Pebbles and sand on asteroid (162173) Ryugu: In situ observation and particles returned to Earth.

The Hayabusa2 spacecraft investigated the C-type (carbonaceous) asteroid (162173) Ryugu. The mission performed two landing operations to collect samples of surface and subsurface material, the latter exposed by an artificial impact. We present images of the second touchdown site, finding that ejecta from the impact crater was present at the sample location. Surface pebbles at both landing sites show morphological variations ranging from rugged to smooth, similar to Ryugu's boulders, and shapes from quasi-spherical to flattened. The samples were returned to Earth on 6 December 2020. We describe the morphology of >5 grams of returned pebbles and sand. Their diverse color, shape, and structure are consistent with the observed materials of Ryugu; we conclude that they are a representative sample of the asteroid.

44Ca doped remineralization study on dentin by isotope microscopy.

OBJECTIVE: The dental caries is developed as a result of an alternative course of mineral gain and loss. In order to distinguish between intrinsic Ca (tooth-derived mineral) and extrinsic Ca (solution-derived mineral) uptakes, a 44Ca doped pH-cycling was performed using 44Ca (a stable calcium isotope) remineralization solution. METHODS: The natural abundance of 40Ca and 44Ca is 96.9% and 2.1%, respectively. The remineralization solution was prepared using 44Ca to contain 1.5mmol/L CaCl2 (44Ca), 0.9mmol/L KH2PO4, 130mmol/L KCl, 20mmol/L HEPES at pH 7.0. The pH-cycling was conducted on bovine root dentin daily by demineralization (pH 5.0) for 2h, incubation in 0% (control) and 0.2% NaF (900ppm fluoride) for 2h and 44Ca doped remineralization for 20h. After 14days pH-cycling, the specimens were sectioned longitudinally. On the sectioned surface, isotope imaging of 40Ca and 44Ca labeled mineral distribution was observed by a high mass-resolution stigmatic secondary ion 77 (Camera IMS 1270, Gennevilliers Cedex, France). RESULTS: Uptake of 44Ca was greater in intensity for the 0.2% fluoride group than the control, especially in the superficial lesions. The control group showed 40Ca (intrinsic) distribution in the subsurface lesions and in the superficial lesions, meanwhile the fluoride group showed 40Ca distribution limited in subsurface lesions. The total Ca (44Ca+40Ca) image revealed more homogeneously for the control than the fluoride group. SIGNIFICANCE: Since the fluoride-treated surface is more acid-resistant than intrinsic dentin, alternative minerals were dissolved from the intact intrinsic lesion in the demineralization cycle.

The fifth epidermal growth factor-like region of thrombomodulin exerts cytoprotective function and prevents SOS in a murine model.

The present study found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) possesses the cytoprotective function in association with an increase in levels of anti-apoptotic myeloid cell leukemia-1 protein in an activated protein C-independent manner in human umbilical vein endothelial cells (HUVECs). Importantly, TME5 counteracted calcineurin inhibitor-induced vascular permeability and successfully prevented monocrotaline-induced sinusoidal obstruction syndrome (SOS) in a murine model. Taken together, TME5 may be useful for preventing or treating lethal complications that develop after hematopoietic stem cell transplantation such as SOS and thrombotic microangiopathy in which endothelial cell damage has a role.

Neutron activation analysis of a particle returned from asteroid Itokawa.

A single grain (~3 micrograms) returned by the Hayabusa spacecraft was analyzed by neutron activation analysis. This grain is mainly composed of olivine with minor amounts of plagioclase, troilite, and metal. Our results establish that the Itokawa sample has similar chemical characteristics (iron/scandium and nickel/cobalt ratios) to chondrites, confirming that this grain is extraterrestrial in origin and has primitive chemical compositions. Estimated iridium/nickel and iridium/cobalt ratios for metal in the Itokawa samples are about five times lower than CI carbonaceous chondrite values. A similar depletion of iridium was observed in chondrule metals of ordinary chondrites. These metals must have condensed from the nebular where refractory siderophile elements already condensed and were segregated.

Cis-acting elements sufficient for induction of FDH1 expression by formate in the methylotrophic yeast Candida boidinii.

The FDH1 gene of Candida boidinii encodes an NAD+-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5' non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.

Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance.

In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.

Characterization and high-level production of D-amino acid oxidase in Candida boidinii.

D-Amino acid oxidase (DAO, EC 1.4.3.3) from a methylotrophic yeast, Candida boidinii, was produced at a high level under the control of the alcohol oxidase gene promoter in the original host. The enzyme was a peroxisomal and monomeric enzyme, and contained noncovalently-bound FAD as a cofactor. The enzyme was active toward several D-amino acids such as D-Ala, D-Met, and D-Ser. An alcohol oxidase-depleted strain (aod1delta) was found to be a more suitable host for DAO production than the wild-type strain. Several post-translational effects may be responsible for the improvement of the DAO productivity by the aod1delta strain. Finally, an aod1delta strain transformant having multi-copies of an expression plasmid on its chromosome could produce DAO amounting up to 30% of the total soluble proteins.

