RSS[Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI].
Source
Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China.
Abstract
Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptidesynthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
- PMID:
- 22034819
- [PubMed - in process]
In vivo targeting of HER2-positive tumor using 2-helix affibody molecules.
Source
Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University, California, Stanford, CA, 94305-5344, USA.
Abstract
Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, (68)Ga and (18)F, with relatively short half-life (t (1/2) < 2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with (64)Cu or (111)In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I(2) oxidation. DOTA-MUT-DS was then radiolabeled with (64)Cu or (111)In to prepare the HER2 imaging probe ((64)Cu/(111)In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with (64)Cu or (111)In. Biodistribution study showed that tumor uptake value of (64)Cu or (111)In-labeled DOTA-MUT-DS was 4.66 ± 0.38 or 2.17 ± 0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n = 3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for (64)Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for (111)In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule Z(HER2:342) with (64)Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. (111)In-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with (64)Cu-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, (64)Cu/(111)In DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.
Early-stage retinal melatonin synthesis impairment in streptozotocin-induced diabetic wistar rats.
Source
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Abstract
PURPOSE:
Retinal melatonin synthesis occurs in the photoreceptor layer in a circadian manner, controlling several physiologic rhythmic phenomena, besides being the most powerful natural free radical scavenger. The purpose of the present work was to evaluate the diurnal profile of retinal melatonin content and the regulation of its synthesis in the retina of streptozotocin-induced diabetic rats.
METHODS:
Diabetes was induced in male Wistar rats (12 hour-12 hour light/dark cycle) with streptozotocin. Control, diabetic, and insulin-treated diabetic animals were killed every 3 hours throughout the light-dark cycle. Retinal melatonin content was measured by high-performance liquid chromatography, arylalkylamine N-acetyltransferase (AANAT) activity was analyzed by radiometric assay, Bmal1 gene expression was determined by qPCR, and cyclic adenosine monophosphate (cAMP) content was assessed by ELISA.
RESULTS:
Control animals showed a clear retinal melatonin and AANAT activity daily rhythm, with high levels in the dark. Diabetic rats had both parameters reduced, and the impairment was prevented by immediate insulin treatment. In addition, the Bmal1 expression profile was lost in the diabetic group, and the retinal cAMP level was reduced 6 hours after lights on and 3 hours after lights off.
CONCLUSIONS:
The present work shows a melatonin synthesis reduction in diabetic rats retinas associated with a reduction in AANAT activity that was prevented by insulin treatment. The Bmal1-flattened gene expression and the cAMP reduction seem to be responsible for the AANAT activity decrease in diabetic animals. The melatonin synthesis reduction observed in the pineal gland of diabetic rats is also observed in a local melatonin tissue synthesizer, the retina.
HNA-3a-specific antibodies recognize choline transporter-like protein-2peptides containing arginine, but not glutamine at Position 154.
Source
Platelet & Neutrophil Immunology Lab, BloodCenter of Wisconsin, Milwaukee, Wisconsin 53201-2178, USA. brian.curtis@bcw.edu
Abstract
BACKGROUND:
Antibodies specific for the neutrophil antigen HNA-3a cause severe, sometimes fatal transfusion-related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti-HNA-3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA-3a is carried on choline transporter-like protein-2 (CTL2) and that the HNA-3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA-3a epitope.
STUDY DESIGN AND METHODS:
Preliminary attempts to express recombinant full-length CTL2 (R154) recognized by anti-HNA-3a were unsuccessful. We therefore tested HNA-3a-specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154.
RESULTS:
Nine of 20 HNA-3a antibodies recognized the R154, but not the Q154 version of a cyclic 36-residue CTL2peptide (D131-K166). However, 11 others failed to distinguish between the two versions of this peptide.
CONCLUSION:
The findings provide direct evidence that R154 in the context of CTL2 D131-K166 is necessary to create the HNA-3a epitope but, in the context of cyclic CTL2 peptide D131-K166, is sufficient to detect only about one-half of the HNA-3a-specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti-HNA-3a in donated blood.
