Cell Wall Antibiotics Provoke Accumulation of Anchored mCherry in the Cross Wall of Staphylococcus aureus.
Source
Microbial Genetics, University of Tübingen, Tübingen, Germany.
Abstract
A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SP(lip) and the -YSIRK motif SP(sasF). mCh-hybrids supplied with the +YSIRK motif SP(lip) were always expressed higher than those with -YSIRK motif SP(sasF). To study the location of the anchoring process and also the influence of SP type, mCh-cw was supplied on the one hand with +YSIRK motif (mCh-cw1) and the other hand with -YSIRK motif (mCh-cw2). MCh-cw1 preferentially localized at the cross wall, while mCh-cw2 preferentially localized at the peripheral wall. Interestingly, when treated with sub-lethal concentrations of penicillin or moenomycin, both mCh-cw1 and mCh-cw2 were concentrated at the cross wall. The shift from the peripheral wall to the cross wall required Sortase A (SrtA), as in the srtA mutant this effect was blunted. The effect is most likely due to antibiotic mediated increase of free anchoring sites (Lipid II) at the cross wall, the substrate of SrtA, leading to a preferential incorporation of anchored proteins at the cross wall.
- PMID:
- 22253886
- [PubMed - in process]
- PMCID: PMC3254641
Small surfactant-like peptides can drive soluble proteins into active aggregates.
Abstract
ABSTRACT:
BACKGROUND:
Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property.
RESULTS:
In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6) were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used), Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core.
CONCLUSIONS:
This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in vivo, and can be explored for production of functional biopolymers with detergent or other interfacial activities.
- PMID:
- 22251949
- [PubMed - as supplied by publisher]
Characterization of channel-forming peptide nanostructures.
Abstract
We have prepared fluorescent analogs of known ion-channel-forming synthetic peptide nanostructures. These analogs were designed as probes to gain insight about the mechanism by which self-assembling amphiphilic peptides interact with lipid membranes. Conformational studies demonstrated that the labeled analogs retain their propensity to adopt a strong helical conformation in 2,2,2-trifluoroethanol and lipid bilayers. Attenuated total reflectance results indicated that the fluorescent peptide nanostructures are under an incorporation equilibrium between two forms, adsorbed at the surface or incorporated within the bilayer, similar to their unlabeled counterparts. However, when using a HeLa mimicking membrane, the proportion of peptide nanostructures in the transmembrane orientation decreases significantly. Finally, we were able to show by confocal microscopy studies that fluorescent analogs internalized into HeLa cells and localized into both the membranes of inner organelles and the cell membrane.
Copyright © 2011 Elsevier B.V. All rights reserved.
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.
Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing.
Source
Center for Bio/Molecular Science and Engineering, Code 6900, ‡Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory , Washington DC 20375, United States.
Abstract
The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.
CD26/DPP-4 inhibition recruits regenerative stem cells via stromal cell-derived factor-1 and beneficially influences ischaemia-reperfusion injury in mouse lung transplantation.
Source
Division of Thoracic Surgery, University Hospital Zurich, Zurich, Switzerland.
Abstract
OBJECTIVESThe CD26 antigen is a transmembrane glycoprotein that is constitutively expressed on activated lymphocytes and in pulmonary parenchyma. This molecule is also identified as dipeptidyl peptidase-4 (DPP-4) that cleaves a host of biologically active peptides. Here, we aimed to identify an important substrate of CD26/DPP-4-stromal cell-derived factor-1 (SDF-1/CXCL12)-as a key modulator for stem-cell homing together with its receptor CXCR4 in response to ischaemic injury of the lung.METHODSOrthotopic single lung transplantation (Tx) was performed between syngeneic C57BL/6 mice. Inhibition of CD26/DPP-4 activity in recipients was achieved using vildagliptin (10 mg/kg, every 12 h) subcutaneously, and 6 h ischaemia time was applied prior to implantation. Forty-eight hours after Tx, lung histology, SDF-1 levels (enzyme-linked immunosorbent assay) in lung, spleen and plasma, and expression of the SDF-1 receptor CXCR4 in blood and lung were assessed. Homing of regenerative progenitor cells to the transplanted lung was evaluated using fluorescent-activated cell sorting.RESULTSCompared with untreated lung transplanted mice, systemic DPP-4 inhibition of Tx recipients resulted in an increase in protein concentration of SDF-1 in plasma, spleen and lung. Concordantly, the frequency of cells bearing the SDF-1 receptor CXCR4 rose significantly in the circulation and also in the lungs of DPP-4-inhibited recipients. We found co-expression of CXCR4/CD34 in the grafts of animals treated with vildagliptin, and the stem-cell markers Flt-3 and c-kit were present on a significantly increased number of cells. The morphology of grafts from DPP-4 inhibitor-treated recipients revealed less alveolar oedema when compared with untreated recipients.CONCLUSIONSTargeting the SDF-1-CXCR4 axis through CD26/DPP-4 inhibition increased the intragraft number of progenitor cells contributing to the recovery from ischaemia-reperfusion lung injury. Stabilization of endogenous SDF-1 is achievable and may be a promising strategy to intensify sequestration of regenerative stem cells and thus emerges as a novel therapeutic concept.
