Encapsulation of myoglobin with a mesoporous silicate results in new capabilities

A metmyoglobin (Fe3+), an oxidized form of myoglobin (Fe2+), was confined in nanospaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a metmyoglobin (Fe3+)-FSM nanoconjugate. The spectral characteristics of metmyoglobin (Fe3+)- and myoglobin (Fe2+)-FSM show an absorption curve quite similar to that of native metmyoglobin, indicating that myoglobin retains its higher-order structure in the pores of FSM. The metmyoglobin (Fe3+)-FSM conjugate had not only a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfomic acid (ABTS) or guaiacol (a hydrogen donor) but also an advanced molecular recognition ability enabling it to distinguish between ABTS and guaiacol. Furthermore, the metmyoglobin (Fe3+)-FSM showed the peroxidase-like activity even in an organic media using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The simple immobilization of metmyoglobin (Fe3+) into FSM results in enhanced catalytic activity in organic media compared to that of native metmyoglobin (Fe3+).     

Carbofuran accelerates the cellular senescence and declines the life span of spns1 mutant zebrafish

Carbofuran is a carbamate pesticide, widely used in agricultural practices to increase crop productivity. In mammals, carbofuran is known to cause several untoward effects, such as apoptosis in the hippocampal neuron, oxidative stress, loss of memory and chromosomal anomalies. Most of these effects are implicated with cellular senescence. Therefore, the present study aimed to determine the effect of carbofuran on cellular senescence and biological ageing. Spinster homolog 1 (Spns1) is a transmembrane transporter, regulates autolysosomal biogenesis and plays a role in cellular senescence and survival. Using senescence-associated β-galactosidase staining, we found that carbofuran accelerates the cellular senescence in spns1 mutant zebrafish. The yolk opaqueness, a premature ageing phenotype in zebrafish embryos, was accelerated by carbofuran treatment. In the survival study, carbofuran shortened the life span of spns1 mutant zebrafish. Autophagy is the cellular lysosomal degradation, usually up-regulated in the senescent cells. To know the impact of carbofuran exposure on autophagy progress, we established a double-transgenic zebrafish line, harbouring EGFP-tagged LC3-II and mCherry-tagged Lamp1 on spns1 mutant background, whereas we found, carbofuran exposure synergistically accelerates autolysosome formation with insufficient lysosome-mediated degradation. Our data collectively suggest that carbofuran exposure synergistically accelerates the cellular senescence and affects biological ageing in spns1 defective animals.     

Identification of an internal cavity in the PhoQ sensor domain for PhoQ activity and SafA-mediated control

The PhoQ/PhoP two-component signal transduction system is conserved in various Gram-negative bacteria and is often involved in the expression of virulence in pathogens. The small inner membrane protein SafA activates PhoQ in Escherichia coli independently from other known signals that control PhoQ activity. We have previously shown that SafA directly interacts with the sensor domain of the periplasmic region of PhoQ (PhoQ-SD) for activation, and that a D179R mutation in PhoQ-SD attenuates PhoQ activation by SafA. In this study, structural comparison of wild-type PhoQ-SD and D179R revealed a difference in the cavity (SD (sensory domain) pocket) found in the central core of this domain. This was the only structural difference between the two proteins. Site-directed mutagenesis of the residues surrounding the SD pocket has supported the SD pocket as a site involved in PhoQ activity. Furthermore, the SD pocket has also been shown to be involved in SafA-mediated PhoQ control.     

The genus Ponticulomyces (Physalacriaceae, Agaricales) from Japan

Two species of the genus Ponticulomyces collected from Japan for the first time are described and illustrated. Ponticulomyces kedrovayae is characterized by its lamellae staining yellow when bruised and in age, stipe lacking an annulus and a pseudorhiza, scattered pileal hairs, and large amygdaliform basidiospores. It mainly occurs on dead wood of Fagus crenata. Ponticulomyces orientalis is characterized by its scattered pileal hairs and broadly ellipsoid to ellipsoid basidiospores. It was collected mostly on dead wood of Cameria japonica.     

Genetic population structure of the fluvial eight-barbel loach <Emphasis Type="Italic">Lefua</Emphasis> sp. 1 in the three river systems in central Honshu,Japan, revealed by microsatellite DNA markers

The fluvial eight-barbel loach Lefua sp. 1 is an undescribed species distributed from the Kinki to Chugoku districts, Honshu, and also on Shikoku Island, Japan. Genetic relationships among local populations are unclear and management units remain undetermined. To aid conservation, we determined genetic population structures from microsatellite loci for 20 populations from three river systems on Honshu. The genetic diversity within populations is relatively low; the majority has experienced genetic bottlenecks. Statistical analysis revealed significant divergence among river systems suggesting that each should be recognized as a management unit. Any conservation program should consider the populations’ genetic uniqueness.     

