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A cloud identification and profiling algorithm is being developed for the multi-spectral imager (MSI), which is one of the four instruments that the Earth Clouds, Aerosols, and Radiation Explorer (EarthCARE) spacecraft will feature. During recent work, we noticed that the MSI response function could shift substantially among some wavelengths (0.67 and 1.65 µm bands) owing to the spectral misalignment (SMILE), in which a shift in the center wavelength appears as a distortion in the spectral image. We evaluated how SMILE affects the cloud retrieval product qualitatively and quantitatively. We chose four detector pixels from bands 1 and 3 with the nadir pixel as the reference to elucidate how the SMILE error affects the cloud optical thickness (τ) and effective cloud droplet radius (re) by simulating the MSI forward radiation with Comprehensive Analysis Program for Cloud Optical Measurement (CAPCOM). We also evaluated the error in simulated scenes from a global cloud system-resolving model and a satellite simulator to measure the effect on actual observation scenes. For typical shallow warm clouds (τ = 8, re = 8 µm), the SMILE error on the cloud retrieval was not significant in most cases (up to 6 % error). For typical deep convective clouds (τ = 8, re = 40 µm), the SMILE error on the cloud retrieval was even less significant in most cases (up to 4 % error). Moreover, our results from two oceanic scenes using the synthetic MSI data agreed well with the forward radiation simulation, indicating that the SMILE error was generally within 10 %. Generally, this negligible impact of the SMILE is true for water surfaces, but it still needs to be investigated further for land surfaces in future works.

Surface downward radiation (SDR), including shortwave downward radiation (SWDR) and longwave downward radiation (LWDR), is of great importance to energy and climate studies. Considering the lack of reliable SDR data with a high spatiotemporal resolution in the East Asia–Pacific (EAP) region, we derived SWDR and LWDR at 10-min and 0.05° resolutions for this region from 2016 to 2020 based on the next-generation geostationary satellite Himawari-8 (H-8). The SDR product is unique in terms of its all-sky features, high accuracy, and high-resolution levels. The cloud effect is fully considered in the SDR product, and the influence of high aerosol loadings and topography on the SWDR are considered. Compared to benchmark products of the radiation, such as Clouds and the Earth’s Radiant Energy System (CERES) and the European Centre for Medium-Range Weather Forecasts (ECMWF) next-generation reanalysis (ERA5), and the Global Land Surface Satellite (GLASS), not only is the resolution of the new SDR product notably much higher, but the product accuracy is also higher than that of those products. In particular, hourly and daily root-mean-square errors of the new SWDR are 104.9 and 31.5 W m−2, respectively, which are much smaller than those of CERES (at 121.6 and 38.6 W m−2, respectively), ERA5 (at 176.6 and 39.5 W m−2, respectively), and GLASS (daily of 36.5 W m−2). Meanwhile, RMSEs of hourly and daily values of the new LWDR are 19.6 and 14.4 W m−2, respectively, which are comparable to that of CERES and ERA5, and even better over high-altitude regions.

A cloud identification and profiling algorithm is being developed for the multi-spectral imager (MSI), which is one of the four instruments that the Earth Clouds, Aerosols, and Radiation Explorer (EarthCARE) spacecraft will feature. During recent work, we noticed that the MSI response function could shift substantially among some wavelengths (0.67 and 1.65 µm bands) owing to the spectral misalignment (SMILE), in which a shift in the center wavelength appears as a distortion in the spectral image. We evaluated how SMILE affects the cloud retrieval product qualitatively and quantitatively. We chose four detector pixels from bands 1 and 3 with the nadir pixel as the reference to elucidate how the SMILE error affects the cloud optical thickness (τ) and effective cloud droplet radius (re) by simulating the MSI forward radiation with Comprehensive Analysis Program for Cloud Optical Measurement (CAPCOM). We also evaluated the error in simulated scenes from a global cloud system-resolving model and a satellite simulator to measure the effect on actual observation scenes. For typical shallow warm clouds (τ = 8, re = 8 µm), the SMILE error on the cloud retrieval was not significant in most cases (up to 6 % error). For typical deep convective clouds (τ = 8, re = 40 µm), the SMILE error on the cloud retrieval was even less significant in most cases (up to 4 % error). Moreover, our results from two oceanic scenes using the synthetic MSI data agreed well with the forward radiation simulation, indicating that the SMILE error was generally within 10 %. Generally, this negligible impact of the SMILE is true for water surfaces, but it still needs to be investigated further for land surfaces in future works.