- PMID:
- 22219460
- [PubMed - as supplied by publisher]
Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines.
Source
Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore, Singapore.
Abstract
Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9(IRES-EGFP)) or the F2A (Sox9(F2A-EGFP)) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome 'skipping' mechanism. To investigate if the discrepancy in the 'skipping' process was locus-dependent, we further analyzed the FLAG(3)-Bapx1(F2A-EGFP) mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the 'skipping' mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1(F2A-Cre-F2A-EGFP)). While the 'skipping' was highly efficient between Bapx1 and Cre, the 'skipping' between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the 'skipping' at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides.
A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity.
Source
Division of Nephrology and Clinical Immunology, University Hospital of the RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany.
Abstract
The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H(14)CO(3)(-) into synthetic peptides and subsequent quantification using liquid scintillation counting. We present a nonradioactive assay using a fluorescein isothiocyanate-labeled short peptide that can be readily detected in its unmodified and γ-glutamyl carboxylated form by reversed-phase HPLC. This method offers a convenient alternative to the established radioactive labeling techniques.
Copyright © 2011 Elsevier Inc. All rights reserved.
Remodeling of hepatic metabolism and hyperaminoacidemia in mice deficient in proglucagon-derived peptides.
Source
Corresponding author: Yoshitaka Hayashi, hayashiy@riem.nagoya-u.ac.jp.
Abstract
Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon-green fluorescent protein (gfp) knock-in mice (Gcg(gfp/gfp): GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action.
Baupain, a plant cysteine proteinase that hinders thrombin-induced human platelet aggregation.
Source
Universidade Federal de São Paulo Departamento de Bioquímica Rua Três de Maio 100, 04044-020 São Paulo, SP, Brazil. olivaml.bioq@epm.br.
Abstract
Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.
- PMID:
- 22185503
- [PubMed - as supplied by publisher]
A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides.
Source
Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien 97401, Taiwan.
Abstract
Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84-93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis.
Copyright © 2011 Elsevier B.V. All rights reserved.
Short peptides as biosensor transducers.
Source
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, via Giorgieri 1, 34127, Trieste, Italy.
Abstract
This review deals with short peptides (up to 50 amino acids) as biomimetic active recognition elements in sensing systems. Peptide-based sensors have been developed in recent years according to different strategies. Syntheticpeptides have been designed on the basis of known interactions between single or a few amino acids and targets, with attention being paid to the presence of peptide motifs known to allow intermolecular self-organization of the sensingpeptides over the sensor surface. Sensitive and sophisticated sensors have been obtained in this way, but the use of designed peptides is limited by severe difficulties in their in silico design. Short peptides from random phage display have been selected in a random way from large, unfocussed, and often preexisting and commercially available phage display libraries, with no design elements. Such peptides often perform better than antibodies, but they are difficult to select when the target is a small molecule because of the need to immobilize it with considerable modifications of its structure. Artificial, miniaturized receptors have been obtained from the reduction of the known sequence of a natural receptor down to a synthesizable and yet stable one. Alternatively, binding sites have been created over a designed, stable peptide scaffold. Short peptides have also been used as active elements for the detection of their own natural receptors: pathogenic bacteria have been detected with antimicrobial and cell-penetrating peptides, but key challenges such as detection of bacteria in real samples, improved sensitivity, and improved selectivity have to be faced. Peptide substrates have been conjugated to fluorescent quantum dots to obtain disposable sensors for protease activity with high sensitivity. Ferrocene-peptide conjugates have been used for electrochemical sensing of protease activity.
Synergistic effects of conjugating cell penetrating peptides and thiomers on non-viral transfection efficiency.
Source
Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck, Innrain 52, Josef Möller Haus, 6020 Innsbruck, Austria.
