Discovery of evocalcet,a next-generation calcium-sensing receptor agonist for the treatment of hyperparathyroidism

The calcium-sensing receptor (CaSR) plays an important role in sensing extracellular calcium ions and regulating parathyroid hormone secretion by parathyroid gland cells, and the receptor is a suitable target for the treatment of hyperparathyroidism. Cinacalcet hydrochloride is a representative CaSR agonist which widely used for the hyperparathyroidism. However, it has several issues to clinical use, such as nausea/vomiting and strong inhibition of CYP2D6. We tried to improve these issues of cinacalcet for a new pharmaceutical agent as a preferable CaSR agonist. Optimization from cinacalcet resulted in the identification of pyrrolidine compounds and successfully led to the discovery of evocalcet as an oral allosteric CaSR agonist. Evocalcet, which exhibited highly favorable profiles such as CaSR agonistic activity and good DMPK profiles, will provide a novel therapeutic option for secondary hyperparathyroidism.     

Structure-based drug design of 1,3,6-trisubstituted 1,4-diazepan-7-ones as selective human kallikrein 7 inhibitors

A novel series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were investigated as human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Based on the X-ray co-crystal structure of compound 1 bound to human KLK7, the derivatives of this scaffold were designed, synthesized, and evaluated. Through structure-activity relationship studies focused on the side chain located in the prime site region of the enzyme, representative compounds 15, 33a, and 35a were identified as highly potent and selective inhibitors of human KLK7.     

Age,growth, and age at sexual maturity of the commercially landed skate species,Dipturus chinensis (Basilewsky, 1855), in the northern East China Sea

Age, growth, and age at sexual maturity of the polkadot skate Dipturus chinensis, in the northern East China Sea were determined for a total of 614 specimens collected from April 2009 to December 2014. Vertebral centrum analysis was used to calculate the age of the skates. Annual band deposition was determined by marginal increment analysis. The von Bertalanffy growth model was fitted to the observed length‐at‐age data for each sex (males, L_∞ = 76.8, k = 0.109, t₀ = ?1.28; females, L_∞ = 83.1, k = 0.103, t₀ = ?1.20). Growth patterns of females and males were similar until the age of 6; thereafter, females grew larger than males. Maximum age recorded was 13 years for males and 15 years for females. Age at 50% sexual maturity was 8.22 years for males and 9.39 years for females. These results indicate that D. chinensis is slow growing, relatively long‐lived, and late maturing, and therefore vulnerable to exploitation.     

Co-precipitation molecules hemopexin and transferrin may be key molecules for fibrillogenesis in TTR V30M amyloidogenesis

The disease model of familial amyloidotic polyneuropathy—7.2-hMet30 mice—manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21–24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.     

Relationships between daytime sleepiness and sleep quality,duration, and phase among school-aged children: a cross-sectional survey

A cross-sectional survey was conducted to simultaneously evaluate sleep quality, duration, and phase in school-aged children and correlations between each dimension of sleep and daytime sleepiness were comprehensively examined. A cross-sectional survey was conducted with school-aged children enrolled in four public elementary schools in Joetsu city, Niigata prefecture in Japan (n = 1683). Among the collected responses (n = 1290), 1134 valid responses (547 boys and 587 girls) were analyzed (valid response rate was 87.90%). Data on daytime sleepiness, sleep quality (problems in sleeping at night), sleep duration (the average sleeping time during a week), and sleep phase (sleep timing: bedtime and rising time on weekdays, and sleep regularity: differences in bedtime and rising time between on weekdays and weekends) were collected. The results of multivariate logistic regression analysis indicated that the following dimensions were significantly correlated with daytime sleepiness: the decline in sleep quality [adjusted odds ratio (AOR) = 2.62, 95% confidence interval (CI) = 1.71–4.00], bedtime after 21:30 on weekdays (AOR = 1.58, 95% CI = 1.15–2.18), bedtime delay on weekends, compared to weekdays (AOR = 1.75, 95% CI = 1.27–2.41), and bedtime advance on weekends, compared to weekdays (AOR = 3.33, 95% CI = 1.78–6.20). Sleep dimensions that significantly affected daytime sleepiness in school-aged children are sleep quality, bedtime-timing, and regularity of bedtime. It is important to detect problems in night sleep and establish treatments, as well as to provide support for early bedding on weekdays and for a regular bedtime both on weekdays and on weekends to prevent daytime sleepiness in school-aged children.

     

High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.     

Distribution of peptidergic nerve fibres in bullfrog lingual papillae demonstrated by a combination of double immunofluorescence labelling and a multiple dye filter

Summary Immunoreactivity of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and galanin is localized in nerve fibres distributed in the fungiform and filiform papillae of the tongue of the bullfrog,Rana catesbeiana. A combination of indirect double immunofluorescence labelling and a multiple dye filter system clearly demonstrated that all substance P fibres in the connective tissue core of the fungiform and filiform papillae, and within the rim of ciliated cells located on the top of the fungiform papillae showed coexistence with calcitonin gene-related peptide. A few fibres in the epithelial discs, which are located in the centre of the top of the fungiform papillae, showed the immunoreactivity of calcitonin gene-related peptide alone. There were no substance P fibres which showed coexistence with vasoactive intestinal polypeptide, galanin, and neuropeptide Y. In high magnification images, substance P and vasoactive intestinal polypeptide, and substance P and galanin fibres were recognized as two interwined fibres within the same thin nerve bundle. No immunoreactivity of leucine- and methionine-enkephalins can be detected. These findings suggest that the chemoreceptor function of the bullfrog gustatory organ may be under the control of complicated peptidergic innervation.     

Preparation of monoclonal antibodies against planarian organs and the effect of fixatives

Monoclonal antibodies were obtained against all part of the body of the planarian Dugesia japonica japonica except tthe nerve cord, though parts of the enteric canal were also non reactive. The effect of a variety of fixatives (acetone, Bouin, formalin) used on planarian tissues prior to screening with antibodies, was assessed.     

Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase-generated fibrin (retention of fibrinopeptide B) and thrombin-generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)-beta 1. Fibroblasts seeded on reptilase-generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin-generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t-PA) increased procollagen mRNA and TGF-beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down-regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin. © 1995 Wiley-Liss, Inc.     

Bradyrhizobium elkanii Induces Outer Cortical Root Swelling in Soybean

When soybean roots were inoculated with Bradyrhizobium elkaniiUSDA76, USDA94 or USDA31, a localized swelling of the root outercortical cells was observed within 10 days after inoculation.This phenotype was designated outer cortical swelling (OCS).OCS was not observed in roots inoculated with B.japonicum USDA110or USDA122. Therefore, the OCS response appears to be specificfor B. elkanii. To determine the involvement of the nodulationgenes in OCS formation, we constructed a nod^- mutant, strainUSDA94