共查询到20条相似文献,搜索用时 15 毫秒
1.
Igor Meerovich Nandhini MuthukrishnanGregory A. Johnson Alfredo Erazo-OliverasJean-Philippe Pellois 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl–CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process.Methods
In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles.Results
The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence.Conclusions
These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently.General significance
Understanding the mechanism behind Fl–CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds. 相似文献2.
Margarida Rodrigues Beatriz G. de la Torre David Andreu Nuno C. Santos 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Nucleolar targeting peptides (NrTPs), resulting from structural minimization of the rattlesnake toxin crotamine, are a novel family of cell-penetrating peptides (CPPs) shown to internalize and deliver cargos into different cell types.Methods
In this study, we address NrTP kinetics of translocation into primary cells. We used flow cytometry to measure the intracellular uptake of rhodamine B-labeled NrTPs in peripheral blood mononucleated cells (PBMCs).Results
The kinetic profiles for each peptide are concentration-independent but significantly different among NrTPs, pointing out for the amino acid sequence importance. Arginine-containing peptides (NrTP7 and Tat48–60, used for comparison) were found to be more toxic than lysine-containing ones, as expected. On the other hand, one same peptide behaves differently in each of the lymphocyte and monocyte cell populations, suggesting differences in entry mechanism that in turn reflect diversity in cell functionality. Uptake results obtained at 4 °C or using chemical endocytosis inhibitors support the importance of non-endocytic mechanisms in the cellular internalization of NrTP1 and NrTP5, while confirming endocytosis as the main mechanism of NrTPs entry.Conclusion
Overall, both direct translocation and endocytosis mechanisms play a role in NrTP entry. Yet, there is predominance of endocytosis-mediated mechanisms. NrTPs (especially NrTP6) are an excellent intracellular delivery tool, with efficient internalization and no toxicity.General significance
This work validates NrTPs as potential therapeutic tools for, e.g., cancer or inhibition of viral replication and establishes a new comparative and quantitative method to test CPP efficiency. 相似文献3.
Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine 总被引:1,自引:0,他引:1
Srinivasan D Muthukrishnan N Johnson GA Erazo-Oliveras A Lim J Simanek EE Pellois JP 《PloS one》2011,6(3):e17732
Background
Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).Methodology/Principal Findings
We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.Conclusions/Significance
Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents. 相似文献4.
5.
Apostolos Koutsokeras Panagiotis S. Kabouridis 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes.Methods
Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy.Results
Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi.Conclusions
Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved.General significance
These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues. 相似文献6.
Alexandra A. Kuznetsova Nikita A. Kuznetsov Alexander A. Ishchenko Murat K. Saparbaev Olga S. Fedorova 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases.Methods
Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4cat and UNG from different structural superfamilies were used.Results
We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1.Conclusions
We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4cat, AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions.General significance
Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. 相似文献7.
8.
9.
Masaki Uchida Xiong Wei Li Peter Mertens H. Oya Alpar 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment.Methods
gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay.Results
This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun's helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles.Conclusions
This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 μm gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 μg DNA/mg gold particles.General significance
These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination. 相似文献10.
Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour 总被引:1,自引:0,他引:1
Maria F. Mazzeo Roberta BonavitaFrancesco Maurano Paolo BergamoRosa A. Siciliano Mauro Rossi 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity.Methods
SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC–ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour.Results
Gliadin and glutenin yields decreased to 7.6 ± 0.5% and 7.5 ± 0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC–ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31–49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31–49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics.Conclusions
The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties.General significance
Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients. 相似文献11.
Eun Jeong Sohn Dae Won Kim Mi Jin Kim Hoon Jae Jeong Min Jea Shin Eun Hee Ahn Soon Won Kwon Young Nam Kim Duk-Soo Kim Kyu Hyung Han Jinseu Park Hyun Sook Hwang Won Sik Eum Soo Young Choi 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult.Methods
A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1–MT-III protein. PEP-1–MT-III protein was purified using affinity chromatograph. Transduced PEP-1–MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry.Results
Purified PEP-1–MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1–MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1–MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1–MT-III protein protects against oxidative stress induced cell death in vitro and in vivo.General significance
Transduced PEP-1–MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1–MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease. 相似文献12.
13.
