sequences were confirmed by flow cytometry, cytotoxicity assay, and analytical RP-HPLC with MALDI-TOF mass. All investigated LOAPs penetrated the cell efficiently with low cellular toxicity. The LOAPs having D-Arg residues at their N-termini seemed to have better metabolic stability than the LOAPs having C-terminal D-Arg residues. The metabolic degradation patterns were similar among all investigated LOAPs. The major hydrolytic site was between lauroyl group and β-Ala residue. Without the lipid chain, the oligoarginine peptide was pumped out ofcells easily. The results presented in this study suggest that structurally modified LOAPs could be used as a novel CPP design toward improved therapeutic application. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 772-779, 2011.
Copyright © 2011 Wiley Periodicals, Inc.
Association between psychological measures and brain natriuretic peptide in heart failure patients.
Abstract
ABSTRACT Objective: Brain natriuretic peptide (BNP) is a promising marker for heart failure diagnosis and prognosis. Although psychological factors also influence heart failure (HF) prognosis, this might be attributed to confounding by BNP. Our aim was to examine the association between multiple psychological markers using a prospective studydesign with repeated NT-proBNP measurements. Design: The sample comprised 94 outpatients with systolic HF (80% men; mean age=62.2±9.32). The psychological markers (i.e., anxiety, depression and Type D personality), assessed with the Hospital Anxiety and Depression Scale (HADS), the Beck Depression Inventory (BDI), and the Type D Scale (DS14) were assessed only at baseline. Plasma NT-proBNP levels were measured at baseline and at 9 months. Results: The prevalence of anxiety, depression and Type D personality at baseline was 23.4% (HADS-A), 17.0% (HADS-D), 46.6% (BDI) and 21.3% (DS14), respectively. At baseline, none of the psychological risk markers were associated with NT-proBNP levels (all ps≥.05). In the subset of patients with scores on psychological risk markers both at baseline and at 9 months there were no association between anxiety (p=0.44), depression (HADS-D: p=0.90; BDI: p=0.85), and Type D (p=0.63) with NT-proBNP levels using ANOVA for repeated measures. Conclusions: Our findings indicate that measures frequently used in HF to assess psychological risk markers are unconfounded by NT-proBNP. Further studies are warranted that replicate these findings and examine whether psychological risk markers are independent predictors of prognosis in HF or an artifact that may be attributed to other biological or behavioral mechanisms.
The use of 2,2'-dithiobis(5-nitropyridine) (DTNP) for deprotection and diselenide formation in protected selenocysteine-containing peptides.
Source
Department of Chemistry, Saint Michael's College, One Winooski Park, Colchester, VT, 05439, USA.
Abstract
In contrast to the large number of sidechain protecting groups available for cysteine derivatives in solid phase peptidesynthesis, there is a striking paucity of analogous selenocysteine Se-protecting groups in the literature. However, the growing interest in selenocysteine-containing peptides and proteins requires a corresponding increase in availability of synthetic routes into these target molecules. It therefore becomes important to design new sidechain protection strategies for selenocysteine as well as multiple and novel deprotection chemistry for their removal. In this paper, we outline the synthesis of two new Fmoc selenocysteine derivatives [Fmoc-Sec(Meb) and Fmoc-Sec(Bzl)] to accompany the commercially available Fmoc-Sec(Mob) derivative and incorporate them into two model peptides. Sec-deprotection assays were carried out on these peptides using 2,2'-dithiobis(5-nitropyridine) (DTNP) conditions previously described by our group. The deprotective methodology was further evaluated as to its suitability towards mediating concurrent diselenide formation in oxytocin-templated target peptides. Sec(Mob) and Sec(Meb) were found to be extremely labile to the DTNP conditions whether in the presence or absence of thioanisole, whereas Sec(Bzl) was robust to DTNP in the absence of thioanisole but quite labile in its presence. In multiple Sec-containing model peptides, it was shown that bis-Sec(Mob)-containing systems spontaneously cyclize to the diselenide using 1 eq DTNP, whereas bis-Sec(Meb) and Sec(Bzl) models required additional manipulation to induce cyclization. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Hindbrain leptin and glucagon-like-peptide-1 receptor signaling interact to suppress food intake in an additive manner.
