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251.
Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-l-maurocalcine analog
Céline Tisseyre Mitra Ahmadi Sandrine Bacot Lucie Dardevet Pascale Perret Michel Ronjat Daniel Fagret Yves Usson Catherine Ghezzi Michel De Waard 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014
L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to 125I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9 × 10−7 μl. 125I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of 125I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5 min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24 h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. 相似文献
252.
Stephen O. Amoo Adeyemi O. Aremu Mack Moyo Lucie Szüčová Karel Doležal Johannes Van Staden 《Plant Cell, Tissue and Organ Culture》2014,116(1):17-26
Globally, there is a continuous effort geared toward improving micropropagation protocols with much emphasis on the type of cytokinins (CKs). We investigated the effect of meta-topolin tetrahydropyran-2-yl [mTTHP—a novel derivative of the aromatic cytokinin meta-topolin (mT)] on shoot proliferation, photosynthetic pigment content, phytochemicals and antioxidant activity of two widely used medicinal plants, Aloe arborescens and Harpagophytum procumbens. In terms of shoot proliferation, besides mTTHP and mT (at equimolar level) showing comparable effects, both topolins were significantly better than the control and benzyladenine riboside (BAR) in micropropagated A. arborescens. In H. procumbens, mT-treated cultures were the most responsive treatment at 2.5 μM when compared to the control. At 5.0 μM concentration, mTTHP and meta-topolin riboside (mTR) demonstrated a comparable effect on shoot proliferation. To a certain extent (particularly at low concentrations), mTTHP had a better rooting stimulatory effect when compared to the other CKs in both investigated species. In micropropagated A. arborescens, 2.5 μM mTTHP showed the best antioxidant activity (ferric reducing power) which was significantly higher than the control and all BAR-treated plants. Although 2.5 μM mTTHP exhibited a significant antioxidant activity (in β-carotene based assay) when compared to mTR at equimolar level in H. procumbens, it was not significantly different from the treatment with 2.5 μM mT and the control. As evidenced in the current study, the efficacy of the novel cytokinin may be species-specific and beneficial for few physiological parameters. It is conceivable that mTTHP is another viable alternative topolin with the extra advantage of inducing rooting at a low concentration. 相似文献
253.
Milan Kuchař Lucie Vaňková Hana Petroková Jiří Černý Radim Osička Ondřej Pelák Hana Šípová Bohdan Schneider Jiří Homola Peter Šebo Tomáš Kalina Petr Malý 《Proteins》2014,82(6):975-989
Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4+ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. 相似文献
254.
Ivo Chamrád Radim Simerský Lucie Bérešová Miroslav Strnad Marek Šebela René Lenobel 《Journal of Plant Growth Regulation》2014,33(4):896-902
Our knowledge on the principle mechanisms of cytokinin action has been significantly deepened over the last years, but several weakly explored areas still remain on the map of cytokinin cellular physiology. Cytokinin-binding proteins could also be included in this pending field of cytokinin research. Probably, the best explored representative of this group is the wheat cytokinin-binding protein 1 (CBP-1). The role of this germ-allocated protein as a presumable regulator of free aromatic cytokinin levels during grain germination has been discussed intensively. To dig deeper into this interesting protein, this study was aimed at the identification of the CBP-1 amino acid sequence. A combination of in silico BLAST search, classical biochemical CBP-1 purification based on isoelectric point precipitation and ion-exchange chromatography, and proteomic analysis of the isolated protein by ultra-high resolution tandem mass spectrometry allowed us to uncover and validate two CBP-1 subunit candidate sequences with molecular masses of 56.2 and 55.0 kDa, respectively. Interestingly, we found the latter sequence alternated in two amino acids in the putative cytokinin-binding motive in comparison to the composition of this domain reported in the original studies. A BLAST search for the amino acid sequence of the binding region among plant proteomes revealed several highly related protein sequences, all originating from the Poaceae family. This piece of information could give support to the elucidation of the role of CBP-1 in physiological processes mediated by aromatic cytokinins. 相似文献
255.
Julie Jaquiéry Solenn Stoeckel Chloé Larose Pierre Nouhaud Claude Rispe Lucie Mieuzet Jo?l Bonhomme Frédérique Mahéo Fabrice Legeai Jean-Pierre Gauthier Nathalie Prunier-Leterme Denis Tagu Jean-Christophe Simon 《PLoS genetics》2014,10(12)
Although evolutionary transitions from sexual to asexual reproduction are frequent in eukaryotes, the genetic bases of such shifts toward asexuality remain largely unknown. We addressed this issue in an aphid species where both sexual and obligate asexual lineages coexist in natural populations. These sexual and asexual lineages may occasionally interbreed because some asexual lineages maintain a residual production of males potentially able to mate with the females produced by sexual lineages. Hence, this species is an ideal model to study the genetic basis of the loss of sexual reproduction with quantitative genetic and population genomic approaches. Our analysis of the co-segregation of ∼300 molecular markers and reproductive phenotype in experimental crosses pinpointed an X-linked region controlling obligate asexuality, this state of character being recessive. A population genetic analysis (>400-marker genome scan) on wild sexual and asexual genotypes from geographically distant populations under divergent selection for reproductive strategies detected a strong signature of divergent selection in the genomic region identified by the experimental crosses. These population genetic data confirm the implication of the candidate region in the control of reproductive mode in wild populations originating from 700 km apart. Patterns of genetic differentiation along chromosomes suggest bidirectional gene flow between populations with distinct reproductive modes, supporting contagious asexuality as a prevailing route to permanent parthenogenesis in pea aphids. This genetic system provides new insights into the mechanisms of coexistence of sexual and asexual aphid lineages. 相似文献
256.
