Immunohistochemical localization of fibromodulin in the periodontium during cementogenesis and root formation in the rat molar

Background: Cementum is essential for periodontal regeneration, as it provides anchorage between the root surface and the periodontal ligament. A variety of macromolecules present in the extracellular matrix of the periodontium, including proteoglycans, are likely to play a regulatory role in cementogenesis. Recently, the small leucine-rich proteoglycan, fibromodulin, has been isolated from bovine periodontal ligament and localized in bovine cementum, as well as in human periodontal ligament. Objective: The aim of this study was to examine the distribution of fibromodulin during cementogenesis and root formation. Methods: A standard indirect immunoperoxidase technique was employed, using an antifibromodulin polyclonal antibody on sections of molar teeth from rats aged 3, 5 and 8 weeks. Results: Immunoreactivity to fibromodulin was evident in the periodontal ligament in all sections. An intense positive stain was observed in the extracellular matrix where the periodontal ligament fibers insert into the alveolar bone and where the Sharpey's fibers insert into the cementum. There was no staining evident in the mineralized cellular and acellular cementum. The intensity of immunoreactivity to the antifibromodulin antibody increased proportionally with increasing tissue maturation. Conclusion: The results from this study suggest that fibromodulin is a significant component of the extracellular matrix in the periodontal ligament during development, and may play a regulatory role in the mineralization process or maintaining homeostasis at the hard-soft tissue interface during cementogenesis.

C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. (C) 2003 Elsevier Inc. All rights reserved.

Increased TUNEL positive cells in human alcoholic brains

Alcohol-sensitive neuronal cell loss, which has been reported in the superior frontal cortex and hippocampus, may underlie the pathogenesis of subsequent cognitive deficits. In the present study, we have used the TUNEL labeling to detect the DNA damage in human alcoholic brains. Seven out of eleven alcoholics exhibited TUNEL-positive cells in both superior frontal cortex and hippocampus, which were co-localized with GFAP immunoreactivity. In contrast, almost no positive cells were detected in the non-alcoholic controls. None of the TUNEL-positive cells showed any typical morphological features of apoptosis or necrosis. TUNEL-positive cells observed in the present study may indicate DNA damage induced by ethanol-related overproduction of reactive oxygen species. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

Localization of the Epstein-Barr virus protein LMP 1 to exosomes

The Epstein-Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane, Co-labelling of cells with both an LIMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LIMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LIMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin's disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system.

A molecular mechanism for mRNA trafficking in neuronal dendrites

Specific neuronal mRNAs are localized in dendrites, often concentrated in dendritic spines and spine synapses, where they are translated. The molecular mechanism of localization is mostly unknown. Here we have explored the roles of A2 response element (A2RE), a cis-acting signal for oligodendrocyte RNA trafficking, and its cognate trans-acting factor, heterogeneous nuclear ribonucleoprotein ( hnRNP) A2, in neurons. Fluorescently labeled chimeric RNAs containing A2RE were microinjected into hippocampal neurons, and RNA transport followed using confocal laser scanning microscopy. These RNA molecules, but not RNA lacking the A2RE sequence, were transported in granules to the distal neurites. hnRNP A2 protein was implicated as the cognate trans-acting factor: it was colocalized with RNA in cytoplasmic granules, and RNA trafficking in neurites was compromised by A2RE mutations that abrogate hnRNP A2 binding. Coinjection of antibodies to hnRNP A2 halved the number of trafficking cells, and treatment of neurons with antisense oligonucleotides also disrupted A2RE - RNA transport. Colchicine inhibited trafficking, whereas cells treated with cytochalasin were unaffected, implicating involvement of microtubules rather than microfilaments. A2RE-like sequences are found in a subset of dendritically localized mRNAs, which, together with these results, suggests that a molecular mechanism based on this cis-acting sequence may contribute to dendritic RNA localization.

The Lake Tekapo experiment (LTEX): An investigation of atmospheric boundary layer processes in complex terrain

A research program on atmospheric boundary layer processes and local wind regimes in complex terrain was conducted in the vicinity of Lake Tekapo in the southern Alps of New Zealand, during two 1-month field campaigns in 1997 and 1999. The effects of the interaction of thermal and dynamic forcing were of specific interest, with a particular focus on the interaction of thermal forcing of differing scales. The rationale and objectives of the field and modeling program are described, along with the methodology used to achieve them. Specific research aims include improved knowledge of the role of surface forcing associated with varying energy balances across heterogeneous terrain, thermal influences on boundary layer and local wind development, and dynamic influences of the terrain through channeling effects. Data were collected using a network of surface meteorological and energy balance stations, radiosonde and pilot balloon soundings, tethered balloon and kite-based systems, sodar, and an instrumented light aircraft. These data are being used to investigate the energetics of surface heat fluxes, the effects of localized heating/cooling and advective processes on atmospheric boundary layer development, and dynamic channeling. A complementary program of numerical modeling includes application of the Regional Atmospheric Modeling System (RAMS) to case studies characterizing typical boundary layer structures and airflow patterns observed around Lake Tekapo. Some initial results derived from the special observation periods are used to illustrate progress made to date. In spite of the difficulties involved in obtaining good data and undertaking modeling experiments in such complex terrain, initial results show that surface thermal heterogeneity has a significant influence on local atmospheric structure and wind fields in the vicinity of the lake. This influence occurs particularly in the morning. However, dynamic channeling effects and the larger-scale thermal effect of the mountain region frequently override these more local features later in the day.

GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the traps-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of Hela cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the traps-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

Contextual binding of p120ctn to E-cadherin at the basolateral plasma membrane in polarized epithelia

E-cadherin-catenin complexes mediate cell-cell adhesion on the basolateral membrane of epithelial cells. The cytoplasmic tail of E-cadherin supports multiple protein interactions, including binding of beta-catenin at the C terminus and of p120(ctn) to the juxtamembrane domain. The temporal assembly and polarized trafficking of the complex or its individual components to the basolateral membrane are not fully understood. In Madin-Darby canine kidney cells at steady state and after treatment with cycloheximide or temperature blocks, E-cadherin and beta-catenin localized to the Golgi complex, but p120ctn was found only at the basolateral plasma membrane. We previously identified a dileucine sorting motif (Leu(586)-Leu(587), termed S1) in the juxtamembrane domain of E-cadherin and now show that it is required to target full-length E-cadherin to the basolateral membrane. Removal of S1 resulted in missorting of E-cadherin mutants (EcadDeltaS1) to the apical membrane; beta-catenin was simultaneously missorted and appeared at the apical membrane. p120(ctn) was not mistargeted with EcadDeltaS1, but could be recruited to the E-cadherin-catenin complex only at the basolateral membrane. These findings help define the temporal assembly and sorting of the E-cadherin-catenin complex and show that membrane recruitment of p120(ctn) in polarized cells is contextual and confined to the basolateral membrane.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs. (C) 2002 Wiley-Liss, Inc.