Merkel cell-nerve cell interaction undergoes formation of a synapse-like structure in a primary culture

Merkel cells have been assumed to guide nerve fibers to the skin. However, there has been little in vitro evidence that supports this hypothesis, because there is no suitable established culture system of Merkel cells. Here we show that Merkel cells isolated from rat footpad skin were successfully cultured in a monolayer with keratinocytes. Keratinocytes did not affect any structural changes in Merkel cells. When nerve cells (NG108-15 or PC12) were added to the culture system, both nerve fibers and cytoplasmic processes of Merkel cells outgrew and cooperatively organized synapse-like structures at their contact points. Nerve cells promoted Merkel cell survival, compared with keratinocytes only. Merkel cell proliferation was not detected in all conditions, even with nerve growth factor, neurotrophin-3, interleukin-6 and tumor necrosis factor-alpha. The data suggest, firstly, that Merkel cells may guide nerve fibers to the skin by interacting with nerve cells; and, secondly, that nerve cells, but not keratinocytes, may produce some survival factors other than the cytokines above for Merkel cells, although Merkel cells may be a terminally differentiated cell type. Our method could open a way to study Merkel cell biology.     

Cell type-specific activation of metabolism reveals that beta-cell secretion suppresses glucagon release from alpha-cells in rat pancreatic islets

Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.     

A novel plant-specific family gene, ROOT PRIMORDIUM DEFECTIVE 1, is required for the maintenance of active cell proliferation

Hypocotyl segments of Arabidopsis (Arabidopsis thaliana) produce adventitious roots in response to exogenously supplied auxin. root primordium defective 1 (rpd1) is a temperature-sensitive mutant isolated on the basis of impairment in this phenomenon. This study describes further phenotypic analysis of the rpd1 mutant and isolation of the RPD1 gene. When adventitious root formation was induced from the rpd1 explants at the restrictive temperature, cell proliferation leading to root promordia formation was initiated at the same time as in wild-type explants. However, development of the root primordia was arrested thereafter in the mutant. Temperature-shift experiments indicated that RPD1 exerts its function before any visible sign of root primordium formation. The expression patterns of the auxin-responsive gene DR5:beta-glucuronidase and the cytodifferentiation marker gene SCARECROW suggest that the rpd1 mutation interferes with neither axis formation nor cellular patterning at the initial stage of root primordium development. Taken together with the effect of the rpd1 mutation on callus cell proliferation, these data imply a role for RPD1 in prearranging the maintenance of the active cell proliferation during root primordium development. Positional cloning of the RPD1 gene revealed that it encodes a member of a novel protein family specific to the plant kingdom. Disruption of the RPD1 gene by a T-DNA insertion caused embryogenesis arrest at the globular to transition stages. This phenotype is consistent with the hypothesized function of RPD1 in the maintenance of active cell proliferation.     

Establishment of a novel method for enriching osteoblast progenitors from adipose tissues using a difference in cell adhesive properties

In the clinical field, cell-based therapies are used to treat bone defects. Adipose tissues contain many osteoblast progenitors, among other cell types. We separated mouse adipose tissue-derived stromal cells (ATSCs) according to their cell adhesive properties. Cells in a fraction adherent to the culture dishes 0.5h after inoculation (AF-0.5) had a potent ability to differentiate into both osteoblasts and adipocytes in vitro. Their differentiation pathways depended on the culture conditions. In these cells, the expression of marker genes for osteoblast differentiation was induced in osteogenic medium. Moreover, the AF-0.5 cells, which were induced to differentiate into osteoblasts in vitro, formed abundant bone tissues in vivo. These results suggest that the AF-0.5 cells have been enriched with bi-potential progenitor cells destined for either osteoblasts or adipocytes. This simple and efficient method for preparing osteoblast progenitor cells from ATSCs may be utilized for bone defect treatment clinically.     

Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.     

Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22

Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo or seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison with the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signaling should include MPK11 as a fourth PAMP-activated MAPK.     

Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites

Neurons develop dendritic arbors in cell type-specific patterns. Using growing Purkinje cells in culture as a model, we performed a long-term time-lapse observation of dendrite branch dynamics to understand the rules that govern the characteristic space-filling dendrites. We found that dendrite architecture was sculpted by a combination of reproducible dynamic processes, including constant tip elongation, stochastic terminal branching, and retraction triggered by contacts between growing dendrites. Inhibition of protein kinase C/protein kinase D signaling prevented branch retraction and significantly altered the characteristic morphology of long proximal segments. A computer simulation of dendrite branch dynamics using simple parameters from experimental measurements reproduced the time-dependent changes in the dendrite configuration in live Purkinje cells. Furthermore, perturbation analysis to parameters in silico validated the important contribution of dendritic retraction in the formation of the characteristic morphology. We present an approach using live imaging and computer simulations to clarify the fundamental mechanisms of dendrite patterning in the developing brain.     

The human apoptosis inhibitor NAIP induces pyroptosis in macrophages infected with Legionella pneumophila

Human nucleotide oligomerization domain-like receptor family apoptosis inhibitory protein (NAIP) prevents apoptosis by inhibiting caspase-3, -7, and -9. Four functional Naip exist in the murine genome, each of which is equally similar to human NAIP. Among them, Naip5 induces pyroptosis by promoting caspase-1 activation in response to Legionella pneumophila infection in macrophages. However, the contribution of human NAIP to this response is unclear. To investigate the role of human NAIP in macrophage survival, we stably expressed human NAIP in RAW264.7 macrophages. Human NAIP inhibited camptothecin-induced apoptosis in macrophages; however, it promoted cytotoxicity in L. pneumophila-infected cells. This cytotoxicity was associated with caspase-1. In addition, human NAIP restricted the intracellular growth of L. pneumophila. L. pneumophila flagellin was required for cytotoxicity, caspase-1 activation, and restriction of intracellular bacterial growth. Expression of murine Naip5 produced comparable results. These data indicate that human NAIP regulates the host response to L. pneumophila infection in a manner similar to that of murine Naip5 and that human NAIP and murine Naip5 regulate cell survival by inhibiting apoptosis or by promoting pyroptosis in response to specific cellular signals.     

Satellite glial cell P2Y12 receptor in the trigeminal ganglion is involved in lingual neuropathic pain mechanisms in rats

Background

Interstitial cystitis/painful bladder syndrome (IC/PBS), is a severely debilitating chronic condition that is frequently unresponsive to conventional pain medications. The etiology is unknown, however evidence suggests that nervous system sensitization contributes to enhanced pain in IC/PBS. In particular, central nervous system plasticity of glutamatergic signaling involving NMDA and metabotropic glutamate receptors (mGluRs) has been implicated in a variety of chronic pain conditions. Here, we test the hypothesis that mGluR5 mediates both non-inflammatory and inflammatory bladder pain or nociception in a mouse model by monitoring the visceromotor response (VMR) during graded bladder distention.

Results

Using a combination of genetic and pharmacologic approaches, we provide evidence indicating that mGluR5 is necessary for the full expression of VMR in response to bladder distention in the absence of inflammation. Furthermore, we observed that mice infected with a uropathogenic strain of Escherichia coli (UPEC) develop inflammatory hyperalgesia to bladder distention, and that the selective mGluR5 antagonist fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl) urea], reduces the VMR to bladder distention in UPEC-infected mice.

Conclusions

Taken together, these data suggest that mGluR5 modulates both inflammatory and non-inflammatory bladder nociception, and highlight the therapeutic potential for mGluR5 antagonists in the alleviation of bladder pain.