Novel Soybean Variety Lacking Raffinose Synthase 2 Activity.

Variation in the raffinose family oligosaccharide (RFO) content in soybean is advantageous for livestock farming and health science. In this study, a soybean variety (GmJMC172) with a significantly low stachyose content in its seeds was identified in the NARO Genebank core collection. The results of the single-nucleotide polymorphism (SNP) analysis suggested that this phenomenon was related to a single-base deletion, inducing a frameshift mutation in raffinose synthase 2 (RS2), rather than the polymorphisms in the RS3, RS4, and stachyose synthase (STS) sequences. Differences in the enzymatic properties between the native RS2 and truncated RS2 were examined by using a three-dimensional model predicted using Alphafold2. In addition to revealing the missing active pocket in truncated RS2, the modeled structure explained the catalytic role of W331* and suggested a sufficient space to bind both sucrose and raffinose in the ligand-binding pocket. The soybean line, with seeds available from the NARO Genebank, could serve as breeding materials for manipulating the RFO content.

Three-dimensional structure database of natural metabolites (3DMET): a novel database of curated 3D structures.

A database of 3D structures of natural metabolites has been developed called 3DMET. During the process of structure conversion from 2D to 3D, we found many structures were misconverted at chiral atoms and bonds. Several popular converters were tested in regard to their conversion accuracy. For verification, three canonical strings were also tested. No procedure could satisfactorily cover all the structures of the natural products. The misconverted structures had to be corrected manually. However, a nonnegligible number of mistakes were also observed even after manual curation, so a self-checking system was developed and introduced to our work flow. Thus, the 3D structures in our 3DMET database were evaluated in two steps: automatically and manually. The current version includes most of the natural products of the KEGG COMPOUND collection [ http://www.genome.jp/kegg/compound/ ] and is searchable by string, value range, and substructure. 3DMET can be accessed via http://www.3dmet.dna.affrc.go.jp/ , which also has detailed manuals.

Antagonizing microRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice.

Postmenopausal bone loss often leads to osteoporosis and fragility fractures. Bone mass can be increased by the first 34 amino acids of human parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), or by a monoclonal antibody against sclerostin (Scl-Ab). Here, we show that PTH and Scl-Ab reduce the expression of microRNA-19a and microRNA-19b (miR-19a/b) in bone. In bones from patients with lower bone mass and from osteoporotic mice, miR-19a/b expression is elevated, suggesting an inhibitory function in bone remodeling. Indeed, antagonizing miR-19a/b in vivo increased bone mass without overt cytotoxic effects. We identified TG-interacting factor 1 (Tgif1) as the target of miR-19a/b in osteoblasts and essential for the increase in bone mass following miR-19a/b inhibition. Furthermore, antagonizing miR-19a/b augments the gain in bone mass by PTH and restores bone loss in mouse models of osteoporosis in a dual mode of action by supporting bone formation and decreasing receptor activator of NF-κB ligand (RANKL)-dependent bone resorption. Thus, this study identifies novel mechanisms regulating bone remodeling, which opens opportunities for new therapeutic concepts to treat bone fragility.

[Effects of aging and menopause on women's vascular systems].

AIM: The morbidity of hypertension increases with aging although the exact relationship between hypertension and menopause has not been clearly elucidated. Therefore we set out to clarify the effects of aging and menopause upon women's vascular systems. METHODS: We divided 151 elderly and middle-aged women into 3 groups (premenopause group, menopause group and post-menopause group). We measured height, weight, blood pressure (BP), total cholesterol (T-chol), HDL-cholesterol (HDL-chol), LDL-cholesterol (LDL-chol), high sensitivity C-reactive protein (hsCRP), creatinine (Cr), triglyceride (TG), fasting blood glucose (FPG), IRI, HOMA-R, brachial-ankle pulse wave velocity (baPWV), augmentation index (AI), percentage of flow-mediated dilatation (%FMD), and echocardiography. RESULTS: In the post-menopause and menopause groups systolic BP and AI were significantly higher than those in the pre-menopause group. There was a significant difference in systolic BP between the post-menopause group and menopause group. In the post-menopause group, baPWV, Cr, and hsCRP were significantly higher than those in the pre-menopause group. There was significant difference in baPWV between the post-menopause group and menopause group. In the post-menopause group, %FMD and eGFR were significantly lower than those in other 2 groups. In the post-menopause group and the menopause group, E/A was significantly lower than in the pre-menopause group. There was also a significant difference in E/A between the post-menopause group and the menopause group. CONCLUSIONS: Elevated blood pressure, atherosclerosis and endothelial dysfunction were associated with aging and menopause in their present study. These results suggest that understanding women's cardiovascular changes which accompany aging are important for women's health care and prevention of cardiovascular events.

Synthesis of Tetramic Acid Derivatives via a Tandem Umpolung Alkylation/Reduction/Cyclization Reaction of γ-Hydrazono ß-Ketoester.

An efficient method for the one-pot synthesis of tetramic acid derivatives was developed utilizing tandem umpolung N-alkylation/reduction/cyclization of γ-hydrazono ß-ketoester. By using this reaction as a key step, the total synthesis of the 3-spiro 7-hydroxamic acid tetralin which possesses an HDAC inhibitory activity was also achieved.

TG-interacting factor 1 (Tgif1)-deficiency attenuates bone remodeling and blunts the anabolic response to parathyroid hormone.

