Morphological,ultrastructural and histochemical investigation of epipodial sensory structures of Haliotis tuberculata (Gastropoda: Haliotidae)

In this study, we describe the microstructure and ultrastructure of the epipodial papillae and epipodial tentacles of Haliotis tuberculata using light and electron microscopy. The epipodial papillae vary morphologically; they are subdivided into several subpapillae whose surface is covered by small micropapillae. The epipodial tentacles are large extendable conically elongated structures whose surface is differentiated in two regions: the dorsal region with long corrugated folds, and a ventral region composed of three parts, a basal part with the same structure as the dorsal, a middle part with shorter corrugated folds and an apical part with large micropapillae. Although the thin sections and ultrastructure examination show that the epithelium of both organs is morphologically similar and composed of supporting cells, sensory cells and different types of secretory cells, there is a certain specialization in their secretory product. Although the epithelium of both structures was positive for acidic glycoconjugates, the tentacle epithelium was also positive for neutral sugars. Further specific differences were revealed by lectin histochemistry. Because papillae and tentacles can be extended or retracted depending on environmental conditions, they probably have tactile and olfactory functions.     

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.     

AFLP Analysis Confirms Exclusive Maternal Genomic Contribution of Meiogynogenetic Sea Bass (Dicentrarchus labrax L.)

Meiogynogenesis was induced in the European sea bass Dicentrarchus labrax L. by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division by a cold shock. Putative gynogenetic progeny derived from three groups of breeders were analyzed for maternal inheritance using amplified fragment length polymorphism (AFLP) markers. Discrimination of fingerprints was based on male-specific bands, which were absent in females. Four of 64 MseI/EcoRI primer pairs used to analyze parental polymerase chain reaction products were selected to screen progeny for paternal AFLP markers in each group. Four to 11 diagnostic bands per fish confirmed the gynogenetic origin of the progeny. AFLP analysis determined that 89.5%, 100%, and 100% of the sea bass from groups 1, 2, and 3, respectively, were gynogenetic. Our results show that AFLP analysis is suitable for verification of gynogenesis in fish.     

Gnathostomulid phylogeny inferred from a combined approach of four molecular loci and morphology

The phylogeny of the obscure metazoan phylum Gnathostomulida has previously only been addressed with cladistic analyses of morphological data. In the present study DNA sequence data from four molecular loci, including 18S rRNA, 28S rRNA, histone H3 and cytochrome c oxidase subunit I, are added to a revised morphological data matrix. The data set represents 23 gnathostomulid species that are analyzed under direct optimization using parsimony as the optimality criterion. The results obtained from analyzing the four molecular loci and combined morphological and molecular data under different parameter sets are generally very congruent, and differ only on minor points. The results clearly support gnathostomulid monophyly, as well as the basal division of Gnathostomulida into Filospermoidea and Bursovaginoidea. Filospermoidea were represented by species of Haplognathia and Cosmognathia, and generic monophyly is supported for both groups. Within Bursovaginoidea, Conophoralia (= Austrognathiidae) and Scleroperalia appear as sister groups. Monophyly of Mesognathariidae was confirmed as well, whereas the relationships between species of Gnathostomulidae and Onychognathiidae were contradicted by the molecular data when compared to morphological observations. ©The Willi Hennig Society 2006.     

Release of d-[3H]aspartic acid from the rat substantia nigra: effect of veratridine-evoked depolarization and cortical ablation

The spontaneous and veratridine-evoked release of radioactive d-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 μM, 1 min) markedly produced a 14-fold increase in d-[³H]aspartic acid release from nigral slices. Omission of Ca²⁺ and increasing Mg²⁺ concentration to 12 mM in the superfusion medium did substantially block d-[³H]aspartate release induced by veratridine depolarization. Nevertheless, veratridine was able to evoke [³H]amino acid release which seemed to be, at least, 30% Ca²⁺-independent. Additional experiments showed that tetrodotoxin (0.01–0.1 μM), a blocker of voltage-dependent Na⁺ channels, totally abolished veratridine-evoked release of d-[³H]aspartate from nigral slices.Lesion studies were performed in order to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce d-[³H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral d-[³H]aspartate and by a large loss from ipsilateral nigral slices in d-[³H]aspartate release evoked by veratridine. In contrast, both the accumulation and veratridine-evoked release of [³H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion.The findings reported suggest that d-[³H]aspartic acid may be taken up and then released, in a Ca²⁺-dependent manner, by nerve terminals located in the substantia nigra. In addition, the results shown provide support to the view that l-glutamate and/or l-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.     

