Necdin is required for terminal differentiation and survival of primary dorsal root ganglion neurons

Necdin is expressed predominantly in postmitotic neurons and serves as a growth suppressor that is functionally similar to the retinoblastoma tumor suppressor protein. Using primary cultures of dorsal root ganglion (DRG) of mouse embryos, we investigated the involvement of necdin in the terminal differentiation of neurons. DRG cells were prepared from mouse embryos at 12.5 days of gestation and cultured in the presence of nerve growth factor (NGF). Immunocytochemistry revealed that necdin accumulated in the nucleus of differentiated neurons that showed neurite extension and expressed the neuronal markers microtubule-associated protein 2 and synaptophysin. Suppression of necdin expression in DRG cultures treated with antisense oligonucleotides led to a marked reduction in the number of terminally differentiated neurons. The antisense oligonucleotide-treated cells did not attempt to reenter the cell cycle, but underwent death with characteristics of apoptosis such as caspase-3 activation, nuclear condensation, and chromosomal DNA fragmentation. Furthermore, a caspase-3 inhibitor rescued antisense oligonucleotide-treated cells from apoptosis and significantly increased the population of terminally differentiated neurons. These results suggest that necdin mediates the terminal differentiation and survival of NGF-dependent DRG neurons and that necdin-deficient nascent neurons are destined to caspase-3-dependent apoptosis.     

Identification of protein functions from a molecular surface database,eF-site

A bioinformatics method was developed to identify the protein surface around the functional site and to estimate the biochemical function, using a newly constructed molecular surface database named the eF-site (electrostatic surface of Functional site. Molecular surfaces of protein molecules were computed based on the atom coordinates, and the eF-site database was prepared by adding the physical properties on the constructed molecular surfaces. The electrostatic potential on each molecular surface was individually calculated solving the Poisson–Boltzmann equation numerically for the precise continuum model, and the hydrophobicity information of each residue was also included. The eF-site database is accessed by the internet (http://pi.protein.osaka-u.ac.jp/eF-site/). We have prepared four different databases, eF-site/antibody, eF-site/prosite, eF-site/P-site, and eF-site/ActiveSite, corresponding to the antigen binding sites of antibodies with the same orientations, the molecular surfaces for the individual motifs in PROSITE database, the phosphate binding sites, and the active site surfaces for the representatives of the individual protein family, respectively. An algorithm using the clique detection method as an applied graph theory was developed to search of the eF-site database, so as to recognize and discriminate the characteristic molecular surfaces of the proteins. The method identifies the active site having the similar function to those of the known proteins.     

Termination of parental care due to small clutch size in the temperate damselfish,Chromis notata

Synopsis Reproductive behavior of the temperate damselfish, Chromis notata, was investigated on the island of Mukaishima, Japan, almost daily during the breeding season in 1982. Both males and females repeated reproductive cycles many times during the breeding season. Females had a strong tendency to spawn a whole clutch on one nest during a few hours. The average number of eggs which a male gained per reproductive cycle was estimated at 38560 (480–131100 eggs). Males ordinarily cared for eggs until just prior to hatching, but abandoned more than half of the nests with the eggs numbering less than 11568. Contribution 207 from the Mukaishima Marine Biological Station.     

Bioluminescence characteristics of the fruiting body of Mycena chlorophos

Bioluminescent fungi are widely distributed on land and most belong to the class Basidomycetes. Light of about 530 nm wavelength maximum is emitted continuously. The molecular basis for the light‐emitting process remains unclear. We investigated the characteristics of the bioluminescence using cultivated fruiting bodies of M. chlorophos. Only fresh fruiting bodies exhibited long‐lasting light emission; rapid decay of light emission was observed with frozen and freeze‐dried samples. Freeze‐dried samples can be stored at room temperature under dry conditions and may be useful for the isolation of luciferin. The light emission of the fresh fruiting bodies was maintained in various buffers at varying pH; it could be stopped with pH 4 acetate buffer and could be recovered at pH 6. The isolation of luciferin from the fresh fruiting bodies might be possible by the control of buffer pH. The effect of temperature on the light emission of fruiting bodies indicated that bioluminescence in M. chlorophos may involve enzymatic reaction(s). The solubilization of bioluminescent components from the fruiting bodies could not be achieved with various surfactants. Copyright © 2011 John Wiley & Sons, Ltd.     