© 2011 American Association of Blood Banks.
Total synthesis of spiruchostatin B aided by an automated synthesizer.
Source
Department of Applied Chemistry, Tokyo Institute of Technology, Tokyo, Japan.
Abstract
The total synthesis of a natural product HDAC inhibitor, spiruchostatin B, was successfully achieved. A 5-step synthesis that included an asymmetric aldol reaction was carried out in an automated synthesizer to provide an (E)-(S)-3-hydroxy-7-thio-4-heptenoic acid segment that is the crucial structure of cysteine-containing, depsipeptidic natural products such as spiruchostatins, FK228, FR901375, and largazole for their inhibitory activity against HDACs.
Fully automated preparation and conjugation of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) with RGD peptide using a GE FASTlab™synthesizer.
Source
Cyclotron Research Center, Liege University, Liege, Belgium. dthonon@ulg.ac.be
Abstract
PURPOSE:
The aim of this work was to automate the radiosynthesis of [(18)F]SFB, a widely used reagent for the labeling of biomolecules with (18)F on a new generation commercial synthesis module (FASTLab™, GE Healthcare).
PROCEDURES:
Two synthesis approaches were implemented on this module: the classical "two-pot radiosynthesis" and the more recently described "one-pot" method.
RESULTS:
The "two-pot" approach affords [(18)F]SFB with a 42% decay-corrected yield in 57 min (n = 24) with a chemical purity sufficient to avoid an intermediate HPLC purification. The recently established "one-pot" method, afforded a product with a lower chemical purity, in the conditions used in this report. The lower d.c. yield obtained (32% (n = 15)) was related to the low (18)F labeling yields obtained in MeCN compared with DMSO. The subsequent conjugation step with a RGD (PRGD2) peptide was also successfully automated.
CONCLUSIONS:
The formulated [(18)F]FPRGD2 was obtained without any operator manipulation with a d.c. yield of 13% ± 3% (n = 13) in 130 min, a radiochemical purity >98% and a specific activity of 140 ± 40 TBq/mmol.
Diagnostic significance of measuring antibodies to cyclic type 3 muscarinic acetylcholine receptor peptides in primary Sjogren's syndrome.
Source
Department of Rheumatology and Immunology, People's Hospital Peking University, 11 Xizhimen South St, Beijing 100044, China.
Abstract
OBJECTIVE:
SS is an autoimmune disease characterized by salivary and lacrimal gland dysfunction leading to dry mouth (xerostomia) and dry eyes (xerophthalmia). Anti-muscarinic acetylcholine type-3 receptor (anti-M3R) autoantibodies have been shown to be a good serum marker in primary SS (pSS). The aim of this study was to assess the clinical correlations of anti-M3R-derived peptide antibodies in patients with pSS.
METHODS:
Sequences of the first to fourth cycle-M3R (c1M3R-c4M3R)-derived peptide was synthesized by a solid-phase technique on an Applied Biosytems Peptide Synthesizer. Synthesized cM3R peptide (cM3RP) was used as substrate in an ELISA to detect IgG anti-cM3RP antibodies in serum samples of patients and controls. The clinical and biological parameters of the diseases were also evaluated. The EULAR SS disease activity index (ESSDAI) score was used to measure disease activity in patients with primary SS.
RESULTS:
(i) Anti-c2M3RP antibodies were highly prevalent in pSS patients, and the titre is much higher than anti-c1,3,4M3RP antibodies. (ii) The prevalence of anti-c2M3RP antibodies in pSS, SLE, RA and healthy controls was 62.2, 7.1, 5.3 and 1.6%, respectively. The prevalence of anti-linear-2-M3RP antibodies in pSS, SLE and RA patients and healthy controls were 56.1, 20.0, 14.7 and 9.4%. (iii) The specificity of anti-c2M3RP antibodies was 95.1%, much higher than that of linear polypeptide (84.7%) for pSS diagnosis. (iv) In pSS patients, anti-c2M3RP positivity had significantly increased frequency in patients who were RF or ANA positive, and had several haematological abnormalities, such as leucopenia, anaemia and thrombocytopenia. Furthermore, the ESSDAI score was significantly higher in anti-c2M3RP-positive pSS patients (P < 0.05).