Arundifungin, a novel antifungal compound produced by fungi: biological activity and taxonomy of the producing organisms

Echinocandins, the lipopeptide class of glucan synthase inhibitors, are an alternative to ergosterol-synthesis inhibitors to treat candidiasis and aspergillosis. Their oral absorption, however, is low and they can only be used parenterally. During a natural product screening program for novel types of glucan synthesis inhibitors with improved bioavailability, a fungal extract was found that inhibited the growth of both a wild-type Saccharomyces cerevisiae strain and the null mutant of the FKS1 gene (fks1::HIS). The mutant strain was more sensitive to growth inhibition, suggesting that the fungal extract could contain an inhibitor of glucan synthesis. A novel acidic steroid, named arundifungin, was purified from a fungal extract obtained from a liquid culture of Arthrinium arundinis collected in Costa Rica. Arundifungin caused the same pattern of hallmark morphological alterations in Aspergillus fumigatus hyphae as echinocandins, further supporting the idea that arundifungin belongs to a new class of glucan synthesis inhibitors. Moreover, its antifungal spectrum was comparable to those of echinocandins and papulacandins, preferentially inhibiting the growth of Candida and Aspergillus strains, with very poor activity against Cryptococcus. Arundifungin was also detected in nine other fungal isolates which were ecologically and taxonomically unrelated, as assessed by sequencing of the ITS1 region. Further, it was also found in two more Arthrinium spp from tropical and temperate regions, in five psychrotolerant conspecific isolates collected on Macquarie Island (South Pacific) and belonging to the Leotiales, and in two endophytes collected in central Spain (a sterile fungus belonging to the Leotiales and an undetermined coelomycete).     

A new model Gondwanan taxon: systematics and biogeography of the harvestman family Pettalidae (Arachnida, Opiliones, Cyphophthalmi), with a taxonomic revision of genera from Australia and New Zealand

The phylogeny of the temperate Gondwanan harvestman family Pettalidae is investigated by means of a new morphological matrix of 45 characters, and DNA sequence data from five markers, including two nuclear ribosomal genes (18S rRNA and 28S rRNA), one nuclear protein coding gene (histone H3), and two mitochondrial genes–one protein coding (cytochrome c oxidase subunit I) and one ribosomal (16S rRNA). Phylogenetic analyses using an array of homology schemes (dynamic and static), criteria (parsimony and maximum likelihood), and sampling strategies (optimal trees versus Bayesian phylogenetics) all agree on the monophyly of Pettalidae as well as several of its subclades, each of which is restricted to a modern landmass. While most genera as traditionally defined are monophyletic, Rakaia and Neopurcellia, distributed across Queensland (Australia) and New Zealand, are not. Instead, the species from Queensland, previously described under three genera, constitute a well‐supported clade, suggesting that in this case biogeography prevails over traditional taxonomy. A taxonomic emendation of the genera from Queensland and New Zealand is presented, and the new genus Aoraki is erected to include the species of the New Zealand denticulata group. A biogeographical hypothesis of the relationships of the former temperate Gondwana landmasses (with the exception of Madagascar) is presented, although ambiguity in the deep nodes of the pettalid tree renders such inference provisional. The data suggest that neither the South African fauna, the New Zealand fauna nor the Australian fauna is monophyletic but instead monophyly is found at smaller geographic scales (e.g., Western Australia, Queensland, NE South Africa). © The Willi Hennig Society 2007.     

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.     

Characterization of the NADP malic enzyme gene family in the facultative,single-cell C<Subscript>4</Subscript> monocot <Emphasis Type="Italic">Hydrilla verticillata</Emphasis>

Hydrilla verticillata has a facultative single-cell system that changes from C₃ to C₄ photosynthesis. A NADP⁺-dependent malic enzyme (NADP-ME) provides a high [CO₂] for Rubisco fixation in the C₄ leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C₄ leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C₄ species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C₃ and C₄ species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The K _m (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C₄ system in H. verticillata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.