Proteomic analysis of the heat shock response in Synechocystis PCC6803 and a thermally tolerant knockout strain lacking the histidine kinase 34 gene

Proteomic analysis of the heat shock response of wild type and a mutant of the histidine kinase 34 gene (Deltahik34), which shows increased thermal tolerance, has been performed in the cyanobacterium Synechocystis sp. PCC6803. In vivo radioactive labelling demonstrates that major proteomic changes occur within 1 h of heat shock. 2-D DIGE and MS have been used to quantify changes in specific proteins following heat shock in the wild type and the mutant. Over 100 spots, corresponding to 65 different proteins alter following heat shock. Changes occur not only in the classical heat shock proteins but also in the protein biosynthetic machinery, amino acid biosynthetic enzymes, components of the light and dark acts of photosynthesis and energy metabolism. The Deltahik34 cells have elevated levels of heat shock proteins under both non-heat shock and heat shock conditions, in comparison to the wild type, consistent with Hik34, or a down stream component, being a negative regulator of heat shock-responsive genes.     

Characterization of the dimerization process of a domain-swapped dimeric variant of human pancreatic ribonuclease

It has been previously reported that the structure of a human pancreatic ribonuclease variant, namely PM8, constitutes a dimer by the exchange of an N-terminal domain, although in an aqueous solution it is found mainly as a monomer. First, we investigated the solution conditions that favour the dimerization of this variant. At 29 degrees C in a 20% (v/v) ethanol buffer, a significant fraction of the protein is found in dimeric form without the appearance of higher oligomers. This dimer was isolated by size-exclusion chromatography and the dimerization process was studied. The dissociation constant of this dimeric form is 5 mm at 29 degrees C. Analysis of the dependence of the dimerization process on the temperature shows that unlike bovine pancreatic ribonuclease, a decrease in the temperature shifts the monomer-dimer equilibrium to the latter form. We also show that a previous dissociation of the exchangeable domain from the main protein body does not take place before the dimerization process. Our results suggest a model for the dimerization of PM8 that is different to that postulated for the dimerization of the homologous bovine pancreatic ribonuclease. In this model, an open interface is formed first and then intersubunit interactions stabilize the hinge loop in a conformation that completely displaces the equilibrium between nonswapped and swapped dimers to the latter one.     

Enzymatic antioxidant response of a labrid fish (Coris julis) liver to environmental caulerpenyne

Exposure of marine animals to certain toxic compounds can enhance reactive oxygen species production with subsequent damage to macromolecules and alterations in oxidant defenses levels. Caulerpenyne is the major metabolite synthesized by Caulerpa species, used as chemical defense affecting several cellular and molecular targets. We assessed the changes produced by the presence of Caulerpa spp. in the activities of antioxidant enzymes as well as lipid peroxidation levels in liver of the teleost Coris julis. Fish were captured at two stations with Caulerpa species-Caulerpa taxifolia and Caulerpa prolifera-and at a region with the seagrass Posidonia oceanica as negative control. Caulerpenyne concentration was significantly higher in C. prolifera than in C. taxifolia (p<0.05). Glutathione S-transferase, glutathione peroxidase and glutathione reductase activities were significantly higher in both Caulerpa stations compared to the P. oceanica (p<0.05). No statistical difference (p>0.05) existed in catalase activity between groups. Glutathione reductase activity is significantly higher in C. prolifera station than in C. taxifolia (p<0.05). Despite the variations in the antioxidant enzyme activities, there was no significant difference in malondialdehyde concentration. In conclusion, the production of caulerpenyne by Caulerpa species could induce an antioxidant adaptation in the liver of C. julis in order to prevent oxidative damage.     

Recognition of Apoptotic Cells by Epithelial Cells: CONSERVED VERSUS TISSUE-SPECIFIC SIGNALING RESPONSES*

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.     

Rapid odorant release in mammalian odour binding proteins facilitates their temporal coupling to odorant signals

We have measured the effect of rat odorant-binding protein 1 on the rates of ligand uptake and liquid-to-air transfer rates with a set of defined odorous compounds. Comparison of observed rate constants (k_obs) with data simulated over a wide range of different kinetic and thermodynamic regimes shows that the data do not agree with the previously held view of a slow off-rate regime (k_off <  0.0004 s^− 1). We propose that a rapid k_off would be a necessary requirement for such a system, since slow odorant-release rates would result in significant decorrelation between the olfactory world and odour perception.     

Methyl-beta-cyclodextrin induces mitochondrial cholesterol depletion and alters the mitochondrial structure and bioenergetics

There is growing evidence of mitochondrial membrane raft-like microdomains that are involved in the apoptotic pathway. The aim of this study was to investigate the effect of methyl-beta-cyclodextrin (MβCD), being a well-known lipid microdomain disrupting agent and cholesterol chelator, on the structure and bioenergetics of rat liver mitochondria (RLM). We observed that MβCD decreases the function of RLM, induces changes in the mitochondrial configuration state and decreases the calcium chloride-induced swelling. These data suggest that disruption of mitochondrial raft-like microdomains by cholesterol efflux on one hand impairs mitochondrial bioenergetics, but on the other hand it protects the mitochondria from swelling.     

Pollen from tree and shrub taxa in the atmosphere of Caxias do Sul (Rio Grande do Sul, Brazil)

An aerobiological study has been carried out in the region of Caxias do Sul in southern Brazil. Pollen monitoring was performed from January 1, 2001 through to December 31, 2002. A total of 30,469 pollen grains were collected during this period, and 40 pollen types were identified; of these, 23,389 pollen grains, representing 29 pollen types, originated from tree and shrub taxa. The maximum pollen concentration was registered in August 2001 and October 2002. In the study area, the pollen type Mimosa scabrella (18.8%) was much more abundant than all of the pollen types from tree and shrub taxa, such as: Urticaceae (18.4%), Myrtaceae (10.2%), Cupressaceae (7.7%), Myrsine (4.8%), Sorocea (3.9%), Pinaceae (2.9%), Asteraceae (2.2%) and Ricinus (2.1%). These nine pollen types accounted for the largest pollen concentrations of all the tree and shrub taxa. The pollen types Carya, Melastomataceae, Mimosa scabrella, Myrsine and Sorocea are reported for the first time in an aerobiological study in Brazil.     

Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana

De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.     

A solar-powered transmitting video camera for monitoring cliff-nesting raptors

ABSTRACT.   We designed a system of solar-powered video cameras that transmitted images via telemetry to a monitor. This system allowed us to study the breeding behavior of the Bearded Vulture ( Gypaetus barbatus ) in the Pyrenees (northeastern Spain). From 2000 to 2004, 12 nests in seven territories were equipped with video cameras. To avoid disturbing the birds, equipment was installed 3–8 weeks before egg-laying. The acceptance rate was 75%. No decline in productivity was observed for nests monitored with video cameras compared to control nests. The cameras enabled us to document egg-laying, hatching asynchrony, the nestlings' diet, and the parents' breeding behavior from distances of 2–3 km, although technical problems temporarily interrupted the transmission of images. Video cameras can be used successfully to study the Bearded Vultures, and probably other cliff-nesting raptors, without reducing productivity.