CONCLUSION:
Anti-c2M3RP antibody was highly specific for patients with pSS. The presence of anti-c2M3RP antibody in pSS indicates that c2M3RP may act as an autoantigen that may play a role in the pathogenesis of pSS.
Synthesis, gene-silencing activity and nuclease resistance of 3'-3'-linked double short hairpin RNA.
Source
Nippon Shinyaku Co., Ltd, Tsukuba, Ibaraki, Japan. h.toyoshima@po.nippon-shinyaku.co.jp
Abstract
To improve the nuclease resistance of siRNA while reducing its induction of an innate immune response and maintaining its biological activity for possible therapeutic application, we designed and synthesized a series of double short hairpin RNAs (dshRNAs). Each dshRNA consisted of two identical short hairpin RNAs (shRNAs) linked at their 3' ends by glycerol. The dshRNAs were synthesized on a glycerol-derivatized solid support from amidites with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group. Synthesis was carried out in a single run on a DNA/RNAsynthesizer, without the need for enzymatic ligation. The dshRNAs showed structure-dependent gene-silencing activity at the protein level, and dshRNAs in which the 3' end of the two sense regions were linked showed especially high activity. Inclusion of 2'-O-methyluridine residues in the loop region was associated with 1.6- to 2.4-fold lower induction of interferon-α than was siRNA, without loss of gene-silencing activity. dshRNA also showed higher exonuclease resistance than siRNA or canonical shRNA. Our studies provide a new approach to gene silencing based on the concept of linking the 3' end of the sense regions of two shRNA molecules to form a double shRNA.
Copyright © 2010 Elsevier Ltd. All rights reserved.
Facile synthesis of peptide nucleic acids and peptide nucleic acid-peptideconjugates on an automated peptide synthesizer.
Source
High-Field Magnetic Resonance Center, Max Planck Institute for Biological Cybernetics, Spemannstrasse 41, 72076 Tuebingen, Germany. rajendra.joshi@tuebingen.mpg.de
Abstract
Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence-specific manner. Therefore, they are widely used in molecular diagnosis of antisense-targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost-effective semi-automated synthesis of PNAs and PNA-peptide constructs on an automatedpeptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high-throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure.
Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.
Possible key residues that determine left gastric artery blood flow response to PACAP in dogs.
Source
Department of Traditional Chinese Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China. weimuxin@njmu.edu.cn
Abstract
AIM:
To determine the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on left gastric artery (LGA) flow and to unveil the structural or functional important sites that may be critical for discrimination of different receptor subtypes.
METHODS:
Peptides, including PACAP-27, PACAP-38, amino acid substituted PACAP-27 and C-terminus truncated analogues PACAP (27-38), were synthesized by a simultaneous multiple solid-phase peptide synthesizer. Flow probes of an ultrasound transit-time blood flowmeter were placed around the LGA of beagle dogs. When peptides were infused intravenously, the blood flow was measured.
RESULTS:
[Ala4, Val5]-PACAP-27 caused a concentration-dependent vasodepressor action which was similar to that caused by PACAP-27. The LGA blood flow response to [Ala4, Val5]-PACAP-27 was significantly higher than that to PACAP-27, which was similar to that to vasoactive intestinal polypeptide (VIP) at the same dose. [Ala6]-PACAP-27 did not increase the peak LGA flow. [Gly8]-PACAP-27 showed a similar activity to VIP. [Asn24, Ser25, Ile26]-PACAP-27 did not change the activity of peptides at all doses.
CONCLUSION:
NH2 terminus is more important to biological activity of peptides and specific receptor recognition than COOH-terminus.
[Detecting EGFR autoantibodies in serums of NSCLC patients with peptidearray].
Source
Department of Cellular and Molecular Biology, Beijing TB and Thoracic Tumor Research Institute, Beijing 101149, China.