Abstract
Nanoparticles generated by complex coacervation of plasmid DNA (pDNA) and modified chitosans namely chitosan-thioglycolic acid (TGA) conjugate and chitosan-HIV-1 Tat peptide conjugate were evaluated as gene delivery systems. In order to optimize transfection efficiency, chitosan-HIV-1 Tat peptide conjugate was combined with chitosan-TGA before its complexation with pDNA. Particle size and zeta potential measurements were performed to characterize the generated nanoparticles. The nanoparticles transfection efficiencies were assessed by exploitation of the greenfluorescent protein (GFP) reporter gene. HEK293 cells were incubated for 24 h with the nanoparticles and the GFP positive cells were observed by fluorescence microscopy. The nanoparticles in the size range of 200-300 nm could transfect HEK293 cells as a model cell line with different transfection efficiencies. Unlike chitosan-TGA, chitosan-HIV-1 Tat peptide led to increased zeta potential of nanoparticles as compared to unmodified chitosan. The transfection efficiency of the nanoparticles generated by combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was comparatively higher than that of the nanoparticles generated by either chitosan-TGA or the combination of chitosan-HIV-1 Tat peptide with unmodified chitosan. After 72 h of incubation, the combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was found to be 7.12- and 67.37 times more efficient than unmodified chitosan and pDNA alone, respectively and showed a synergistic effect in transfection of pDNA into the cells. Moreover, none of the nanoparticles showed any severe cytotoxicity. Accordingly, this strategy might result in a potent carrier for gene delivery.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Detection of Edwardsiella tarda by fluorometric or biosensor methods using a peptide ligand.
Source
Department of Chemistry, Sejong University, Seoul 143-747, Republic of Korea.
Abstract
In this study, we identified a peptide ligand for Edwardsiella tarda from a phage peptide library and tested two approaches for sensitive detection of the bacteria with the peptide labeled with fluorescein or biotin. At first, thefluorescent peptide was proved to be advantageous in the fluorescence polarization (FP) assay because sensitivity of the assay is maximized when a fluorophore is linked to a small molecule. The FP assay using the fluorescent peptide enabled detection of E. tarda in a range from 5.2×10(3) to 2.1×10(5) cells. Second, we devised a new assay method using a quartz crystal microbalance (QCM) biosensor connected to a filter module. When a mixture of E. tarda and the biotinylated peptide was injected into the filter module, the E. tarda-peptide complex was separated from the unbound peptide by a filter and detected with a streptavidin-coated QCM sensor chip. On injection of samples containing the biotinylated peptide and E. tarda, concentration-dependent frequency change was observed in a range from 8×10(2) to 8×10(6) cells. The two approaches are expected to facilitate development of assay methods using other bacteria-bindingpeptides.
Copyright © 2011 Elsevier Inc. All rights reserved.
Screening of soy and milk protein hydrolysates for their ability to activate the CCK1 receptor.
Source
Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium; Laboratory of Food Chemistry and Human Nutrition, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Abstract
The cholecystokinin receptor-type 1 (CCK1R) is a G-protein coupled receptor localized in the animal gastrointestinal tract. Receptor activation by the natural peptide ligand CCK leads to a feeling of satiety. In this study, hydrolysates from soy and milk proteins were evaluated for their potential to activate the CCK1R, assuming that bioactive peptides with a satiogenic effect can be used as an effective therapeutic strategy for obesity. Different protein hydrolysates were screened with a cell-based bioassay, which relies on the generation of a fluorescent signal upon receptor activation. Fluorescence was monitored using a fluorescence plate reader and confocal microscopy. Results from the fluorescence plate reader were biased by background autofluorescence of the protein hydrolysate matrices, which makes the fluorescence plate reader inappropriate for the evaluation of complex formulations. Measurements with the confocal microscope resulted in reliable and specific results. The latter approach showed that the gastrointestinal digested 7S fraction of soy protein demonstrates CCK1R activity.
Copyright © 2011 Elsevier Inc. All rights reserved.
In vivo characterization of a new abdominal aortic aneurysm mouse model with conventional and molecular magnetic resonance imaging.
Source
Translational and Molecular Imaging Institute, Mount Sinai School of Medicine, 1428 Madison Avenue, New York, NY 10029, USA.
Abstract
OBJECTIVES:
The goal of this study was to use noninvasive conventional and molecular magnetic resonance imaging (MRI) to detect and characterize abdominal aortic aneurysms (AAAs) in vivo.
