Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.     

Protection of Coral Larvae from Thermally Induced Oxidative Stress by Redox Nanoparticles

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNP^O) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33 °C, RNP^O treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNP^O treatment, whereas these proteins were absent in RNP^O-treated larvae, which suggested that RNP^O effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNP^O treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from thermal stress.     

Structure–activity relationships of bisphenol A analogs at estrogen receptors (ERs): Discovery of an ERα-selective antagonist

Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures, has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton can serve as a surrogate for a steroid skeleton. Here, on the basis of those ideas, we hypothesized that the diphenylmethane derivative bisphenol A (BPA) would bind to the ligand-binding domain of estrogen receptors (ERs) in a similar manner to estradiol and act as a steroid surrogate. To test this idea, we synthesized a series of BPA analogs and evaluated their structure-activity relationships, focusing on agonistic/antagonistic activities at ERs and ERα/ERβ subtype selectivity. Among the compounds examined, 18 was found to be a potent ERα-antagonist with high selectivity over ERβ and androgen receptor under our assay conditions. A computational docking study suggested that 18 would bind to the antagonistic conformation of ERα. ERα-selective antagonists, such as 18, are candidate agents for treatment of breast cancer.     

Induction of migration of periodontal ligament cells by selective regulation of integrin subunits

The recruitment of tissue‐resident stem cells is important for wound regeneration. Periodontal ligament cells (PDL cells) are heterogeneous cell populations with stemness features that migrate into wound sites to regenerate periodontal fibres and neighbouring hard tissues. Cell migration is regulated by the local microenvironment, coordinated by growth factors and the extracellular matrix (ECM). Integrin‐mediated cell adhesion to the ECM provides essential signals for migration. We hypothesized that PDL cell migration could be enhanced by selective expression of integrins. The migration of primary cultured PDL cells was induced by platelet‐derived growth factor‐BB (PDGF‐BB). The effects of blocking specific integrins on migration and ECM adhesion were investigated based on the integrin expression profiles observed during migration. Up‐regulation of integrins α3, α5, and fibronectin was identified at distinct localizations in migrating PDL cells. Treatment with anti‐integrin α5 antibodies inhibited PDL cell migration. Treatment with anti‐integrin α3, α3‐blocking peptide, and α3 siRNA significantly enhanced cell migration, comparable to treatment with PDGF‐BB. Furthermore, integrin α3 inhibition preferentially enhanced adhesion to fibronectin via integrin α5. These findings indicate that PDL cell migration is reciprocally regulated by integrin α3‐mediated inhibition and α5‐mediated promotion. Thus, targeting integrin expression is a possible therapeutic strategy for periodontal regeneration.     

Detection and distribution of six linear mitochondrial plasmids in the shiitake mushroom,Lentinula edodes

The presence of plasmids was surveyed in 90 wild isolates ofLentinula edodes collected from geographically different world regions. DNA plasmids of different sizes were found in about 80% of the isolates. The plasmids detected were of six kinds, designated as pLE1 (9.0 kb), pLE2 (11.1 kb,=pLLE1 described by other authors), pLE3A (9.8 kb), pLE3B (10.8 kb), pLE3C (12.1 kb), and pLE3D (12.3 kb). Hybridization analysis suggested that pLE1 and pLE2 were distinct plasmid types of different homology groups to each other, and the four other plasmids were variant types belonging to a third homology group. These plasmids had no homology with their host's and non-host's nuclear and mitochondrial genome DNAs. Restriction analysis and electron microscopy indicated that the plasmids are linear in form. Since all six plasmids were transmitted uniparentally in sexual crosses and were consistently associated with the DNA preparations from mitochondria fractionated from mycelia of representative isolates, they were suggested to be located in mitochondria, similar to many other known fungal DNA plasmids. Geographically, pLE1 and pLE2 were widely distributed in natural populations ofL. edodes, while the remaining four plasmids were uniquely present in delimited natural populations. Contribution No. 322 from the Tottori Mycological Institute.     