Abstract
BACKGROUND AND OBJECTIVE:
Autoantibodies as new tumor markers may play an important role in the early diagnosis and evaluating the prognosis of lung cancer. In this study, we detect epidermal growth factor receptor (EGFR) autoantibodies using peptide array and screen the epitopes which are recognized by EGFR autoantibodies.
METHODS:
Peptide array covering the extracellular domain of EGFR protein was synthesized by a synthesizer(ASPSL) made by Intavis company. EGFR autoantibodies in the serums of non-small cell lung cancer patients was detected using peptide array.
RESULTS:
Six of 20 patients were found to have EGFR autoantibodies. The positive rate is 30%. Nine high frequency spots were found in the 6 positive patients and 8 high frequency spots clustered in the III and IV domains.
CONCLUSIONS:
These findings will offer new clues for the further studies of EGFR and EGFR autoantibodies.
Automated maskless photolithography system for peptide microarray synthesis on a chip.
Source
School of Chemical and Biological Engineering and School of Electrical Engineering and Computer Science, Seoul National University, Seoul 151-744, Korea.
Abstract
Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide arraysynthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptidesequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.
Solid-phase synthesis of the lipopeptide Myr-HBVpreS/2-78, a hepatitis B virus entry inhibitor.
Source
Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany.
Abstract
Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). Synthetic peptidesderived from the N-terminus of the large HBV envelope protein (L-protein) have been shown to efficiently block HBV entry. Myr-HBVpreS/2-78, the parent compound of these drugs, inhibits human HBV infection in vitro and in vivo. An efficient synthesis is required, as these peptides constitute a novel class of anti HBV drugs. Consequently, the solid phase synthesis of the N-terminal 77 amino acids of the viral L-protein was studied in detail. The peptide was N-terminally myristoylated to resemble the natural, postranslationally modified protein. The synthesis was monitored using the Fmoc cleavage pattern of the solid phase synthesis on a standard peptide synthesizer and by LC-MS analyses of the arising side products. "Difficult sequences" in the positions 42-47 of the peptide sequence complicate the efficient synthesis of the 77-mer peptide HBVpreS/2-78. Attempts were undertaken to optimize the synthesis by heating, double coupling or the use of pseudoproline dipeptides. HPLC-MS analyses showed that the efficiency of the synthesis could be increased best by temperature elevation. This resulted in a higher purity of the crude product after solid phase synthesis. It was possible to minimize the occurrence of side products due to the positive effects related to higher reaction temperature. In conclusion, the peptide is accessible by stepwise SPPS without the necessity of segment coupling.
- PMID:
- 20657392
- [PubMed - indexed for MEDLINE]
Automated synthesis, characterization and biological evaluation of [(68)Ga]Ga-AMBA, and the synthesis and characterization of (nat)Ga-AMBA and [(67)Ga]Ga-AMBA.
Source
Ernst Felder Laboratories, Bracco Research USA Inc., 305 College Road East, Princeton, NJ 08540, USA.
Abstract
Ga-AMBA (Ga-DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is a bombesin-like agonist with high affinity for gastrin releasing peptide receptors (GRP-R). Syntheses for (nat)Ga-AMBA, [(67)Ga]Ga-AMBA and [(68)Ga]Ga-AMBA were developed. The preparation of HPLC-purified and Sep-Pak purified [(68)Ga]Ga-AMBA were fully automated, using the built-in radiodetector of the Tracerlab FX F-N synthesizer to monitor fractionated (68)Ge/(68)Ga generator elution and purification. The total synthesis time, including the fractional elution of the generator, was 20 min for Sep-Pak purified material and 40 min for HPLC-purified [(68)Ga]Ga-AMBA. Both [(67)Ga]Ga-AMBA and [(177)Lu]Lu-AMBA showed comparable high affinity for GRP-R in the human prostate cancer cell line PC-3 in vitro (k(D)=0.46+/-0.07; 0.44+/-0.08 nM), high internalization (78; 77%) and low efflux from cells at 2 h (2.4+/-0.7; 2.9+/-1.8%). Biodistribution results in PC-3 tumor-bearing male nude mice showed comparable uptake for [(177)Lu]Lu-, [(111)In]In-, [(67)Ga]Ga- and [(68)Ga]Ga-AMBA.
