Genetic variation of resistance to mercury poisoning in steelhead (Oncorhynchus mykiss) alevins

Newly hatched steelhead alevins were obtained from the factorial breeding of 24 male and 10 female steelhead trout, Oncorhynchus mykiss. Each set of offspring were in a separate cell. They were tested for resistance to intoxication by methylmercuric chloride (CH3HgCl) in water at a nearly constant mercury concentration of 8 microg l(-1). High mortality (81% of the tested alevins) occurred within 2 weeks. Resistance to intoxication, as measured by the time to death, as well as by the survival rate, shared high paternal and maternal variation with negligible interaction. Heritability of time to death was 0.59 +/- 0.17; heritability of survival (all-or-none trait) was lower (0.26 +/- 0.09). Mercury in dead alevins increased with time to death, exhibiting a large environmental variation and (comparatively) negligible genetic influence. At the end of the bioassay, the mercury content in survivors varied widely (3-21 microg g(-1) wet weight). The content was greater than, but correlated with that of dead alevins from the same cells, and it showed little relation with survival rate. Thus, it seems that resistance to poisoning implies a tolerance to high levels of mercury rather than a limitation of its accumulation.     

Molineus torulosus (Nematoda, Trichostrongylina, Molineoidea) a parasite of Neotropical primates: new morphological and histological data

Molineus torulosus (Molin, 1861) parasite of Cebus spp. from South America is redescribed in Cebus apella and C. olivecaeus (new host) from French Guyana with emphasis on the synlophe. During the maturation process, the larvae dwelt in the cysts carved alongside the external part of the small intestine. The turn-out of the mature worms and the laid eggs depended on the tissular organisation of cyst walls as the inflammatory process waned and fibrosis progressed to seal the cystic lumen. Adult worms entwine themselves in the cysts, live there permanently as their presence has never been evidenced in the intestinal lumen. They copulated, laid eggs, degenerated and died once entrapped by the fibrotic process. Laid eggs released in the intestinal lumen through a narrow channel ensured the continuation of the developmental cycle. However, erratic migration was possible via the vascular channels surrounding the cysts.     

Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.     

Activation of a nuclear-localized SIPK in tobacco cells challenged by cryptogein, an elicitor of plant defence reactions

When a plant cell is challenged by a well-defined stimulus, complex signal transduction pathways are activated to promote the modulation of specific sets of genes and eventually to develop adaptive responses. In this context, protein phosphorylation plays a fundamental role through the activation of multiple protein kinase families. Although the involvement of protein kinases at the plasma membrane and cytosolic levels are now well-documented, their nuclear counterparts are still poorly investigated. In the field of plant defence reactions, no known study has yet reported the activation of a nuclear protein kinase and/or its nuclear activity in plant cells, although some protein kinases, e.g. MAPK (mitogen-activated protein kinase), are known to be translocated into the nucleus. In the present study, we investigated the ability of cryptogein, a proteinaceous elicitor of tobacco defence reactions, to induce different nuclear protein kinase activities. We found that at least four nuclear protein kinases are activated in response to cryptogein treatment in a time-dependent manner, some of them exhibiting Ca(2+)-dependent activity. The present study focused on one 47 kDa protein kinase with a Ca(2+)-independent activity, closely related to the MAPK family. After purification and microsequencing, this protein kinase was formally identified as SIPK (salicyclic acid-induced protein kinase), a biotic and abiotic stress-activated MAPK of tobacco. We also showed that cytosolic activation of SIPK is not sufficient to promote a nuclear SIPK activity, the latter being correlated with cell death. In that way, the present study provides evidence of a functional nuclear MAPK activity involved in response to an elicitor treatment.     

Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes

It is often anticipated that many of today's diploid plant species are in fact paleopolyploids. Given that an ancient large-scale duplication will result in an excess of relatively old duplicated genes with similar ages, we analyzed the timing of duplication of pairs of paralogous genes in 14 model plant species. Using EST contigs (unigenes), we identified pairs of paralogous genes in each species and used the level of synonymous nucleotide substitution to estimate the relative ages of gene duplication. For nine of the investigated species (wheat [Triticum aestivum], maize [Zea mays], tetraploid cotton [Gossypium hirsutum], diploid cotton [G. arboretum], tomato [Lycopersicon esculentum], potato [Solanum tuberosum], soybean [Glycine max], barrel medic [Medicago truncatula], and Arabidopsis thaliana), the age distributions of duplicated genes contain peaks corresponding to short evolutionary periods during which large numbers of duplicated genes were accumulated. Large-scale duplications (polyploidy or aneuploidy) are strongly suspected to be the cause of these temporal peaks of gene duplication. However, the unusual age profile of tandem gene duplications in Arabidopsis indicates that other scenarios, such as variation in the rate at which duplicated genes are deleted, must also be considered.     

