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BBA - General Subjects (v.1800, #5)
Radiolabelled proteins for positron emission tomography: Pros and cons of labelling methods by Vladimir Tolmachev; Sharon Stone-Elander (pp. 487-510).
Dynamic biomedical research is currently yielding a wealth of information about disease-associated molecular alterations on cell surfaces and in the extracellular space. The ability to visualize and quantify these alterations in vivo could provide important diagnostic information and be used to guide individually-optimized therapy. Biotechnology can provide proteinaceous molecular probes with highly specific target recognitions. Suitably labelled, these may be used as tracers for radionuclide-based imaging of molecular disease signatures. If the labels are positron-emitting radionuclides, the superior resolution, sensitivity and quantification capability of positron emission tomography (PET) can be exploited.This article discusses different approaches to labelling proteins with positron-emitting nuclides with suggestions made depending on the biological features of the tracers.Factors such as matching biological and physical half-lives, availability of the nuclide, labelling yields, and influences of labelling on targeting properties (affinity, charge and lipophilicity, cellular processing and retention of catabolites) should be considered when selecting a labelling strategy for each proteinaceous tracer.The labelling strategy used can make all the difference between success and failure in a tracer application. This review emphasises chemical, biological and pharmacological considerations in labelling proteins with positron-emitting radionuclides.
Keywords: Positron emission tomography; Radionuclide; Labelling; Peptide; Protein; Antibody
Influence of SelS gene silence on β-Mercaptoethanol-mediated endoplasmic reticulum stress and cell apoptosis in HepG2 cells by Shaoqing Du; Hongmei Liu; Kaixun Huang (pp. 511-517).
Selenoprotein S (SelS), a transmembrane selenoprotein, may be related to the response of endoplasmic reticulum (ER) stress. In this report, the influence of selenite supplementation and SelS gene silence on β-mercaptoethanol (β-ME)-mediated ER stress and cell apoptosis in HepG2 cells were examined. The results showed that SelS protein expression was markedly increased by 10mM β-ME and 100nM sodium selenite in HepG2 cells. GRP78 protein level was significantly increased after treatment with 10mM β-ME in HepG2 cells, which suggested that β-ME was also an ER stress inducer. Meanwhile, β-ME (10mM) was found to induce cell apoptosis, which was alleviated obviously when cells were pretreated with 100nM selenite before exposure to β-ME. Moreover, the suppression of SelS gene by siRNA could aggravate HepG2 cell apoptosis induced by β-ME significantly. In conclusion, these results suggested that β-ME, also an ER stress agent, could induce cell apoptosis, and SelS may play an important role in protecting cells from apoptosis induced by ER stress in HepG2 cells.
Keywords: Selenium; Selenoprotein S; β-mercaptoethanol; siRNA; Apoptosis
PKC activation contributes to caspase-mediated eIF2α phosphorylation and cell death by Pendyala Pushpanjali; Kolluru V.A. Ramaiah (pp. 518-525).
Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α), involved in translation, promotes cell suicide or survival. Since multiple signaling pathways are implicated in cell death, the present study has analyzed the importance of PKC activation in the stress-induced eIF2α phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda ( Sf9) and in their extracts.Cell death is analyzed by flow cytometry. Caspase activation is measured by Ac-DEVD-AFC hydrolysis and also by the cleavage of purified recombinant PERK, an endoplasmic reticulum-resident eIF2α kinase. Status of eIF2α phosphorylation and cytochrome c levels are analyzed by western blots.PMA, an activator of PKC, does not promote cell death or affect eIF2α phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2α phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2α phosphorylation in cell extracts. These changes are mitigated more efficiently by caspase inhibitor, z-VAD-fmk, than by calphostin, an inhibitor of PKC. In contrast, tunicamycin-induced eIF2α phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.While caspase activation is a cause and consequence of eIF2α phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2α phosphorylation, and cell death in Sf9 cells.Caspases can activate multiple signaling pathways to enhance cell death.
Keywords: Abbreviations; PMA; Phorbol myristate acetate; PKC; Protein kinase C; UV; Ultraviolet; ER; Endoplasmic reticulum; eIF2α; alpha-subunit of eukaryotic initiation factor-2; z-VAD-fmk; Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone; PERK; ER-resident eIF2α kinase; Sf9; Spodoptera frugiperdaeIF2α phosphorylation; Apoptosis; Sf9; cell; PKC; eIF2α kinases
A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity by Ajit Kumar; Mala Rao (pp. 526-536).
Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (AsparticProteaseInhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.The peptide has an amino acid sequence N-Ile1-Cys2-Glu3-Ala4-Glu5-His6-Lys7-Trp8-Gly9-Asp10-Tyr11-Leu12-Asp13-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I50=600nM and Ki=510nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)2]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k5=8.7±1×10−3s−1 and k6=7.3±0.6×10−5s−1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.
Keywords: Abbreviations; Af; ChiB1; chitinase from; Aspergillus fumigatus; API; aspartic protease inhibitor; ChiA; chitinase A; [p-NP-(GlcNAc); 2; ]; p-nitrophenyl-; N,N; ′-diacetyl-β-chitobioside; OPTA; o-phthalaldehyde; rp-HPLC; reverse phase high performance liquid chromatography; Sc; CTS1; chitinase from; Saccharomyces cerevisiae; TFA; trifluoroacetate; TNBS; 2,4,6-trinitrobenzenesulphonic acid; v; 0; initial velocity; v; s; steady-state velocity; WRK; Woodward's reagent K,; N; -Ethyl-5-phenylisoxazolium-3′-sulfonic acidAspartic protease inhibitor; Chitinase A; Bacillus licheniformis; Enzyme kinetics; Slow tight binding inhibition
Muscle transfection and permeabilization induced by electrotransfer or pluronic® L64 by Michel F. Bureau; Juge Lauriane Jugé; Johanne Seguin; Marie-Noëlle Rager; Daniel Scherman; Nathalie Mignet (pp. 537-543).
Muscle transfection by electrotranfer is an efficient currently used procedure. Recently, the block copolymer pluronic L64 has been reported to improve muscle transfection. Both procedures are known to permeabilize muscle fibres. Relation between muscle transfection and permeabilization by electrotransfer and L64 was investigated herein.Muscle transfection was evaluated by optical detection of the luciferase reporter gene activity. Muscle permeabilization was evaluated by the uptake of the T1 contrast agent gadolinium-Dotarem (Gd-DOTA) using Magnetic resonance imaging (MRI). Histological examination of muscle sections was also performed.Electrotransfer and L64 (at a 0.25% concentration) similarly improved muscle transfection, although the interindividual variability was higher for pluronic. On the same animals, the permeabilized volume to the Gd-DOTA was significantly increased after electrotransfer, and L64 from 0.1% to 1%. The concentration of the Gd-DOTA in the permeabilized volume was significantly increased after electrotransfer and L64 at 0.5% and 1%. By histological observation, the inflammation was maximum at day 3 after electrotransfer or L64 injection, and mostly reversed after 7days. The permeabilized volume and the transfection level correlated for the set of all the conditions tested. However, no significant correlation was observed between Gd-DOTA concentration and transfection.It is possible to use successively on the same animals MRI and optical imaging for paired studies of muscle transfection and permeabilization. Permeabilization is possibly not related to gene transfer but it indicates membrane modification related to transfection by the electrotransfer or co-injection of DNA with the L64.
Keywords: Block copolymer; Permeability; Transfection; Electroporation; MRI; Optical imaging