Copyright 2010 Elsevier Ltd. All rights reserved.
Automated 'X-Y' robot for peptide synthesis with microwave heating: application to difficult peptide sequences and protein domains.
Source
Faculty of Life Sciences, IGM, Section for Bioorganic Chemistry, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Abstract
Precise microwave heating has emerged as a valuable method to aid solid-phase peptide synthesis (SPPS). New methods and reliable protocols, as well as their embodiment in automated instruments, are required to fully use this potential. Here we describe a new automated robotic instrument for SPPS with microwave heating, report protocols for its reliable use and report the application to the synthesis of long sequences, including the beta-amyloid 1-42 peptide. The instrument is built around a valve-free robot originally developed for parallel peptide synthesis, where the robotic arm transports reagents instead of pumping reagents via valves. This is the first example of an 'X-Y' robotic microwave-assisted synthesizer developed for the assembly of long peptides. Although the instrument maintains its capability for parallel synthesis at room temperature, in this paper, we focus on sequential peptide synthesis with microwave heating. With this valve-free instrument and the protocols developed for its use, fast and efficient syntheses of long and difficultpeptide sequences were achieved.
Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.
Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-alpha production inhibitors.
Source
Nara Research & Development Center, Santen Pharmaceutical Co., Ltd, Ikoma, Nara, Japan. hiroshi.enomoto@santen.co.jp
Abstract
A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-alpha production. We focused on the 1,1-substituted moiety (R(1) and R(2)) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-alpha production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.
Copyright 2010 Elsevier Ltd. All rights reserved.
Evaluating the role of rheumatoid factors for the development of rheumatoid arthritis in a mouse model with a newly established ELISA system.
Source
Department of Pathogenomics, Ehime University Graduate School of Medicine, Ehime, Japan.
Abstract
Enzyme-linked immunosorbent assays (ELISA) have been widely used to determine quantitatively autoantibodies. However, the processes for the purification and immobilization of antigens in conventional ELISA methods include multiple steps, which have hampered the application for screening of autoantibodies. Here, we have developed a novel ELISA system using the plates pre-coated with glutathione casein to capture recombinant proteins fused to N-terminal glutathione S-transferase (GST). The GST-fused proteins were synthesized with the wheat germ cell-free protein production system. Thus, the present system combined the GST-capture ELISA with the cell-free protein production system, which allowed immobilization of the recombinant proteins with one-step purification. Using this ELISA method, we determined whether rheumatoid factors (RF), which have been considered as one of the representative disease-specific autoantibodies for rheumatoid arthritis (RA), were genetically associated with severity of arthritis in a mouse model for RA, MRL/Mp-lpr/lpr (MRL/lpr). GST-fused human IgG1-Fc (GST-Fc), synthesized with the robotic proteinsynthesizer, were used as reactants for RF. Serum samples for RF were prepared from 11 lines of a recombinant inbred mouse strain, MXH/lpr, which was established from intercrosses between MRL/lpr and non-arthritic C3H/HeJ-lpr/lpr (C3H/lpr) strains, composed of a different genomic recombination derived from the parental strains in each line. A correlation of RF titers with the severity of the arthritis in these lines was not significant, indicating genetic dissociation of RF from arthritis and that RF is not necessarily required for the development of RA. The present method may provide high-throughput screening for determining the disease-specific autoantibodies in autoimmune diseases.
Synthesis, radiolabeling and biological evaluation of a neutral tripeptide and its derivatives for potential nuclear medicine applications.
