The effect of superoxide dismutase alleles on aging inDrosophila

The effects of superoxide dismutase on aging were tested using two differt experimental approaches. In the first, replicated populations with postponed aging were compared with their controls for frequencies of electrophoretic alleles at the SOD locus. Populations with postponed aging had consistently greater frequencies of the allele coding for more active SOD protein. This allele was not part of a segregating inversion polymorphism. The second experimental approach was the extraction ofSOD alleles from different natural populations followed by the construction of differentSOD genotypes on hybrid genetic backgrounds. This procedure did not uncover any statistical effect ofSOD genotype on hybrid genetic backgrounds. This effects on longevity and fecundity due to the family from which a particularSOD genotype was derived. To detect the effects ofSOD genotypes on longevity with high probability would require a ten-fold increase in the number of families used.     

Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon

Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.     

Phylogenetic reconstruction of the Drosophila obscura group, on the basis of mitochondrial DNA

We have constructed restriction-site maps of the mtDNAs in 13 species and one subspecies of the Drosophila obscura group. The traditional division of this group into two subgroups (affinis and obscura) does not correspond to the phylogeny of the group, which shows two well- defined clusters (the Nearctic affinis and pseudoobscura subgroups) plus a very heterogeneous set of anciently diverged species (the Palearctic obscura subgroup). The mtDNA of Drosophila exhibits a tendency to evolve toward high A+T values. This leads to a "saturation" effect that (1) begets an apparent decrease in the rate of evolution as the time since the divergence of taxa increases and (2) reduces the value that mtDNA restriction analysis has for the phylogenetic reconstruction of Drosophila species that are not closely related.      

K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant.

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3',5'-monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K(+)-channel blockers 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3,4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.     

Functional reconstitution of a chloride channel protein from bovine trachea.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.     

Engineering d‐limonene synthase down‐regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus‐growing areas worldwide. The pathogen may infect the fruit for 20–24 weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d ‐Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d ‐limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d ‐limonene content, an over‐accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up‐regulation of different pathogenesis‐related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.     

Comorbidities are associated with different features of severe asthma

Background

According to ATS/ERS document on severe asthma (SA), the management of these patients requires the identification and proper treatment of comorbidities, which can influence the control of asthma.

Methods

The aim of this study was to assess the independent effect of different comorbidities on clinical, functional and biologic features of SA. Seventy-two patients with SA according to GINA guidelines were examined. We collected demographic data, smoking habit, asthma history, and assessment of comorbidities. Pulmonary function, inflammatory biomarkers, upper airway disease evaluation, asthma control and quality of life were carefully assessed.

Results

The mean age of patients was 59.1 years (65.3% female, 5.6% current smokers). Comorbidities with higher prevalence were: chronic rhinosinusitis with or without nasal polyps (CRSwNP or CRSsNP), obesity and gastro-esophageal reflux (GERD), with some overlapping among them. In an univariate analysis comparing patients with single comorbidities with the other ones, asthmatics with CRSwNP had lower lung function and higher sputum eosinophilia; obese asthmatics had worse asthma control and quality of life, and tended to have lower sputum eosinophils; asthmatics with GERD showed worse quality of life. In multivariate analysis, obesity was the only independent factor associated with poor asthma control (OR 4.9), while CRSwNP was the only independent factor associated with airway eosinophilia (OR 16.2). Lower lung function was associated with the male gender and longer duration of asthma (OR 3.9 and 5.1, respectively) and showed a trend for the association with nasal polyps (OR 2.9, p?=?0.06).

Conclusion

Our study suggests that coexisting comorbidities are associated with different features of SA.

     

Alkali metal ion selectivity of the hemocyanin channel

Summary The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K⁺ measured from biionic potentials were: NH ₄ ⁺ (1.52)>Rb⁺ (1.05)>K⁺ (1.0)>Cs⁺ (0.89)>Na⁺ (0.81)>Li⁺ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K⁺, Rb⁺, NH ₄ ⁺ , Cs⁺, Na⁺ and Li⁺, respectively. Current-voltage curves for the different monovalent cations were measured and described using a threebarrier model previously used to explain the voltage dependence of the instantaneous channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH ₄ ⁺ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.     

Patterns of puffing activity and chromosomal polymorphism in Drosophila subobscura

Summary The effect of inbreeding on the puffing patterns of polytene chromosomes of Drosophila subobscura was analysed. Puffing activity was studied in two strains of D. subobscura: one which had been subject to inbreeding for 288 generations, and in the hybrids from a cross between them. A strong overall decrease in puffing activity was found in the inbred line. In general, hybrids behaved in a similar way to the inbred line or showed activity intermediate between the two lines. The fertility and viability of the two homozygous lines and of the hybrids were also determined. These parameters of fitness are very low in the inbred line. Hybrids displayed intermediate behaviour.Publication No. 109, Departamento de Genética, Universidad de Valencia, Spain     

Microbial metabolism of chloroanilines: enhanced evolution by natural genetic exchange

2-, 3-, and 4-chloroaniline degrading bacteria were obtained by natural genetic exchange between an aniline or toluidine degrading Pseudomonas strain and the chlorocatechol assimilating Pseudomonas sp. B13. Hybrid organisms were isolated through cocultivation of the parent strains in the chemostat as well as through conjugation on solid media in presence of chloroanilines as the selective substrates. Biochemical analysis of the gene products in the hybrid strains clearly showed that the genes coding for the aniline dioxygenase or the genes for the chlorocatechol assimilatory sequence had been transferred.