Phenotype‐based analysis of circulating tumor cells (CTCs) is a promising approach to identification of new therapeutic targets and to elucidation of the biological properties. Nonetheless, ex vivo ...culturing of CTCs is still a technical challenge. Here, we develop a novel ex vivo culture method for CTCs using a fibroblast feeder layer and a magnetic coculture protocol. CTCs in the blood of a mouse metastasis model are labeled magnetically with magnetite nanoparticles. The labeled CTCs are isolated by a magnetic capture column and a size‐selective capture filter. The isolated CTCs are positioned on a fibroblast feeder layer by the magnetic force. As a result, we observe adhesion and proliferation of the CTCs under the conditions of the fibroblast feeder layer and the magnetic force, whereas no adhesion or proliferation is observed without the feeder layer. After that, we culture the CTCs and obtain three CTC‐derived cell lines. Using these cell lines, we perform phenotype‐based analyses of invasiveness and drug resistance and find that the CTC‐derived cell lines are more malignant than the original cells. Thus, the proposed method would be a promising approach to ex vivo culture of CTCs for phenotype‐based analysis, and possibly used in cancer treatment.
Phenotype‐based analysis of circulating tumor cells (CTCs) is a promising approach to identification of new therapeutic targets and to elucidation of the biological properties. Nonetheless, ex vivo culturing of CTCs is still a technically challenging. Here, we developed a novel ex vivo culture method for CTCs using a fibroblast feeder layer and a magnetic coculture protocol. We obtained 3 CTC‐derived cell lines and performed phenotype‐based analyses of invasiveness and drug resistance. This article is part of an AFOB (Asian Federation of Biotechnology) Special issue. To learn more about the AFOB, visit www.afob.org.
Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient ...conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles. Biotechnol. Bioeng. 2010;106: 845-851.
Transcriptional analysis has been investigated to detect the genes involved in organic solvent tolerance. A time course of gene expression profiles of Escherichia coli after exposure to organic ...solvents revealed that the expression levels of manX, manY, and manZ genes were strongly upregulated by 13.2-, 10.0-, and 7.0-folds, respectively, after 2 min and then decreased after 10 min. Organic solvent tolerance of E. coli was investigated by inducing overexpression of manX, manY, and manZ genes and manXYZ operon that encode a sugar transporter of the phosphotransferase system. Although the expression of manX, manY, and manZ alone was not effective, the organic solvent tolerance level was increased by the expression of manXYZ. The intracellular hexane level was kept lower in E. coli cells overexpressing manXYZ after 4 h of incubation with hexane.
Detecting and analyzing circulating tumor cells (CTCs) in the blood of cancer patients is a promising approach for the early diagnosis of metastasis. Previously, we developed a size-selective filter ...for capturing CTCs, but its use was time consuming, particularly for capturing CTCs from large volumes of blood. In the present study, we describe the use of a magnetic capture column for rapid and efficient isolation of CTCs, which were magnetically labeled with magnetite cationic liposomes. In the capturing process, large volumes of blood containing magnetically labeled cancer cells were introduced into the column at a high flow rate to capture the cells, which were then added into the filter at a low flow rate. Our results show that the combined use of the column and filter decreased the required time for the spiked cancer cell capture, and the recovery rate of the spiked cancer cells from blood was significantly higher using the combination process (80.7 %) than that using the filter alone (64.7 %). Moreover, almost twice the number of CTCs could be captured from the blood of metastatic model mice using the combination process. These results suggest that the developed process would be useful for the rapid and efficient isolation of CTCs.