Aims
Cellular senescence is an important tumor suppression process in vivo. Tamoxifen is a well-known anti-breast cancer drug; however, its molecular function is poorly understood. Here, we examined whether tamoxifen promotes senescence in breast cancer and colon cancer cells for the first time.Main methods
Human breast cancer MCF-7, T47D, and MDA-MB-435 and colorectal cancer HCT116 cells were treated with tamoxifen. Cellular senescence was measured by SA-β-gal staining and based on the protein expression of p53 and p21Cip1/WAF1. The production of reactive oxygen species (ROS) was determined by staining with CM-H2DCFDA and dihydroethidium (DHE). CK2 activity was assessed with a specific peptide substrate.Key findings
Tamoxifen promoted senescence phenotype and ROS generation in MCF-7 and HCT116 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), and the NADPH oxidase inhibitor, apocynin, almost completely abolished this event. Tamoxifen inhibited the catalytic activity of CK2. Overexpression of CK2α antagonized senescence mediated by tamoxifen, indicating that tamoxifen induced senescence via a CK2-dependent pathway. A well-known CK2 inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), also stimulated ROS production and senescence in MCF-7 cells. Finally, experiments using T47D (wild-type p53) and MDA-MB-435 (mutant p53) cell lines suggested that tamoxifen induces p53-independent ROS production as well as p53-dependent senescence in breast cancer cells.Significance
These results demonstrate that tamoxifen promotes senescence through a ROS–p53–p21Cip1/WAF1 dependent pathway by inhibiting CK2 activity in breast cancer and colon cancer cells. 相似文献14.
Hiroshi Tsujikawa Takehiro Shoda Toshiyuki Mizota Kazuhiko Fukuda 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Morphine has been shown to affect the function of immune system, but the precise mechanism remains to be elucidated. The present study was aimed to clarify the mechanism for the morphine-induced immune suppression by analyzing the direct effect of morphine on human CD3+ T cells.Methods
To identify genes up-regulated by action of morphine on the opioid receptor expressed in CD3+ T cells, PCR-select cDNA subtraction was performed by the use of total RNA from human CD3+ T cells treated with morphine in the presence and absence of naloxone.Results
We show that p53 and damage-specific DNA binding protein 2 (ddb2) genes are up-regulated by morphine in a naloxone-sensitive manner. Furthermore, the results indicate that DNA damage, quantified by apurinic–apyrimidinic site counting assay and phosphorylation of Ser-15 in P53 protein, is induced in CD3+ T cells by morphine in a naloxone-sensitive manner.General significance
Because it was shown that only the κ opioid receptor gene is expressed in CD3+ T cells in the opioid receptor family, the present study suggests that morphine induces DNA damage through the action on the κ opioid receptor, which leads to immune suppression by activation of P53-mediated signal transduction. 相似文献15.
Bennett L. Ibey Andrei G. Pakhomov Betsy W. Gregory Vera A. Khorokhorina Caleb C. Roth Mikhail A. Rassokhin Joshua A. Bernhard Gerald J. Wilmink Olga N. Pakhomova 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.Methods
Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.Results
10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.Conclusions
1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD50 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD50 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.General significance
Nanosecond EP can selectively target certain cells in medical applications like tumor ablation. 相似文献16.
Jure Pohleven Nataša Obermajer Jerica Sabotič Sabina Anžlovar Kristina Sepčić Janko Kos Bogdan Kralj Borut Štrukelj Jože Brzin 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.Methods
Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.Results
A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.Conclusions
The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.General significance
CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties. 相似文献17.
18.
Zhaojing Zheng Ru-en Yao Juan Geng Xingming Jin Yongnian Shen Daming Ying Qihua Fu Yongguo Yu 《Gene》2013
Background
Microduplication at 17p13.3 and microdeletion at 21q22 are both rare chromosomal aberrations. The presence of both genomic imbalances in one patient has not been previously reported in literature. In this study, we performed a molecular diagnostic testing with a whole genome microarray on a 3-year-old boy with developmental delay, mental retardation and multiple malformations.Methods
A routine G-banding karyotype analysis was performed using peripheral lymphocytes. Chromosome microarray analysis (CMA) was done using Affymetrix CytoScan™ HD array. Genomic imbalances were further confirmed by multiple ligation-dependent probe amplification (MLPA).Results
The result of karyotyping was normal but CMA detected a 9.8 Mb microduplication at 17p13.3–13.1 (chr17: 1–9,875,545) and a 2.8 Mb microdeletion involving 21q22.3–qter (chr21: 45,239,077–48,097,372). The imbalances were due to a balanced translocation present in patient's mother. The patient was characterized with short stature, profound developmental delay, non-verbal, intellectual disability as well as craniofacial dysmorphism, subtle brain structural anomaly and sparse scalp hair.Conclusions
This is the first patient reported with a combination of a microduplication at 17p13.3–13.1 and a microdeletion at 21q22.3–qter. Both genomic imbalances were undetected by conventional karyotyping but were delineated with CMA test. Synergistic effect from the two rare genomic imbalances is likely responsible for the severe clinical phenotypes observed in this patient. 相似文献19.
Kiyoshi Yamaguchi Kohsuke Takeda Hisae Kadowaki Ikumi Ueda Yoshio Namba Yasuyoshi Ouchi Hideki Nishitoh Hidenori Ichijo 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013