Competitive inhibition evidence for specific intermolecular interactions in hyaluronate solutions

Intermolecular associations in hyaluronate systems have been investigated by a competitive inhibition approach, monitored by low deformation oscillatory measurements of dynamic viscosity and rigidity. Solutions of sodium hyaluronate isolated by a mild procedure from rooster combs showed, under physiological conditions of pH and ionic strength, coupling behaviour typical of a transient polymer network. On addition of an equal concentration of hyaluronate segments (~60 disaccharide units), all evidence of coupling is lost and the solution rheology approximates closely to that typical of isolated chains. Such behaviour is clearly incompatible with entanglement coupling, such as occurs between synthetic polymers in solution, but parallels the behaviour of gelling polysaccharides, where co-operative interchain association is known to occur. Similar inhibition is observed in hyaluronate viscoelastic “putties” and “gels”, where further intermolecular association is promoted by suppression of interchain electrostatic repulsion and reduction of water activity. The effects are particularly pronounced for hyaluronate of lower molecular weight, where crosslinking in putties and gels may approach the minimum requirements for a cohesive network. No inhibition is observed with very short chain segments (~4 disaccharide residues) nor with longer segments (~400 disaccharides). On the basis of this evidence we suggest that hyaluronate chains interact by formation of specific co-operative junctions analogous to those characterised for plant structural polysaccharides, but having substantially shorter lifetimes. The magnitude of rheological changes on suppression of these fleeting associations by competitive inhibition suggests that they are likely to dominate the physical properties of hyaluronate in vivo.     

Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.     

First images of a glutamate receptor ion channel: oligomeric state and molecular dimensions of GluRB homomers.

We have expressed, purified, and characterized glutamate receptor ion channels (GluR) assembled as homomers of the subunit GluRB. For the first time, single-milligram quantities of biochemically homogeneous GluR have been obtained. The protein exhibits the expected pharmacological profile and a high specific activity for ligand binding. Density-gradient centrifugation reveals a uniform oligomeric assembly and a molecular mass suggesting that the channel is a tetramer. On the basis of electron microscopic images, the receptor appears to form an elongated structure that is visualized in several orientations. The molecular dimensions of the molecule are approximately 11 x 14 x 17 nm, and solvent-accessible features can be seen; these may contribute to formation of the ion-conducting pathway of the channel. The channel dimensions are consistent with an overall 2-fold symmetric assembly, suggesting that the tetrameric receptor may be a dimer of dimers.     

Pulmonary endocrine cells in plexogenic pulmonary arteriopathy

A study of the numbers of pulmonary endocrine cells per cm2 of section of lung obtained at combined heart-lung transplantation in 25 cases of plexogenic pulmonary arteriopathy demonstrated that the peptide which may become unduly prominent in pulmonary arterial disease is bombesin. The type of vascular disease in which bombesin becomes prominent is plexogenic pulmonary arteriopathy, be this primary or secondary to congenital heart disease. The increased prominence of bombesin appears to be related to the stage reached in the arteriopathy. Increased numbers of pulmonary endocrine cells are found in association with classic cellular plexiform lesions with narrow vascular channels. Their numbers are within normal limits when the plexiform lesions are mature with wide vascular channels and narrow intervening septa. The pulmonary endocrine cells are most prominent in the pre-plexiform stage when smooth muscle cells in the inner half of the media of the pulmonary artery show increased electron density, and migrate through gaps in the inner elastic lamina to reach the intima. Here they are transformed into myofibroblasts and proliferate. The migration of muscle cells may be related in some way to long-acting trophic factors released from the pulmonary endocrine cells into the surrounding tissues from which they reach the blood and hence the pulmonary arteries.