We developed a new aerosol retrieval algorithm combining a numerical aerosol forecast. In the retrieval algorithm, the short-term forecast from an aerosol data assimilation system was used for a priori estimate instead of spatially and temporally constant values. This method was demonstrated using the Advanced Himawari Imager onboard the Japan Meteorological Agency’s geostationary satellite Himawari-8, and the results showed spatially finer distributions than the model forecast and less noisy distributions than the original algorithm. We validated the new algorithm using ground observation data and found that the aerosol parameters detectable by satellite sensors were retrieved more accurately than a priori model forecast by adding satellite information. Moreover, the retrieval accuracy was improved by using the model forecast as compared with using constant a priori estimates. By using the assimilated forecast for a priori estimate, information from previous observations can be propagated to future retrievals, thereby leading to better retrieval accuracy. Observational information from the satellite and aerosol transport by the model is incorporated cyclically to effectively estimate the optimum field of aerosol.

Background Vitamin D deficiency (VDD) is associated with an increased risk for lifestyle-related diseases. In Japan, VDD is quite prevalent in all age groups, with its high risk in young women. Furthermore, its association during pregnancy with gestational hypertension and low birth weight has also been reported. VDD can be diagnosed by serum 25-hydroxyvitamin D [25(OH)D] levels, which, however, is not suited for screening. Therefore, we will create a predictive model for serum 25(OH)D concentration and prevalence of VDD based on such data as region, sun exposure habit, and vitamin D intake in young women. Methods From 2020 to 2022, we conduct a cross-sectional study of 600 young women in four regions of Japan, identify the indices associated with serum 25(OH)D concentrations such as sun exposure habits, habitual vitamin D intake, ultraviolet-B irradiation, seasons (summer and winter) and latitude, and construct prediction models for serum 25(OH)D concentrations and VDD risk. This study has been registered with UMIN-CTR (ID: UMIN000041527). Results One hundred and fifteen subjects have been collected from 6 institutions in winter as of May 2021. When data from more than 200 subjects have become available, we will conduct the interim analysis, summarize the data by region and facility, review the inclusion criteria for analysis, and check for missing values and outliers. Prediction models for serum 25(OH)D concentration and VDD will be determined in the final analysis when all cases have been collected. Conclusions A screening tool for VDD risk to be developed in our study based on the predictive model would help the public and medical professionals prevent lifestyle-related diseases through improving VDD. Additionally, the results may serve as the scientific basis for determining the appropriate vitamin D intake and sun exposure standards.

形態: カラー図版あり, Physical characteristics: Original contains color illustrations, 資料番号: PA1810011000

To prevent worsening of diabetes mellitus, we conducted a developmental research of personal health record (PHR), whereby affected individuals, medical staff and an insurer cooperate and manage effective treatment. Medical expenses can be suppressed by preventing the onset of lifestyle-related diseases. Companies can benefit from improved employee productivity by promoting healthy and efficient working styles. We conducted a health and medical consciousness survey to examine PHR models that are attractive for both employers and employees.

Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, the inhibitory activity of 1-arylimidazole-2(3 H )-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled 3 H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID 50 values range from 0.02 to 2511 nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25 °C during a 30 min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect’s population.

The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (TA) by catalysis of tyramine-β-hydroxylase (TβH). In this study, the Drosophila 70 kDa tyramine-β-hydroxylase (DmTβH) protein was purified after the recombinant nucleopolyhedrovirus isolated from Bombyx mori (BmNPV) containing the TβH gene was injected into the hemocoel of the fifth instar larvae from the d17 B. mori strain. Western blot analysis revealed an immunoreactive band with a molecular mass of 70 kDa. The products formed by incubating the enzyme reaction mixture were separated and detected by reverse phase high-performance liquid chromatography. The optimum pH, temperature, and incubation time for the conversion of TA to OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3 H )-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.

EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine), a nucleoside reverse transcriptase inhibitor with extremely potent anti-HIV activity, was concisely synthesized from (R)-glyceraldehyde acetonide in an 18% overall yield by a 12-step sequence involving highly diastereoselective ethynylation of an α-alkoxy ketone intermediate. The present synthesis is superior, both in overall yield and in the number of steps, to the previous one which required 18 steps from an expensive starting material and resulted in a modest overall yield of 2.5%.

A 70-year-old Japanese man with chronic kidney disease under treatment with oral prednisolone for organizing pneumonia developed pulmonary aspergilloma. The patient was started on micafungin (MCFG), with no addition of any other new drug. About 5 weeks later, aggravation of his normocytic anemia associated with a low reticulocyte count was observed. Bone marrow puncture and biopsy revealed intense hypoplasia of the erythroblasts. As there was no evidence of malignancy, human parvovirus B19 infection, autoimmune diseases or hemorrhage, the patient was diagnosed as having acquired pure red cell aplasia (PRCA). The anemia improved along with an increase of the reticulocyte count to the normal level within 12 weeks of discontinuation of the MCFG therapy. The patient showed no evidence subsequently of any recurrence of the normocytic normochromic anemia or relapse of the PRCA. This is the first reported case of PRCA associated with MCFG.

Human herpesvirus 6 (HHV-6) glycoprotein Q1 (gQ1), a unique gene in HHV-6, forms a complex with glycoproteinH (gH) and gL, which is the viral ligand for its cellular receptor, CD46. However, whether gQ1 is essential for virus growth is unknown, because a system is lacking for making gene knockouts for HHV-6. Recently, bacterial artificial chromosome (BAC) and E. coli mutagenesis techniques have been applied to herpesvirus investigation. Here we successfully inserted the HHV-6A genome into a BAC, and obtained reconstituted infectious virus from the HHV-6A-containing BAC DNA. Using this system, we generated a gQ1 mutant virus genome, which failed to yield reconstituted infectious virus, whereas its revertant virus could be produced, indicating that the HHV-6 gQ1 gene is essential for virus growth. Therefore, we successfully applied BAC and E. coli mutagenesis techniques to the study of HHV-6, and discovered that HHV-6 gQ1 is an essential gene for virus growth.

The storage glucans of Cyanidioschyzon merolae [clade L-1 (cyanidian algae), order Porphyridiales, subclass Bangiophycidae], which is considered to be one of the most primitive rhodophytes, were analyzed to understand the early evolution of the glucan structure in the Rhodophyta. Chain-length distribution analysis of the glucans of cyanidian algae demonstrated that while the glucans of Cyanidium caldarium and Galdieria sulphuraria are of the glycogen type, those of C. merolae are of the semiamylopectin type, as in other lineages of the Rhodophyta. Gel permeation chromatography, however, showed that the glucans of C. merolae do not include amylose, being different from those of other Bangiophycidae species. Identifi cation by MALDI–TOF-MS and enzyme assaying of glucan granulebound proteins indicated that phosphorylase, but not starch synthase, is included. Thus, C. merolae has an unusual glucan and bound-protein composition for the Bangiophycidae, appearing to be a member of the Florideophycidae. The fi nding that the alga does not contain amylose or the related enzyme, granule-bound starch synthase, is, however, consistent with previously reported results of molecular phylogenetic analysis of starch synthases. Our results support an evolutionary scenario defi ned by the loss of starch and reversion to glycogen synthesis during the evolution of cyanidian algae, and suggest the possibility that a C. merolae like primitive rhodophyte might have evolved into the Florideophycidae.

Purpose Differentiation of endometrial stromal cells into decidual cells occurs during embryo implantation and pregnancy. Recently, it has been reported that relaxin affects the decidualization of cultured human endometrial cells in vitro; however, there has been no study on the decidualization of the Mongolian gerbil (Meriones unguiculatus). The authors demonstrated artificially induced decidualization, and the effect of relaxin on decidualization in gerbils.

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP < or = 13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The alpha-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B(2-3) chains of amylopectin in the PUL mutant was approximately 3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP < or = 7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities.

