Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway.

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.     

Lysosomal accumulation and recycling of lipopolysaccharide to the cell surface of murine macrophages, an in vitro and in vivo study.

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway of B. abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages with B. abortus strain 19. After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection. Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward, LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.     

Temperature dependence of temporal resolution in an insect nervous system

The vast majority of animals are poikilotherms, and thus face the problem that the temperature of their nervous systems rather smoothly follows the temperature changes imposed by their environment. Since basic properties of nerve cells, e.g., the time constants of ion channels, strongly depend on temperature, a temperature shift likely affects the processing of the temporal structure of sensory stimuli. This can be critical in acoustic communication systems in which time patterns of signals are decisive for recognition by the receiver. We investigated the temperature dependence of the responses of locust auditory receptors and interneurons by varying the temperature of the experimental animals during intracellular recordings. The resolution of fast amplitude modulations of acoustic signals was determined in a gap detection paradigm. In auditory receptors and local (second order) interneurons, temporal resolution was improved at higher temperatures. This gain could be attributed to a higher precision of spike timing. In a third-order neuron, a rise in temperature affected the interactions of inhibition and excitation in a complex manner, also resulting in a better resolution of gaps in the millisecond range.     

Isolation and characterisation of an aryl-β-D-glucoside uptake and utilisation system ( abg ) from the gram-positive ruminal Clostridium species C. longisporum

A phosphotransferase-dependent aryl-β-glucoside uptake and utilisation system (abg) was isolated from the ruminal Clostridium (“C. longisporum”). The system is composed of three genes, abgG, abgF and abgA, and a number of regulatory regions, including terminator/antiterminator type stem-loop structures preceding the abgG and abgF genes. Similarity analysis of the proteins encoded by these genes indicated that they were responsible for the regulation of the abg system through antitermination (AbgG), the uptake and phosphorylation of aryl-β-glucosides (AbgF) and the hydrolysis of the intracellular phosphorylated glycosides (AbgA). Experimental evidence for the functions of AbgF and AbgA was obtained. Although it was not possible to demonstrate any function for AbgG, a promoter 5′ to the abgG gene was identified which was responsible for expression of the downstream genes. The abg system is remarkably similar to operons from the gram negative Enterobacteriaceae, both in the coding and non-coding regulatory regions. Received: 3 April 1997 / Accepted: 8 September 1997     

Linoleic acid hydroperoxide favours hypochlorite- and myeloperoxidase-induced lipid peroxidation

Liposomes composed of soybean phosphatidylcholine were peroxidized using the reagent sodium hypochlorite or the myeloperoxidase-hydrogen peroxide-Cl^- system. Linoleic acid hydroperoxide previously prepared from linoleic acid by means of lipoxidase was incorporated into liposomes. The yield of thiobarbituric acid reactive substances (TBARS) continuously increased with higher amounts of hydroperoxide groups after the initiation of lipid peroxidation by hypochlorous acid producing systems. The accumulation of TBARS was inhibited by scavengers of free radicals such as butylated hydroxytoluene and by the scavengers of hypochlorous acid, taurine and methionine. Lipid peroxidation was also prevented by sodium azide or chloride free medium in the myeloperoxidase-hydrogen peroxide-Cl^- system. Here we show for the first time that the reaction of hypochlorous acid with a biologically relevant hydroperoxide yields free radicals able to cause further oxidation of lipid molecules.     

Subtle re-location of dendritic spine branches containing membrane with fast spiking mechanisms can alter synaptic efficacy

The potential physiological impact of morphological changes in the active dendritic spines, which are believed to be associated with altered synaptic efficacy, was investigated in a computer simulation study using the NEURON package [1]. A compartmental model of a simplified neuron was built, which included 30 complex spines (neck, head, and active zone) and accommodating AMPA-type synaptic inputs with alpha-function conductances. Hodgkin-Huxley type excitable membranes were inserted into the spine heads. It was shown that arranging spines in dense clusters, as opposed to a uniformly random spine distribution, has a negligible effect on the synaptic signal transfer (other model conditions, including synaptic input and spine density, remained unchanged). However, if a proportion (e.g., 3–20%) of the spines partly fuse with their neighbors forming branched spines, this could increase dramatically the cell response to the unchanged synaptic input. Results of this pilot study provide the basis for a more detailed investigation of the relationship between the spine arrangement and synaptic function, considering dual-component synaptic currents and mechanisms controlling ion fluxes in the dendritic compartments.     

Steady-state kinetics of F1-ATPase. Mechanism of anion activation

The kinetic behaviour of the ATPase activity of beef heart F1 depends largely on the exposure of the enzyme to some anionic ligands such as sulphate and/or EDTA. F1 prepared in the presence of such anions exhibited a triphasic kinetic pattern whereas F1 from which those anions were removed by dialysis exhibited only two Km values for ATP. Conversely to what has been previously reported, bicarbonate did not linearize F1-ATPase kinetics. Moreover, anion activation cannot be simply explained by promotion of ADP release but mainly by an increase in affinity of the third catalytic site for ATP.     

Two-dimensional crystallization of bovine rhodopsin

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.