Methylguanidine reduces the development of non septic shock induced by zymosan in mice

In the present study we evaluate the effect of methylguanidine (MG), a product of protein catabolism, in a model of acute inflammation (zymosan induced inflammation) in mice where oxyradical and nitric oxide (NO) play a crucial role. Our data show that MG, given intraperitoneally at the dose of 30 mg/Kg, inhibits the inflammatory response reducing significantly (P < 0.05) peritoneal exudates formation, mononuclear cell infiltration and histological injury in mice. Furthermore, our data suggests that there is a significant (P < 0.05) reduction in kidney, liver and pancreas injury as demonstrated by the reduction in amylase, lipase, creatinine, AST, ALT, bilirubine and alkaline phosfatase levels. MG is also able to reduce the appearance of nitrotyrosine and of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) synthase (PARS) immunoreactivity in the inflamed intestinal and lung tissues. The histological examination revealed a significant reduction in zymosan-induced intestinal and lung damage in MG-treated mice. Taken together, the present results demonstrate that MG exerts potent anti-inflammatory effects on zymosan-induced shock.     

Multiple and distinct effects of mutations of Tyr122, Glu123, Arg324, and Arg334 involved in interactions between the top part of second and fourth transmembrane helices in sarcoplasmic reticulum Ca2+-ATPase: changes in cytoplasmic domain organization during isometric transition of phosphoenzyme intermediate and subsequent Ca2+ release

We explored, by mutational substitutions and kinetic analysis, possible roles of the four residues involved in the hydrogen-bonding or ionic interactions found in the Ca2+-bound structure of sarcoplasmic reticulum Ca2+-ATPase, Tyr(122)-Arg(324), and Glu(123)-Arg(334) at the top part of second transmembrane helix (M2) connected to the A domain and fourth transmembrane helix (M4) in the P domain. The observed substitution effects indicated that Glu(123), Arg(334), and Tyr(122) contributed to the rapid transition between the Ca2+-unbound and bound states of the unphosphorylated enzyme. Results further showed the more profound inhibitory effects of the substitutions in the M4/P domain (Arg(324) and Arg(334)) upon the isomeric transition of phosphorylated intermediate (EP) (loss of ADP sensitivity) and those in M2/A domain (Tyr(122) and Glu(123)) upon the subsequent processing and hydrolysis of EP. The observed distinct effects suggest that the interactions seen in the Ca2+-bound structure are not functionally important but indicate that Arg(334) with its positive charge and Tyr(122) with its aromatic ring are critically important for the above distinct steps. On the basis of the available structural information, the results strongly suggest that Arg(334) moves downward and forms new interactions with M2 (likely Asn(111)); it thus contributes to the inclination of the M4/P domain toward the M2/A domain, which is crucial for the appropriate gathering between the P domain and the largely rotated A domain to cause the loss of ADP sensitivity. On the other hand, Tyr(122) most likely functions in the subsequent Ca2+-releasing step to produce hydrophobic interactions at the A-P domain interface formed upon their gathering and thus to produce the Ca2+-released form of EP. During the Ca2+-transport cycle, the four residues seem to change interaction partners and thus contribute to the coordinated movements of the cytoplasmic and transmembrane domains.     

Responses induced by high concentration of cadmium in Phragmites australis roots

Cadmium is an important environmental pollutant with high toxicity to plants. We report the effects of high-dose Cd (100 µ M for 21 days) on the root apparatus of Phragmites australis plants, which are characterized by elevated water detoxification capacity and widely used in phytoremediation programmes. The examination of root sections by light and electron microscopy failed to reveal any significant cadmium-induced structural or ultra-structural modifications. However, histochemical localization of Cd disclosed accumulation of the metal in the parenchyma cells below the exodermis. Phytochelatins (PC) are thiol-rich peptides whose synthesis is induced by a range of metals. Our results indicate that total PC production increases after exposure to Cd, which suggests a pivotal role for phytochelatins in the sequestration of metal. Cd treatment also induced lignin deposition and marked stimulation of root antioxidant systems, suggesting that, because of its ability to adopt different strategies against the harmful effects of cadmium, Phragmites australis is a plant with high detoxification potential.     

Abscisic acid signaling through cyclic ADP-ribose in hydroid regeneration

Cyclic ADP-ribose (cADPR) is an intracellular calcium (Ca(2+)(i)) mobilizer involved in fundamental cell functions from protists to higher plants and mammals. Biochemical similarities between the drought-signaling cascade in plants and the temperature-sensing pathway in marine sponges suggest an ancient evolutionary origin of a signaling cascade involving the phytohormone abscisic acid (ABA), cADPR, and Ca(2+)(i). In Eudendrium racemosum (Hydrozoa, Cnidaria), exogenously added ABA stimulated ADP-ribosyl cyclase activity via a protein kinase A (PKA)-mediated phosphorylation and increased regeneration in the dark to levels observed under light conditions. Light stimulated endogenous ABA synthesis, which was conversely inhibited by the inhibitor of plant ABA synthesis Fluridone. The signal cascade of light-induced regeneration uncovered in E. racemosum: light --> increasing ABA --> PKA --> cyclase activation --> increasing [cADPR](i) --> increasing [Ca(2+)](i) --> regeneration is the first report of a complete signaling pathway in Eumetazoa involving a phytohormone.     

Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase

The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the beta-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal alpha-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal betaalphabeta flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.     

Otodectes cynotis (Acari: Psoroptidae): examination of survival off-the-host under natural and laboratory conditions

The biological and environmental factors affecting survival off-the-host of Otodectes cynotis (Acari: Psoroptidae) ear mites were investigated under natural and laboratory conditions. From November 2000 to November 2002 mites were collected monthly from cats and divided into four groups according to sex and stage. In laboratory conditions, the mites were placed in an incubator with a steady 95% relative humidity (r.h.), a 10 degrees C. All the plates were examined by stereomicroscopy every 24 h until all the mites had died. The data were analysed statistically by multiple linear regression and survival analysis. At 10 degrees C, the maximum survival time of mites was between 15 and 17 days, while at 34 degrees C, it was between 5 and 6 days. The maximum survival time of adult females was significantly longer than that of other stages. No differences were observed in maximum survival times of mites that had been offered food and those that had not, or in the time (in days) to reach 50% mortality (LT50). When exposed to environmental conditions, the maximum survival time (12 days) was observed at temperatures ranging from 12.3 to 14.2 degrees C and r.h.s between 57.6 and 82.9%. Multiple regression analysis showed that temperature alone influenced the maximum survival time and LT50 of mites, and that the rate of survival declined linearly with increasing mean temperature. This basic understanding of off-host survival suggests that, places which have been inhabited by infected animals may need to be disinfected or remain vacated for at least 12 days before occupancy by clean cats or dogs.