Both relaxin and insulin-like growth factor (IGF) are members of the insulin super family. This study aimed to investigate the effect of relaxin and IGF-I on the pre-implantation of Mongolian gerbil of blastocyst development in vitro. Blastocysts and eight-cell stage embryos were collected from female gerbils. Eight-cell embryos and blastocysts were cultured in mM16 medium supplemented with or without relaxin or IGF-I for 24 h. Blastocysts were counted for total, inner cell mass (ICM) and trophectoderm (TE) cell numbers, and assessed apoptosis incidence. In addition, to measure incorporation of 3H-methionine, blastocysts were cultured for 3 h with relaxin or IGF-I, washed with trichloroacetic acid and measured by liquid scintiration counter. Relaxin (200 ng/ml) increased total, TE and ICM cell numbers of blastocyst (P

A reliable quantitative assay method for starch branching enzyme (BE) remains to be established whereas it has been required to characterize BE towards understanding the regulatory mechanism for the synthesis of starch in plant tissues. We describe a new quantitative assay method for BE activities with malto-oligosaccharides, amylose and amylopectin as substrates by using bicinchoninic acid (BCA) for measuring directly the reducing terminals of linear glucans formed after debranching the reaction products. The BCA method not only can be performed by simple procedures and easy handling with the use of cheap toxic-free reagents, but also shows highly color-stable properties after the treatment of the reaction mixture with the color-yielding reagents compared with the modified Park-Johnson method (Carbohydr. Res., 94, 205-213(1981)). The intensity and the spectrum of the purple color generated in the treated assay mixture are maintained in sugars and glucans in the range of glucose and amylose with degree of polymerization up to at least 1658. The BCA method can be also applied for characterization of BE when amylopectin is used as glucan substrate. In this paper we report this efficient, reproducible and quantitative BCA method by showing some experimental results obtained in an attempt to determine the kinetic parameters of BEIIb from rice endosperm.

SSI accounts for 60% of the total SS activity in the soluble fraction in the developing rice en- dosperm. Rice SSI-deficient mutants were identified by using reverse genetics, and the chain-length analysis of the endosperm starch showed that SSI distinctly synthesizes DP 8―12 chains from short DP 6―7 chains emerg- ing from the A chains and the branch points in the B1 chains of amylopectin. 1) In this study, to evaluate by in vitro study the function of recombinant SSI of rice (rSSI), the change in the chain-length distribution of glyco- gen or amylopectin in the activity band after rSSI enzymatic reactions in native-PAGE gel was examined. When glycogen was used as the substrate, the -glucan produced in the rSSI activity band on the native- PAGE gel had specifically fewer DP 6 chains and more DP 8 chains than unmodified glycogen did. When rice amylopectin was used as the substrate, the -glucan produced in the rSSI activity band showed the oscillation of a short turn in the range of DP 20 compared with the unmodified amylopectin; the chains with DP 6, 7, 10, 11, 13 and 17 decreased, while the extent of decrease was reduced in the chains with DP 8, 12 and 15. These results suggest that rSSI preferentially elongates the chains up to DP 8 from the short and long B1 and B2 chains with DP 6 or 7 from branch points to the non-reduced end as well as the A chains with DP 6 or 7 by adding one or two glucose moieties.

The general transcription factor TFII-I, with the corresponding gene name GTF2I, is an unusual transcriptional regulator that associates with both basal and signal-induced transcription factors. TFII-I consists of six GTF2I repeat domains, called I-repeats R1-R6. The structure and function of the GTF2I domain are not clearly understood, even though it contains a helix-loop-helix motif, which is considered to be the protein-protein interaction area, based on biochemical analyses. Here, we report the solution structure of the fifth repeat of the six GTF2I repeat domains from murine TFII-I, which was determined by heteronuclear multidimensional NMR spectroscopy (PDB code 1Q60). The three-dimensional structure of the GTF2I domain is classified as a new fold, consisting of four helices (residues 8-24, 34-39, 63-71, and 83-91), two antiparallel beta strands (residues 44-47 and 77-80), and a well-defined loop containing two beta-turns between sheet 1 and helix 3. All of the repeats probably have similar folds to that of repeat 5, because the conserved residues in the GTF2I repeat domains are assembled on the hydrophobic core, turns, and secondary structure elements, as revealed by a comparison of the sequences of the first through the sixth GTF2I repeats in TFII-I.

