Analysis of non-Saccharomyces yeast populations isolated from grape musts from Sicily (Italy).

AIMS: The aim of this study was to identify the non-Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. METHODS AND RESULTS: Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5.8S-internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. CONCLUSIONS: We isolated non-Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation is a first step in understanding the distribution of non-Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non-Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.

Toxicity of recombinant beta-amyloid prefibrillar oligomers on the morphogenesis of the sea urchin Paracentrotus lividus.

A distinctive feature of Alzheimer's disease is the deposition of amyloid beta-protein (Abeta) in senile or diffuse plaques. The 42 residue beta-peptide (Abeta42) is the predominant form found in plaques. In the present work we report a high-yield expression and purification method of production of a recombinant Abeta42. The purified recombinant peptide shows characteristics similar to the synthetic human peptide. Different size aggregates, either small oligomers or larger aggregates, were obtained upon dissolving the recombinant Abeta42 peptide under different conditions at pH 7.2 or pH 3, respectively. We report a new toxicity assay on the morphogenic development of the sea urchin Paracentrotus lividus and study the toxicity of the two kinds of aggregates. Despite the difference between the ionic strength of human extracellular fluid (0.154 mol/l) and artificial sea water (0.48 mol/l), toxicity data collected in this system have an intrinsic relevance. The different ionic strength, in fact, could change the kinetics of oligomer formation, but the effect of morphogenic development reported here is related to the final oligomer sizes. Results of the toxicity assay of Abeta42 on sea urchin development also show a dose-dependent effect. After only 4 h of embryo development, one can note morphological defects in the cell membrane. Retardation of the embryo's development, along with cellular disorders visible inside the blastocoele, can be observed after 1 day of development. Cellular degeneration in two different pathological phenotypes-the occluded blastulae and the occluded prism-is present after 48 h of development. Results show that a greater effect on cell death is induced by the small oligomers stabilized under physiological conditions than at acid pH. In this case only occluded blastulae are found after 48 h of development.

[Molecular characterization of variable tandem repeat sequence determined during RAPD analysis among Posidonia oceanica insular and coastline populations]. / Molekuliarnye kharakteristiki variabel'noi tandemno povtoriaiushcheisia posledovatel'nosti, vyiavlennoi v khode RAPD-analiza ostrovnoi i pribrezhnoi populiatsii.

The seagrass Posidonia oceanica plays a multifunctional role in the coastal area as an important and productive component of ecosystems in the Mediterranean Sea. We detected by RAPD analysis with two arbitrary primers genetic differences in P. oceanica collected from several sites in the Southern Mediterranean. By AMOVA analysis we observed a level of about 20% genetic difference among individuals within a population and 80% among populations. A common band of 200 bp was found in all the amplified samples. Cloning and sequencing analysis of this band revealed the presence of a simple tandem repeat sequence (minisatellite) that we called PoTR (Posidonia oceanica tandem repeat). Finally, the ability of PoTR to detect genetic variability in P. oceanica genome was demonstrated by the presence of amplification products of different lengths utilizing primers internal to this sequence.

Identification of an antigen related to the sea urchin RNA-binding protein LP54 in mammalian central nervous system.

LP54 is an RNA-binding protein involved in localization of maternal messengers in sea urchin egg and embryos. Using a polyclonal antibody directed against Paracentrotus lividus LP54 we detected a 66-kDa cross-reacting antigen in undifferentiated and differentiated SH-SY5Y human neuroblastoma cells. After treatment of undifferentiated cells with detergent, the 66-kDa antigen was found to be enriched in the cytoskeletal fraction. By Western blot the expression of this antigen was also analyzed in regions of the CNS and in tissues of the adult rat and its exclusive presence in the hippocampus and thalamus was revealed. The immunoreactivity with P. lividus antibody against LP54 in hippocampal lysate was also confirmed throughout anti-LP54 immunoaffinity column and competition experiments. The results indicates that a related protein to the sea urchin LP54 is evolutionary conserved in mammalian CNS.

Bep4 protein is involved in patterning along the animal-vegetal axis in the Paracentrotus lividus embryo.

