Neuronal interactions between neuropeptide W- and orexin- or melanin-concentrating hormone-containing neurons in the rat hypothalamus

Neuropeptide W (NPW) was recently discovered as the endogenous ligand for GPR7 and GPR8, which are orphan G protein-coupled receptors isolated from the porcine brain. These receptors are assumed to be involved in feeding regulation and/or energy homeostasis. Recent anatomical studies have revealed that high levels of GPR7 mRNA are distributed in the brain, including the hypothalamus and amygdala. However immunohistochemical studies on the distribution and localization of NPW have revealed differing results concerning whether or not NPW-containing cell bodies and their processes are present in the hypothalamus. Only a few immunohistochemical reports have been published concerning the presence of NPW-containing neurons in the brains of rodents, while there have been no anatomical studies of the co-localization of this neuropeptide with other transmitters. On this basis, we used a specific antiserum against NPW to determine immunohistochemically the presence of NPW-containing neurons in the rat hypothalamus. Many NPW-like immunoreactive cell bodies and their processes could be detected in the caudal region of the lateral hypothalamus but not in its anterior or middle regions. Given this positive identification of NPW-containing neurons in the lateral hypothalamus, we further studied the nature of interaction between NPW-containing neurons and neurons containing feeding regulating peptides such as orexin- and melanin-concentrating hormone (MCH). Very close interactions between NPW-containing nerve processes and orexin- and MCH-containing neuronal cell bodies and processes could be observed. These morphological findings strongly suggest that NPW is involved in the regulation of feeding and/or sleep/arousal behavior through orexin- and/or MCH-mediated neuronal pathways.     

Nuclear DNA microsatellites reveal genetic variation but a lack of phylogeographical structure in an endangered species, Fraxinus mandshurica, across North-east China

Background and Aims

The widely accepted paradigm that the modern genetic structure of plant species in the northern hemisphere has been largely determined by recolonization from refugia after the last glacial maximum fails to explain the presence of cold-tolerant species at intermediate latitudes. Another generally accepted paradigm is that mountain ridges act as important barriers causing genetic isolation of species, but this too has been challenged in recent studies. The aims of the work reported here were to determine the genetic diversity and distribution patterns of extant natural populations of an endangered cool temperate species, Faxinus mandshurica, and to examine whether these two paradigms are appropriate when applied to this species over a wide geographical scale.

Methods

1435 adult individuals were sampled from 30 natural populations across the main and central range of the species, covering major mountain ranges across North-east China (NEC). Genetic variation was estimated based on nine polymorphic nuclear microsatellite loci. Phylogeographical analyses were employed using various approaches, including Bayesian clustering, spatial analysis of molecular variance, Monmonier''s algorithm, neighbor-joining trees, principal co-ordinate analysis and isolation by distance.

Key Results

Genetic diversity within populations was relatively high, and no significant recent bottlenecks were detected in any of the populations. A significant negative correlation between intra-population genetic diversity and latitude was identified. In contrast, genetic differentiation among all the populations examined was extremely low and no clear geographic genetic structure was identified, with the exception of one distinct population.

Conclusions

The modern genetic structure in this species can be explained by extensive gene flow, an absence of mountains acting as barriers, and the presence of a wide refuge across NEC rather than multiple small refugia. Intra-population genetic variation along latitudes is probably associated with the systematically northward shifts of forest biomes in eastern China during the mid-Holocene. To determine important genetic patterns and identify resources for conservation, however, it will be necessary to examine differentially inherited genetic markers exposed to selection pressures (e.g. chloroplast DNA) and to investigate different generations.Key words: Fraxinus mandshurica, nuclear microsatellites, latitude variation, historical migration, fossil pollen, spatial genetic structure, genetic barriers     

Plant DNA polymerase lambda, a DNA repair enzyme that functions in plant meristematic and meiotic tissues.

Little is known about the functions of DNA polymerase lambda (Pol lambda) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol lambda may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol lambda in a higher plant, rice (Oryza sativa L. cv. Nipponbare) (OsPol lambda). OsPol lambda had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol lambda. Northern blot analysis showed that the level of OsPol lambda expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol lambda is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol beta and terminal deoxyribonucleotidyl transferase.     

A novel site-specific recombination system derived from bacteriophage phiMR11

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.     

Physical interaction between Tbx6 and mespb is indispensable for the activation of bowline expression during Xenopus somitogenesis

Crk is a member of a family of adaptor proteins that are involved in intracellular signal pathways altering cell adhesion, proliferation, and migration. Increased expression of Crk has been described in lung cancer and associated with increased tumor invasiveness. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21–25 nt long) that are capable of targeting genes for either degradation of mRNA or inhibition of translation. Crk is a predicted putative target gene for miR-126. Over-expression of miR126 in a lung cancer cell line resulted in a decrease in Crk protein without any alteration in the associated mRNA. These lung cancer cells exhibit a decrease in adhesion, migration, and invasion. Decreased cancer cell invasion was also evident following targeted knockdown of Crk. MiR-126 alters lung cancer cell phenotype by inhibiting adhesion, migration, and invasion and the effects on invasion may be partially mediated through Crk regulation.     

Ineffectiveness of Lipopolysaccharide for Preventing the Tolerance Induction in Bone Marrow-Derived Lymphoid Cells with Dinitrophenyl-Poly-l-(Glutamic Acid,Lysine)

The effect of endotoxin or lipopolysaccharide (LPS) on tolerance induction in bone marrow-derived lymphoid cells (B cells) was investigated. Dinitrophenylated amino acid copolymer-l-(glutamic acid, lysine) (DNP-GL) acts as a potent tolerogen on normal and DNP-primed B cells. LPS significantly enhanced the anti-sheep red blood cell plaque-forming cell (anti-SRBC PFC) response that occurred after the immunization with a low dose of SRBC. LPS did not induce the primary anti-DNP PFC response after the injection of DNP-GL, nor did it prevent the tolerance induction in normal and DNP-primed B cells that occurred after the administration of DNP-GL.     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.