Starch synthase IIIa (SSIIIa)-deficient rice (Oryza sativa) mutants were generated using retrotransposon insertion and chemical mutagenesis. The lowest migrating SS activity bands on glycogen-containing native polyacrylamide gel, which were identified to be those for SSIIIa, were completely absent in these mutants, indicating that they are SSIIIa null mutants. The amylopectin B2 to B4 chains with degree of polymerization (DP) ≥ 30 and the M r of amylopectin in the mutant were reduced to about 60% and 70% of the wild-type values, respectively, suggesting that SSIIIa plays an important part in the elongation of amylopectin B2 to B4 chains. Chains with DP 6 to 9 and DP 16 to 19 decreased while chains with DP 10 to 15 and DP 20 to 25 increased in the mutants amylopectin. These changes in the SSIIIa mutants are almost opposite images of those of SSI-deficient rice mutant and were caused by 1.3- to 1.7-fold increase of the amount of SSI in the mutants endosperm. Furthermore, the amylose content and the extralong chains (DP ≥ 500) of amylopectin were increased by 1.3- and 12-fold, respectively. These changes in the composition in the mutants starch were caused by 1.4- to 1.7-fold increase in amounts of granules-bound starch synthase (GBSSI). The starch granules of the mutants were smaller with round shape, and were less crystalline. Thus, deficiency in SSIIIa, the second major SS isozyme in developing rice endosperm affected the structure of amylopectin, amylase content, and physicochemical properties of starch granules in two ways: directly by the SSIIIa deficiency itself and indirectly by the enhancement of both SSI and GBSSI gene transcripts.

Four starch synthase I (SSI)-deficient rice (Oryza sativa) mutant lines were generated using retrotransposon Tos17 insertion. The mutants exhibited different levels of SSI activities and produced significantly lower amounts of SSI protein ranging from 0% to 20% of the wild type. The mutant endosperm amylopectin showed a decrease in chains with degree of polymerization (DP) 8 to 12 and an increase in chains with DP 6 to 7 and DP 16 to 19. The degree of change in amylopectin chain-length distribution was positively correlated with the extent of decrease in SSI activity in the mutants. The structural changes in the amylopectin increased the gelatinization temperature of endosperm starch. Chain-length analysis of amylopectin in the SSI band excised from native-polyacrylamide gel electrophoresis/SS activity staining gel showed that SSI preferentially synthesized DP 7 to 11 chains by elongating DP 4 to 7 short chains of glycogen or amylopectin. These results show that SSI distinctly generates DP 8 to 12 chains from short DP 6 to 7 chains emerging from the branch point in the A or B1 chain of amylopectin. SSI seemingly functions from the very early through the late stage of endosperm development. Yet, the complete absence of SSI, despite being a major SS isozyme in the developing endosperm, had no effect on the size and shape of seeds and starch granules and the crystallinity of endosperm starch, suggesting that other SS enzymes are probably capable of partly compensating SSI function. In summary, this study strongly suggested that amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa, and SSIIIa isoforms.

Techniques involving fluorescein-5-isothiocyanate-conjugated gelatin injection, immunohistochemistry, and in situ reverse transcription/polymerase chain reaction (RT-PCR) revealed a close relationship between vascular endothelial growth factor (VEGF)-A-expressing cells and microvessels in the hypothalamic-pituitary axis of the rat. In situ RT-PCR clearly indicated the presence of VEGF-A mRNA-expressing cells in the pars tuberalis and in the pars distalis both at embryonic day 15.5 (E15.5) and in later developmental stages. The primary capillaries extended along the developing pars tuberalis, whereas the portal vessels penetrated into the pars distalis at E15.5 and subsequently expanded into the lobe to connect with the secondary capillary plexus, emerging in the pars distalis. At the same time, several VEGF-A-positive cells appeared in the pars distalis. These VEGF-A-positive cells were found to correspond to a portion of adrenocorticotropin (ACTH) cells by dual-staining for in situ RT-PCR and immunohistochemistry, suggesting that some ACTH cells have the potential to produce VEGF-A. Thus, the present study suggests that VEGF-A is involved in the development of the primary capillaries and in the vascularization of the pars distalis, but not in the portal vessels since the formation of portal vessels begins at E13.5, before the appearance of VEGF-A in the rostral region of the pars distalis.