Dorthea M. Grégoire Dan T. Quiring Lucie Royer Éric Bauce 《Arthropod-Plant Interactions》2014,8(4):361-371
The balsam woolly adelgid (Adelges piceae) is a gout-inducing hemipteran native to the silver fir forests of Europe. Introduced to eastern North America approximately 100 years ago, it is now found in most balsam fir forests in Atlantic Canada. When A. piceae feed, they trigger a reaction in the host branch that alters both xylem and phloem morphology. We conducted a field survey to examine the relationship between A. piceae gout density and balsam fir foliar chemistry and shoot growth in naturally unthinned and precommercially thinned stands. A. piceae gout density negatively affected branch growth and was related to changes in the chemistry of older, but not current-year foliage. Older foliage experienced decreases in camphene and bornyl acetate, while foliar concentrations of camphene, myrcene, phenolics, potassium and water differed between thinned and unthinned stands. Foliar chemistry was also influenced by interactions between thinning and A. piceae gout density in old foliage. This study suggests that changes in balsam fir associated with A. piceae gout density may force native defoliators that feed in highly gouted trees to adapt to diets of different chemical compositions and that thinning may alter these interactions. 相似文献
257.
Menno Pruijm Lucie Hofmann Maciej Piskunowicz Marie-Eve Muller Carole Zweiacker Isabelle Bassi Bruno Vogt Matthias Stuber Michel Burnier 《PloS one》2014,9(4)
Experimentally renal tissue hypoxia appears to play an important role in the pathogenesis of chronic kidney disease (CKD) and arterial hypertension (AHT). In this study we measured renal tissue oxygenation and its determinants in humans using blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) under standardized hydration conditions. Four coronal slices were selected, and a multi gradient echo sequence was used to acquire T2* weighted images. The mean cortical and medullary R2* values ( = 1/T2*) were calculated before and after administration of IV furosemide, a low R2* indicating a high tissue oxygenation. We studied 195 subjects (95 CKD, 58 treated AHT, and 42 healthy controls). Mean cortical R2 and medullary R2* were not significantly different between the groups at baseline. In stimulated conditions (furosemide injection), the decrease in R2* was significantly blunted in patients with CKD and AHT. In multivariate linear regression analyses, neither cortical nor medullary R2* were associated with eGFR or blood pressure, but cortical R2* correlated positively with male gender, blood glucose and uric acid levels. In conclusion, our data show that kidney oxygenation is tightly regulated in CKD and hypertensive patients at rest. However, the metabolic response to acute changes in sodium transport is altered in CKD and in AHT, despite preserved renal function in the latter group. This suggests the presence of early renal metabolic alterations in hypertension. The correlations between cortical R2* values, male gender, glycemia and uric acid levels suggest that these factors interfere with the regulation of renal tissue oxygenation. 相似文献
258.
Ilona Hromadnikova Katerina Kotlabova Lucie Hympanova Jindrich Doucha Ladislav Krofta 《PloS one》2014,9(12)
Objective
The objective of the study was to evaluate risk assessment for gestational hypertension based on the profile of circulating placental specific C19MC microRNAs in early pregnancy.Study design
The prospective longitudinal cohort study of women enrolled at first trimester screening at 10 to 13 weeks was carried out (n = 267). Relative quantification of placental specific C19MC microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) was determined in 28 normal pregnancies and 18 pregnancies which developed gestational hypertension using real-time PCR and a comparative Ct method relative to synthetic C. elegans microRNA (cel-miR-39).Results
Increased extracellular C19MC microRNA plasmatic levels (miR-516-5p, p<0.001; miR-517*, p = 0.007; miR-520h, p<0.001; miR-518b, p = 0.002) were detected in patients destined to develop gestational hypertension. MiR-520h had the best predictive performance with a PPV of 84.6% at a 7.1% false positive rate. The combination of miR-520h and miR-518b was able to predict 82.6% of women at the same false positive rate. The overall predictive capacity of single miR-518b (73.3% at 14.3% FPR), miR-516-5p (70.6% at 17.9% FPR) and miR-517* (57.9% at 28.6% FPR) biomarkers was lower.Conclusion
The study brought interesting finding that the up-regulation of miR-516-5p, miR-517*, miR-520h and miR-518b is associated with a risk of later development of gestational hypertension. First trimester screening of extracellular miR-520h alone or in combination with miR-518b identified a significant proportion of women with subsequent gestational hypertension. 相似文献259.
Stancho Stanchev Zbigniew Zawada Lenka Monincová Lucie Bednárová Jiřina Slaninová Vladimír Fučík Václav Čeřovský 《Journal of peptide science》2014,20(9):725-735
The antimicrobial 40‐amino‐acid‐peptide lucifensin was synthesized by native chemical ligation (NCL) using N‐acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid‐phase synthesis is imcompatible. Two models of ligation were developed: [15 + 25] Ala‐Cys and [19 + 21] His‐Cys. The [19 + 21] His‐Cys method gives lower yield because of the lower stability of 18‐peptide‐His‐Nbz‐CONH2 peptide, as suggested by density functional theory calculation. Acetamidomethyl‐deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1?6], [C3?5], and [C2?4], which limits its ability to form α‐helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one‐pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
260.
Lucie Sancey Vincent Motto-Ros Shady Kotb Xiaochun Wang Fran?ois Lux Gérard Panczer Jin Yu Olivier Tillement 《Journal of visualized experiments : JoVE》2014,(88)
Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1 to 24 hr after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 μm. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response: elemental, molecular, or cellular. 相似文献