Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC–RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE–RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC–RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE–RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.     

Function and induction of the microsomal heme oxygenase

Molecular and Cellular Biochemistry - The microsomal heme oxygenase system consists of heme oxygenase and NADPH-cytochrome P-450 reductase, and is considered to play a key role in the physiological...     

TGF-β1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition

Epithelial–mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-β1) and whether EMT could be induced through TGF-β1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-β1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-β1. Analysis of EMT markers showed that mRNA levels changed with TGF-β1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-β1 treatment. Invasion assays showed that TGF-β1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-β1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.     

Purification and Some Properties of Plant Serine Acetyltransferase

Serine acetyltransferase (SATase) that catalyzes the conversionof L-serine to O-acetyl-L-serine (OAS) in the presence of acetyl-CoAwas highly purified from rape leaf extract, using a coupledassay system in which OAS is converted to cysteine by enzymaticaction of exogenous cysteine synthase. Through purificationprocedures including heat treatment, ammonium sulfate fractionationand successive column chromatographies on DEAE-Toypearl, Blue-Toyopearland Toyopearl HW-55, the specific activity was raised to about4,800-fold over that in the crude extract. The molecular weightof rape enzyme was estimated to be about 350,000 by gel filtrationcolumn chromatography. The cysteine-forming activity of thefinal preparation was completely dependent on L-serine, acetyl-CoAand sulfide. However, this preparation had low activity forL-cysteine synthesis from L-serine even in the absence of exogenouscysteine synthase, suggesting that plant SATase exists as ahigh-molecular weight enzyme complexed with cysteine synthase. (Received November 6, 1987; Accepted March 25, 1988)     

Cholecystokinin tetrapeptide,proglumide and open-field behavior in rats

The effect of cholecystokinin tetrapeptide (CCK-4) was studied in an open field situation. CCK-4 increased locomotion and rearing and the effect was enhanced by proglumide, a selective antagonist of CCK-8. This is in sharp contrast to our earlier findings that CCK-8 decreased the open-field behavior and that proglumide completely blocked the effect. Thus, the effects of CCK-4 and CCK-8 appear to be opposite to each other in that one is excitatory and the other inhibitory to open-field responses.     

The narQP genes for a two-component regulatory system from the deep-sea bacterium Shewanella violacea DSS12.

Shewanella violacea DSS12 is facultative piezophile isolated from the deep-sea. The expression of cydDC genes (required for d-type cytochrome maturation) of the organism is regulated by hydrostatic pressure. In this study, we analyzed the nucleotide sequence upstream of cydDC in detail and found that there are putative binding sites for the NarL protein which is part of a two-component regulatory system also containing the sensor protein NarX. Furthermore, we identified the narQP genes (homologues of narXL) from S. violacea DSS12 and demonstrated the heterologous expression of narP in Escherichia coli. These results will be helpful in examining pressure regulation of gene expression in S. violacea at the molecular level.     

Antioxidant effect of caeruloplasmin on microsomal lipid peroxidation

Microsomal NADPH-dependent lipid peroxidation catalyzed by ADP-Fe³⁺ was inhibited by the addition of caeruloplasmin. The antioxidant effect of caeruloplasmin was independent of the superoxide anion (O^?₂ scavenging activity. Since caeruloplasmin enhanced the function of ADP-Fe³⁺ acting as electron acceptor for microsomal electron transport system, the antioxidant effect of caeruloplasmin is considered to depend on the ferroxidase activity.     

Effect of sulfur and nitrogen nutrition on derepression of ferredoxin-sulfite reductase in leek seedlings

The ferredoxin-sulfite reductase (Fd-SiR; hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1) activities of shoot and root of leek (Allium tuberosum) were increased by sulfate limitation in the early stage of growth. Western blot analysis demonstrated an increased amount of SiRs in root under sulfate limitation, suggesting that SiRs were derepressed. The derepression was observed in shoot when 1.5 mM nitrate was supplied to the plants under sulfate limitation, and clearly in root when 15 mM nitrate was supplied under sulfate limitation. When nitrate was absent from the nutrient solution, the SiR activity in both tissues was very low. Combined with the results of the sulfate- or nitrate-limitation experiments, it is suggested that the degree of the derepression of SiR in both tissue under sulfate limitation is affected by the concentration of nitrate, and further that the mechanism of regulation of the SiR activity is different in each tissue. The decreases in the ratios of the total SiR activities (shoot/root) in the latter stage of seedling growth indicate that root play a very important role in sulfate assimilation.