Nitrogen uptake and assimilation in Enteromorpha intestinalis (L.) Link (Chlorophyta): using N to determine preference during simultaneous pulses of nitrate and ammonium

We investigated the ability of Enteromorpha intestinalis (L.) Link to take up pulses of different species of nitrogen simultaneously, as this would be an important mechanism to enhance bloom ability in estuaries. Uptake rates and preference for NH₄⁺ or NO₃⁻ following 1, 3, 6, 9, 12 or 24 h of exposure to either ¹⁵NH₄NO₃ or NH₄¹⁵NO₃ were determined by disappearance of N from the medium. Differences in assimilation rates for NH₄⁺ or NO₃⁻ were quantified by the accumulation of NH₄⁺, NO₃⁻, and atom % ¹⁵N in the algal tissue. NH₄⁺ concentration was reduced more quickly than water NO₃⁻ concentration. Water column NH₄⁺ concentration after the longest time interval was reduced from 300 to 50 μM. Water NO₃⁻ was reduced from 300 to 150 μM. The presence of ¹⁵N or ¹⁴N had no effect on uptake of either NH₄⁺ or NO₃⁻. ¹⁵N was removed from the water at an almost identical rate and magnitude as ¹⁴N. Differences in accumulation of ¹⁵NH₄⁺ and ¹⁵NO₃⁻ in the tissue reflected disappearance from the water; ¹⁵N from NH₄⁺ accumulated faster and reached an atom % twice that of ¹⁵N from NO₃⁻. This outcome suggested that when NH₄⁺ and NO₃⁻ were supplied in equal concentrations, more NH₄⁺ was taken up and assimilated. The ability to take up high concentrations of NH₄⁺, and NO₃⁻ simultaneously is important for bloom-forming species of estuarine macroalgae subject to multiple nutrient species from various sources.     

Cytokeratin 18 is a specific marker of bovine intestinal M cell

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.     

Linking floral symmetry genes to breeding system evolution

Understanding the genetic basis of ecologically important traits is a major focus of evolutionary research. Recent advances in molecular genetic techniques should significantly increase our understanding of how regulatory genes function. By contrast, our understanding of the broader macro-evolutionary implications of developmental gene function lags behind. Here we review published data on the floral symmetry gene network (FSGN), and conduct phylogenetic analyses that provide evidence of a link between floral symmetry and breeding systems in angiosperms via dichogamy. Our results suggest that known genes in the FSGN and those yet to be described underlie this association. We posit that the integration of floral symmetry and the roles of other regulatory genes in plant breeding system evolution will provide new insights about macro-evolutionary patterns and processes in flowering plants.     

Nicotine stimulates prolactin-releasing peptide (PrRP) cells and non-PrRP cells in the solitary nucleus

Nicotine has been reported to regulate food intake and body weight. But the mechanisms underlying these roles have not been fully elucidated. In the present study, we showed that acute administration of nicotine (0.5 mg/kg s.c.) could activate prolactin-releasing peptide (PrRP)-bearing neurons in the A2 area of the NTS of rats, suggesting that PrRP may be associated with nicotine-induced effects in the central nervous system (CNS). We next treated rats with nicotine chronically (4 mg/kg/day for 7 days i.p.), and the results showed that the body weight was strongly reduced and food intake was greatly suppressed compared to the vehicle control group (p<0.01). Immunocytochemical studies revealed that PrRP-bearing neurons in the NTS were evidently activated after chronic administration of nicotine, suggesting that PrRP was involved in the regulation of nicotine-mediated body weight loss and food intake suppression in rats. We also found that acute/chronic administration of nicotine activated PrRP-negative neurons in the NTS, and the majority of these neurons were shown to be TH-negative, suggesting that noncatecholaminergic, PrRP-negative neurons in the NTS are associated with the roles of nicotine. Nicotine has also been shown to stimulate the secretion of ACTH, a stress responsive hormone. In the present study, rats received nicotine (0.5 mg/kg s.c.) or saline followed by restraint stress for 30 min. The immunocytochemical results showed that nicotine/stress and saline/stress both activated the majority of the PrRP neurons in the NTS, there being no significant difference between the two treatments (p>0.05). Nicotine/stress also greatly activated PrRP/TH-negative neurons in the NTS. Saline/stress, however, caused much lower effect on the activation of PrRP/TH-negative neurons. In addition, the activation effect of nicotine/stress on PrRP/TH-negative neurons was much stronger than that of nicotine alone (p<0.01). These results indicated that PrRP was associated with stress responses, but it had little effect on nicotine-mediated stress responses. On the other hand, nicotine and restraint stress may synergistically activate PrRP/TH-negative neurons in the NTS. Taken together, our data show that PrRP is involved in the nicotine-induced regulation of body weight and food intake, but may not be involved in the mediation of nicotine on stress responses. PrRP/TH-negative neurons in the NTS are also associated with the roles of nicotine in the CNS.