Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm

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.     

Application of magnetic force-based cell patterning for controlling cell-cell interactions in angiogenesis

To investigate the effects of cell-cell interactions on cellular function, the microenvironment surrounding cells should be precisely controlled. Here, we describe a cell patterning technique, which utilizes magnetic force and magnetite nanoparticles. This method was used to develop cell culture arrays for investigation of cell behaviors in angiogenesis. Pin holder devices that contain more than 6,000 pillars on the surface are used for fabricating the cell culture arrays by setting it on a magnet. The magnetically labeled cells were arranged by magnetic distribution. When the human umbilical vein endothelial cells are arranged at 250 microm intervals (5.9 cells/spot), the cells spread toward other cell cluster on adjacent spots in 4.5 h, and formed cord-like structures in 8.5 h. It was shown that cell-cell interactions were successfully investigated using magnetic cell arrangement.     

NSC-87877, inhibitor of SHP-1/2 PTPs, inhibits dual-specificity phosphatase 26 (DUSP26)

Protein phosphorylation plays critical roles in many regulatory mechanisms controlling cell activities and thus involved in various diseases. The cellular equilibrium of phosphorylation is regulated through the actions of protein kinases and phosphatases. Therefore, these regulatory proteins have emerged as promising targets for drug development. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), a potent inhibitor of SHP-1 and SHP-2 PTPs. Phosphatase activity of dual-specificity protein phosphatase 26 (DUSP26) was decreased by the inhibitor in a dose-dependent manner. Kinetic studies with NSC-87877 and DUSP26 revealed a competitive inhibition. NSC-87877 effectively inhibited DUSP26-mediated dephosphorylation of p38, a member of mitogen-activated protein kinase (MAPK) family. Since DUSP26 is involved in survival of anaplastic thyroid cancer (ATC) cells, NSC-87877 could be a therapeutic reagent for treating ATC.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Association of a reactive intermediate derived from 1′,6‐dihydroxy metabolite with benzbromarone‐induced hepatotoxicity

Treatment with benzbromarone can be associated with liver injury, but the detailed mechanism remains unknown. Our recent studies demonstrated that benzbromarone was metabolized to 1′,6‐dihydroxybenzbromarone and followed by formation of reactive intermediates that were trapped by glutathione, suggesting that the reactive intermediates may be responsible for the liver injury. The aim of this study was to clarify whether the reactive intermediates derived from 1′,6‐dihydroxybenzbromarone is a risk factor of liver injury in mice. An incubation study using mouse liver microsomes showed that the rates of formation of 1′,6‐dihydroxybenzbromarone from benzbromarone were increased by pretreatment with dexamethasone. Levels of a hepatic glutathione adduct derived from 1′,6‐dihydroxybenzbromarone were increased by pretreatment with dexamethasone. Furthermore, plasma alanine amino transferase activities were increased in mice treated with benzbromarone after pretreatment with dexamethasone. The results suggest that the reactive intermediate derived from 1′,6‐dihydroxybenzbromarone may be associated with liver injury.     

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.