BACKGROUND:
Collagen is an essential constituent of aneurysms. Noninvasive MRI of collagen may represent an opportunity to help detect and better characterize AAAs and initiate intervention.
METHODS:
We used an AAA C57BL/6 mouse model in which a combination of angiotensin II infusion and transforming growth factor-β neutralization results in AAA formation with incidence of aortic rupture. High-resolution, multisequence MRI was performed to characterize the temporal progression of an AAA. To allow molecular MRI of collagen, paramagnetic/fluorescent micellar nanoparticles functionalized with a collagen-binding protein (CNA-35) were intravenously administered. In vivo imaging results were corroborated with immunohistochemistry and confocal fluorescence microscopy.
RESULTS:
High-resolution, multisequence MRI allowed the visualization of the primary fibrotic response in the aortic wall. As the aneurysm progressed, the formation of a secondary channel or dissection was detected. Further analysis revealed a dramatic increase of the aortic diameter. Injection of CNA-35 micelles resulted in a significantly higher magnetic resonance signal enhancement in the aneurysmal wall compared with nonspecific micelles. Histological studies revealed the presence of collagen in regions of magnetic resonance signal enhancement, and confocal microscopy proved the precise co-localization of CNA-35 micelles with type I collagen. In addition, in a proof-of-concept experiment, we reported the potential of CNA-35 micelles to discriminate between stable AAA lesions and aneurysms that were likely to rapidly progress or rupture.
CONCLUSIONS:
High-resolution, multisequence MRI allowed longitudinal monitoring of AAA progression while the presence of collagen was visualized by nanoparticle-enhanced MRI.
Copyright © 2011 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
Effects of Tat peptide on intracellular delivery of arsenic trioxide albumin microspheres.
Source
aCollege of Biomedical Engineering, South-Centeral University for Nationalities, Wuhan bDepartment of Immunology, Guangxi Medical University, Nanning, China.
Abstract
The current study was designed to evaluate the ability of cell-penetrating peptides to deliver arsenic trioxide albumin microspheres (AsAMs) into bladder cancer cells. The transactivating transcriptional activator (Tat) peptide was labeled with the enhanced green fluorescent protein (EGFP) using eukaryotic vector construction and fusion gene expression techniques. Arsenic trioxide albumin mirospheres were prepared using the chemical crosslink and solidification method. The conjugate, Tat-EGFP-As2O3-AMs (TEAsAMs), was synthesized using the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio) propionate and verified by electrophoresis under reducing conditions and fluorescence microscopy. The intracellular delivery of TEAsAMs was evaluated by laser confocal microscopy and transmission electron microscopy. The arsenic content in the bladder cancer EJ cells was assayed to evaluate the efficiency of delivery. Gene sequencing showed that the pET-Tat-EGFP expression vector was constructed successfully. The expression of the Tat-EGFP fusion protein was verified by matrix-assisted laser desorption/ionization-time of flight analysis, and the protein was transduced into cell cytoplasm as observed under a fluorescence microscope. Electrophoresis under reducing conditions demonstrated the covalent linkage between Tat-EGFP and AsAMs. Under a laser confocal microscope and a transmission electron microscope, TEAsAMs surrounded by green fluorescence were shown to enter the cells faster than EGFP-As2O3-AMs, with an increase in the intracellular arsenic content being observed in cells treated with TEAsAMs compared with those treated with EGFP-As2O3-AMs. These results suggest that Tat peptide promotes the cellular uptake of large albumin microspheres with encapsulated arsenicals.
[Influence of SGHWJN particles on mediators of inflammation in esophageal tissues of rat with reflux esophagitis].
Source
Institute of Combined Traditional Chinese and Western Medicine, Lanzhou University, Lanzhou 730000, China. qiyf1999@126.com
Abstract
OBJECTIVE:
To study the influence of SGHWJN particles on inflammation and the mediators of inflammation in esophageal tissues of rat with reflux esophagitis.
METHOD:
Fifty SD rats were randomly divided into 5 groups, namely, a control group, a sham-operated group, a model group, a SGHWJN particles group and a PPI group. Reflux esophagitis was induced by adopting partial pyloric ligation plus cardiomyotomy. One week later, the rats were orally administered twice daily for 28 days. Pathological changes of esophagus mucous membrane were evaluated by using HE staining and Harry S. Cooper's method in every groups. MDA and SOD contents in esophageal tissues were measured by colorimetric method. Expression of TNF-alpha in esophageal tissues were examined by real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-FQ-PCR) with SYBR Green.
