Effects of LPA1 and LPA6 on the regulation of colony formation activity in colon cancer cells treated with anticancer drugs

Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits a variety of cellular responses via the activation of G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA₁) to LPA₆). The aim of our study was to investigate effects of LPA receptors on soft agar colony formation in colon cancer cells treated with anticancer drugs. DLD1 cells were treated with fluorouracil (5-FU) or cisplatin (CDDP) for at least six months (DLD-5FU and DLD-CDDP cells, respectively). LPAR1 gene expression was markedly elevated in DLD-5FU cells. In contrast, DLD-CDDP cells showed the high expression of LPAR6 gene. In colony formation assay, DLD-5FU cells formed markedly large-sized colonies, while no colony formation was observed in DLD1 and DLD-CDDP cells. The large-sized colonies formed in DLD-5FU cells were suppressed by LPA₁ knockdown. In contrast, LPA₆ knockdown increased the size of colonies. In addition, DLD-5FU cells were further treated with CDDP for three months (DLD-C-F cells). DLD-CDDP cells were also treated with 5-FU (DLD-F-C cells). DLD-C-F cells formed large-sized colonies, but not DLD-F-C cells, correlating with LPAR1 and LPAR6 gene expression levels. These results suggest that LPA₁ and LPA₆ may regulate the colony formation activity in DLD1 cells treated with anticancer drugs.     

Molecular characterization of a bean α-amylase inhibitor that inhibits the α-amylase of the Mexican bean weevil Zabrotes subfasciatus

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an α-amylase inhibitor (αAI-1) that inhibits porcine pancreatic α-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of αAI-1, called αAI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant αAI-2 inhibits Z. subfasciatus α-amylase (ZSA), but not PPA. We purified αAI-2 and studied its interaction with ZSA. The formation of the αAI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode αAI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes αAI-2. The inhibitors αAI-1 and αAI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the αAI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction. Received: 14 April 1997 / Accepted: 10 May 1997     

Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.     

Fine mapping and development of DNA markers for the <Emphasis Type="Italic">qPDH1</Emphasis> locus associated with pod dehiscence in soybean

Pod dehiscence (shattering) is a major cause of yield loss in mechanical harvesting of soybeans. To develop useful selection markers, we conducted a high-resolution mapping of a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1. The progeny of a residual heterozygous line, which was a recombinant inbred line segregating only for the genomic region around qPDH1, was screened for flanking markers to obtain various recombinants in the vicinity of the QTL. Analysis of the relationship between degree of pod dehiscence and graphical genotype of these lines confined the location of qPDH1 to a 134-kb region on chromosome 16 (formerly linkage group J), where ten putative genes were predicted to be present. None of these genes showed significant sequence homology with the Arabidopsis genes that have previously been reported to be associated with pod dehiscence, suggesting the presence of a novel gene and mechanism underlying pod dehiscence in soybean. Sequencing analysis of the parental shattering-resistant and -susceptible cultivars for the candidate genes revealed a high-frequency nucleotide polymorphism in this genomic region between the cultivars. Three markers were developed using insertion/deletion variations in the region. Polymorphism at these marker loci was basically conserved between diverse shattering-resistant and -susceptible cultivars/lines, suggesting the versatility and usefulness of these markers for marker-assisted selection.     

Structural basis for androgen receptor agonists and antagonists: interaction of SPEED 98-listed chemicals and related compounds with the androgen receptor based on an in vitro reporter gene assay and 3D-QSAR

The androgen receptor (AR) activity of listed chemicals, so called SPEED 98, by the Ministry of the Environment, Japan, and structurally related chemicals was characterized using MDA-kb2 human breast cancer cells stably expressing an androgen-responsive luciferase reporter gene, MMTV-luc. Since our results suggested that chemicals with diverse chemical structures were capable of disrupting the endocrine systems mediated by AR, a comparative molecular field analysis (CoMFA) model was developed to analyze the structural requirements necessary to disrupt AR function. A significant CoMFA model with r(2)=0.825 and q(2)=0.332 was developed for AR antagonist activity of 35 pure antagonists excluding procymidone. On the other hand, a good CoMFA model with r(2)=0.983 and q(2)=0.555 was obtained for antagonist activity of 13 chemicals with both agonist and antagonist activities. The steric and electrostatic properties were sufficient to describe the structural requirements for AR antagonist activity. In addition, the structural difference of AR agonists and antagonists was explained based on CoMFA results and the AR-LBD crystal structure. As several ERalpha agonists such as diethylstilbestrol (DES) acted as AR antagonists, the surface area of the AR ligand-binding domain (LBD) was compared with that of the ERalpha-LBD based on their reported crystal structures to analyze how those ligands interact with LBDs. The surface area of AR-LBD was shown to be smaller than that of ERalpha-LBD and therefore compounds with both estrogenic and antiandrogenic activities can fit well into the ERalpha-LBD but may protrude from the AR-LBD. It is likely that this subtle difference of the surface areas of the LBDs determines whether an ERalpha agonist acts as an AR antagonist or an agonist.     

High-resolution CT scoring system-based grading scale predicts the clinical outcomes in patients with idiopathic pulmonary fibrosis

Background

The 2011 idiopathic pulmonary fibrosis (IPF) guidelines are based on the diagnosis of IPF using only high-resolution computed tomography (HRCT). However, few studies have thus far reviewed the usefulness of the HRCT scoring system based on the grading scale provided in the guidelines. We retrospectively studied 98 patients with respect to assess the prognostic value of changes in HRCT findings using a new HRCT scoring system based on the grading scale published in the guidelines.

Methods

Consecutive patients with IPF who were diagnosed using HRCT alone between January 2008 and January 2012 were evaluated. HRCT examinations and pulmonary function tests were performed at six-month intervals for the first year after diagnosis. The HRCT findings were evaluated using the new HRCT scoring system (HRCT fibrosis score) over time. The findings and survival rates were analyzed using a Kaplan-Meier analysis.

Results

The HRCT fibrosis scores at six and 12 months after diagnosis were significantly increased compared to those observed at the initial diagnosis (p < 0.001). The patients with an elevated HRCT fibrosis score at six months based on a receiver operating characteristic (ROC) curves analysis had a poor prognosis (log-rank, hazard ratio [HR] 2.435, 95% CI 1.196-4.962; p = 0.0142). Furthermore, among the patients without marked changes in %FVC, those with an elevated score above the cut-off value had a poor prognosis (HR 2.192, 95% CI 1.003-4.791; p = 0.0491).

Conclusions

Our data demonstrate that the HRCT scoring system based on the grading scale is useful for predicting the clinical outcomes of IPF and identifying patients with an adverse prognosis when used in combination with spirometry.