Porcine relaxin is a peptide hormone belonging to the insulin super family that has a variety of biological functions. The present experiment was designed to investigate the effects of relaxin on sperm function and on in vitro fertilization (IVF) of porcine oocytes. Porcine spermatozoa were washed, swum-up, and incubated for 1-4 h in mTALP medium supplemented with 0, 20 or 50 ng/ml porcine relaxin. Motility was determined by observing the type of forward movement of the spermatozoa, and acrosome status was evaluated by applying the triple staining technique. Immature oocytes were aspirated from antral follicles and matured in IVM medium (modified NCSU-37). Matured oocytes were co-cultured with spermatozoa in IVF medium (mTALP) supplemented with 0, 5, 10, 15 or 20 ng/ml relaxin. After 6 h of sperm-oocyte co-incubation, putative zygotes were cultured for 18 h in oocyte culture medium NCSU-37 and then assessed for the rates of monospermy, polyspermy, and male pronucleus formation after acetic orcein staining. Relaxin improved (P

In mammals, the circadian and stress systems (both centers of which are located in the hypothalamus) are involved in adaptation to predictable and unpredictable environmental stimuli, respectively. Although the interaction and relationship between these two systems are intriguing and have been studied in different ways since the "pre-clock gene" era, the molecular interaction between them remains largely unknown. Here, we show by systematic molecular biological analysis that acute physical stress elevated only Period1 (Per1) mRNA expression in mouse peripheral organs. Although behavioral rhythms in vivo and peripheral molecular clocks are rather stable against acute restraint stress, the results of a series of promoter analyses, including chromatin immunoprecipitation assays, indicate that a glucocorticoid-responsive element in the Per1 promoter is indispensable for induction of this mRNA both in vitro and in vivo. These results suggest that Per1 can be a potential stress marker and that a third pathway of Per1 transcriptional control may exist in addition to the clock-regulated CLOCK-BMAL1/E-box and light-responsive cAMP-responsive element-binding protein/cAMP-responsive element pathways.

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

EI-1941-1 ( 1a ) and EI-1941-2 ( 2a ) accompanied by EI-1941-3 ( 3 ) have been isolated from culture broth of Farrowia sp. E-1941 as the inhibitors of interleukin-1β converting enzyme. The structures of 1a , 2a , and 3 were elucidated by the analysis of NMR and MS data, and finally the absolute stereochemistries of 1a and 2a were confirmed by optical rotation data, or X-ray crystallographic analysis of p -bromobenzoate, 2b , respectively.

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.

The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.

Ascorbic acid (vitamin C) is a cofactor required in catecholamine synthesis for conversion of dopamine to norepinephrine by dopamine beta-hydroxylase. Mutant mice lacking the plasma membrane ascorbic acid transporter (SVCT2) have severely reduced tissue levels of ascorbic acid and die after birth. We therefore investigated whether these mice might have impaired synthesis of catecholamines. Levels of catecholamines in brain were unaffected by SVCT2 deficiency. In heart, the only evidence for impaired dopamine beta-hydroxylase activity was a twofold increase in tissue dopamine. An influence of the deficiency on tissue catecholamines was most prominent in the adrenals where norepinephrine was decreased by 50% and epinephrine, by 81%. On the ultrastructural level, adrenal chromaffin cells in SVCT2 null mice showed depletion of catecholamine storage vesicles, increased amounts of rough endoplasmic reticulum, signs of apoptosis, and increased glycogen storage. Decreased plasma levels of corticosterone indicated additional effects of the deficiency on adrenal cortical function. These data show that deranged catecholamine system function in SVCT2 null mice is largely restricted to the adrenal medulla and cannot account for the lethality in these animals. The data, however, establish a crucial role for ascorbic acid in adrenal chromaffin cell function.

