DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA

Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.     

Evaluation of immunochromatographic test kits for food allergens using processed food models

It has been mandatory to label five allergenic substances (AS; egg, milk, wheat, buckwheat and peanut) in all processed foods, since April 2002 in Japan. Two kinds of ELISA kits have been provided as screening test kits for the Japanese official method. The kits have many advantages but some disadvantages, i.e., the kits are not necessarily suitable for daily monitoring in food manufacturing plants, because they require various analytical equipments and the use of complicated procedures. To overcome these drawbacks, we have developed other diagnostic kits based on immunochromatography that should enable more rapid and simple screening for food allergens. Then we examined the performance of these immunochromatographic test kits (IC kits) in terms of sensitivity, repeatability and cross-reactivity to AS proteins in 11 kinds of food models with various heating conditions and physical properties. We also examined processed food models including AS protein of constant concentration, using the IC kits and ELISA kits, and compared the results. The IC kits detected AS proteins at 5 microg/g in the extracts from processed food models, and provided highly reproducible results. Cross-reactivity among the AS proteins was not observed. The results obtained using the IC kits showed performance equivalent to that of the ELISA kits we examined in unheating processed food models including AS proteins of constant concentration. The IC kits should be more suitable for daily monitoring in food manufacturing plants.     

Simultaneous determination of pesticides in agricultural products by LC/MS/MS using clean-up with ultrafiltration

A method for simultaneous determination of multiple pesticide residues in agricultural products was developed by using a pretreatment with ultrafiltration, followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pretreatment process (extraction of pesticides from agricultural products with methanol, dilution of the extract with water, and ultrafiltration) gave recoveries in the range of 50-150% for 63 of 83 pesticides spiked at 0.25 microg/ g into 6 agricultural products. The detection limits of pesticides by LC/MS/MS were below 0.0005-0.05 micro/g. This method is useful for screening purposes and for multiresidue analysis of pesticides in agricultural products. Pesticide residues in 50 domestic crops were investigated by this method, and residues of 14 pesticides were detected in 30 crops.     

Analysis of essential amino acid residues for catalytic activity of glutaminase from Micrococcus luteus K-3

Structural-based mutational analysis of salt-tolerant glutaminase from Micrococcus luteus K-3 (Micrococcus glutaminase) revealed that three amino acid residues, S64, K67, and E160, were essential to a catalytic reaction. The result suggested that Micrococcus glutaminase had a possible catalytic mechanism similar to class A beta-lactamase rather than glutaminase-asparaginase from Pseudomonas 7A.     

Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system

Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically aminoacylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.     

In vivo visual reporter system for detection of estrogen-like substances by transgenic medaka

Detection of endocrine disrupting chemicals, in particular, environmental estrogens with living organisms, has many advantages if compared to chemical analysis. The screening of novel pollutants with meaningful endpoints, the integration of uptake, bioconcentration, and excretion as well as the evaluation of endocrine disrupting effects with respect to toxicity require in vivo biotests for estrogen-like substances (ELSs). Critical disadvantages of whole organism biotests are their low sensitivity and the need for laborious and time-consuming work. To overcome these problems, we have developed a transgenic medaka strain harboring the green fluorescence protein (GFP) gene driven by choriogenin H gene regulatory elements. Choriogenin H is an egg envelope protein induced by estrogens in the liver. With yolk sac larvae of this strain, GFP induction in liver was observed 24 h after onset of aqueous exposure to 0.63 nM 17beta-estradiol (E2), 0.34 nM ethynylestradiol, or 14.8 nM estrone. Furthermore, concentrated sewage treatment effluent induced GFP expression. Comparison of E2 equivalents estimated by GFP-induction in transgenic medaka, a YES assay, and GC/MS showed detection limits in the same order of magnitude. These results indicated that the sensitivity of the transgenic medaka strain was sufficient for application as an alternative model in monitoring environmental water samples for ELSs.     

Colorimetric microdetermination of sulphites in foods by use of the modified rankine apparatus. IV.

