Molecular determinants that convert hormone sensitive lipase into gibberellin receptor

Gibberellins (GAs) are tetracyclic, diterpenoid plant hormones, essential for many developmental processes in higher plants. Plants perceive GA through a nuclear-localized GA receptor, GA INSENSITIVE DWARF1 (GID1). From sequence similarity, it is suggested that GID1 evolved from a hormone-sensitive lipase (HSL), and recent x-ray crystallography of the GA-GID1 complex has given insights into how GID1 recognizes GA. Analyses of the GA signaling pathway in several plant species further suggest that the GID1-mediated GA signaling pathway emerged in the vascular plant lineage and since then regulation of GA recognition specificity seems to have been fine tuned to strictly regulate the on-off GA signal.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.     

The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity

When the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF 1 (GID1) binds to GA, GID1 interacts with DELLA proteins, repressors of GA signaling. This interaction inhibits the suppressive function of DELLA protein and thereby activates the GA response. However, how DELLA proteins exert their suppressive function and how GID1s inhibit suppressive function of DELLA proteins is unclear. By yeast one-hybrid experiments and transient expression of the N-terminal region of rice DELLA protein (SLR1) in rice callus, we established that the N-terminal DELLA/TVHYNP motif of SLR1 possesses transactivation activity. When SLR1 proteins with various deletions were over-expressed in rice, the severity of dwarfism correlated with the transactivation activity observed in yeast, indicating that SLR1 suppresses plant growth through transactivation activity. This activity was suppressed by the GA-dependent GID1-SLR1 interaction, which may explain why GA responses are induced in the presence of GA. The C-terminal GRAS domain of SLR1 also exhibits a suppressive function on plant growth, possibly by directly or indirectly interacting with the promoter region of target genes. Our results indicate that the N-terminal region of SLR1 has two roles in GA signaling: interaction with GID1 and transactivation activity.     

Carbon sequestration in soil in a semi‐natural Miscanthus sinensis grassland and Cryptomeria japonica forest plantation in Aso,Kumamoto, Japan

Although Miscanthus sinensis grasslands (Misc‐GL) and Cryptomeria japonica forest plantations (Cryp‐FP) are proposed bioenergy feedstock systems, their relative capacity to sequester C may be an important factor in determining their potential for sustainable bioenergy production. Therefore, our objective was to quantify changes in soil C sequestration 47 years after a Misc‐GL was converted to a Cryp‐FP. The study was conducted on adjacent Misc‐GL and Cryp‐FP located on Mt. Aso, Kumamoto, Japan. After Cryp‐FP establishment, only the Misc‐GL continued to be managed by annual burning every March. Mass C and N, δ¹³C, and δ¹⁵N at 0–30 cm depth were measured in 5 cm increments. Carbon and N concentrations, C:N ratio, δ¹³C, and δ¹⁵N were measured in litter and/or ash, and rhizomes or roots. Although C input in Misc‐GL by M. sinensis was approximately 36% of that in Cryp‐FP by C. japonica, mean annual soil C sequestration in Misc‐GL (503 kg C ha^?1 yr^?1) was higher than that in Cryp‐FP (284 kg C ha^?1 yr^?1). This was likely the result of larger C input from aboveground litter to soil, C‐quality (C:N ratio and lignin concentration in aboveground litter) and possibly more recalcitrant C (charcoal) inputs by annual burning. The difference in soil δ¹⁵N between sites indicated that organic C with N had greater cycling between heterotrophic microbes and soil and produces more recalcitrant humus in Misc‐GL than in Cryp‐FP. Our data indicate that in terms of soil C sequestration, maintenance of Misc‐GL may be more advantageous than conversion to Cryp‐FP in Aso, Japan.     

Jacaric acid,a linolenic acid isomer with a conjugated triene system,has a strong antitumor effect in vitro and in vivo

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.     

Frequency and aetiology of dermatophytosis in children age 12 and under in the state of Amazonas,Brazil

BackgroundFew scientific studies have evaluated dermatophytosis among children in the state of Amazonas or in the greater northern region of Brazil.AimsThe aim of this study was to research the frequency and aetiology of dermatophytosis in children age 12 and under, who were seen between March 1996 and November 2005 at the Mycology Laboratory of the National Institute of Amazonian Research.MethodsFor mycological diagnoses, epidermal scales and/or hairs were used. A portion of this material was treated with potassium hydroxide for direct examination, and another portion was cultivated in Mycobiotic Agar for the isolation of dermatophytes.ResultsOf the 590 samples analysed, 210 showed positive diagnoses by direct examination and cultivation. Tinea capitis (153 cases) was the most frequent type of dermatophytosis, and Trichophyton tonsurans (121 cases) was the most frequently isolated fungal agent. Tinea corporis was observed in 48 cases where the most frequently isolated fungal agent was also T. tonsurans (17 cases), and the corporal regions most affected were the face, arms and trunk. The laboratory confirmed tinea pedis in 6 cases, and the principal fungal agents isolated were Trichophyton rubrum (3) and Trichophyton mentagrophytes (3). The presence of tinea cruris was confirmed in 3 cases, and T. rubrum, T. tonsurans and Epidermophyton floccosum were isolated from these cases.ConclusionsThe children examined were primarily affected by tinea capitis, and the main fungal agent for this dermatophytosis was T. tonsurans.     

Redesign of coenzyme B(12) dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols

Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract ? Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).