An Insertion Peptide in Yeast Glycyl-tRNA Synthetase Facilitates both Productive Docking and Catalysis of Cognate tRNAs

The yeast Saccharomyces cerevisiae possesses two distinct glycyl-tRNA synthetase (GlyRS) genes: GRS1 and GRS2. GRS1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while GRS2 is dysfunctional and not required for growth. The protein products of these two genes, GlyRS1 and GlyRS2, are much alike but are distinguished by an insertion peptide of GlyRS1, which is absent from GlyRS2 and other eukaryotic homologues. We show that deletion or mutation of the insertion peptide modestly impaired the enzyme''s catalytic efficiency in vitro (with a 2- to 3-fold increase in K_m and a 5- to 8-fold decrease in k_cat). Consistently, GRS2 can be conveniently converted to a functional gene via codon optimization, and the insertion peptide is dispensable for protein stability and the rescue activity of GRS1 at 30°C in vivo. A phylogenetic analysis further showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently, with GRS1 being the predecessor. These results indicate that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. Our study suggests that the insertion peptide represents a novel auxiliary domain, which facilitates both productive docking and catalysis of cognate tRNAs.     

Theory of filtration of mixed blood suspensions

A theory is developed for the flow of suspensions of blood cells through filters in which the properties of the cells are defined by statistical distributions. It is shown that conditions are generally transient, and computational procedures are developed to compute the pressure drop and the fraction of the pores of the filter containing cells of various types as a function of time. The computations show a large influence of very small concentrations of stiff cells which gradually collect in the filter and effectively plug the filter during the time of a typical test. It is also shown that the mean value of the resistance offered by a cell population with a limited distribution of resistances is more important than dispersion of resistances about the mean in determining the observable pressure curve. Experimental data are presented demonstrating that the drug pentoxifylline reduces the stiffness of leukocytes.     

Velocity distribution on the membrane of a tank-treading red blood cell

The kinematics of an area-conserving tank-treading disk-shaped red blood cell membrane is studied using the stream function method suggested by Secomb and Skalak (Q. Jl Mech. appl. Math. 35, Pt 2, 233–247, 1982). Two simple area-conserving velocity fields are superimposed to satisfy the continuity condition at the curved edges of the disk. A differential equation for the trajectory of any material point of the membrane is derived. The requirement of synchrony of the cycle for all membrane points leads to an integral equation which determines a magnitude function. An approximate solution is made possible by assuming small trajectory deflections.     

Membrane protein phosphorylation during stomatocyte-echinocyte transformation of human erythrocytes

The normal, discoid shape of red blood cells represents an equilibrium between two opposing factors, i.e., stomatocytic and echinocytic transformations. Most stomatocytic agents were found to be inhibitors of calmodulin, a regulator of the phosphorylation of membrane proteins. We determined whether red cell shape transformations could be caused by changes in phosphorylation of membrane proteins, specifically the cAMP-dependent phosphorylation of ankyrin and band 4.1. Red blood cells were incubated with 32P and 100 microM chlorpromazine (stomatocytic transformation) or 30 mM sodium salicylate (echinocytic transformation) for various time intervals. Ghost membrane proteins were examined by polyacrylamide gel electrophoresis and autoradiography. Spectrin (beta-chain), ankyrin, band 3, band 4.1 and 4.9 were phosphorylated. No change was found in the degree and pattern of phosphorylation after stomatocytic transformation. Salicylate caused a reversible inhibition of transmembranous phosphate transport in both directions. The results indicate that the stomatocytic transformation induced by chlorpromazine and the echinocytic transformation induced by salicylate do not involve a change in phosphorylation, but that the echinocytic transformation induced by salicylate is associated with an inhibition of transmembranous transport of phosphate. Studies with salicylate suggest that the phosphorylation sites of band 3 are found mainly on the endofacial side of the membrane.     

Differential interleukin 1 elaboration by unfractionated and density fractionated human alveolar macrophages and blood monocytes: relationship to cell maturity

The elaboration of interleukin 1 (IL 1) by mononuclear phagocytes is important in the regulation of human inflammatory and fibrotic reactions. Mononuclear phagocytes are morphologically and functionally heterogeneous cells. To further understand the processes controlling inflammation and fibrosis, in particular that in the human lung, we studied the elaboration of IL 1 by unfractionated and density-fractionated human alveolar macrophages and blood monocytes. Stimulated blood monocytes elaborated more IL 1 than stimulated alveolar macrophages. In addition, denser alveolar macrophages and blood monocytes elaborated more IL 1 than less dense alveolar macrophages and monocytes. Lastly, as monocytes matured in vitro, they lost their ability to elaborate IL 1 and became less dense. Thus, there is variability between and within mononuclear phagocyte cell populations in their ability to elaborate IL 1. These differences may result in part from differences in cell maturation.     

The bulk rheology of close-packed red blood cells in shear flow

A theoretical analysis is made of the dynamical behavior and bulk rheology of close-packed red blood cell suspensions subjected to simple shear flow. The model for the polyhedral cell shapes and tank-treading membrane motion developed in the companion paper (1) is used. The flow in the thin lubricating plasma layers between cells is analyzed taking into account the mechanical properties of the membrane at the corner regions of sharp membrane curvature. This leads to predictions for the apparent viscosity as a function of hematocrit and shear rate. Good agreement with experimental results is obtained at moderate and high shear rates (above 20 s-1). At lower shear rates, a rapid rise in apparent viscosity has been found experimentally, and the mechanisms leading to this behavior are examined.