Regulation of bidirectional melanosome transport by organelle bound MAP kinase

Regulation of intracellular transport plays a role in a number of processes, including mitosis, determination of cell polarity, and neuronal growth. In Xenopus melanophores, transport of melanosomes toward the cell center is triggered by melatonin, whereas their dispersion throughout the cytoplasm is triggered by melanocyte-stimulating hormone (MSH), with both of these processes mediated by cAMP-dependent protein kinase A (PKA) activity [1, 2]. Recently, the ERK (extracellular signal-regulated kinase) pathway has been implicated in regulating organelle transport and signaling downstream of melatonin receptor [3, 4]. Here, we directly demonstrate that melanosome transport is regulated by ERK signaling. Inhibition of ERK signaling by the MEK (MAPK/ERK kinase) inhibitor U0126 blocks bidirectional melanosome transport along microtubules, and stimulation of ERK by constitutively active MEK1/2 stimulates transport. These effects are specific because perturbation of ERK signaling has no effect on the movement of lysosomes, organelles related to melanosomes [5]. Biochemical analysis demonstrates that MEK and ERK are present on melanosomes and transiently activated by melatonin. Furthermore, this activation correlates with an increase in melanosome transport. Finally, direct inhibition of PKA transiently activates ERK, demonstrating that ERK acts downstream of PKA. We propose that signaling of organelle bound ERK is a key pathway that regulates bidirectional, microtubule-based melanosome transport.     

Humoral immune response against soluble and fractionate antigens in experimental sporotrichosis

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii, which is widely distributed in nature, and presents a saprophytic mycelial form on plant debris and soil. The immunological mechanisms involved in the prevention and control of sporotrichosis are not yet fully understood. In this study, mice were studied after infection with Sporothrix schenckii. In the first week after infection, fungal loading increased and thence decreased drastically 14 days after infection. Analysis by immunoblotting showed that the sera of all mice tested had antibodies reacting only with a 70 kDa antigen, with predominance of IgG1 and IgG3. Taken together, our results show that antigens from S. schenckii induced a specific humoral response in infected mice.     

EPR studies of chlorocatechol 1,2-dioxygenase: evidences of iron reduction during catalysis and of the binding of amphipatic molecules

Chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is a dioxygenase responsible for ring cleavage during the degradation of recalcitrant aromatic compounds. We determined the zero-field splitting of the Fe(III) cofactor (|D| = 1.3 +/- 0.2 cm(-1)) by electron paramagnetic resonance (EPR) experiments that along with other structural data allowed us to infer the Fe(III) coordination environment. The EPR spectrum of the ion shows a significantly decrease of the g = 4.3 resonance upon substrate binding. This result is rationalized in terms of a mechanism previously proposed, where catechol substrate is activated by Fe(III), yielding an exchange-coupled Fe(II)-semiquinone (pair). The Pp 1,2-CCD capacity of binding amphipatic molecules and the effects of such binding on protein activity are also investigated. EPR spectra of spin labels show a protein-bound component, which was characterized by means of spectral simulations. Our results indicate that Pp 1,2-CCD is able to bind amphipatic molecules in a channel with the headgroup pointing outwards into the solvent, whereas the carbon chain is held inside the tunnel. Protein assays show that the enzyme activity is significantly lowered in the presence of stearic-acid molecules. The role of the binding of those molecules as an enzyme activity modulator is discussed.     

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.     

Synergic interaction between pomegranate extract and antibiotics against Staphylococcus aureus

We evaluated the interaction between Punica granatum (pomegranate) methanolic extract (PGME) and antibiotics against 30 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). Susceptibility testing of the isolates to PGME and antibiotics was performed by the broth dilution method. Synergic activity was detected between PGME and the 5 antibiotics tested, chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin, ranging from 38% to 73%. For some isolates, PGME did not interfere with the action of any of the antibiotics tested. The bactericidal activity of PGME (0.1 x MIC) in combination with ampicillin (0.5 x MIC) was assessed using chosen isolates by time-kill assays, and they confirmed the synergic activity. Using this combination, cell viability was reduced by 99.9% and 72.5% in MSSA and MRSA populations, respectively. PGME increased the post-antibiotic effect (PAE) of ampicillin from 3 to 7 h. In addition, PGME demonstrated the potential to either inhibit the efflux pump NorA or to enhance the influx of the drug. The detection of in vitro variant colonies of S. aureus resistant to PGME was low and they did not survive. In conclusion, PGME dramatically enhanced the activity of all antibiotics tested, and thus, offers an alternative for the extension of the useful lifetime of these antibiotics.     

Behavioral mediators of cyclical oligogyny in the Amazonian swarm-founding wasp Asteloeca ujhelyii (Vespidae, Polistinae, Epiponini)

Summary In the Epiponini, queen number may vary from many to few during the colonial cycle. This cyclical oligogyny may be important for the maintenance of high genetic relatedness found in several taxa of epiponines. Even though queen reduction in accord with the colony cycle has either been demonstrated or inferred for many taxa, the factors responsible for queen number reduction remain largely unknown. In this study, behavioral interactions (queen-queen and queen-worker) were analyzed in Asteloeca ujhelyii. Conflicts among queens leading to queen elimination were more intense during colony establishment, and queens behavioral performances were significantly correlated with their potential reproductive capacity. Frequency of workers testing behaviors toward queens did not relate as a function of the colony cycle. Thus, it seems that in Asteloeca ujhelyii cyclical oligogyny is indeed behaviorally mediated.Received 16 January 2003; revised 1 June 2003; accepted 3 July 2003.     

Genotoxicity of diphenyl diselenide in bacteria and yeast

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.