Three-dimensional (3D) cell culture arrays of melanoma cell spheroids were assembled to evaluate the combined effect of a melanogenesis-targeting drug, N-propionyl-4-cysteaminylphenol (NPrCAP), and ...heat treatment. An array-like multicellular pattern of mouse melanoma B16F1 cells in a collagen gel was established by magnetic cell labeling using a pin-holder device to exert a magnetic force. The cellular spheroids were exposed to NPrCAP and heat (42°C for 1 h) as a model of anti-cancer treatment. As a result, melanogenesis of B16F1 cells was 29-fold higher in this 3D array than in conventional two-dimensional (2D) monolayer cultures. Because the spheroid size was linearly correlated with the cell number within a spheroid, the antiproliferative effect could be evaluated in a non-destructive manner. Moreover, the half-maximal inhibitory concentration of NPrCAP coupled with heat treatment calculated from the spheroid size was 2-fold higher in the 3D array (0.30mM) than in 2D culture (0.15 mM). These results indicate that spheroid formation decreases the chemosensitivity of cancer cells, and this model would be suitable as a susceptibility assay for melanogenesis-targeting drugs. Therefore, this 3D culture model provides a better screening format to evaluate drug and physical treatments for cancer therapy than 2D formats.
Migrasomes are extracellular vesicles that form on the retraction fibers of migrating cells. In this study, we report the formation of migrasome‐like vesicles enriched in tetraspanin 4 and containing ...cytoplasmic components in response to hypoosmotic stress. When migrating cells were subjected to hypoosmotic stress, vesicles with a size distribution of 0.5 to 2 μm formed on the retraction fibers, and vanished in a few minutes. The vesicles are rich in cholesterol, and their number was reduced when cells were pretreated with lipoprotein‐deficient serum. The formation of migrasome‐like vesicles upon hypoosmotic stress may provide biophysical cues regarding the cellular response to this external stimulus in cells and tissues.
When migrating cells were subjected to hypoosmotic stress, the formation of migrasome‐like vesicles enriched in tetraspanin 4 and containing cytoplasmic components was observed. These vesicles are rich in cholesterol and their number is reduced when cells are pretreated with lipoprotein‐deficient serum.
Increased generation of reactive oxygen species (ROS) is recognized as a prominent feature of vascular dysfunction with diabetes. For self-monitoring of the risk of diabetic complications, we ...proposed a simple adhesion test of erythrocytes using positively charged surfaces and its adhesion property was found to correlate HbA
1c level in diabetic patients.
Inhibition of dipeptidyl peptidase IV (DPP-IV) is an effective pharmacotherapy for the management of type 2 diabetes. Recent findings have suggested that various dietary proteins can serve as ...precursors to peptides that inhibit DPP-IV. Although several DPP-IV inhibitory peptides derived from food materials have been reported, more effective inhibitory peptides remain to be discovered. This study aimed to identify potent DPP-IV inhibitory peptides that earlier approaches had overlooked by employing a screening method that combined peptide arrays and neutralizing antibodies. Octa-peptides covering the complete amino acid sequences of four casein proteins and two whey proteins were synthesized on arrays via a solid-phase method. These peptides were then reacted with a monoclonal antibody specifically engineered to recognize glucagon-like peptide 1 (GLP-1), a substrate of DPP-IV. The variable region of the anti-GLP-1 monoclonal antibody is utilized to mimic the substrate-binding region of DPP-IV, enabling the antibody to bind to peptides that interact with DPP-IV. Based on this feature, 26 peptides were selected as DPP-IV inhibitory peptide candidates, 11 of which showed strong DPP-IV inhibitory activity. Five of these peptides consistently contained cysteines positioned two to four residues from the N-terminus. Treatment with disulfide formation decreased the DPP-IV inhibitory activity of these cysteine-containing peptides, while the inhibitory activity of α-lactalbumin hydrolysates increased with reducing treatment. These results revealed that the thiol group is important for DPP-IV inhibitory activity. This study provides a useful screen for DPP-IV inhibitory peptides and indicates the importance of reductive cysteine residues within DPP-IV inhibitory peptides.
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•The DPP-IV inhibitory peptides were identified using a peptide array-based assay from milk proteins.•The monoclonal antibody that recognized GLP-1 was utilized to mimic the substrate-binding region of DPP-IV.•Eleven novel DPP-IV inhibitory peptides were identified by employing an anti-GLP-1 antibody and peptide arrays.•Five of these peptides consistently contained cysteines positioned two to four residues from the N-terminus.•This study revealed that the thiol group is important for DPP-IV inhibitory activity.
We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even ...limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.
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