Detection of adiponectin and monocyte chemoattractant protein-1 using a calixcrown derivatives-coated protein chip.

We used a ProteoChip coated with a calixcrown derivative protein linker to measure adiponectin and monocyte chemoattractant protein-1 (MCP-1) levels and compared the results with commercial enzyme-linked immunosorbent assay (ELISA) kits. Adiponectin and MCP-1 levels in normal human serum and RAW264 cell supernatants, respectively, were measured. The ProteoChip quantification results correlated with those from the ELISA kits; however, the ProteoChip required less sample volume, exhibited higher sensitivity, and had a wider detection range. The ProteoChip was capable of detecting and quantifying small amounts of protein, possibly replacing ELISA kits in evaluating the levels of adiponectin and MCP-1.

Regulatory mechanism for the stimulatory action of genistein on glucose uptake in vitro and in vivo.

Genistein, an isoflavone, is known to possess diverse biological functions such as antioxidative and anti-inflammatory actions. It also acts like estrogen and inhibits several tyrosine kinases. Genistein was reported to suppress insulin-mediated glucose uptake in adipocytes. In this study, we investigated the effects of genistein on glucose uptake in vitro and in vivo as well as the mechanisms associated with the glucose uptake. We found that genistein decreased nonfasting blood glucose levels in KK-Ay/Ta Jcl mice, a type 2 diabetic animal model. It also dose-dependently induced insulin secretion by Rin-5F cells. In L6 myotubes, it directly stimulated glucose uptake independently of insulin under normal and high glucose conditions in dose-dependent manners. It promoted the translocation of glucose transporter 4 to the cell membrane under both glucose conditions. Based on studies using inhibitors of signaling molecules related to glucose uptake, the stimulatory effect of genistein on glucose uptake appeared to be dependent on the phosphatidylinositol 3-kinase, mammalian target of rapamycin, protein kinase C and 5'-adenosine-monophosphate-activated protein kinase pathway under both glucose conditions. In addition, O-GlcNAcylation by O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate, an inhibitor of N-acetylglucosaminidase, reduced the stimulatory effect of genistein on glucose uptake under both glucose conditions. Taken together, genistein may regulate glucose uptake by increasing the phosphorylation and decreasing the O-GlcNAcylation of proteins related to glucose homeostasis.

Enzymatic hydrolysis of structurally diverse phthalic acid esters by porcine and bovine pancreatic cholesterol esterases.

A weak hydrolyzing activity against bis (2-ethylhexyl) phthalate (DEHP) was discovered in a commercial crude lipase (EC 3.1.1.3) preparation from porcine pancreas. DEHP was hydrolyzed to mono (2-ethylhexyl) phthalate (MEHP) not by a pancreatic lipase but by a cholesterol esterase (CEase, EC 3.1.1.13), a trace contaminant in the crude lipase preparation. Enzymatic hydrolysis of phthalic acid esters (PAEs), suspected to be endocrine-disrupting chemicals, was investigated using CEases from two species of mammals and a microorganism. Eight structurally diverse PAEs, namely diethyl phthalate (DEP), di-n-propyl phthalate (DPrP), di-n-butyl phthalate (DBP), di-n-pentyl phthalate (DPeP), di-n-hexyl phthalate (DHP), DEHP, n-butyl benzyl phthalate (BBP), and dicyclohexyl phthalate (DCHP), were hydrolyzed to their corresponding monoesters by both porcine and bovine pancreatic CEases, while a microbial CEase from Pseudomonas sp. had no hydrolyzing activity against these PAEs. The hydrolysis experiments with bovine pancreatic CEase (50 U) indicated complete hydrolysis of every PAE (5 µmole) except for BBP and DCHP within 15 min; BBP and DCHP were hydrolyzed within 30 min and 6h, respectively. The rates of PAE hydrolysis could be affected by the bulkiness of alkyl side chains in the PAEs. This study provides important evidence that mammalian pancreatic CEases, such as those from porcine and bovine sources, are potential enzymes for nonspecific degradation of structurally diverse PAEs.

[Highly sensitive method of detecting verotoxins produced by enterohaemorrhagic Escherichia coli O-157 : H7 using a protein chip].

Enterohaemorrhagic Escherichia coli O-157 : H7 produces verotoxins called Shiga toxin or Shiga-like toxin. The type 1 and type 2 toxins present different immunological and physicochemical characteristics, and cause symptoms such as watery diarrhea, bloody diarrhea, severe stomach ache, fever and vomiting. The toxins can be assayed by several methods, including ELISA and PCR, with some limitations as to sensitivity. In the present study, we used a ProteoChip, which requires smaller volumes of reagents and allows detection of lower concentrations of the toxins, compared with the conventional assay.