Occurrence of a nonsulfated chondroitin proteoglycan in the dried saliva of Collocalia swiftlets (edible bird's-nest)

Despite their wide occurrence, proteoglycans (PGs) have never been isolated from the saliva of higher animals. We found that the Collocalia glycoproteins isolated from edible birds'-nests (the dried forms of regurgitated saliva of male Collocalia swiftlets) were rich in a PG containing nonsulfated chondroitin glycosaminoglycans (GAGs). We have devised a method to isolate a PG from the water extract of the white nest built by Aerodramus fuciphagus (white nest swiftlets) with a yield of 2-mg PG per gram nest. This PG contained 83% of carbohydrates, of which 79% were GalNAc and GlcUA (D-glucuronic acid) in an equimolar ratio. By using chondroitin AC lyase, the structure of GAGs in this PG was established to be chondroitin ( --> 4GlcUAbeta1 --> 3GalNAcbeta1 --> )(n) chains. The average molecular mass of the chondroitin chain was estimated to be 49 kDa by gel filtration. We have isolated a linkage region hexasaccharide, DeltaHexUAalpha1 --> 3GalNAcbeta1 --> 4GlcUAbeta1 --> 3Galbeta1 --> 3Galbeta1 --> 4Xyl, from this PG by chondroitinase ABC digestion to show that the GAGs in this PG are also linked to the core protein through the common tetrasaccharide linker, GlcUAbeta1 --> 3Galbeta1 --> 3Galbeta1 --> 4Xyl, found in various PGs. As water was not effective in extracting uronic acid-containing glycoconjugates from the black nest built by black nest swiftlets (A. maximus), we used 4 M guanidium chloride and anion-exchange chromatography in the presence of urea to extract and isolate about 30 mg of a chondroitin PG preparation from 10 g of the desialylated black nest. As the biological significance of chondroitin is still not well understood, bird's nest should become a convenient source for preparing this unique GAG to study its biological functions.     

IDH2 stabilizes HIF‐1α‐induced metabolic reprogramming and promotes chemoresistance in urothelial cancer

Drug resistance contributes to poor therapeutic response in urothelial carcinoma (UC). Metabolomic analysis suggested metabolic reprogramming in gemcitabine‐resistant urothelial carcinoma cells, whereby increased aerobic glycolysis and metabolic stimulation of the pentose phosphate pathway (PPP) promoted pyrimidine biosynthesis to increase the production of the gemcitabine competitor deoxycytidine triphosphate (dCTP) that diminishes its therapeutic effect. Furthermore, we observed that gain‐of‐function of isocitrate dehydrogenase 2 (IDH2) induced reductive glutamine metabolism to stabilize Hif‐1α expression and consequently stimulate aerobic glycolysis and PPP bypass in gemcitabine‐resistant UC cells. Interestingly, IDH2‐mediated metabolic reprogramming also caused cross resistance to CDDP, by elevating the antioxidant defense via increased NADPH and glutathione production. Downregulation or pharmacological suppression of IDH2 restored chemosensitivity. Since the expression of key metabolic enzymes, such as TIGAR, TKT, and CTPS1, were affected by IDH2‐mediated metabolic reprogramming and related to poor prognosis in patients, IDH2 might become a new therapeutic target for restoring chemosensitivity in chemo‐resistant urothelial carcinoma.     

Direct interaction between substrates and endogenous steroids in the active site may change the activity of cytochrome P450 3A4

CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrate including endogenous steroids and some drugs that coexist at the active site. To clarify the mechanism of the effect of endogenous steroids on the drug metabolism, the interaction between substrates, nevirapine (NVP) and carbamazepine (CBZ), and endogenous steroids was investigated by theoretical calculations. When the activities of NVP 2-hydroxylation and CBZ 10,11-epoxidation by expressed CYP3A4 were measured in the presence of steroids, NVP 2-hydroxylation was found to be remarkably increased by aldosterone and inhibited by estradiol. CBZ 10,11-epoxidation was increased by androstenedione. Three-dimensional computer modeling has shown that the active site of CYP3A4 is especially large, permitting access of two substrate molecules. The interactions between NVP and aldosterone and between CBZ and androstenedione were estimated by theoretical calculations assuming the substrate and steroids to be present in the active site at the same time. It was shown that NVP or CBZ would be stably fixed close to the oxygen atom at the sixth ligand of heme by interaction with steroids, suggesting that NVP and CBZ may be hydroxylated more easily due to the interaction with steroids. Estradiol was also expected to interact with NVP via a pi/pi interaction between a benzene ring, in which the NVP hydroxylation site is located, and a benzene ring of estradiol, suggested to inhibit the reaction. From these results, interactions between the substrate and endogenous steroids in the active site may change the activity of CYP3A4.     

The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering

The structure of a histidine kinase (ThkA) complexed with a response regulator (TrrA) in the two-component regulatory system from hyperthermophile Thermotoga maritima was determined by a combination of X-ray crystallography at a resolution of 4.2 A and small-angle X-ray scattering (SAXS). The boundary of the three component domains (PAS-sensor, dimerization and catalytic domains) of ThkA and the bound TrrA molecule were unambiguously assigned in the electron density map at 4.2 A resolution. ThkA forms a dimer with crystallographic 2-fold symmetry and two monomeric TrrAs bind to the ThkA dimer. SAXS experiments also confirmed this association state in solution and specific binding between ThkA and TrrA (Kd=8.2x10(-11) M(-2)). The association interface between ThkA and TrrA contains the phosphotransfer His residue in the ThkA, indicative of an efficient receipt of the phosphoryl group. One Per-Arnt-Sim (PAS) domain does not interact with the other PAS domain, but with the catalytic domain of the same polypeptide chain and with one TrrA molecule. Observed inter-domain and inter-molecular interactions reveal a definite pathway of signal transduction in the kinase/regulator complex. In addition, we propose a responsible role of TrrA for the feedback regulation of sensing and/or kinase activities of ThkA.