RESULT:
Model group, esophageal inflammation scores, expression of TNF-alpha in esophageal tissues and MDA contents compared with the normal group and sham operation group were significantly higher (P < 0.05). SOD contents in the esophageal tissues of the model group was significantly lower than that of control group and sham-operated group (P < 0.05). SGHWJN particles group and PPI group of esophageal tissue inflammation scores, expression of TNF-a in esophageal tissues and MDA levels than those in model group decreased significantly (P < 0.05). SOD content was significantly higher than that of model group (P < 0.05), SGHWJN particles group and PPI group showed no statistically significant difference between the above-mentioned indicators. The above-mentioned indicators showed no statistically significant difference between the normal group and sham-operated group. MDA content and expression of TNF-alpha in esophageal tissue was positively correlated with inflammatory scores of model group (r = 0.813). Model group esophageal tissue SOD content and inflammation scores were negatively correlated (r = -0.847). Esophageal tissue SOD levels were negatively correlated with MDA levels (r = -0.863).
CONCLUSION:
SGHWJN particles can effectively inhibit inflammation in rat with reflux esophagitis through regulating TNF-alpha, SOD and MDA.
- PMID:
- 22121815
- [PubMed - indexed for MEDLINE]
Immobilization of aptamer-based molecular beacons onto optically-encoded micro-sized beads.
Source
School of Chemical and biological Engineering, Seoul National University, Seoul 151-747, Korea.
Abstract
This paper presents a method for the novel immobilization of aptamer-based molecular beacons (apta-beacons) onto optically-encoded micro-sized beads (apta-beacon beads). To immobilize apta-beacons onto flourescently-encoded micro-sized beads, core-shell type beads containing a fluorescent dye-encoded core and apta beacon-coupled shell were prepared. The fluorescent dye-encoded beads were prepared from TentaGel resins by coupling RITC to the amino groups of the core region, after partial protection of amino groups with Fmoc-OSu in a diffusion-controlled manner. After deprotection of the Fmoc-amino groups, FITC-coupled molecular beacons (MBs) were immobilized to the beads together with a quencher by covelent bonding. Briefly, aspartic acid (Asp) was coupled to the shell part of the beads. Then, the quencher was coupled to the N-terminal amino group of Asp and the MBs were coupled to the side chain carboxyl group. In a model study, thrombin was directly detected using this apta-beacon bead method. The thrombin-bound apta-beacon beads were easily recognized by the appearance of fluorescence without any further labeling step.
- PMID:
- 22121695
- [PubMed - indexed for MEDLINE]
Molecular imaging of breast tumors using a near-infrared fluorescently labeled clusterin binding peptide.
Source
Receptors, Signaling and Proteomics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal (Quebec) Canada H4P 2R2; Bio-NMR and Protein Research Group, Biotechnology Research Institute, 1200 Montreal Road, M-54, Ottawa, Ontario, K1A 0R6, Canada.
Abstract
Several reports have shown that secreted clusterin (sCLU) plays multiple roles in tumor development and metastasis. Here, we report on a 12-mer sCLU binding peptide (designated P3378) that was identified by screening a phage-display peptide library against purified human sCLU. Differential resonance perturbation (DRP) nuclear magnetic resonance (NMR) using P3378 and a scrambled control peptide (designated P3378R) confirmed the P3378-sCLU interaction and demonstrated that it was sequence specific. P3378 and P3378R peptides were conjugated to an Alexa680 near infrared fluorophore (NIRF) and assessed for their tumor homing abilities in in vivo time-domain fluorescence optical imaging experiments using living 4T1 tumor bearing BALB/c mice. When injected in separate animals, both peptidesaccumulated at the tumor site, however the NIRF-labeled P3378 peptide was retained for a significant longer period of time than the P3378R peptide. Similar observations were made after simultaneously injecting the same tumor-bearing animal with a peptide mixture of P3378 DyLight (DL)680 and the P3378R-DL800. Co-injection of P3378-DL680 with excess unlabeled P3378 blocked tumor accumulation of fluorescent signal while excess P3378R control peptide did not confirming the sequence specificity of the tumor accumulation. Finally, ex vivo fluorescence microscopy of these tumors confirmed the presence of P3378-DL680 in the tumor and its colocalization with CLU. These results confirm the tumor targeting specificity of the P3378 CLU-binding peptide and suggest its usefulness for the in vivo monitoring of solid tumors secreting detectable levels of CLU. © 2011 Wiley-Liss, Inc.
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