Source
1] Department of Physiology and Pathophysiology, School of Medicine, Xi'an Jiaotong University, Xi'an, People's Republic of China [2] Department of Psychology, School of Art and Science University of Pennsylvania, Philadelphia, PA, USA.
Abstract
Background:The physiological control of feeding behavior involves modulation of the intake inhibitory effects of gastrointestinal satiation signaling via endogenous hindbrain leptin receptor (LepR) and glucagon-like-peptide-1 receptor (GLP-1R) activation.Design and Results:Using a variety of dose-combinations of hindbrain delivered (4th intracerebroventricular; i.c.v.) leptin and the GLP-1R agonist exendin-4, experiments demonstrate that hindbrain LepR and GLP-1R signaling interact to control food intake and body weight in an additive manner. In addition, the maximum intake suppressive response that could be achieved by 4th i.c.v. leptin alone in non-obese rats (∼33%) was shown to be further suppressed when exendin-4 was co-administered. Importantly, it was determined that the interaction between hindbrain LepR signaling and GLP-1R signaling is relevant to endogenous food intake control, as hindbrain GLP-1R blockade by the selective antagonist exendin-(9-39) attenuated the intake inhibitory effects of hindbrain leptin delivery.Conclusions:Collectively, the findings reported here show that hindbrain LepR and GLP-1R activation interact in at least an additive manner to control food intake and body weight. As evidence is accumulating that combination pharmacotherapies offer greater sustained food intake and body weight suppression in obese individuals when compared with mono-drug therapies or lifestyle modifications alone, these findings highlight the need for further examination of combined central nervous system GLP-1R and LepR signaling as a potential drug target for obesity treatment.International Journal of Obesity advance online publication, 17 January 2012; doi:10.1038/ijo.2011.265.
Overexpression of a modified protein from amaranth seed in Escherichia coli and effect of environmental conditions on the protein expression.
Source
Centro de Investigación para el Desarrollo Integral Regional, CIDIIR-IPN, Sinaloa, Mexico.
Abstract
Amaranth seeds are considered as an excellent complementary source of food protein due to their balanced amino acid composition. Amarantin acidic subunit has the potential as a functional and nutraceutical protein, and it is structurally a good candidate for modification. The aim of this work was to improve its functionality, then the primary structure was modified into the third variable region of 11S globulins, by inserting antihypertensive peptides: four Val-Tyr in tandem and Arg-Ile-Pro-Pro in the C-terminal region. Modified protein was expressed in Escherichia coli Origami (DE3) and was purified. The culture conditions, including the culture media, temperature, agitation speed and air flow were tested in order to obtain an increased expression levels of the modified protein. A 2(3) factorial design was used for evaluate the effect of environmental conditions on modified protein production. The results indicated that the yield of modified protein could be increased by up 3-fold in bioreactor as compared with flask. In addition, the temperature, the agitation speed and the oxygen were significant factors on the expression of the antihypertensive protein. The maximum production was 99mg protein-L(-1). The hydrolyzed protein showed a high inhibitory activity of the angiotensin converting enzyme (IC(50)=0.047mgmL(-1)).
Copyright © 2012 Elsevier B.V. All rights reserved.
A Synthetic C34 Trimer of HIV-1 gp41 Shows Significant Increase in Inhibition Potency.
Source
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062 (Japan).
Abstract
Supramolecular interference: A synthetic peptide mimetic of the trimeric form of gp41 showed significantly increased inhibitory activity against the HIV-1 fusion mechanism. This study demonstrates a useful strategy for the design of effective inhibitors against viral infections that proceed by membrane fusion with host cells.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Immobilizing CC chemokine receptor 4's N-terminal extracellular tail on a capillary to study its potential ligands by capillary electrophoresis.
Source
Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, People's Republic of China.
Abstract
ML40 is the equivalent peptide derived from the N terminal of CCC4 (CC chemokine receptor 4), which plays a pivotal role in allergic inflammation. A new capillary electrophoresis method was developed to study the interactions between ML40 and its potential ligands in which ML40 was immobilized on the inner wall of capillary as the stationary phase based on the covalent linking technique. The interaction between S009, a known CCR4 antagonist, and the immobilized ML40 was studied to validate the bioactivity of ML40. The electropherogram of S009 showed that the peak height was reduced and the peak width was broadened in the ML40 immobilized capillary. Otherwise, 25 computer-aided designand drafting compounds were screened out using this method. Four compounds' peak widths were broadened and their peak heights were reduced, as with S009. Meanwhile, nonlinear chromatography was used to calculate the constants for the ligand-receptor complex formation. Furthermore, the tertiary amine compounds belonging to the chiral tertiary amines of the type NRR'R″, which are optically inactive resulting from rapid pyramide inversion, were chiral separated by our protein immobilization method for the first time. In general, the methodology presented would be applicable to study compound-ML40 interactions as a reliable and robust screening method for CCR4 antagonist discovery.
