Opioidergic effects of nonopioid analgesics on the central nervous system

1. The analgesic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is partly due to the fact that they act upon the periaqueductal gray matter (PAG) and the rostral ventromedial medulla of the brain stem and thus activate the descending pain-control system, which inhibits nociceptive transmission at the spinal dorsal horn.2. The analgesic action of dipyrone (metamizol) and of lysine-acetylsalicylate (LASA), two well-known NSAIDs, whether microinjected into the PAG or given systemically, can be reverted by naloxone. Repeated administration of dipyrone or LASA induces tolerance to their antinociceptive effect, with cross-tolerance to morphine, and a withdrawal syndrome upon naloxone administration. Dipyrone tolerance can be reverted by proglumide, a cholecystokinin antagonist.3. These findings reveal a close association between the central action of NSAIDs and endogenous opioids.     

Novel cytotoxic chelators that bind iron(II) selectively over zinc(II) under aqueous aerobic conditions

To achieve cellular iron deprivation by chelation, it is important to develop chelators with selective metal-binding properties. Selectivity for iron has long been the province of certain oxygen-donor chelators such as desferrioxamine, which target Fe(III) and exploit the strength of a relatively ionic Fe(III)-O interaction. We have been studying novel chelators that possess mechanisms to selectively chelate +2 biometals, particularly tachpyr [N,N',N"-tris(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane] and derivatives from N,N',N"-trialkylation and pyridine ring alkylation. Metal-exchange and metal-binding competition reactions have been conducted at pH 7.4, 37 degrees C and time periods until no further change was observed (generally 24-48 h). Under anaerobic conditions, tachpyr is strongly selective for iron, binding 95+/-5% Fe(II) versus 5+/-5% Zn(II) in the forms [Fe(tachpyr)](2+) and [Zn(tachpyr)](2+) respectively. Under aerobic conditions, tachpyr complexes Fe(II) more effectively than Fe(III), forming iminopyridyl complexes [Fe(tachpyr-ox-n)](2+) (n=2, 4) by O(2)-induced and iron-mediated oxidative dehydrogenation. Complexes [Fe(tachpyr-ox-n)](2+) are also strongly bound forms of iron that are unaffected by an excess of Zn(II) (75 mol zinc:1 mol iron complex). The preference of tachpyr for iron over zinc under aerobic conditions appears to be hindered by oxidation of Fe(II) to Fe(III), such that the proportions bound are 44+/-10% Fe(II) versus 56+/-10% Zn(II), in the respective forms [Fe(tachpyr-ox-n)](2+) and [Zn(tachpyr)](2+). However, upon addition of the reducing agent Na(2)S(2)O(4) that converts Fe(III) to Fe(II), the binding proportions shift to 76+/-10% Fe(II) versus 24+/-10% Zn(II), demonstrating a clear preference of tachpyr for Fe(II) over Zn(II). Iron(II) is in the low-spin state in [Fe(tachpyr)](2+) and [Fe(tachpyr-ox-n)](2+) (n=2, 4), which is a likely cause of the observed selectivity. N-methylation of tachpyr [giving (N-methyl)(3)tachpyr] results in the loss of selectivity for Fe(II), which is attributed to the steric effect of the methyl groups and a resulting high-spin state of Fe(II) in [Fe(N-methyl)(3)tachpyr)](2+). The relationship of chelator selectivity to cytotoxicity in the tach family will be discussed.     

An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan

The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.     

Peroxynitrite flux-mediated LDL oxidation is inhibited by manganese porphyrins in the presence of uric acid

We have studied the role of three Mn(III)porphyrins differing in charge, alkyl substituent length and reactivity, on LDL exposed to low fluxes of peroxynitrite (PN) in the presence of uric acid. Mn(III)porphyrins (5 microM, MnTE-2-PyP(5+), MnTnOct-2-PyP(5+), and MnTCPP(3-)) plus uric acid (300 microM) inhibited cholesteryl ester hydroperoxide formation, changes in REM as well as spared alpha- and gamma-tocopherol. MnTnOct-2-PyP(5+), the more lipophilic compound, was the most effective in protecting LDL lipids, while MnTCPP(3-) exerted the lesser protection. Mn(III)porphyrins react fast with PN ( approximately 10(5)-10(7) M(-1) s(-1)) to yield a O=Mn(IV) complex. The stoichiometry of uric acid consumption was approximately 1.7 moles per mol of PN, in agreement with reactions with both the O=Mn(IV) complex and nitrogen dioxide. A shift from an anti- to a pro-oxidant action of the Mn(III)porphyrin was observed after uric acid was significantly consumed, supporting competition reactions between LDL targets and uric acid for the O=Mn(IV) complex. Overall, the data is consistent with the catalytic reduction of PN in a cycle that involves a one electron oxidation of Mn(III) to Mn(IV) by PN followed by the reduction back to Mn(III) by uric acid. These antioxidant effects should predominate under in vivo conditions having plasma uric acid concentration range between 150 and 500 microM.     