Source
Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Kolkata-32, West Bengal 700032, India. susmita2506@yahoo.com
Abstract
Peptides are important regulators of growth and cellular functions not only in normal tissue but also in tumors. So they are becoming radioligands of increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. So development of new peptide radiopharmaceuticals is becoming one of the most important areas in nuclear medicine research. A small tripeptide derivative NH(2)PhePheCys was synthesized by Fmoc solid phase peptide synthesis using an automated synthesizer. The oxidized form, i.e. NH(2)PhePheCysCysPhePheNH(2,) was also prepared by iodine oxidation method from NH(2)PhePheCys(ACM). The ligands were analyzed by HPLC and mass spectroscopy. They were radiolabeled with (99m)Tc using SnCl(2). In vitro analytical studies and biological characterizations were performed using the peptide radiopharmaceuticals. Images taken under gamma camera showed very high uptake in the liver, lung and spleen. Significant uptake was also observed in bone marrow and brain for (99m)Tc-NH(2)PhePheCys. Metabolites were produced in vivo when the radiopharmaceuticals were injected intravenously and were identified from rat brain and liver homogenate studies. Clearance through kidney did not show any evidence of breaking of the labeled compounds and formation of free (99m)Tc. Radiopharmaceuticals prepared using tripeptide and hexapeptide ligands were transported into the brain through blood brain barrier depending on the size and sequence characteristics. Using this property of peptide new derivatives can be prepared to develop (99m)Tc radiopharmaceuticals for imaging normal brain tissues as well as for diagnosing various brain disorders.
Isolation, identification, and characterization of small bioactive peptides from Lactobacillus GG conditional media that exert both anti-Gram-negative and Gram-positive bactericidal activity.
Source
University of Maryland School of Medicine, Mucosal Biology Research Center, Health Science Facility II, 20 Penn St, Baltimore, MD 21201, USA. rlu@mbrc.umaryland.edu
Abstract
OBJECTIVES:
Diarrheal diseases remain a major human plague that still claim millions of lives every year. Probiotics, including Lactobacillus GG (LGG), are known to have a beneficial effect on diarrheal diseases, but their mechanism of action has not yet been completely established. Therefore, the main objective of this work was to identify and characterize moieties elaborated by LGG that exert antibacterial activity.
MATERIALS AND METHODS:
Lactobacillus GG conditional media was subjected to liquid chromatography/mass spectrometry. The identified peptides were synthesized by Symphony peptide synthesizer and purified by HPLC using Dynamax reverse-phase C18 column. Using A600 measurement and tested for their antibacterial activity.
RESULTS:
We identified 7 small peptides from LGG cultured media, 2 of which are NPSRQERR and PDENK, retained the antibacterial activity detected with LGG conditional media. The antibacterial activity was exerted against both Gram-negative (Escherichia coli EAEC 042 and Salmonella typhi) and, with less potency, Gram-positive (Staphylococcus aureus) bacteria.
CONCLUSIONS:
Lactobacillus GG elaborates small peptides showing various degrees of antibacterial activity. NPSRQERR showed the most potent antibacterial effect that was detected both in Gram-negative and Gram-positive microorganisms. These synthetic peptides may represent novel tools for the treatment of bacterial infectious diseases.
Fully automated synthesis procedure of 4-[18F]fluorobenzaldehyde by commercial synthesizer: amino-oxi peptide labelling prosthetic group.
Source
IBB-CNR, Edificio 10, Via Pansini 5, Napoli, Italy. antonio.speranza@hotmail.com
Abstract
Automatic synthesis of 4-[18F]fluorobenzaldehyde has been developed by a commercially available TRACERlab FX(F-N) synthesis module to be used as prosthetic group for amino-oxy functionalized peptide labelling in clinical routine application. In addition a handmade purification device (HPD) has been setup to perform automatic cartridge purification as well as to back-up the reactor where one-pot synthesis is not applicable. Cartridges for solid phase extraction such as C18, C8, phenyl has been tested to best perform purification as well as activity recovery. Radiochemical yield (RCY) at end of synthesis (EOS) was in average 67% after about 45 min (90% decay corrected at EOB). The RCY of the entire procedure was 54% with a radiochemical purity above 99%.
- PMID:
- 19443231
- [PubMed - indexed for MEDLINE]
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