Snapshots of DsbA in action: detection of proteins in the process of oxidative folding

DsbA, a thioredoxin superfamily member, introduces disulfide bonds into newly translocated proteins. This process is thought to occur via formation of mixed disulfide complexes between DsbA and its substrates. However, these complexes are difficult to detect, probably because of their short-lived nature. Here we show that it is possible to detect such covalent intermediates in vivo by a mutation in DsbA that alters cis proline-151. Further, this mutant allowed us to identify substrates of DsbA. Alteration of the cis proline, highly conserved among thioredoxin superfamily members, may be useful for the detection of substrates and intermediate complexes in other systems.     

Magmatic dike resonances inferred from very-long-period seismic signals

Very-long-period (VLP) signals showing simple decaying harmonic oscillations with periods near 10 seconds and lasting for about 300 seconds were observed in association with an earthquake swarm that occurred beneath Hachijo Island, Japan. Results from the source-mechanism analysis and waveform simulation based on a fluid-filled crack model consistently point to the resonance of a dike filled with a basaltic magma as the source of the VLP signals. Thus, VLP signals can be used to probe the state of the fluid and dynamic processes within a volcanic system.     

Oxygen isotopic compositions of asteroidal materials returned from Itokawa by the Hayabusa mission

Meteorite studies suggest that each solar system object has a unique oxygen isotopic composition. Chondrites, the most primitive of meteorites, have been believed to be derived from asteroids, but oxygen isotopic compositions of asteroids themselves have not been established. We measured, using secondary ion mass spectrometry, oxygen isotopic compositions of rock particles from asteroid 25143 Itokawa returned by the Hayabusa spacecraft. Compositions of the particles are depleted in (16)O relative to terrestrial materials and indicate that Itokawa, an S-type asteroid, is one of the sources of the LL or L group of equilibrated ordinary chondrites. This is a direct oxygen-isotope link between chondrites and their parent asteroid.     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Lipogenic gene expression in abdominal adipose and liver tissues of diet-induced overweight cats

The effect of overweight status on the expression of SREBP-1c and downstream lipogenic genes, such as ATP citrate lyase (ACL) and fatty acid synthase (FAS), in abdominal adipose and liver tissues was determined in cats using a diet-induced weight gain model. ACL and SREBP-1c mRNA expression was significantly reduced (～65% and 20%, respectively) in liver tissue, whereas FAS and SREBP-1c expression was significantly increased (～80% and 45%, respectively) in abdominal omental adipose tissue of overweight animals as compared to healthy animals. Additionally, ACL, FAS, and SREBP-1c expression was significantly reduced by ～50%, 75%, and 70%, respectively, in abdominal subcutaneous adipose tissue of overweight animals. Omental adipose tissue appeared to foster, whereas subcutaneous adipose and liver tissues appeared to defer lipid storage based on differences in SREBP-1c mRNA expression. Overall, reduced lipogenic gene mRNA expression patterns support the hypothesis that SREBP-1c expression is reduced in overweight and possibly obese cats, reflecting down-regulation of the lipogenic pathway to prevent further fat accumulation and weight gain.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.     

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.