A possible relation between the stereostructure of (−)-(13aS)-tetrahydroberberine (1) and its enzymatic/chemical N-methylation, an important biosynthetic reaction to isoquinoline alkaloids in plants, was examined by CD spectroscopic, X-ray crystallographic, and energy calculation methods. The CD measurements indicated that 1 has two conformers (cis and trans) concerning the ring junction of the quinolizidine skeleton, and exist with a cis/trans ratio of about 1/4 in a diethyl ether : 2-methylbutane : ethanol (5 : 5 : 2) mixture. The dimensional/conformational difference between these cis and trans conformers was clarified by the X-ray crystal-structure analyses of two stereoisomers of N-methylated 1 (3 and 4). By using these structural parameters, the progress of N-methylation was simulated by energy profile calculations, suggesting that the cis and trans conformers are the major substrate for the enzymatic and chemical N-methylation reactions, respectively. Taking these results and the simulation of N-me...

The in vitro metabolism of fenthion and its sulfoxide (fenthion sulfoxide) in sea bream (Pagrus major) and goldfish (Carassius auratus) was investigated and compared with that in rats. Fenthion was oxidized to fenthion sulfoxide and the oxon derivative, but not to its sulfone, in the presence of NADPH by liver microsomes of sea bream, goldfish, and rats. These liver microsomal activities of the fish were lower than those of rats but were of the same order of magnitude. The NADPH-linked oxon- and sulfoxide-forming activities of liver microsomes of the fish and rats were inhibited by SKF 525-A, metyrapone, alpha-naphthoflavone, and carbon monoxide. The oxidizing activity to fenthion sulfoxide was also inhibited by alpha-naphthylthiourea. Several cytochrome P450 isoforms and flavin-containing monooxygenase 1 exhibited these oxidase activities. Fenthion sulfoxide was reduced to fenthion with liver cytosol of the fish and rats upon addition of 2-hydroxypyrimidine, N(1)-methylnicotinamide, or butyraldehyde, each of which is an electron donor of aldehyde oxidase, under anaerobic conditions. The activity was inhibited by menadione, beta-estradiol, and chlorpromazine, which are inhibitors of aldehyde oxidase. The activities in the fish livers were similar to those of rat liver. Aldehyde oxidase purified from the livers of sea bream and rats exhibited the reducing activity. Thus, fenthion and fenthion sulfoxide are interconvertible in fish and rats through the activities of cytochrome P450, flavin-containing monooxygenase, and aldehyde oxidase.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD.

Cellular RNA in Schizosaccharomyces pombe cells drastically decreases in amount during nitrogen starvation. Previously, we found and purified a soluble RNA-degrading enzyme whose activity drastically increased in the cells of S. pombe undergoing nitrogen starvation. The enzyme was a nuclease encoded by pnu1(+). In this study, the increase in the RNA-degrading activity and the decrease in cellular RNA level are examined in a null-mutant of pnu1(+) (pnu1Delta). During nitrogen starvation, wild-type cells show an apparent increase in RNA-degrading activity, whereas the pnu1Delta cells do not. The wild-type cells show a drastic decrease in cellular RNA amount, whereas the pnu1Delta cells show only a slight decrease. These results suggest that Pnu1 nuclease is implicated in the decrease in cellular RNA amount during nitrogen starvation, probably via the RNA-degrading activity. The increase in the RNA-degrading activity is independent of both the Wis1 stress-activated MAP kinase cascade and Tor1 signaling pathway, but it is strongly dependent on isp6(+), a gene for a possible protease, whose expression is induced during nitrogen starvation. A disruption mutant for isp6(+) (isp6Delta) is deficient in both the increase in the RNA-degrading activity and the drastic decrease in the cellular RNA amount during nitrogen starvation, which suggests that isp6(+) is involved in the RNA degradation via regulating the RNA-degrading activity of Pnu1.

Soybean extracts (SBE) containing isoflavone glycosides were cultured with Ganoderma lucidum mycelia producing beta-glucosidase. The anti-angiogenic effects of the cultivated product, containing rich in genistein, named GCP (genistein combined polysaccharide), were assessed with chick chorioallantoic membranes (CAM) and a mouse dorsal air-sac model. Beta-glucosidase produced by the mycelia converted the isoflavone glycosides into aglycons. A test of volunteers showed that serum concentrations of genistein in the subjects treated with GCP (n = 4) at 3 h after administration were significantly higher than those in the subjects treated with SBE (n = 4). GCP inhibited angiogenesis in CAM, and the activity of GCP was greater than that of SBE. GCP inhibited the formation of new vessels induced by colon carcinoma cells in vivo.

