Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.     

Molecular identification of GHS-R and GPR38 in Suncus murinus

We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus.     

Purification and properties of phenolic acid decarboxylase from Candida guilliermondii

A heat-labile phenolic acid decarboxylase from Candida guilliermondii (an anamorph of Pichia guilliermondii) was purified to homogeneity by simple successive column chromatography within 3 days. The molecular mass was 20 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 36 kDa by gel-filtration chromatography, suggesting that the purified enzyme is a homodimer. The optimal pH and temperature were approximately 6.0 and 25°C. Characteristically, more than 50% of the optimal activity was observed at 0°C, suggesting that this enzyme is cold-adapted. The enzyme converted p-coumaric acid, ferulic acid, and caffeic acid to corresponding products with high specific activities of approximately 600, 530, and 46 U/mg, respectively. The activity was stimulated by Mg²⁺ ions, whereas it was completely inhibited by Fe²⁺, Ni²⁺, Cu²⁺, Hg²⁺, 4-chloromericuribenzoate, N-bromosuccinimide, and diethyl pyrocarbonate. The enzyme was inducible and expressed inside the cells moderately by ferulic acid and p-coumaric acid and significantly by non-metabolizable 6-hydroxy-2-naphthoic acid.     

Absorption of Radionuclides from the Fukushima Nuclear Accident by a Novel Algal Strain

Large quantities of radionuclides have leaked from the Fukushima Daiichi Nuclear Power Plant into the surrounding environment. Effective prevention of health hazards resulting from radiation exposure will require the development of efficient and economical methods for decontaminating radioactive wastewater and aquatic ecosystems. Here we describe the accumulation of water-soluble radionuclides released by nuclear reactors by a novel strain of alga. The newly discovered green microalgae, Parachlorella sp. binos (Binos) has a thick alginate-containing extracellular matrix and abundant chloroplasts. When this strain was cultured with radioiodine, a light-dependent uptake of radioiodine was observed. In dark conditions, radioiodine uptake was induced by addition of hydrogen superoxide. High-resolution secondary ion mass spectrometry (SIMS) showed a localization of accumulated iodine in the cytosol. This alga also exhibited highly efficient incorporation of the radioactive isotopes strontium and cesium in a light-independent manner. SIMS analysis showed that strontium was distributed in the extracellular matrix of Binos. Finally we also showed the ability of this strain to accumulate radioactive nuclides from water and soil samples collected from a heavily contaminated area in Fukushima. Our results demonstrate that Binos could be applied to the decontamination of iodine, strontium and cesium radioisotopes, which are most commonly encountered after nuclear reactor accidents.     

Thermotomaculum hydrothermale gen. nov., sp. nov., a novel heterotrophic thermophile within the phylum Acidobacteria from a deep-sea hydrothermal vent chimney in the Southern Okinawa Trough

A novel heterotrophic, thermophilic bacterium, designated strain AC55^T, was isolated from a deep-sea hydrothermal vent chimney at the Hatoma Knoll in the Okinawa Trough, Japan. Cells of strain AC55^T were non-motile, long rods (2.0- to 6.8-μm long and 0.3- to 0.6-μm wide). The strain was an obligatory anaerobic heterotroph capable of fermentative growth on complex proteinaceous substances. Elemental sulfur was reduced to hydrogen sulfide but did not stimulate growth. Growth was observed between 37 and 60°C (optimum 55°C), pH 5.5 and 8.5 (optimum pH 6.6), and in the presence of 1.5–4.5% (w/v) NaCl (optimum 2.5%, w/v). Menaquinone-7 and -8 were the major respiratory quinones. The G + C content of the genomic DNA from strain AC55^T was 51.6 mol%. The 16S rRNA gene sequence analysis revealed that strain AC55^T was the first cultivated representative of Acidobacteria subdivision 10. Based on the physiological and phylogenetic features of the novel isolate, the genus name Thermotomaculum gen. nov. is proposed, with Thermotomaculum hydrothermale sp. nov. as the type species. The type strain is AC55^T (=JCM 17643^T = DSM 24660^T = NBRC 107904^T).     

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.     

MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.     

Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity

Neural circuits are shaped by experience in early postnatal life. Distinct GABAergic connections within visual cortex determine the timing of the critical period for rewiring ocular dominance to establish visual acuity. We find that maturation of the parvalbumin (PV)-cell network that controls plasticity onset is regulated by a selective re-expression of the embryonic Otx2 homeoprotein. Visual experience promoted the accumulation of non-cell-autonomous Otx2 in PV-cells, and cortical infusion of exogenous Otx2 accelerated both PV-cell development and critical period timing. Conversely, conditional removal of Otx2 from non-PV cells or from the visual pathway abolished plasticity. Thus, the experience-dependent transfer of a homeoprotein may establish the physiological milieu for postnatal plasticity of a neural circuit.