A petal‐specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal‐specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal‐specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal‐specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β‐glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal‐specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA‐like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal‐specific promoter in molecular breeding of floricultural crops.     

LC–MS/MS-based metabolome analysis detected changes in the metabolic profiles of small and large intestinal adenomatous polyps in <Emphasis Type="Italic">Apc</Emphasis><Superscript><Emphasis Type="Italic">Min</Emphasis>/+</Superscript> mice

Introduction

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene that is inactivated in the initiation of colorectal neoplasia. Apc ^Min/+ mice, which possess a heterozygous APC mutation, develop numerous adenomatous polyps, which are similar to those observed in familial adenomatous polyposis (FAP) in humans. However, unlike FAP patients, Apc ^Min/+ mice predominantly develop adenomatous polyps in the small intestine. The metabolic changes associated with the development of polyps in the small and large intestine remain to be investigated.

Objectives

The objective of this study was to elucidate the metabolic changes associated with intestinal polyp formation.

Methods

We compared the metabolite levels of pairs of polyp and non-polyp tissues obtained from the small intestines (n = 12) or large intestines (n = 7) of Apc ^Min/+ mice. To do this, we analyzed the tissue samples using two methods, liquid chromatography-tandem mass spectrometry (1) with a pentafluorophenylpropyl column for cation analysis, and (2) with a C18 reversed phase column coupled to an ion-pair reagent for anion analysis.

Results

Pathway mapping of the metabolites whose levels were significantly altered revealed that the polyp tissue of the small intestine contained significantly higher levels of intermediates involved in glycolysis, the pentose phosphate pathway, nucleotide metabolism, or glutathione biosynthesis than in the equivalent non-polyp tissue. In addition, significantly higher levels of methionine cycle intermediates were detected in the polyp tissues of both the large and small intestines. Organ-dependent (small vs. large intestine) differences were also detected in the levels of most amino acids and urea cycle intermediates.

Conclusion

Our results indicate that various metabolic changes are associated with polyp development, and understanding these alterations could make it possible to evaluate the treatment response of colorectal cancer earlier.

     

Structure-activity relationships of trichothecenes against COLO201 cells and Cochliobolus miyabeanus: The role of 12-epoxide and macrocyclic moieties

The novel trichothecene 12-deoxytrichodermin (3) was isolated from the fungus Trichoderma sp. 1212-03, and included with other known natural trichothecenes in a structure-activity relationship investigation against a human colon cancer cell line (COLO201) and filamentous fungus Cochliobolus miyabeanus. This revealed that the 12-epoxide functionality is critical for the cytotoxicity of simple trichothecenes trichodermin (4) and deoxynivalenol (2), while not critical for the cytotoxicity of roridin J (6) and epiisororidin E (8). In contrast, 12-epoxide is essential for the antifungal activity.     

Quantitative proteomic analysis of rat liver for carcinogenicity prediction in a 28-day repeated dose study

The potential of quantitative proteomic analysis to predict carcinogenicity of chemical compounds was investigated. Using 2D-DIGE, we analyzed the effects of 63 chemical compounds on protein expression in the rat liver after 28 daily doses. Types of carcinogens were categorized depending on the species and organ specificity. The carcinogen characteristic proteins for each classification were identified by Welch's t value. For evaluation of the predictive concordance we used support vector machines. The rat hepatic carcinogen-specific classification gave higher concordance than the other classification. The generalization performance was measured by leave-one-out cross-validation. For genotoxic and non-genotoxic compounds, a concordance of 79.3 and 76.5%, respectively, was obtained by the top 30 ranked proteins with Welch's t value. Furthermore, we found that the increase of the expression level of the stress response proteins as the common feature of poorly predicted chemical compounds in the leave-20%-out cross-validation. Quantitative proteomics could be promising technique for developing biomarker panels that can be used for carcinogenicity prediction. The list of proteins identified in this study and the zoomed gel images of the top ranked proteins in statistic analysis are provided in Supplementary Data.     

Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.     

Koji glycosylceramide commonly contained in Japanese traditional fermented foods alters cholesterol metabolism in obese mice

ABSTRACT

Koji, which is manufactured by proliferating non-pathogenic fungus Aspergillus oryzae on steamed rice, is the base for Japanese traditional fermented foods. We have revealed that koji and related Japanese fermented foods and drinks such as amazake, shio-koji, unfiltered sake and miso contain abundant glycosylceramide. Here, we report that feeding of koji glycosylceramide to obese mice alters the cholesterol metabolism . Liver cholesterol was significantly decreased in obese mice fed with koji glycosylceramide. We hypothesized that their liver cholesterol was decreased because it was converted to bile acids. Consistent with the hypothesis, many bile acids were increased in the cecum and feces of obese mice fed with koji glycosylceramide. Expressions of CYP7A1 and ABCG8 involved in the metabolism of cholesterol were significantly increased in the liver of mice fed with koji glycosylceramide. Therefore, it was considered that koji glycosylceramide affects the cholesterol metabolism in obese mice.     

Rapid and simultaneous detection of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization

This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately denatured at 75°C for 8 min, hybridized for 5 min at 37°C and overall FISH steps were done within a few hours. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig-signal, 99.2% of the sperm nuclei had the biotin-signal and the average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa. © 1996 Wiley-Liss, Inc.     

Purification and characteristics of cytosolic chitinase from Piromyces communis OTS1

Abstract A chitinase was purified from the cytosolic fraction of the anaerobic rumen fungus Piromyces communis OTS1 by affinity chromatography using regenerated chitin, gel filtration and chromatofocusing. The chitinase was most active at pH 6.2 and at 60 °C in a 20-min assay. The molecular mass of the purified protein was estimated by SDS-PAGE to be 42 kDa and its pI was 4.9. The enzyme activity, which was of the 'endo' type, was inhibited by A⁺, Hg²⁺ and allosamidin. N -Acetyl- β -glucosaminidase and 'exo' type chitinase activity were absent from the purified preparation.