A concise enantioselective total synthesis of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), an extremely potent anti-HIV agent, has been accomplished from (R)-glyceraldehyde acetonide in 18% overall yield by a 12-step sequence involving a highly diastereoselective ethynylation of an α-alkoxy ketone intermediate.

The conformational/physicochemical features of corycavidine (1), 5,7,8,15-tetrahydro-3,4-dimethoxy-6,15-dimethyl-[1,3]benzodioxolo[5,6-e] [2]benzazecin-14(6H)-one, an important protopine-type alkaloid that serves as the key intermetabolite in the biosynthesis of isoquinoline alkaloids, were examined by X-ray crystallography, 1H NMR spectroscopy, as well as thermal analysis and energy calculations. Two stable conformations of 1, as proposed by energy calculations, were also observed in both the crystalline and solution states. Racemization of optically active 1, which was caused by heating of the crystal, was analyzed by differential-scanning calorimetry. By taking advantage of the H-2H replacement reaction at the C13 atom of 1, the rate of racemization in the C2H3O2H/NaO2H solution was measured as a function of the temperature; the energy barrier between both enantiomers was estimated to be 4.7 kcal mol-1. The crystallization of racemic 1 led to two different crystal forms, i.e., the racemic crystal and t...

Substrate selectivities of microbial proline 4-hydroxylase and proline 3-hydroxylases, all of which were purified from recombinant Escherichia coli , were investigated. l 2-Azetidine carboxylate, 3,4-dehydro- l -proline and l -pipecolinic acid were hydroxylated by those enzymes in regio- and stereospecific manner.

The in vitro metabolism of p, p'-DDT (p, p'-dichlorodiphenyltrichloroethane), an important environmental pollutant, was examined in fish, focusing on reductive dechlorination. When p, p'-DDT was incubated with hepatopancreas microsomes or the blood of goldfish, Carassius auratus, in the presence of both a reduced pyridine nucleotide and FMN, a dechlorinated metabolite, p, p'-DDD (p, p'-dichlorodiphenyldichloroethane) was formed under anaerobic conditions. These reductase activities were inhibited by carbon monoxide. Although the microsomes or blood was boiled, the dechlorinating activity was not abolished. Hemoglobin and hematin exhibited the reductase activity toward p, p'-DDT with NADH and FMN. The activity of hematin was also exhibited with FMNH2. The reductive dechlorination appears to proceed nonenzymatically by the reduced flavin, catalyzed by the heme group of hemoproteins.

The estrogenic effect of fenthion, an insecticide, in goldfish (Carassius auratus) was examined in terms of the induction of vitellogenin, a biomarker of estrogens in fish. When male goldfish were kept in water containing diethylstilbestrol (0.1mg/l) or nonylphenol (0.5mg/l) for 5 days, a significant level of vitellogenin in the blood was observed. However, vitellogenin was not detected in the blood of male goldfish kept in water containing fenthion (3mg/l) for 5 days. When female goldfish were kept in water containing fenthion for 5 days, the levels of vitellogenin in the blood were not enhanced . Furthermore, fenthion sulfoxide and fenthion sulfone, the oxidized products of fenthion, did not induce vitellogenin in male goldfish. These results suggest that fenthion and its oxidized products do not produce estrogenic activity in goldfish.

The in vitro metabolism of fenthion, an insecticide, was examined in fish, focusing on the interconversion between fenthion and its sulfoxide (fenthion sulfoxide). When fenthion was incubated with hepatopancreas microsomes of goldfish, Carassius auratus, in the presence of NADPH, the oxidized metabolite, fenthion sulfoxide was formed, but not fenthion sulfone. The oxidase activity was inhibited by SKF 525-A, and partly by α-naphthylthiourea. In contrast, fenthion sulfoxide was reduced back to fenthion by the hepatopancreas cytosol of goldfish in the presence of 2-hydroxypyrimidine, an electron donor for aldehyde oxidase. The activity was markedly inhibited by menadione, an inhibitor for aldehyde oxidase. The interconversion appears to be mediated mainly by the cytochrome P450 system and aldehyde oxidase.