Antioxidant system within yeast peroxisome. Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii.

Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using the PMP20-deleted strain of C. boidinii (pmp20Delta strain). The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H(2)O(2). Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding to Cys(53). The pmp20Delta strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Delta strain had a more severe growth defect than the cta1Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, the cta1Delta strain accumulated H(2)O(2), whereas the pmp20Delta strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose H(2)O(2). In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.

Analysis of alcohol oxidase isozymes in gene-disrupted strains of methylotrophic yeast Pichia methanolica.

A cell-free extract of methanol-grown Pichia methanolica cells was found to contain nine alcohol oxidase (AOD) isozymes by active staining of a native polyacrylamide electrophoresis gel. Our previous study revealed that AOD in P. methanolica was encoded by two genes, MOD1 and MOD2, and the results of an experiment involving Candida boidinii as an expression host suggested that the AOD isozymes observed in P. methanolica were due to random association of Mod1p and Mod2p into an active octamer [Nakagawa et al., Yeast, 15, 1223-1230 (1999)]. This study was conducted using P. methanolica MOD1- and/or MOD2-gene disrupted strains to confirm a previous hypothesis. While the cell-free extract of the wild-type strain gave nine ladder bands, the mod1delta and mod2delta strains gave a single active AOD band corresponding to the mobilities of Mod2p and Mod1p on a native electrophoresis gel, respectively. The cell-free extract of glyceorl-grown wild-type cells gave a single band corresponding to Mod1p, showing that only MOD1 is expressed in glycerol-grown cells. While the expression of both MOD1 and MOD2 was induced by methanol, this finding and our previous observations indicated that the expression of MOD1 and MOD2 was controlled by a distinct regulatory mechanism in P. methanolica.

Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii.

A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii. The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C. boidinii. We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes.

A methylotrophic pathway participates in pectin utilization by Candida boidinii.

The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii.

We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.

Chalcone and stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro.

Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different cyclization reactions of the common tetraketide to give different products, naringenin chalcone and resveratrol, respectively. We have previously observed in vitro cross-reaction of CHS and STS overexpressed in Escherichia coli, resveratrol production by CHS and chalcone production by STS. When expressed in eucaryotic cells, or in E. coli as thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction. STS refolded from inclusion bodies also showed reduced cross-reaction. While addition of bovine serum albumin and pH in the reaction were without noticeable effect, addition of glycerol decreased the cross-reaction of CHS likely due to its stabilizing effect on enzyme conformation. These results were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi T. et al., FEBS Lett., 460, 457-4 61 (1999)) that the in vitro cross-reaction of CHS and STS is due to intrinsic capability of these enzymes to catalyze different types of cyclization, which, in turn, is endowed by conformational flexibility of their active sites.

Primary structure and expression of peroxisomal acetylspermidine oxidase in the methylotrophic yeast Candida boidinii.

Acetylspermidine oxidase (ASOD) belongs to a family of FAD-containing amine oxidases and catalyzes the oxidation of N-acetylated spermidine in polyamine metabolism. ASOD was purified to apparent homogeneity from cells of the methylotrophic yeast Candida boidinii grown on spermidine as the sole nitrogen source. C. boidinii ASOD catalyzed the oxidation of only N(1)-acetylspermidine. Based on partial amino acid sequences, oligonucleotide primers were designed for polymerase chain reaction, and the ASOD-encoding gene, ASO1, was cloned. The open reading frame encoding ASO1 was 1530 bp long and corresponded to a protein of 509 amino acid residues (calculated molecular mass=57167 Da). ASO1 contained a FAD-binding motif of G-A-G-I-A-G in the N-terminal region and carried an amino acid sequence of -S-K-L at the C-terminal, representing a typical peroxisome targeting signal 1. ASOD was localized in the peroxisomes in overexpressed C. boidinii. To our knowledge, this is the first report on the gene coding for ASOD that can catalyze the oxidation of N-acetylated polyamine as a substrate, from any type of organism.

Peroxisomal membrane protein Pmp47 is essential in the metabolism of middle-chain fatty acid in yeast peroxisomes and Is associated with peroxisome proliferation.