Copyright © 2012. Published by Elsevier Inc.
Engineering Domain-Swapped Binding Interfaces by Mutually Exclusive Folding.
Abstract
Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein-protein recognition, and it can also potentially be exploited as a mechanism for controlled self-assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the "mutually exclusive folding" design to apply conformational stress to the monomeric state. Ubiquitin (Ub) is inserted into one of six surface loops of barnase (Bn). The 38-Å amino-to-carboxy-terminal distance of Ub stresses the Bn monomer, causing it to split at the point of insertion. The 2.2-Å X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically unfolded Bn domain, resulting in a domain-swapped polymer. All six constructs oligomerize, suggesting that inserting Ub into each surface loop of Bn results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate "smart" biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Role of lipids in the interaction of antimicrobial peptides with membranes.
Source
CIQ(UP), Departamento de Química e Bioquímica, FCUP, R. do Campo Alegre, Porto 4169-007, Portugal; REQUIMTE, Departamento de Química e Bioquímica, FCUP, R. do Campo Alegre, Porto 4169-007, Portugal.
Abstract
Antimicrobial peptides (AMPs) take part in the immune system by mounting a first line of defense against pathogens. Recurrent structural and functional aspects are observed among peptides from different sources, particularly the net cationicity and amphipathicity. However, the membrane seems to be the key determinant of their action, either as the main target of the peptide action or by forming a barrier that must be crossed by peptides to target core metabolic pathways. More importantly, the specificity exhibited by antimicrobial peptides relies on the different lipid composition between pathogen and host cells, likely contributing to their spectrum of activity. Several mechanisms of action have been reported, which may involve membrane permeabilization through the formation of pores, membrane thinning or micellization in a detergent-like way. AMPs may also target intracellular components, such as DNA, enzymes and even organelles. More recently, these peptides have been shown to produce membrane perturbation by formation of specific lipid-peptide domains, lateral phase segregation of zwitterionic from anionic phospholipids and even the formation of non-lamellar lipid phases. To countermeasure their activity, some pathogens were successful in developing effective mechanisms of resistance to decrease their susceptibility to AMPs. The functional and integral knowledge of such interactions and the clarification of the complex interplay between molecular determinants of peptides, the pathogen versus host cells dichotomy and the specific microenvironment in which all these elements convene will contribute to an understanding of some elusive aspects of their action and to rationally design novel therapeutic agents to overcome the current antibiotic resistance issue.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Oral biodrug delivery using cell-penetrating peptide.
Source
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal University, Ismailia 415-22, Egypt.
Abstract
During the past few decades, the novel biotherapeutic agents such as peptides and proteins have been contributed to the treatment of several diseases. However, their oral absorption is significantly limited due to their poor delivery through the intestinal mucosa. Therefore, the feasible approaches are needed for improving the oral bioavailability of biodrugs. Recently, cell-penetrating peptides (CPPs) such as HIV-1 Tat, penetratin and oligoarginine are considered as a useful tool for the intracellular delivery of therapeutic macromolecules. Hence, it was expected that the ability of CPPs may be applicable to enhance the absorption of biodrugs through intestinal epithelial membrane. CPPs are likely to become powerful tools for overcoming the low permeability of therapeutic peptides and proteins through the intestinal membrane, the major barrier to their oral delivery. Further advantage of this promising strategy is that this successful intestinal absorption could be achieved by more convenient methodology, coadministration of CPP with drugs via intermolecular interaction among them. Hereafter, the further establishment of delivery system based on CPPs is required to realize the development of the oral forms of therapeutic peptides and proteins. The aim here is to introduce our vision focusing on oral biodrug delivery by the use of CPPs as potential peptide carrier in order to provide new information in the design and development of new oral delivery systems for novel biotherapeutics.
Copyright © 2011. Published by Elsevier B.V.
No comments:
Post a Comment