Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.     

Bilingualism Accentuates Children's Conversational Understanding

Background

Although bilingualism is prevalent throughout the world, little is known about the extent to which it influences children''s conversational understanding. Our investigation involved children aged 3–6 years exposed to one or more of four major languages: English, German, Italian, and Japanese. In two experiments, we examined the children''s ability to identify responses to questions as violations of conversational maxims (to be informative and avoid redundancy, to speak the truth, be relevant, and be polite).

Principal Findings

In Experiment 1, with increasing age, children showed greater sensitivity to maxim violations. Children in Italy who were bilingual in German and Italian (with German as the dominant language L1) significantly outperformed Italian monolinguals. In Experiment 2, children in England who were bilingual in English and Japanese (with English as L1) significantly outperformed Japanese monolinguals in Japan with vocabulary age partialled out.

Conclusions

As the monolingual and bilingual groups had a similar family SES background (Experiment 1) and similar family cultural identity (Experiment 2), these results point to a specific role for early bilingualism in accentuating children''s developing ability to appreciate effective communicative responses.     

Ninety-year-, but not single, application of phosphorus fertilizer has a major impact on arbuscular mycorrhizal fungal communities

Background and aims

Arbuscular mycorrhizal (AM) fungi play a significant role in P nutrition of crops in agriculture, but P accumulation in the soil, e.g., application of P-fertilizer, generally reduces AM fungal colonization. The impact of long-term application of chemical fertilizer on AM fungal communities was investigated with respect to the time scale.

Methods

Soils were collected from four plots with different fertilizer management in the long-term experimental field established in 1914. Lotus japonicus was grown in the soils in a greenhouse, while Glycine max was grown in the plots in the field. DNA was extracted from their roots, and the diversity and community compositions were analyzed based on occurrence of the AM fungal phylotypes defined by sequence similarity in the LSU rDNA.

Results

The 90-year-application of N and K in the absence of P increased AM fungal diversity and resulted in formation of a distinctive fungal community compared with those in the other treatments. This effect was not cancelled by single application of P. Whereas the impact of balanced application of N, P, and K was ambiguous.

Conclusion

These observations suggest that the presence/absence of P-fertilizer has a major impact on AM fungal communities, but the action may appear only on a long time scale.     

Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles

-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin.     

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning,biochemical properties and developmentally regulated expression

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

Age-independent constancy of mineral contents in human ribs

On age relationships of mineral contents in human bones, the contents of the sixth rib and a piece of its compact bone were determined by inductively coupled plasma atomic emission spectrometry (ICPS). The ribs were resected from 21 subjects (14 men and 7 women) who died in age ranging from 65 to 93 yr. There were no age-dependent decreases in Ca and P contents of the ribs in the age range on ICPS. It was found that there were no age-dependent decreases in Ca and P in compact bones of ribs.