Pmp47 of the methylotrophic yeast Candida boidinii belongs to a mitochondrial family of solute transporters and is localized in peroxisomal membranes. Its human homolog, Pmp34, is also known. In this study, we characterized the role of Pmp47 in fatty acid metabolism and peroxisome proliferation using the PMP47-deleted strain of C. boidinii (strain pmp47Delta). The wild-type strain grew well on a middle-chain fatty acid, laureate, as the single carbon source, and mild peroxisome proliferation was observed during its growth. The pmp47Delta strain could not grow on laureate but could grow on long-chain fatty acids including palmitate, myristate, and oleate. The levels of laureate oxidation activity in intact cells and in semi-permeabilized cells of strain pmp47Delta were lower than the respective level in the wild-type strain, although the level of laureate oxidation activity in the cell lysate and the level of lauroyl-CoA oxidation in semi-permeabilized cells of strain pmp47Delta were indistinguishable from the respective level in the wild-type strain. When lauroyl-CoA was provided in the cytosol of strain pmp47Delta through expression of Saccharomyces cerevisiae Faa2p (lauroyl-CoA synthetase) in which its peroxisome targeting signal was deleted, the growth of strain pmp47Delta on laureate was recovered to the level of growth of the wild-type strain. Laureate is converted to its CoA form in peroxisomes by the action of lauroyl-CoA synthetase. These results suggested that Pmp47 is involved in the transport of a small molecule (possibly ATP) required in the conversion of laureate to its CoA form in peroxisomes and that the absence of Pmp47 causes impairment of laureate metabolism, which results in the inability of pmp47Delta cells to grow on laureate. In addition, Pmp47 may be involved in peroxisome proliferation, because the pmp47Delta strain contained a reduced number of peroxisomes, as judged from the fluorescence analysis of cells expressing green fluorescent protein tagged with the peroxisome targeting signal 1 (GFP-AKL).

Production of fungal fructosyl amino acid oxidase useful for diabetic diagnosis in the peroxisome of Candida boidinii.

A high-level production of fructosyl amino acid oxidase (FAOD), whose production was toxic in Escherichia coli, was investigated through attempts to utilize the peroxisome of Candida boidinii as the place for protein accumulation. The alcohol oxidase-depleted strain (strain aod1Delta) produced FAOD at a four to five times higher level than the wild type strain in terms of protein amount and enzyme activity, although the transcriptional level was similar. As a result of this study, we could improve FAOD productivity approximately 47-fold from the original transformant, and FAOD accumulated within membrane-bound peroxisomes up to 18% of the total soluble proteins.

Alcohol oxidase hybrid oligomers formed in vivo and in vitro.

Cell-free extract prepared from methanol-grown cells of the methylotrophic yeast Pichia methanolica showed nine multiple alcohol oxidase (AOD) bands on active staining in native polyacrylamide gel electrophoresis. Their molecular basis was investigated and two AOD-encoding genes, MOD1 and MOD2, were cloned from P. methanolica genome. When the two genes were expressed in a heterologous host, an alcohol oxidase-depleted strain of Candida boidinii(aod1Delta strain), both MOD1 and MOD2 partially complemented growth defect of the host strain on methanol. While expression of either MOD1 or MOD2 in C. boidinii aod1Delta strain gave a single AOD band corresponding to the band with the largest and smallest mobility among the nine AOD bands, respectively, co-expression of MOD1 and MOD2 resulted in multiple band formation. Mixed oligomerization of Mod1p and Mod2p in vitro also gave nine multiple bands. From these results, we concluded that the nine multiple forms of AOD observed on native-PAGE represent two homo-octamers and seven hetero-octamers of Mod1p and Mod2p. Using this zymogram analysis, we also found that Mod1p was preferably produced at low methanol concentrations in the media, while Mod2p was produced at higher methanol concentrations. This shows distinct regulatory features of the two AOD-encoding genes in this methylotrophic yeast.

A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli.

A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.

Subcellular localization of fructosyl amino acid oxidases in peroxisomes of Aspergillus terreus and Penicillium janthinellum.

Fructosyl amino acid oxidase (FAOD) is the enzyme catalyzing the oxidative deglycation of Amadori compounds, such as fructosyl amino acids, yielding the corresponding amino acids, glucosone, and H(2)O(2). In a previous report, we determined the primary structures of cDNAs coding for FAODs from two fungal strains Aspergillus terreus AP1 and Penicillium janthinellum and we found that both fungal FAODs included the putative peroxisome targeting signal 1 (PTS1) at the carboxyl terminal (Yoshida, N. et al., Eur. J. Biochem., 242, 499-505, 1996). In this study, we determined the intracellular localization of FAODs in these two fungi. Subcellular fractionation experiments and immuno-electronmicroscopic observations, together with the previous findings indicated that the FAODs were localized in peroxisomes of A. terreus AP1 and P. janthinellum. These FAODs were also found to belong to a new member of "peroxisomal sarcosine oxidase family protein" in eucaryotic cells.

Oxygen isotope exchange between refractory inclusion in allende and solar nebula Gas

A calcium-aluminum-rich inclusion (CAI) from the Allende meteorite was analyzed and found to contain melilite crystals with extreme oxygen-isotope compositions ( approximately 5 percent oxygen-16 enrichment relative to terrestrial oxygen-16). Some of the melilite is also anomalously enriched in oxygen-16 compared with oxygen isotopes measured in other CAIs. The oxygen isotopic variation measured among the minerals (melilite, spinel, and fassaite) indicates that crystallization of the CAI started from oxygen-16-rich materials that were probably liquid droplets in the solar nebula, and oxygen isotope exchange with the surrounding oxygen-16-poor nebular gas progressed through the crystallization of the CAI. Additional oxygen isotope exchange also occurred during subsequent reheating events in the solar nebula.