Summary Detection and determination of traces of sulphites in foods was attempted by use of the modified Rankine apparatus and pararosaniline colorimetry. Replacement of alkaline titration reported previously by pararosaniline colorimetry lowered the absolute detection limit from 30 g (titration method) to 2 g. In view of clean analysis, in the color developing system, 0.1 N-sodium hydroxide was used in place of mercuric chloride solution commonly used as an absorbant of sulphites. In order to prevent oxidative decomposition of sulphites during operation, nitrogen gas was used as carrier instead of air. Dimedone and sodium azide were used for the elimination of aldehydes and nitrites, respecitvely, in the sample, which will disturb the color development of sulphites with pararosaniline-formaldehyde reagents. With this improved method, it was possible to determine the residual sulphites in frozen peeled shrimps, sugared beans and other foods with low sulphite contents accurately.

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Colorimetrische Mikrobestimmung von Sulfiten in Lebensmitteln bei Anwendung der modifizierten IV. Rankine Apparatur

Zusammenfassung Geringe Sulfitmengen in Lebensmitteln (geschälte Garnelen, gezuckerte Bohnen) können colorimetrisch bestimmt werden. Die neuentwickelte Methode beruht auf einer Kombination von colorimetrischer Bestimmung mittels p-Rosanilin und der Bestimmungsmethode nach Rankine. Auf diese Weise lassen sich Gehalte von 2 g noch genau bestimmen. Bei der Farbentwicklung wurde das giftige Quecksilbertetrachlorid durch 0.1 n-NaOH ersetzt, anstelle von Luft Stickstoff als Trägergas verwendet und somit eine Oxydation des Sulfits während der Bestimmung vermieden. Da Nitrit und Aldehyde die Farbentwicklung stören, wurde ihr Einfluß durch Dimedon und Natriumazid ausgeschaltet.

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Studies on the Analyses of Sulphites in Foods (IV)     

Establishment of a modified rankine method for the separate determination of free and combined sulphites in foods. III

Summary Considerable improvements were made to the original Rankine method. Replacement of aspiration with an injection system contributed a great deal to the simplification of procedure, being accompanied with an increase in reproducibility. Air (flow rate 1.01/min) was used for injection because the use of inert gas gave little increase in recovery rate.Sodium bisulphite (free sulphite) and three kinds of combined sulphite compound (bisulphite adducts of acetaldehyde, pyruvic acid and D-mannose) were used to find the most suitable conditions for the separate determination of free and combine sulphites.Free sulphite was expelled from the sample by bubbling at 0 °C for 30 min. It was confirmed that no combined sulphite was dissociated under these conditions. The phosphoric acid concentration had an important role in the liberation of sulphite. When 25% phosphoric acid was used, more than 99% of free sulphite was expelled by cold bubbling and more than 99% of combined sulphite was recovered by heating afterwards for 10 min.The scope of the modified Rankine method was also extended to the determination of sulphite in concentrated orange juice.

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Verwendung der modifizierten Rankine-Methode zur getrennten Bestimmung von Sulfiten in Lebensmitteln. III

Zusammenfassung Die Rankine-Methode wurde bedeutend verbessert. Ein Umtausch der Aspiration mit Blasensystem trug beträchtlich zur Vereinfachung des Bestimmungsverfahrens bei, und die Reproduzierbarkeit wurde verbessert. Luft (Fließrate 1,01/min) wurde als Blasengas benutzt, da der Gebrauch von Inertgas für die Wiederfindungsrate unbedeutend ist.Natriumhydrogensulfit (freies Sulfit) und drei Arten gebundener Sulfite (Acetaldehydhydrogensulfit, Pyruvathydrogensulfit undd-Mannosehydrogensulfit) dienten dazu, die geeignetsten Bedingungen für die getrennte Bestimmung der freien und gebundenen Sulfite zu ermitteln.Freies Sulfit wurde bei 0 °C durch 30 min Durchblasen vertrieben. Hierbei ging kein gebundenes Sulîit verloren. Die Phosphorsäurekonzentration war wichtig für die Freisetzung des Sulfites. Wenn man 25%ige Phosphorsäure verwendet, werden > 99% freien Sulfites beim Durchblasen in der Kälte vertrieben, während > 99% gebundenen Sulfites durch nachheriges 10 min langes Erhitzen wiedergewonnen werden.Die modifizierte Rankine-Methode wurde weiterhin für konzentrierte Säfte verwendet.

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Studies on the Analyses of Sulphites in Foods (III)