Carbohydrate metabolism and respiration after harvest of the cultivated mushroom, Agaricus bisporus

The carbohydrate metabolism of the mushroom and the respiration of the sporophore after harvest at different stages of growth have been studied. Mannitol and trehalose were found to be the major soluble carbohydrates in the sporophore and mycelium respectively. Mannitol and trehalose levels fall during sporophore storage, and feeding experiments with¹⁴C-labelled sugars indicate that they are metabolised. The results were discussed in relation to the post-harvest development of the sporophore.     

Effect of Temperature and Salinity Stress on Growth and Lipid Composition of Shewanella gelidimarina

The maximum growth temperature, the optimal growth temperature, and the estimated normal physiological range for growth of Shewanella gelidimarina are functions of water activity (a_w), which can be manipulated by changing the concentration of sodium chloride. The growth temperatures at the boundaries of the normal physiological range for growth were characterized by increased variability in fatty acid composition. Under hyper- and hypoosmotic stress conditions at an a_w of 0.993 (1.0% [wt/vol] NaCl) and at an a_w of 0.977 (4.0% [wt/vol] NaCl) the proportion of certain fatty acids (monounsaturated and branched-chain fatty acids) was highly regulated and was inversely related to the growth rate over the entire temperature range. The physical states of lipids extracted from samples grown at stressful a_w values at the boundaries of the normal physiological range exhibited no abrupt gel-liquid phase transitions when the lipids were analyzed as liposomes. Lipid packing and adaptational fatty acid composition responses are clearly influenced by differences in the temperature-salinity regime, which are reflected in overall cell function characteristics, such as the growth rate and the normal physiological range for growth.     

An evolutionary comparison of Acinetobacter calcoaceticus trpF with trpF genes of several organisms

The deduced amino acid sequence of Acinetobacter calcoaceticus N-(5'-phosphoribosyl) anthranilate isomerase (PRAI), which is coded by trpF, was compared with TrpF of Caulobacter crescentus, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. Sixty percent of identical or similar amino acids were located in alpha/beta TIM (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. In addition, the analysis of trpF genes presented here supports a model by which fusion between separate trpC and trpF genes arose in some cases by in-frame deletions.     

Clinical and environmental isolates of Cryptococcus gattii from Australia that retain sexual fecundity

Cryptococcus gattii is a primary pathogenic yeast that causes disease in both animals and humans. It is closely related to Cryptococcus neoformans and diverged from a common ancestor approximately 40 million years ago. While C. gattii has a characterized sexual cycle dependent upon a dimorphic region of the genome known as the MAT locus, mating has rarely been observed in this species. In this study, we identify for the first time clinical (both human and veterinary) and environmental isolates from Australia that retain sexual fecundity. A collection of 120 isolates from a variety of geographic locations was analyzed for molecular type, mating type, and the ability to develop mating structures when cocultured with fertile tester strains. Nine isolates produced dikaryotic filaments with paired nuclei, fused clamp connections, and basidiospores. DNA sequence analysis of three genes (URA5, the MATalpha-specific SXI1alpha gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MATalpha strains sequenced, the SXI1alpha gene was found to exist as 11 different alleles. Phylogenetic analysis found most variation to occur in the more fertile genotypes. Although some lineages of Australian C. gattii have retained the ability to mate, the majority of isolates were sterile, suggesting that asexuality is the dominant mode of propagation in these populations.     

Effects of pipette geometry on the time course of solution change in patch clamp experiments.

The time course of change in current through KATP channels in inside-out membrane patches, after step change of permeant ion (K+) concentration, was measured. A simple model of the patch as a membrane disc at the base of a cone with the apex removed, was able to describe the time course of channel activity after step change of [K+]. By measuring pipette geometry and using jumps of [permeant ion], it was then possible to estimate the time course of concentration at the membrane for jumps of any other ion or gating ligand. A simple channel block mechanism was used to simulate experiments with concentration jumps of a blocking ligand. The rate constants for ligand-channel interaction were extracted by least-squares fitting of computed mass action responses to those observed in simulated experiments. The simulations showed that even with diffusion delays of hundreds of milliseconds (as may occur in inside-out patch experiments), ligand association and dissociation rates of up to 1,000 s-1 could be accurately extracted by this approach. The approach should be generally applicable to the analysis of ligand concentration jump experiments on any ion channel whose activity is modulated by intracellular ligand.     

DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur

Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.     

Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs

It was recently shown that the structure of the fluorophore attached to the acyl chain of phosphatidylcholine analogs determines their mechanism of transport across the plasma membrane of yeast cells (Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight into the physical properties of these fluorescent phosphatidylcholine (PC) analogs, the rate and mechanism of their intervesicular transport was determined. The rate of spontaneous exchange was measured for PC analogs containing either NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene), Bodipy 530 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene), or Bodipy 581 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene) attached to a five or six carbon acyl chain in the sn-2 position. The rate of transfer between phospholipid vesicles was measured by monitoring the increase in fluorescence as the analogs transferred from donor vesicles containing self-quenching concentrations to unlabeled acceptor vesicles. Kinetic analysis indicated that the transfer of each analog occurred by diffusion through the water phase as opposed to transfer during vesicle collisions. The vesicle-to-monomer dissociation rate constants differed by over four orders of magnitude: NBD-PC (k(dis)=0.115 s(-1); t(1/2)=6.03 s); Bodipy FL-PC (k(dis)=5.2x10(-4); t(1/2)=22.2 min); Bodipy 530-PC (k(dis)=1.52x10(-5); t(1/2)=12.6 h); and Bodipy 581-PC (k(dis)=5.9x10(-6); t(1/2)=32.6 h). The large differences in spontaneous rates of transfer through the water measured for these four fluorescent PC analogs reflect their hydrophobicity and may account for their recognition by different mechanisms of transport across the plasma membrane of yeast.