Linear polysaccharides reduce production of inflammatory cytokines by LPS-stimulated bovine fibroblasts.

Chronic lesions in the limbs of farm animals cause lameness due to chronic infection and inflammation. Exploratory treatments for chronic wounds in humans may be suitable for adaptation into the field of animal care. Specifically, antimicrobial linear polysaccharides like oxidized regenerated cellulose (ORC) and chitin/chitosan are biodegradable hemostats that are being explored to promote healing of chronic wounds but have not been directly compared using the same biological specimen. Despite their current use in humans, linear polysaccharides possess features that may preclude their use as biodegradable bandages. For example, ORC promotes inflammation when it remains in vivo and chitin/chitosan stimulate size-dependent proinflammatory responses. In order to assess the use of these materials to treat chronic wounds we have compared their effects on cellular toxicity and in stimulating the production of proinflammatory cytokines by bovine epidermal fibroblasts. While neither polysaccharide increased cell mortality, on average, they caused minor alterations in expression of proinflammatory cytokines from cells isolated from different animals. Both polysaccharides reduced expression of proinflammatory cytokines stimulated by microbial lipopolysaccharide. We conclude that the polysaccharides used in this study are relatively inert and may improve healing of chronic epidermal wounds in farm animals.

Construction and Composition of the Squid Pen from Doryteuthis pealeii.

The pen, or gladius, of the squid is an internalized shell. It serves as a site of attachment for important muscle groups and as a protective barrier for the visceral organs. The pen's durability and flexibility are derived from its unique composition of chitin and protein. We report the characterization of the structure, development, and composition of pens from Doryteuthis pealeii. The nanofibrils of the polysaccharide ß-chitin are arranged in an aligned configuration in only specific regions of the pen. Chitin is secreted early in development, enabling us to characterize the changes in pen morphology prior to hatching. The chitin and proteins are assembled in the shell sac surrounded by fluid that has a significantly different ionic composition from squid plasma. Two groups of proteins are associated with the pen: those on its surface and those embedded within the pen. Only 20 proteins are identified as embedded within the pen. Embedded proteins are classified into six groups, including chitin associated, protease, protease inhibitors, intracellular, extracellular matrix, and those that are unknown. The pen proteins share many conserved domains with proteins from other chitinous structures. We conclude that the pen is one of the least complex, load-bearing, chitin-rich structures currently known and is amenable to further studies to elucidate natural construction mechanisms using chitin and protein.

Electromigration of cell surface macromolecules in DC electric fields during cell polarization and galvanotaxis.

DC electric fields (EFs) can often induce cellular polarity, and direct migration of cells toward one of the electrical poles. The mechanism(s) by which cells sense weak EFs is not established. We present here a molecular flux model to describe electromigration of plasma membrane macromolecules and compare its predictions to electromigration of a lipid-anchored surface protein, tdTomato-GPI, under different experimental conditions. Gradients of tdTomato-GPI are assembled based on its electrophoretic and electro-osmotic mobilities and collapsed by its own diffusion. The flux model predicts greatest cathodal accumulation for tdTomato-GPI under slightly acidic conditions, and weak cathodal accumulation under alkaline conditions. Predictions by the flux model align closely with measurements of the electromigration of tdTomato-GPI except at pH 6, the only condition examined in which the protein exhibits a net positive surface charge. We use the model to predict the time course and relative steady state concentration difference for asymmetric accumulation of other surface macromolecules based on their physical properties. We also describe a method for identifying the physical properties of the plasma membrane proteins in zebrafish keratocytes, in order to predict likely candidates for the electric field receptor in this model migratory system that exhibits cathodal galvanotaxis, and to predict the asymmetric distribution of proteins in other cell types. We provide a physical basis for predicting the dynamics of electromigration for numerous cell surface macromolecules and provide evidence for supporting the role of electromigration in directing cell polarity, migration and growth in response to weak EFs.

Reversing the direction of galvanotaxis with controlled increases in boundary layer viscosity.

Weak external electric fields (EFs) polarize cellular structure and direct most migrating cells (galvanotaxis) toward the cathode, making it a useful tool during tissue engineering and for healing epidermal wounds. However, the biophysical mechanisms for sensing weak EFs remain elusive. We have reinvestigated the mechanism of cathode-directed water flow (electro-osmosis) in the boundary layer of cells, by reducing it with neutral, viscous polymers. We report that increasing viscosity with low molecular weight polymers decreases cathodal migration and promotes anodal migration in a concentration dependent manner. In contrast, increased viscosity with high molecular weight polymers does not affect directionality. We explain the contradictory results in terms of porosity and hydraulic permeability between the polymers rather than in terms of bulk viscosity. These results provide the first evidence for controlled reversal of galvanotaxis using viscous agents and position the field closer to identifying the putative electric field receptor, a fundamental, outside-in signaling receptor that controls cellular polarity for different cell types.