Ultrastructural description of the spore maturation stages of the clam parasite Minchinia tapetis (Vilela, 1951) (Haplosporida: Haplosporidiidae)

The fine structure of maturing spores of a haplosporidian parasite found in the gill, mantle and foot tissues of Ruditapes decussatus L. (Mollusca, Bivalvia), a species of commercial importance in Portugal, is described. When observed free in suspension, immature spores exhibit one or two epispore cytoplasmic extensions (ECE) which constitute a projection of a portion of the exosporoplasm, sometimes without ultrastructural organisation, surrounded by the plasmalemma. Free spores observed by light microscopy (LM) after 3-5 days of incubation in filtered sea-water exhibit no ECE attached to the spore wall. The mature spore is ovoid to ellipsoid, operculate, uninucleate and measures c. 4.8 microm long and c. 3.9 microm wide. The spore shape and size and the identity of the host living in the same geographical region suggest that this species is the same as previously described using LM observations as Haplosporidium tapetis Vilela, 1951 and later transferred to Minchinia Labbé, 1896.     

Effect of the respiratory activity on photoinhibition of the cyanobacterium Synechocystis sp.

The influence of respiratory activity on photosynthesis in Synechocystis cells that had been exposed to high light intensity was studied using distinct conditions of nitrogen supply. The photoinhibitory rate of N-sufficient cells was not influenced by the presence of different nitrogen sources. In contrast, when N-starved cells were resupplied with ammonium, they were protected from photoinhibition. Although N-starved cells presented a higher rate of dark O₂ uptake than N-sufficient ones, the photoinhibitory rate increased in both cases after addition of sodium azide or sodium azide plus salicylhydroxamic acid in the photoinhibitory treatment. In the absence of the D₁ protein repair mechanism, photodamage to Photosystem II was faster in N-sufficient cells than in N-starved ones. Mitigation of photodamage disappeared when the respiratory activity of N-starved cells was partially suppressed by the addition of sodium azide or sodium azide and salicylhydroxamic acid. Our results suggest that electron flow through cyanobacterial terminal oxidases can assist Photosystem I in removing electrons from the reduced plastoquinone pool, thus contributing to both reopening of Photosystem II reaction centers and avoiding photogeneration of reactive oxygen species under photoinhibitory conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

5-Aminolevulinic acid inhibits [3H]muscimol binding to human and rat brain synaptic membranes

The interaction of 5-aminolevulinic acid (ALA) with GABA_A receptors has been proposed to underlie the neurological dysfunctions of ALA-accumulating disorders, such as acute intermittent porphyria. The effects of ALA on [³H]muscimol binding to human and rat cerebral cortical membranes were compared. ALA (0.1–10 mM) significantly inhibited the binding of [³H]muscimol (12 nM), with a similar potency in rat and human membranes (IC₅₀ = 199 vs. 228 M, respectively). Kinetical analysis revealed that ALA (1 mM) significantly increased the Kd and decreased the Bmax of [³H]muscimol to both rat (100 and 50%, respectively) and human (200 and 40%, respectively) membranes, indicating a mixed-type inhibition. The similarity in the potency and mechanism of the ALA-induced inhibition of muscimol binding in rat and human membranes indicate that rat studies are useful to evaluate the neurotoxic properties of ALA towards the human GABAergic system, and may help to understand the pathophysiology of porphyria.     

Ventricular remodeling and diastolic myocardial dysfunction in rats submitted to protein-calorie malnutrition

The effects of protein-calorie malnutrition (PCM) on heart structure and function are not completely understood. We studied heart morphometric, functional, and biochemical characteristics in undernourished young Wistar rats. They were submitted to PCM from birth (undernourished group, UG). After 10 wk, left ventricle function was studied using a Langendorff preparation. The results were compared with age-matched rats fed ad libitum (control group, CG). The UG rats achieved 47% of the body weight and 44% of the left ventricular weight (LVW) of the CG. LVW-to-ventricular volume ratio was smaller and myocardial hydroxyproline concentration was higher in the UG. Left ventricular systolic function was not affected by the PCM protocol. The myocardial stiffness constant was greater in the UG, whereas the end-diastolic pressure-volume relationship was not altered. In conclusion, the heart is not spared from the adverse effects of PCM. There is a geometric alteration in the left ventricle with preserved ventricular compliance despite the increased passive myocardial stiffness. The systolic function is preserved.     

Tyrosine Hydroxylase Dephosphorylation by Protein Phosphatase 2A in Bovine Adrenal Chromaffin Cells

This study was undertaken to characterise the protein phosphatases in bovine adrenal chromaffin cells acting on tyrosine hydroxylase. Cells were pre-labelled with ³²P_i and permeabilized with digitonin. The extent of dephosphorylation of Ser-8, Ser-19, Ser-31 and Ser-40 on tyrosine hydroxylase was found to be 30%, 38%, 37% and 71% respectively over 5 min. For Ser-19, Ser-31 and Ser-40 the dephosphorylation was entirely due to protein phosphatase 2A, as the dephosphorylation could be completely blocked by microcystin, but not by the protein phosphatase 1 inhibitory peptide. Permeabilization did not change the distribution of protein phosphatase 2A or tyrosine hydroxylase, or the activity of PP2A, from that occurring in intact cells. The dephosphorylation of Ser-8 was not altered by any inhibitor, suggesting the involvement of other protein phosphatases. The method developed here can be used to determine the protein phosphatases acting on substrates in conditions closely approximating those in situ, including the endogenous state of substrate phosphorylation and phosphatase location.     

