Stable-isotope probing with multiple growth substrates to determine substrate specificity of uncultivated bacteria

Stable-isotope probing (SIP) has been used to determine which microorganisms in a complex environmental sample are capable of metabolizing a labeled substrate. We hypothesized that DNA-based stable-isotope probing with a combination of a (13)C-labeled carbon source and a second, unlabeled carbon source could be combined with analyses of the entire gradient of separated DNA to provide information concerning the utilization of a mixture of environmentally relevant compounds by uncultivated organisms. As a test of the method, we evaluated the response of a microbial community in a laboratory bioreactor treating contaminated soil to two polycyclic aromatic hydrocarbons (PAH). The compounds were added either individually as [U-(13)C]phenanthrene or [U-(13)C]pyrene, or as a mixture in which one was labeled with (13)C and the other was unlabeled. After ultracentrifugation of DNA extracted from a given incubation, fractions containing DNA enriched with varying levels of (13)C were examined by denaturing-gradient gel electrophoresis (DGGE) and by real-time quantitative PCR (qPCR) for 16S rRNA genes belonging to organisms in groups of bacteria previously associated with PAH degradation by single-compound SIP. Four groups of bacteria (three uncultivated) were followed in this study. Two of the uncultivated groups showed evidence for simultaneous or sequential utilization of both compounds while the remaining two appeared to assimilate carbon from only one of the compounds. DNA-based SIP therefore appears to be useful to evaluate the selectivity among substrates in a mixture by uncultivated microbes.     

Distribution of TRPC4 in developing and adult murine brain

The transient receptor potential (TRP) superfamily comprises of a group of non-selective cation channels that have been implicated in both receptor and store-operated channel functions. The family of classical TRPs (TRPCs) consists of seven members (TRPC1-7), with TRPC4 possibly playing a role in neuronal signaling. We have examined the distribution pattern of TRPC4 mRNA and protein in the developing and postnatal murine brain by using in situ hybridization, Western blotting, and immunocytochemistry. Expression of TRPC4 mRNA starts at embryonic day 14.5 (E14.5) in the developing septal area and cerebellar anlagen. At E16.5, prominent expression is additionally seen in the hippocampal formation and cortical plate. High densities of cells expressing TRPC4 mRNA occur in the adult olfactory bulb and hippocampus, whereas the cortex and septum display lower densities of cells positive for TRPC4 mRNA. Analysis of the adult hippocampal formation has revealed TRPC4 immunoreactivity in hippocampal areas CA1 to CA3 and in the dentate gyrus. Functions consistent with this spatially restricted pattern of expression remain to be revealed.     

Human connexin26 and connexin30 form functional heteromeric and heterotypic channels

Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts. gap junctions; hearing; fluorescence resonance energy transfer; fluorescence recovery after photobleaching; immunoprecipitation; dye transfer; electrophysiology     

Association of tyrosine phosphatase epsilon with microtubules inhibits phosphatase activity and is regulated by the epidermal growth factor receptor

Protein tyrosine phosphatases (PTPs) are key mediators that link physiological cues with reversible changes in protein structure and function; nevertheless, significant details concerning their regulation in vivo remain unknown. We demonstrate that PTPepsilon associates with microtubules in vivo and is inhibited by them in a noncompetitive manner. Microtubule-associated proteins, which interact strongly with microtubules in vivo, significantly increase binding of PTPepsilon to tubulin in vitro and further reduce phosphatase activity. Conversely, disruption of microtubule structures in cells reduces their association with PTPepsilon, alters the subcellular localization of the phosphatase, and increases its specific activity. Activation of the epidermal growth factor receptor (EGFR) increases the PTPepsilon-microtubule association in a manner dependent upon EGFR-induced phosphorylation of PTPepsilon at Y638 and upon microtubule integrity. These events are transient and occur with rapid kinetics similar to EGFR autophosphorylation, suggesting that activation of the EGFR transiently down-regulates PTPepsilon activity near the receptor by promoting the PTPepsilon-microtubule association. Tubulin also inhibits the tyrosine phosphatase PTP1B but not receptor-type PTPmu or the unrelated alkaline phosphatase. The data suggest that reversible association with microtubules is a novel, physiologically regulated mechanism for regulation of tyrosine phosphatase activity in cells.     

Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation

During cutaneous wound repair, platelets, dermal fibroblasts (DF) and endothelial cells all cooperate. We have presently investigated the regulation of endothelial cell tubulogenesis by human platelet thrombospondin-1 (TSP-1), in comparison to transforming growth factor-beta1 (TGF-beta1) and total platelet lysates (PL), in a fibrin matrix cell culture system incorporating DF. TSP-1, TGF-beta1 and PL all stimulated VEGF expression in DF dose dependently at mRNA and protein level. TSP-1- and PL-treated DF supernatants significantly stimulated capillary-like structure formation (tubulogenesis) by dermal microvascular endothelial cells (HMEC-1 and HDMEC), in part via VEGF, as confirmed with neutralizing anti-VEGF antibodies. In contrast, TGF-beta1-treated DF supernatants did not induce tubulogenesis. This apparent discrepancy could be explained by the differential expression regulation in HMEC-1 of fibrinolysis and metalloproteinase mediators by TSP-1 and TGF-beta1. TSP-1 potently reduced the expression of plasminogen activator inhibitor-1 (PAI-1) (mRNA and protein), whereas TGF-beta1 enhanced it. The crucial role of PAI-1 in tubulogenesis was confirmed via the addition of active recombinant PAI-1, which abrogated tubulogenesis. In contrast, neutralizing PAI-1 antibodies enhanced tubulogenesis. Our results suggest that platelet TSP-1 released in a wound stimulates endothelial cell tubulogenesis through an upregulation of DF VEGF expression and a downregulation of endothelial cell PAI-1 expression.