Skin wounds and severed nerves heal normally in mice lacking tenascin-C.

A large number of functions have been demonstrated for tenascin-C by antibody perturbation assays and in vitro cell culture experiments. However, these results contrast sharply with the lack of any apparent phenotype in mice with a genetic deletion of tenascin-C. A possible explanation for the lack of phenotype would be expression of some altered but functional tenascin-C in the mutant. We report the generation of an independent tenascin-C null mouse and conclude that the original tenascin-C knockout, which is genetically very similar to ours, is also a true null. As found previously, the absence of tenascin-C has no influence on development, adulthood, life span, and fecundity. We have studied in detail two models of wound healing. After axotomy, the regeneration of the sciatic nerve is not altered without tenascin-C. During healing of cutaneous wounds, deposition of collagen I, fibulin-2, and nidogen is identical in mutant and wild-type mice. In contrast. fibronectin appears diminished in wounds of tenascin-C-deficient mice. However, the lack of tenascin-C together with the reduced amount of fibronectin has no influence on the quality of the healing process.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-γ (INF-γ) or tumor necrosis factor-α (TNF-α) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-γ plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-α antibody, suggesting the release and autocrine action of TNF-α. Treatment with INF-γ plus TNF-α also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell–cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18α-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-γ plus LPS or INF-γ plus TNF-α. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

Regulation of Oleoresinosis in Grand Fir (Abies grandis)1 : Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding

Grand fir (Abies grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.