Osteoporosis is caused by increased bone resorption and decreased bone formation. Intermittent administration of a fragment of Parathyroid hormone (PTH) activates osteoblast-mediated bone formation and is used in patients with severe osteoporosis. However, the mechanisms by which PTH elicits its anabolic effect are not fully elucidated. Here we show that the absence of the homeodomain protein TG-interacting factor 1 (Tgif1) impairs osteoblast differentiation and activity, leading to a reduced bone formation. Deletion of Tgif1 in osteoblasts and osteocytes decreases bone resorption due to an increased secretion of Semaphorin 3E (Sema3E), an osteoclast-inhibiting factor. Tgif1 is a PTH target gene and PTH treatment failed to increase bone formation and bone mass in Tgif1-deficient mice. Thus, our study identifies Tgif1 as a novel regulator of bone remodeling and an essential component of the PTH anabolic action. These insights contribute to a better understanding of bone metabolism and the anabolic function of PTH.

Chemical curation to improve data accuracy: recent development of the 3DMET database.

We have developed a three-dimensional structure database of natural metabolites (3DMET). Early development of the 3DMET database relied on content auto-generated from 2D-structures of other chemical databases. From 2009, we began manual curation, obtaining new compounds from published works. In the process of curation, problems of digitizing 3D-structures from structure drawings of documents were accumulated. As the same as auto-generation, structure drawings should be also payed attention about stereochemistry. Our experiences in manual curation of 3DMET, as described herein, may be useful to others in this field of research and for the development of supporting systems of a chemical structure database. Manual curation is still necessary for proper database entry of the 3D-configurations of chiral atoms, a problem encountered frequently among natural products.

Current Challenges in Development of a Database of Three-Dimensional Chemical Structures.

We are developing a database named 3DMET, a three-dimensional structure database of natural metabolites. There are two major impediments to the creation of 3D chemical structures from a set of planar structure drawings: the limited accuracy of computer programs and insufficient human resources for manual curation. We have tested some 2D-3D converters to convert 2D structure files from external databases. These automatic conversion processes yielded an excessive number of improper conversions. To ascertain the quality of the conversions, we compared IUPAC Chemical Identifier and canonical SMILES notations before and after conversion. Structures whose notations correspond to each other were regarded as a correct conversion in our present work. We found that chiral inversion is the most serious factor during the improper conversion. In the current stage of our database construction, published books or articles have been resources for additions to our database. Chemicals are usually drawn as pictures on the paper. To save human resources, an optical structure reader was introduced. The program was quite useful but some particular errors were observed during our operation. We hope our trials for producing correct 3D structures will help other developers of chemical programs and curators of chemical databases.

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study.

INTRODUCTION: We designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis. METHODS: Twenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed. RESULTS: The OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites. CONCLUSION: The peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Development of new indices to evaluate protein-protein interfaces: assembling space volume, assembling space distance, and global shape descriptor.

Protein-protein interaction is fundamental to initiate the cellular functions of proteins, and thus structural analyses of protein interfaces are the first step to understand their functions at the molecular level. Although shape complementarities have been used to evaluate the fitness of interfaces, the conventional method did not distinguish between two main components of complementarity, the well-fitting of the surface shapes and the size of the gap regions between the pair of molecules, and could not evaluate the global shape of the interfaces. Therefore, we now propose three new indices to describe protein interfaces: assembling space volume (ASV), assembling space distance (AS-distance), and global shape descriptor (GS). The ASV is calculated using Delaunay tessellation, and the AS-distance is calculated as the ratio of ASV to delta-ASA (accessible surface area). The GS is calculated as the ratio of the volume of Delaunay tetrahedra with a long edge to that of all tetrahedra in the assembling space. To evaluate the feasibility of the three indices, we applied our method to homo-dimer proteins, and performed systematic comparisons with SURFNET by Laskowski and shape complementarity by Lawrence and Colman. As a result, our indices behave differently from the existing ones, and shed light on new features of protein-protein interfaces, as general trends of AS-distances for all protein interfaces.

Cloning and sequence analysis of D-erythrulose reductase from chicken: its close structural relation to tetrameric carbonyl reductases.

Sequence analysis of a cDNA for D-erythrulose reductase from chicken liver showed that the deduced open reading frame encodes the protein with a molecular mass of 26 kDa consisting of 246 amino acids. Although the reductase shares more than 60% identity in the amino acid sequence with the mouse tetrameric carbonyl reductase, these two enzymes have many biochemical differences; their substrate specificity, subcellular localization, organ distribution, etc. A three-dimensional structure of D-erythrulose reductase was predicted by comparative modeling based on the structure of the tetrameric carbonyl reductase (PDB entry = 1CYD). Most of the residues at the active site (within 4 A from the ligand) of the carbonyl reductase were also conserved in the D-erythrulose reductase. Nevertheless, Val190 and Leu146 in the active site of the tetrameric carbonyl reductase were substituted in the D-erythrulose reductase by Asn192 and His148, respectively. The substitutions in the active sites may be related to the difference in substrate specificity of the two enzymes. The phylogenic analysis of D-erythrulose reductase and the other related proteins suggests that the protein described as a carbonyl reductase D-erythrulose reductase.

Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley.

Two cDNA clones encoding vacuolar H+-inorganic pyrophosphatase (HVP1 and HVP10), one clone encoding the catalytic subunit (68 kDa) of vacuolar H+-ATPase (HvVHA-A), and one clone encoding vacuolar Na+/H+ antiporter (HvNHX1) were isolated from barley (Hordeum vulgare), a salt-tolerant crop. Salt stress increased the transcript levels of HVP1, HVP10, HvVHA-A, and HvNHX1, and osmotic stress also increased the transcript levels of HVP1 and HvNHX1 in barley roots. The transcription of HVP1 in response to salt stress was regulated differently from that of HVP10. In addition, the HVP1 expression changed in a pattern similar to that of HvNHX1 expression. These results indicate that the expression of HVP1 is co-ordinated with that of HvNHX1 in barley roots in response to salt and osmotic stresses.