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141.
Hillairet De Boisferon M Raguin O Thiercelin C Dussaillant M Rostène W Barbet J Pélegrin A Gruaz-Guyon A 《Bioconjugate chemistry》2002,13(3):654-662
Radiolabeled peptides are emerging tools for diagnosis and therapy of tumors overexpressing receptors. However, binding to receptors expressed by nontumor tissues may cause toxicity. The objective of this study was to specifically enhance the binding affinity of labeled peptides to tumor cells, as opposed to receptor-positive nontumor cells, to ensure targeting selectivity. This was achieved by the simultaneous binding of hapten-bearing peptides to their receptor and to a tumor-associated antigen, mediated by a bispecific antibody directed to the tumor antigen and to the hapten. Binding of labeled neurotensin analogues bearing the DTPA(indium) hapten (NT-DTPA(111In)) to human colorectal carcinoma cells (HT29), which express the neurotensin receptor (NTR1) and carcinoembryonic antigen (CEA), was studied in the presence of a bispecific antibody (BsmAb) directed to CEA and to DTPA(indium). In vitro dual binding of NT-DTPA in the presence of BsmAb was about 6.5-fold higher than monovalent binding to NTR1 and 3.5-fold higher than the sum of the monovalent bindings to NTR1 or to CEA, suggesting cooperativity. Increased binding under bivalent conditions translated into increased internalization. In vivo pretargeting with BsmAb enhanced tumor uptake and tumor retention. Hapten bearing peptides binding simultaneously an overexpressed cell-surface receptor and a tumor antigen show increased selectivity to target tumor cells as compared to cells only expressing the cell surface receptor. Better resistance to enzymatic degradation and optimized administration protocols should further enhance in vivo targeting selectivity and may allow the development of radiopharmaceuticals labeled with isotopes suitable for radiotherapy such as 131I or 90Y. 相似文献
142.
Escherichia coli K1 traversal of the human brain microvascular endothelial cells (HBMEC) that constitute the blood-brain barrier (BBB) is a complex process involving E. coli adherence to and invasion of HBMEC. In this study, we demonstrated that human transforming growth factor-beta-1 (TGF-beta1) increases E. coli K1 adherence, invasion, and transcytosis in HBMEC. In addition, TGF-beta1 increases RhoA activation and enhances actin condensation in HBMEC. We have previously shown that E. coli K1 invasion of HBMEC requires phosphatidylinositol-3 kinase (PI3K) and RhoA activation. TGF-beta1 increases E. coli K1 invasion in PI3K dominant-negative HBMEC, but not in RhoA dominant-negative HBMEC, indicating that TGF-beta1-mediated increase in E. coli K1 invasion is RhoA-dependent, but not PI3K-dependent. Our findings suggest that TGF-beta1 treatment of HBMEC increases E. coli K1 adherence, invasion, and transcytosis, which are probably dependent on RhoA. 相似文献
143.
Cellular localisation of a water-soluble fullerene derivative 总被引:6,自引:0,他引:6
Foley S Crowley C Smaihi M Bonfils C Erlanger BF Seta P Larroque C 《Biochemical and biophysical research communications》2002,294(1):116-119
Fullerenes are a new class of compounds with potential uses in biology and medicine and many insights were made in the knowledge of their interaction with various biological systems. However, their interaction with organised living systems as well as the site of their potential action remains unclear. In this work, we have demonstrated that a fullerene derivative could cross the external cellular membrane and it localises preferentially to the mitochondria. We propose that our finding supports the potential use of fullerenes as drug delivery agents as their structure mimics that of clathrin known to mediate endocytosis. 相似文献
144.
Stortelers C van De Poll ML Lenferink AE Gadellaa MM van Zoelen C van Zoelen EJ 《Biochemistry》2002,41(13):4292-4301
Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members. 相似文献
145.
Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers 总被引:19,自引:0,他引:19 下载免费PDF全文
Vandebrouck C Martin D Colson-Van Schoor M Debaix H Gailly P 《The Journal of cell biology》2002,158(6):1089-1096
Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers. 相似文献
146.
Fimbriae and adherence of Stenotrophomonas maltophilia to epithelial cells and to abiotic surfaces 总被引:1,自引:0,他引:1
de Oliveira-Garcia D Dall'Agnol M Rosales M Azzuz AC Alcántara N Martinez MB Girón JA 《Cellular microbiology》2003,5(9):625-636
Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen associated with several infectious diseases and opportunistic infections, especially in immunocompromised patients. These bacteria adhere avidly to medical implants and catheters forming a biofilm that confers natural protection against host immune defences and different antimicrobial agents. The nature of the bacterial surface factors involved in biofilm formation on inert surfaces and in adherence of S. maltophilia to epithelial cells is largely unknown. In this study, we identified and characterized fimbrial structures produced by S. maltophilia grown at 37 degrees C. The S. maltophilia fimbriae 1 (SMF-1) are composed of a 17 kDa fimbrin subunit which shares significant similarities with the N-terminal amino acid sequences of several fimbrial adhesins (G, F17, K99 and 20K) found in Escherichia coli pathogenic strains and the CupA fimbriae of Pseudomonas aeruginosa. All of the clinical S. maltophilia isolates tested produced the 17 kDa fimbrin. Antibodies raised against SMF-1 fimbriae inhibited the agglutination of animal erythrocytes, adherence to HEp-2 cells and biofilm formation by S. maltophilia. High resolution electron microscopy provided evidence of the presence of fimbriae acting as bridges between bacteria adhering to inert surfaces or to cultured epithelial cells. This is the first characterization of fimbriae in this genus. We provide compelling data suggesting that the SMF-1 fimbriae are involved in haemagglutination, biofilm formation and adherence to cultured mammalian cells. 相似文献
147.
In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene. 相似文献
148.
Escherichia coli heat shock protein DnaK: production and consequences in terms of monitoring cooking 总被引:1,自引:0,他引:1
Seyer K Lessard M Piette G Lacroix M Saucier L 《Applied and environmental microbiology》2003,69(6):3231-3237
Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F(70)(10) of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55 degrees C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50 degrees C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55 degrees C, F(70)(10) = 3 min) accompanied by a 12-h recovery (containing 76,786 +/- 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 +/- 6,056 molecules/cell) when later heated to 60 degrees C for 50 min (F(70)(10) = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment. 相似文献
149.
Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation 总被引:22,自引:0,他引:22 下载免费PDF全文
Pujuguet P Del Maestro L Gautreau A Louvard D Arpin M 《Molecular biology of the cell》2003,14(5):2181-2191
Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane. 相似文献
150.
Geuze HJ Murk JL Stroobants AK Griffith JM Kleijmeer MJ Koster AJ Verkleij AJ Distel B Tabak HF 《Molecular biology of the cell》2003,14(7):2900-2907
The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived. 相似文献