Examination of the requirement for an amphiphilic helical structure in beta-endorphin through the design, synthesis, and study of model peptides

In our approach to beta-endorphin modeling, we have proposed that the biological properties of the natural peptide are determined by the combination of three basic structural units: a highly specific opiate recognition sequence at the NH2 terminus (residues 1-5) connected via a hydrophilic peptide link (residues 6-12) to a potential amphiphilic helix in the COOH-terminal residues 13-31. In the alpha-helical conformation the hydrophobic domain twists around the length of the helix and covers almost one-half of its surface. The other distinctive features of the helix include its basicity and the two aromatic residues Phe18 and Tyr27. In contrast to previous models we have studied, peptide 4 is a "negative" model in the sense that it was designed and examined in order to determine how the lack of a well defined amphiphilic structure affects the biological properties of beta-endorphin. For this purpose, peptide 4 retains the three structural units previously postulated for beta-endorphin, but the amino acids of the 13-31 region are arranged in such a way that no definite continuous hydrophobic zone could be formed in an alpha- or pi-helical conformation of this region. In aqueous buffered solutions, peptide 4 showed almost the same amount of alpha-helical structure as beta-endorphin, with a slight tendency toward less helicity in 50% aqueous 2,2,2-trifluoroethanol. In rat brain homogenate, peptide 4 was degraded slightly slower than beta-endorphin, in contrast to the apparently much higher stability of previous models under the same conditions. With regard to opiate receptor binding, peptide 4 was twice as potent as beta-endorphin in mu-receptor assays but half as potent in delta-receptor assays. The opiate potency of peptide 4 on the guinea pig ileum was higher than that of beta-endorphin. In contrast, in the rat vas deferens assay, which is very specific for beta-endorphin, the potency of peptide 4 was very low and could be shown not to be mediated by the same opiate mechanism or by the same opiate receptor. A comparison of these results with those of previous model peptides provides further evidence for the importance of an amphiphilic helical structure in beta-endorphin residues 13-31, which determines the resistance to proteolysis of the natural molecule and contributes to the delta- and mu-opiate receptor interaction. The amphiphilicity of this helical structure must also be essential for high opiate activity on the rat vas deferens (epsilon-receptors), whereas no such structural requirement appears to be necessary for interaction with the opiate receptors on the guinea pig ileum.     

The PPPY motif of human T-cell leukemia virus type 1 Gag protein is required early in the budding process

Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.     

Arginine and glutamine availability and macrophage functions in the obese insulin-resistant Zucker rat

Increased susceptibility to infections in obese patients may be related to decreased availability of arginine and glutamine, which may affect immune cell functions. Our aim was to evaluate the in vitro effects of these amino acids on the function of macrophages from obese insulin-resistant Zucker rats. Macrophages, isolated from male Zucker obese or lean rats by peritoneal lavage, were incubated in Dulbecco's modified Eagle medium (DMEM) without arginine or glutamine. Arginine or glutamine was added to the medium at increasing final concentrations (0, 0.25, 0.5, 1 or 2 mM). After stimulation by lipopolysaccharide (LPS) from E. coli (40 microg/ml), productions of tumour necrosis factor alpha (TNFalpha) and of nitric oxide (NO) were measured after 3 or 48 h incubation, respectively. NO production, lower in macrophages from obese rats, decreased in macrophages from lean rats (0 mM: 2,423 +/- 1,174 vs. 2 mM: 198 +/- 31 microM/mg protein/24 h; P < 0.05), but not in those from obese rats, when glutamine was added. TNFalpha production, lower in macrophages from obese rats, was inversely correlated with glutamine concentration. In the presence of arginine, NO production was constantly higher in macrophages from obese rats. It peaked at 0.5 mM arginine and decreased thereafter in both groups. TNFalpha production in macrophages from lean rats was unaffected by arginine, but decreased in macrophages from obese rats (0 mM: 1920 +/- 450 vs. 2 mM: 810 +/- 90 microM/mg protein/3 h; P < 0.05). These results suggest that abnormalities in cell signalling or in arginine and glutamine metabolism in macrophages of obese rats, resulting in decreased TNFalpha production and increased NO release, may contribute to increased susceptibility to infection in insulin-resistant states.