No abstract

Noise overstimulation will induce or influence intracellular molecular pathways in the cochlea. One of these is the ‘classical’ stress response pathway involving heat shock proteins. Hsp70 is induced in the cochlea by a wide variety of stresses including noise, hyperthermia and ototoxic drugs. When a stress that induces Hsp70 is applied to the cochlea, there is protection from a subsequent noise that would normally cause a permanent hearing loss. An upstream regulator of heat shock protein transcription, heat shock factor 1, is expressed in the cochlea and activated by stress. Mice lacking this heat shock factor have reduced recovery from noise-induced hearing loss. The same noise exposure that induces Hsp70 also increases the level of glial cell line-derived neurotrophic factor in the cochlea. Moreover, when this neurotrophic factor is applied into the perilymph of scala tympani prior to a noise exposure there is a significant reduction in hair cell loss and hearing loss. With the potential for activation of multiple pathways in the response to noise, gene microarrays can be useful to examine global gene expression. Initial studies examined differential gene expression immediately following a mild noise exposure (from which there is complete recovery) versus an intense noise (giving profound permanent deafness). Differential expression of several immediate early genes was found following the intense but not the mild noise exposure.

Understanding the mechanisms of substrate based control of cell function is critical to the design of biomaterials. Cells interact with their extracellular matrix through cell adhesion contacts. We have previously described the self assembly of bone-like mineral onto an organic template and have shown that these biomimetic surfaces lead to an increased volume fraction of bone regenerated in vivo. In the present study, we compared the distribution of cell adhesion contacts, cell spreading, and cell motility of murine bone marrow stromal cells (BMSC) on mineralized vs. nonmineralized substrates. We developed a new approach for quantification of cell-material interactions and demonstrated that cell adhesion contacts on mineralized substrates were distributed throughout the cell surface contacting the substrate, whereas on nonmineralized substrates cell adhesion contacts were present near the cell periphery. We propose that mineralized substrates stimulate the predominant expression of fibrillar contacts, and nonmineralized substrates stimulate expression of focal adhesion contacts. Cell motility assays with colloidal gold demonstrated a statistically significant decrease in the average phagokinetic index of migrating cells on mineralized vs. nonmineralized substrates after 90 min of cell seeding. We propose that the physical-chemical properties of the substrate, altered by mineralization, cause expression of specific types of cell contacts and, as a result, modify molecular mechanisms responsible for cell spreading, motility, and possibly differentiation.

The effects of low doses of cytochalasin B (2 ,ug/ ml) and cytochalasin D (0.2 lag/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors.Substrate-spread fibroblasts and epithelial cells have a special structure, the lamellar cytoplasm, at their periphery. Characteristic features of the lamellar cytoplasm. include a flattened shape, the absence of large organelles, the presence of numerous focal contacts with the substrate at the lower surface, and a distinctive ability of the surface to clear itself from patched membrane receptors (see review in ref. 1). The zones of active edges are usually located at the distal parts of the lamellar cytoplasm; these are the zones where pseudopods, such as lamellopodia and ruffles, are formed. A study of spreading in medium containing cytochalasin B (CB) at 10 ,ug/ml suggested that formation of the lamellar cytoplasm can be regarded as a combination of two functionally different processes-CB-resistant rudimentary pseudopodial reactions leading to the extension and attachment of arborized outgrowths and CB-sensitive lamellization of these outgrowths leading to their transformation into flattened wide lamellas (2). Experiments presented in this paper show that lamellization, in its turn, can be subdivided into components that have different sensitivities to CB: a low concentration of CB (2 ,ug/ml) prevents the formation of ruffling active edges but does not inhibit the outgrowth of lamellas that lack this specialized distal part. MATERIALS AND METHODSMouse embryo fibroblasts were cultured in a 1:1 mixture of Eagle's medium and 0.5% lactalbumin hydrolysate/10% bovine serum. CB (Serva, Heidelberg, Federal Republic of Germany) was added to this medium at 2 pig/ml (low-CB medium) or 10 jkg/ml (high-CB medium). Cytochalasin D (Sigma) was used at 0...