Chronic Powder Diet After Weaning Induces Sleep,Behavioral, Neuroanatomical,and Neurophysiological Changes in Mice

The purpose of this study is to clarify the effects of chronic powder diet feeding on sleep patterns and other physiological/anatomical changes in mice. C57BL/6 male mice were divided into two groups from weaning: a group fed with solid food (SD) and a group fed with powder food (PD), and sleep and physiological and anatomical changes were compared between the groups. PD exhibited less cranial bone structure development and a significant weight gain. Furthermore, these PD mice showed reduced number of neurogenesis in the hippocampus. Sleep analysis showed that PD induced attenuated diurnal sleep/wake rhythm, characterized by increased sleep during active period and decreased sleep during rest period. With food deprivation (FD), PD showed less enhancement of wake/locomotor activity compared to SD, indicating reduced food-seeking behavior during FD. These results suggest that powder feeding in mice results in a cluster of detrimental symptoms caused by abnormal energy metabolism and anatomical/neurological changes.     

Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.     

Role of hydrogen generation by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in the oral cavity

Some gastrointestinal bacteria synthesize hydrogen (H₂) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H₂ or what role H₂ might play in the oral cavity. It was found that a significant amount of H₂ is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H₂-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H₂. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H₂ in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe⁺⁺ and H₂O₂ increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H₂ generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H₂ generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.     

Epigenetic inheritance in rice plants

BACKGROUND AND AIMS: Epigenetics is defined as mechanisms that regulate gene expression without base sequence alteration. One molecular basis is considered to be DNA cytosine methylation, which reversibly modifies DNA or chromatin structures. Although its correlation with epigenetic inheritance over generations has been circumstantially shown, evidence at the gene level has been limited. The present study aims to find genes whose methylation status directly correlates with inheritance of phenotypic changes. METHODS: DNA methylation in vivo was artificially reduced by treating rice (Oryza sativa ssp. japonica) seeds with 5-azadeoxycytidine, and the progeny were cultivated in the field for > 10 years. Genomic regions with changed methylation status were screened by the methylation-sensitive amplified polymorphysm (MSAP) method, and cytosine methylation was directly scanned by the bisulfite mapping method. Pathogen infection with Xanthomonas oryzae pv. oryzae, race PR2 was performed by the scissors-dip method on mature leaf blades. KEY RESULTS: The majority of seedlings were lethal, but some survived to maturity. One line designated as Line-2 showed a clear marker phenotype of dwarfism, which was stably inherited by the progeny over nine generations. MSAP screening identified six fragments, among which two were further characterized by DNA blot hybridization and direct methylation mapping. One clone encoding a retrotransposon gag-pol polyprotein showed a complete erasure of 5-methylcytosines in Line-2, but neither translocation nor expression of this region was detectable. The other clone encoded an Xa21-like protein, Xa21G. In wild-type plants, all cytosines were methylated within the promoter region, whereas in Line-2, corresponding methylation was completely erased throughout generations. Expression of Xa21G was not detectable in wild type but was constitutive in Line-2. When infected with X. oryzae pv. oryzae, against which Xa21 confers resistance in a gene-for-gene manner, the progeny of Line-2 were apparently resistant while the wild type was highly susceptible without Xa21G expression. CONCLUSIONS: These results indicated that demethylation was selective in Line-2, and that promoter demethylation abolished the constitutive silencing of Xa21G due to hypermethylation, resulting in acquisition of disease resistance. Both hypomethylation and resistant trait were stably inherited. This is a clear example of epigenetic inheritance, and supports the idea of Lamarckian inheritance which suggested acquired traits to be heritable.     

Novel thyroid hormone receptor antagonists with an N-alkylated diphenylamine skeleton

Thyroid hormones play important roles in growth, development and homeostasis, and disruption of their functions induces serious disease, so novel synthetic thyroid hormone analogues are candidates for clinical application. We designed and synthesized novel diphenylamine derivatives with a thiazolidinedione moiety as the terminal polar group as thyroid hormone receptor (TR) antagonists. Compounds bearing an appropriately sized N-alkyl group showed antagonistic activities towards both the hTRalpha1 and hTRbeta1 subtypes.     

Nitric oxide (NO) serves as a retrograde messenger to activate neuronal NO synthase in the spinal cord via NMDA receptors.

We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.     

Amphiphysin 1 is important for actin polymerization during phagocytosis

Amphiphysin 1 is involved in clathrin-mediated endocytosis. In this study, we demonstrate that amphiphysin 1 is essential for cellular phagocytosis and that it is critical for actin polymerization. Phagocytosis in Sertoli cells was induced by stimulating phosphatidylserine receptors. This stimulation led to the formation of actin-rich structures, including ruffles, phagocytic cups, and phagosomes, all of which showed an accumulation of amphiphysin 1. Knocking out amphiphysin 1 by RNA interference in the cells resulted in the reduction of ruffle formation, actin polymerization, and phagocytosis. Phagocytosis was also drastically decreased in amph 1 (-/-) Sertoli cells. In addition, phosphatidylinositol-4,5-bisphosphate-induced actin polymerization was decreased in the knockout testis cytosol. The addition of recombinant amphiphysin 1 to the cytosol restored the polymerization process. Ruffle formation in small interfering RNA-treated cells was recovered by the expression of constitutively active Rac1, suggesting that amphiphysin 1 functions upstream of the protein. These findings support that amphiphysin 1 is important in the regulation of actin dynamics and that it is required for phagocytosis.