Characterization and Evolution of mariner Elements from Closely Related Species of Fruit Flies (Diptera: Tephritidae)

Mariner elements were amplified using the polymerase chain reaction from two species of tephritid flies, Ceratitis rosa and Trirhithrum coffeae. The sequences were ∼1.3 kb in length. None of these elements appeared to be functional, as in every case the open reading frame (ORF) was disrupted by the presence of frameshifts or stop codons. These elements, Crmar1 and Tcmar1, are very similar to the Ccmar1 element previously amplified from the closely related tephritid species C. capitata and are members of the mellifera subfamily of mariner elements. The phylogeny and pattern of divergence of these elements were examined in relation to the phylogeny of the host species. It is highly probable that the elements were present in the ancestral lineage prior to the divergence of the three species. The copy numbers of the elements within each species are very different, ranging from about 10 in T. coffeae to 5,000 in C. rosa. The possible mechanisms which determine the copy number of an element in the host genome are discussed. Received: 25 April 1997 / Accepted: 31 July 1997     

Phylogenetic analyses of the genera Pipra, Lepidothrix and Dixiphia (Pipridae, Passeriformes) using partial cytochrome b and 16S mtDNA genes

The piprids (manakins) are small-bodied Neotropical birds characterized by sexual dimorphism of the plumage and elaborate courtship rituals. The phylogenetic relationships of some of piprid genera, in particular Pipra , are not well defined. Recently, Pipra was divided into three distinct groups, and the recognition of two new genera — Lepidothrix and Dixiphia — was suggested, based on the characteristic of the syringial morphology. In the present study, we analyse the phylogenetic relationships among these genera on the basis of data from the mitochondrial genes rRNA 16S and cytochrome b . Bayesian Inference (BI), Maximum Parsimony (MP) and Maximum Likelihood (ML) methods of analyses produced phylogenetic trees with very similar topologies with regard to the groupings formed by the species representing the three genera. These results indicate that the genera Pipra, Lepidothrix and Dixiphia do not constitute a single monophyletic clade, and support the current hypothesis of polyphyletism for the former representatives of the genus Pipra . However, the topologies presented do not coincide exactly with the phylogeny suggested by the morphological data with regard to the relationship between these three groups and the remaining piprid genera analysed here, emphasizing the need for further studies of this group of birds. The agreement between the molecular data presented here and the biogeographical information available for some of the species analysed appears to confirm the potential of using segments of the mitochondrial genome for the study of phylogeographical patterns in this group of organisms.     

The importance of forest patch networks for the conservation of the Thorn-tailed Rayaditos in central Chile

Conservation of forest birds in fragmented landscapes requires not only determining the critical patch characteristics influencing local population persistence but also identifying patch networks providing connectivity and suitable habitat conditions necessary to ensure regional persistence. In this study, we assessed the importance of patch attributes, patch connectivity, and network components (i.e., groups of interconnected patches) in explaining the occupancy pattern of the Thorn-tailed Rayadito (Aphrastura spinicauda), a forest bird species of central Chile. Using a daily movement threshold distance, we identified a total of 16 network components of sclerophyllous forest within the study area. Among those components, patch area and vegetation structure-composition were important predictors of patch occupancy. However, the inclusion of patch connectivity and component size (i.e., the area of a network component) into the models greatly increases the models’ accuracy and parsimony. Using the best-fitted model, a total of 33 patches were predicted to be occupied by rayaditos within the study area, but such occupied patches were distributed in only six network components. These results suggest that persistence of rayaditos in central Chile requires the maintenance of large single patches and patch networks providing habitat and connectivity.