Glycosylation enhances functional stability of the chemotactic cytokine CCL2

The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins. In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro. However, CCL2L could maintain full activity upon prolonged incubation at 37 degrees C, whereas the non-glycosylated chemokine readily lost activity. It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability. The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine. Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological versus physiological conditions.     

Heavy metals in sewage sludges contribute to their adverse effects on the arbuscular mycorrhizal fungus<Emphasis Type="Italic">Glomus mosseae</Emphasis>

Applying sewage sludges to agricultural land is a widespread practice because of the sludges’ agronomic value as a source of plant nutrients and organic matter. Nevertheless, sludges often contain micropollutants that can constitute a menace for health and the environment. Arbuscular mycorrhizal fungi are sensitive to sewage sludges that have been spiked, or not, with metallic trace elements (MTE). Here we have investigated if MTE in sewage sludges could be responsible for effects on mycorrhizal development betweenGlomus mosseae andMedicago truncatula. The impact of a dehydrated or composted urban sewage sludge spiked or not with MTE, was tested on spore germination and root colonization byG. mosseae. The sewage sludges depressed both the presymbiotic andin planta stages of development of the mycorrhizal fungus. This negative effect was more related to the metallic pollutant contents of the sludges than to the presence of antagonistic microorganisms or phosphorus.     

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.     

Conflict as a determinant of rat behavior in three types of elevated plus-maze

Three groups of rats were tested in different types of elevated plus-mazes, a normal one (two closed and two open arms), a totally closed one (four closed arms) and a totally open (four open arms). Closed arms were surrounded by 40-cm high wooden walls and open arms were surrounded by 0.5-cm high transparent Plexiglas ledges. As expected, in the closed maze rats explored equally all the arms, both in terms of time and frequency of entries, as well as in exploration of the extremities. Rats in the totally open maze also presented a similar pattern of exploration, that is, no significant differences were found between the results obtained with the closed and the open mazes in terms of central and extremities exploration. It is suggested that the typical behavior of rats in the conventional elevated plus-maze is caused by the contrasting characteristics of open and closed arms rather than by the physical aversive characteristics of the open arms per se. Results also confirm a prediction made by a computer model simulating rat exploratory behavior in virtual mazes, normal, totally open and totally closed.     

DNA replication: a complex matter

In eukaryotic cells, the essential function of DNA replication is carried out by a network of enzymes and proteins, which work together to rapidly and accurately duplicate the genetic information of the cell. Many of the components of this DNA replication apparatus associate with other cellular factors as components of multiprotein complexes, which act cooperatively in networks to regulate cell cycle progression and checkpoint control, but are distinct from the pre-replication complexes that associate with the origins and regulate their firing. In this review, we summarize current knowledge about the composition and dynamics of these large multiprotein complexes in mammalian cells and their relationships to the replication factories.     

Degradation of the immunogenic peptide gp100(280-288) by the monocyte-like U937 cell line

The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.     

Structural investigation of proapoptotic peptide by CD and NMR spectroscopy

We have performed a systematic investigation of the structural features of the peptides Int (a sequence able to cross cell membranes) and Int-H1(S6A,F8A) (which shows interesting antitumoral properties). After screening in aqueous solution at different ionic strength and pH values, we analyzed the structures of the peptides in different water/trifluoroethanol mixtures by Circular Dichroism and Nuclear Magnetic Resonance techniques.     

Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP-RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 X 10(-5) s(-1), considerably slower than free RhoA. Hydrolysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP-bound G14V-RhoA/RhoGDI complex (but not the GDP form) into serum-starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These results show that GTP-triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.