Participation of host cell actin filaments during interaction of trypomastigote forms of Trypanosoma cruzi with host cells

The involvement of actin filaments from the host cell on the process of invasion of trypomastigote forms of Trypanosma cruzi was analyzed in seven different cell lines. Prior incubation of all cell lines with cytochalasin D, under conditions which interfere with actin filaments, markedly inhibited parasite internalization and increased parasite attachment. Attached parasites were readily ingested following washing of the drug-treated cells. Cytochalasin treatment interfered with the distribution of actin filaments of the host cell as evaluated by visualization of the filaments using confocal laser scanning microscopy of cells incubated in the presence of FITC-phalloidin. Concentration of actin filaments could be observed in most, but not all, parasites in the process of internalization. We also treated LLCMK 2 and macrophage cells with Jasplakinolide, a drug that stabilizes actin filaments, before interaction with the trypomastigote forms. This drug partially inhibits parasite invasion into the cells. Prior incubation of the host cells in the presence of colchicine, which interfere with microtubules, also inhibited parasite internalization into the cells.     

Inferring the history of speciation from multilocus DNA sequence data: the case of Drosophila pseudoobscura and close relatives

The divergence of Drosophila pseudoobscura from its close relatives, D. persimilis and D. pseudoobscura bogotana, was examined using the pattern of DNA sequence variation in a common set of 50 inbred lines at 11 loci from diverse locations in the genome. Drosophila pseudoobscura and D. persimilis show a marked excess of low-frequency variation across loci, consistent with a model of recent population expansion in both species. The different loci vary considerably, both in polymorphism levels and in the levels of polymorphisms that are shared by different species pairs. A major question we address is whether these patterns of shared variation are best explained by gene flow or by persistence since common ancestry. A new test of gene flow, based on patterns of linkage disequilibrium, is developed. The results from these, and other tests, support a model in which D. pseudoobscura and D. persimilis have exchanged genes at some loci. However, the pattern of variation suggests that most gene flow, although occurring after speciation began, was not recent. There is less evidence of gene flow between D. pseudoobscura and D. p. bogotana. The results are compared with recent work on the genomic locations of genes that contribute to reproductive isolation between D. pseudoobscura and D. persimilis. We show that there is a good correspondence between the genomic regions associated with reproductive isolation and the regions that show little or no evidence of gene flow.     

NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos

Exhaustive extraction of the endosperm from the seed of Gleditsia triacanthos using water at room temperature and 50 degrees C left a residue, which was further extracted at 95 degrees C. Precipitation of this extract with 2-propanol yielded major amounts of galactomannan components, while the supernatant was mainly composed of arabinose-rich constituents. Two fractions were obtained by anion-exchange chromatography. The fraction that eluted with water is an arabinan with (1-->5) alpha-L linkages and branching mainly on C-2, accompanied with equal amounts of a low-galactose galactomannan oligosaccharide, and a small proportion of a beta-(1-->4)-galactan. The fraction eluted with an increased ionic strength consists mainly of a similar arabinan, and lower proportions of a high-galactose galactomannan, galactan, and protein. The arabinan moiety in both fractions was characterized by chemical analysis and 1D and 2D NMR spectroscopic techniques.     

Novel isoform of insulin receptor substrate p53/p58 is generated by alternative splicing in the CRIB/SH3-binding region

Insulin receptor substrate p53/p58 (IRSp53) is involved in cytoskeletal dynamics and is a candidate disease sensor in polyglutamine expansion neurodegeneration. It is widely expressed throughout the body, but its levels are dramatically elevated in forebrain regions. IRSp53 functions as a signal transducing adaptor between activated Rho family GTPases and their effectors. There are four known alternatively spliced isoforms of IRSp53 that vary by the identity of the 3'-terminal exon. We report here that there is a fifth alternatively spliced isoform, IRSp53-B, which lacks 40 amino acids abutting the CRIB/SH3 (Cdc42/Rac-interactive binding/Src homology 3)-binding site. We speculate that the novel form has an altered function related to the mechanism of autoinactivation. IRSp53-B has an odd history in the mammalian lineage, which may complicate the use of rodent models to study cytoskeletal reorganization.     

Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila

The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans (9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11; dGalNAc-T2 (CG6394) and GalNAc-T7). dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNAc-transferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964-2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa(HG8), l(2)35Aa(SF12), and l(2)35Aa(SF32). The finding that subfamilies of GalNAc-transferases with distinct catalytic functions are evolutionarily conserved stresses that GalNAc-transferase isoforms may serve unique biological functions rather than providing functional redundancy, and this is further supported by the lethal phenotype of l(2)35Aa.