Pancreatoblastoma. A case report with special emphasis on squamoid corpuscles with optically clear nuclei rich in biotin

BACKGROUND: Pancreatoblastoma (PBL) is a rare neoplasm that generally occurs in the pediatric age group and shows unique histopathology, including squamoid corpuscles that may contain tumor cells with optically clear nuclei (OCN) rich in biotin. In the English-language literature there have been two reports on the cytology of PBL, but neither of them refers to the cytologic features of squamoid corpuscles. CASE: A 3-year-old boy with nausea and general fatigue was referred to our center. Imaging studies showed an approximately 7.5-cm, left-sided abdominal mass and multiple metastases in the lung. The abdominal mass was biopsied, and its histology showed solid cellular nests with occasional acinar differentiation and squamoid corpuscles. Imprint cytology of the biopsied sample displayed cellular epithelial nests with focal acinar structures and foci composed of larger cells with a low nuclear/cytoplasmic ratio. These foci contained a few tumor cells with biotin-rich OCN and were determined to be squamoid corpuscles. CONCLUSION: Detection of occasional squamoid corpuscles with biotin-rich OCN can be useful in making a diagnosis of PBL on cytologic samples.     

Presenilin-1 mutation activates the signaling pathway of caspase-4 in endoplasmic reticulum stress-induced apoptosis

In the previous reports, we showed that the familial Alzheimer's disease (AD)-linked presenilin-1 (PS1) mutation induced the fragility to the endoplasmic reticulum (ER) stress and that caspase-4 mediates ER stress-induced- and beta-amyloid induced-apoptotic signaling in human cells. These results suggest the involvement of ER stress and caspase-4 in the cell death observed in AD. In this report, we studied the activation of caspase-4 in the familial AD-linked PS1 mutation (DeltaE9). Cleavage of caspase-4 under ER stress was enhanced by the overexpression of the familial AD-linked mutation (DeltaE9), showing that caspase-4 is a key caspase involved in the apoptotic signaling of AD. We also showed that the overexpression of caspase-4 induced cleavage of caspase-9 and caspase-3 without releasing cytochrome-c from the mitochondria. Thus, caspase-4 activates downstream caspases independently of mitochondrial apoptotic signaling and this might contribute to the pathogenesis of AD. To sum up our data, the familial AD-linked PS1 mutation accelerates the cleavage of caspase-4 under the ER stress and results in the activation of caspase-9 and caspase-3, apoptosis signal, without releasing cytochrome-c.     

Assessment of the nutritional status of field-caught larval Pacific bluefin tuna by RNA/DNA ratio based on a starvation experiment of hatchery-reared fish

RNA/DNA ratio is a useful and reliable indicator of the nutritional status of fish larvae and juveniles. In order to assess the nutritional status of field-caught larval Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel), starvation experiments of hatchery-reared larvae were conducted and changes in the RNA/DNA ratio of fed and starved larvae were analyzed. Starvation experiments were conducted every 3 days after first feeding. The survival rate of Pacific bluefin tuna larvae ranged 10-50% after 1 day of starved conditions and growth retardation was observed immediately. These results suggest that Pacific bluefin tuna larvae have a very low tolerance to starvation. The RNA/DNA ratios of fed larvae were approximately 2.0-4.0. On the other hand, the value of starved larvae significantly decreased to 1.0-3.0. The nutritional status of 3 cohorts of field-caught tuna larvae collected in the northwestern Pacific Ocean was examined based on the value of the RNA/DNA ratio of the 1 day starved larvae. 4.35-25.77% of the cohorts were regarded as the “starving condition”, which was negatively correlated to the ambient prey densities. These findings suggest that the nutritional condition of larval Pacific bluefin tuna was influenced by the ambient prey density, and starvation itself and starvation-induced predation could greatly contribute to mortality in the larval period of Pacific bluefin tuna.     

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.