Erratum to: Aromatic C–H bond hydroxylation by P450 peroxygenases: a facile colorimetric assay for monooxygenation activities of enzymes based on Russig’s blue formation

Aromatic C–H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450_BSβ (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of 1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed by its further oxidation to yield Russig’s blue. Russig’s blue formation allows us to estimate the peroxygenation activity of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements.     

Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatus.

In this paper, we report MALDI-TOF ms analysis of 2-nitrobenzenesulfenyl (NBS) labeled peptides with the powerful aid of an LC-automatic spotting system. using this approach we analyzed mammalian sera (rat and mouse) as biological samples to demonstrate performance. The labeling was carried out using a binary set of 2-nitrobenzenesulfenyl chloride (heavy and light), which modified tryptophan residues in sample proteins. Approximately 1600 doublet peaks were detected in the mass spectrum, some of which had more than threefold differences in their intensities. systematic separation/spotting followed by mass analysis of the NBS-labeled peptides derived from biological samples is described for the first time. This method has proved to be an effective application of NBS-labeled peptides and can be a powerful technique for quantitative analysis of proteins expressed in biological systems.     

DPPH radical scavengers from dried leaves of oregano (Origanum vulgare)

1,1-Dipehnyl-2-picrylhydrazyl (DPPH) radical scavenging activities were found in the extract of dried leaves of oregano (Origanum vulgare). The water-soluble active ingredients were isolated, and their structures were determined to be 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl 4-O-methylprotocatechuate by (1)H-, (13)C-NMR, DEPT, HMQC, and HMBC spectral analyses, and by NOE experiments. The DPPH radical scavenging activities of these compounds were compared with those of rutin, quercetin and rosmarinic acid at a concentration of 2 x 10(-5) M. The scavenging activity of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was almost the same as that of quercetin and rosmarinic acid, but that of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybennzyl 4-O-methylprotocatechuate was less than that of quercetin, rosmarinic acid and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate. The amount of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was estimated to be 3.8 mg/1 g of dried leaves by an HPLC analysis.     

Natural genetic variation in Arabidopsis: tools, traits and prospects for evolutionary ecology

BACKGROUND: The model plant Arabidopsis thaliana (Arabidopsis) shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, the potential of Arabidopsis for molecular genetic analysis of this natural variation has increased dramatically in recent years. SCOPE: Advanced genomics has accelerated molecular phylogenetic analysis and gene identification by quantitative trait loci (QTL) mapping and/or association mapping in Arabidopsis. In particular, QTL mapping utilizing natural accessions is now becoming a major strategy of gene isolation, offering an alternative to artificial mutant lines. Furthermore, the genomic information is used by researchers to uncover the signature of natural selection acting on the genes that contribute to phenotypic variation. The evolutionary significance of such genes has been evaluated in traits such as disease resistance and flowering time. However, although molecular hallmarks of selection have been found for the genes in question, a corresponding ecological scenario of adaptive evolution has been difficult to prove. Ecological strategies, including reciprocal transplant experiments and competition experiments, and utilizing near-isogenic lines of alleles of interest will be a powerful tool to measure the relative fitness of phenotypic and/or allelic variants. CONCLUSIONS: As the plant model organism, Arabidopsis provides a wealth of molecular background information for evolutionary genetics. Because genetic diversity between and within Arabidopsis populations is much higher than anticipated, combining this background information with ecological approaches might well establish Arabidopsis as a model organism for plant evolutionary ecology.     

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.     

DISAPPEARANCE OF MALE MITOCHONDRIAL DNA AFTER THE FOUR‐CELL STAGE IN SPOROPHYTES OF THE ISOGAMOUS BROWN ALGA SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE,PHAEOPHYCEAE)1

Mitochondrial DNA (mtDNA) of the isogamous brown alga Scytosiphon lomentaria (Lyngb.) Link is inherited maternally. We used molecular biological and morphological analyses to investigate the fate of male mitochondria. Ultrastructural observations showed that the number of 25 mitochondria in a zygote coincided with the number of mitochondria derived from male and female gametes. This number remained almost constant during the first cell division. Strain‐specific PCR in single germlings suggested that mtDNA derived from the female gamete remained in the germling during development, while the male mtDNA gradually and selectively disappeared after the four‐cell stage. One week after fertilization, male mtDNA had disappeared in sporophytic cells. Using bisulfite DNA modification and methylation mapping assays, we found that the degree of methylation on three analyzed sites of mtDNA was not different between male and female gametes, suggesting that maternal inheritance of mtDNA is not defined by its methylation. This study indicates that the mechanism of selective elimination of male mtDNA is present in each cell of a four‐celled sporophyte and that it does not depend on different degrees of DNA methylation between male and female mtDNA.     

The parenting attitudes and the stress of mothers predict the asthmatic severity of their children: a prospective study

Objective  

To examine relationships between a mother's stress-related conditions and parenting attitudes and their children's asthmatic status.     

A potent endocytosis inhibitor Ikarugamycin up-regulates TNF production

Ikarugamycin (IK) is an antibiotic which has been reported to have a variety of functions, such as inhibition of clathrin-mediated endocytosis (CME), anti-tumor effects and regulation of the immune system. Whether IK influences cytokine production is poorly understood. We have investigated the relationship between IK and production of tumor necrosis factor-α (TNF). TNF plays a pivotal role in pathogenesis of many diseases. Although the dynamics of soluble TNF (sTNF) has been widely explored so far, the functions of the membrane form of TNF (mTNF) have not been fully elucidated. We demonstrated that IK increases the amount of mTNF and prolongs the duration of TNF expression. This effect is unrelated to the shedding activity of disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17). Our results revealed that there is a mechanism to terminate inflammation at the cellular level which IK dysregulates. Furthermore, IK can be a tool to study TNF signaling due to its effect of increasing mTNF expression.     

Aromatic C–H bond hydroxylation by P450 peroxygenases: a facile colorimetric assay for monooxygenation activities of enzymes based on Russig’s blue formation

Aromatic C–H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450_BSβ (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of 1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed by its further oxidation to yield Russig’s blue. Russig’s blue formation allows us to estimate the peroxygenation activity of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements.