Cooperative responses of multiple kinesins to variable and constant loads

Microtubule-dependent transport is most often driven by collections of kinesins and dyneins that function in either a concerted fashion or antagonistically. Several lines of evidence suggest that cargo transport may not be influenced appreciably by the combined action of multiple kinesins. Yet, as in previous optical trapping experiments, the forces imposed on cargos will vary spatially and temporally in cells depending on a number of local environmental factors, and the influence of these conditions has been largely overlooked. Here, we characterize the dynamics of structurally defined complexes containing multiple kinesins under the controlled loads of an optical force clamp. While demonstrating that there are generic kinetic barriers that restrict the ability of multiple kinesins to cooperate productively, the spatial and temporal properties of applied loads is found to play an important role in the collective dynamics of multiple motor systems. We propose this dependence has implications for intracellular transport processes, especially for bidirectional transport.     

Simple rules describe bottom-up and top-down control in food webs with alternative energy pathways

Many human influences on the world's ecosystems have their largest direct impacts at either the top or the bottom of the food web. To predict their ecosystem-wide consequences we must understand how these impacts propagate. A long-standing, but so far elusive, problem in this endeavour is how to reduce food web complexity to a mathematically tractable, but empirically relevant system. Simplification to main energy channels linking primary producers to top consumers has been recently advocated. Following this approach, we propose a general framework for the analysis of bottom-up and top-down forcing of ecosystems by reducing food webs to two energy pathways originating from a limiting resource shared by competing guilds of primary producers (e.g. edible vs. defended plants). Exploring dynamical models of such webs we find that their equilibrium responses to nutrient enrichment and top consumer harvesting are determined by only two easily measurable topological properties: the lengths of the component food chains (odd-odd, odd-even, or even-even) and presence vs. absence of a generalist top consumer reconnecting the two pathways (yielding looped vs. branched webs). Many results generalise to other looped or branched web structures and the model can be easily adapted to include a detrital pathway.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes.

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24 degrees C. Within 20 min most of the cell lines destroyed 20% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37 degrees C. Bmax and KD for the different lymphocytes was 5.3--19 . 10(11) M and 1.8--18,5 . 10(11) M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Effects of bovine pancreatic ribonuclease A, S protein, and S peptide on activation of purified rat hepatic glucocorticoid-receptor complexes

Bovine pancreatic ribonuclease (RNase) A and S protein (enzymatically inactive proteolytic fragment of RNase A which contains RNA binding site) stimulate the activation, as evidenced by increasing DNA-cellulose binding, of highly purified rat hepatic glucocorticoid-receptor complexes. These effects are dose dependent with maximal stimulation of DNA-cellulose binding being detected at approximately 500 micrograms (50 units of RNase A/mL). RNase A and S protein do not enhance DNA-cellulose binding via their ability to interact directly with DNA or to increase nonspecific binding of receptors to cellulose. Neither S peptide (enzymatically inactive proteolytic fragment which lacks RNA binding site) nor cytochrome c, a nonspecific basic DNA binding protein, mimics these effects. RNase A and S protein do not stimulate the conformational change which is associated with activation and is reflected in a shift in the elution profile of receptor complexes from DEAE-cellulose. In contrast, these two proteins interact with previously heat-activated receptor complexes to further enhance their DNA-cellulose binding capacity and thus mimic the effects of an endogenous heat-stable cytoplasmic protein(s) which also function(s) during step 2 of in vitro activation [Schmidt, T. J., Miller-Diener, A., Webb, M. L., & Litwack, G. (1985) J. Biol. Chem. 260, 16255-16262]. Preadsorption of RNase A and S protein to an RNase affinity resin containing an inhibitory RNA analogue, or trypsin digestion of the RNA binding site within S protein, eliminates the subsequent ability of these two proteins to stimulate DNA-cellulose binding of the purified receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distributions of polymorphisms among pathways of carbohydrate metabolism in the earthworm Eisenia fetida (Oligochaeta).

1. Activities and genetic banding patterns of 36 isozymes in carbohydrate metabolism were detected by spectrophotometry and starch-gel electrophoresis, respectively, in the earthworm Eisenia fetida. 2. Polymorphisms were not distributed randomly among metabolic pathways, activity levels, or gene copy numbers. 3. In glycolysis and the Krebs cycle, 16% of the loci were polymorphic and polymorphisms occurred only when multiple copies of the loci existed. 4. In other pathways, 45% of the loci were polymorphic and the distribution of polymorphisms was independent of gene copy number. 5. Polymorphisms may affect metabolic phenotypes and natural selection may have led to conserved biochemical activity in glycolysis and the Krebs cycle.     

Effects of dilution and temperature of analysis on blood serum values in rainbow trout, Salmo gairdneri

Albumin, cholesterol, glucose, inorganic phosphorus, and total protein analyses were not affected by temperatures between 20 and 38°C nor by dilution with either 0.65% or 0.85% NaCl. Additionally, blood urea nitrogen, calcium and creatinine were unaffected by dilution. Alkaline phosphotase, cholinesterase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, α-hydroxybutyrate dehydrogenase, lactic dehydrogenase, and phosphohexose isomerase were all affected by temperature. Results of dilution tests were generally inconclusive, while results of enzymatic reactions indicate that reaction temperatures must be closely controlled to produce comparable results.     

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.