In sea urchin embryos, the initial animal-vegetal (AV) axis is specified during oogenesis but the mechanism is largely unknown. By using chemical reagents such as lithium, it is possible to shift the principal embryonic territories toward a vegetal fate. We have investigated the possibility of obtaining the same morphological effect as with lithium by utilizing Fabs against the maternal Bep4 protein that is localized in the animal part of Paracentrotus lividus egg and embryos. Incubation of fertilized eggs with Fabs against Bep4 protein causes exogastrulation at 48 h of development of P. lividus embryos, similar to embryos treated with lithium. This vegetalizing effect was ascertained by utilizing territorial markers such as EctoV, EndoI, and Ig8. The effect of Fabs against Bep4 on gene expression was observed by monitoring spatial expression of the hatching enzyme gene. A decreased expression domain compared to its normal spatial distribution was detected and this effect was again comparable to those obtained with lithium treatment. Association of Bep4 with a cadherin was demonstrated by immunoprecipitation and immunostaining experiments, and an involvement in cell signaling is discussed. In addition, treatment of embryos with anti-Bep4 Fabs causes an enhancement in the level and an expansion in the pattern of nuclear beta-catenin. Moreover, this treatment also provokes a decrease of beta-catenin in adherens junctions. Together, these data indicate that anti-Bep4 Fabs provoke a shift of the animal-vegetal boundary toward the animal pole and suggest an active role of Bep4 protein in patterning along the AV axis.

Localization and association to cytoskeleton of COLL1alpha mRNA in Paracentrotus lividus egg requires cis- and trans-acting factors.

COLL1alpha mRNA is asymmetrically distributed in the Paracentrotus lividus egg. Here we examine the involvement of the cytoskeleton in the localization process of collagen mRNA. The use of drugs such as colchicine and cytochalasin B reveals a perturbation of localization collagen mRNA. Moreover, the presence of specific cis-and trans-acting factors involved in cytoskeleton binding and the localization process was investigated. By Northwestern experiment we found that the 3'UTR of COLL1alpha mRNA is also able to bind two proteins of 54 and 40 kDa in a cellular fraction containing the cytoskeleton. Finally, we found that the protein of 54 kDa is LP54, a protein that binds the 3'UTRs of P. lividus maternal bep messengers and is necessary for their localization.

Isolation of a trans-acting factor involved in localization of Paracentrotus lividus maternal mRNAs.

Localization of Paracentrotus lividus bep maternal mRNAs at the animal pole occurs by association with the cytoskeleton and involves a 54-kDa protein, called LP54, that is able to bind to the 3' untranslated regions (UTRs) of bep mRNAs. We describe here the isolation and purification of this protein. Antibodies raised against purified LP54 allowed us to establish its localization in P. lividus eggs and embryos. This localization coincides with the mRNAs with which it is associated, that is, the animal pole in the egg, and, after fertilization, the regions derived from this part of the egg, and finally the oral ectoderm of the pluteus. Association with the cytoskeleton was shown by the copurification of LP54 in a microtubule preparation. Involvement in bep mRNA localization was demonstrated by microinjection of anti-LP54 antibodies in P. lividus eggs, which caused alteration of spatial distribution of bep3 mRNA.

Asymmetrical localization and segregation of Paracentrotus lividus Bep4 maternal protein.

Asymmetric divisions that produce two distinct cells play a fundamental role in generating different cell types during development. Here we investigate the role of the cortex region and mitotic apparatus in asymmetrical localization and segregation of Bep4 protein in Paracentrotus lividus egg. By centrifugation of eggs with or without drugs we established an involvement of the cortex region in localization of Bep4 protein, confirmed by immunohistochemistry of isolated cortex. Association with the mitotic apparatus during cell division permits selective partitioning of Bep4 protein into the daughter cells. Direct association with spindle was also demonstrated both by Western blot and immunohistochemistry after isolation of the mitotic apparatus.

Folding and binding activity of the 3'UTRs of Paracentrotus lividus bep messengers.

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3'UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lengths of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3'UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.

Involvement of the cytoskeleton in localization of Paracentrotus lividus maternal BEP mRNAs and proteins.

The maternal bep1 and bep4 mRNAs and their protein products are localized at the animal pole of Paracentrotus lividus eggs. We have examined the role of the cytoskeleton in localization both of bep RNAs and BEP proteins in unfertilized and fertilized eggs and in determining the polarity of P. lividus eggs. The use of drugs such as colchicine and cytochalasin B, which depolymerize microtubules and microfilaments respectively, revealed a perturbation of localization of bep1 and bep4 mRNAs. In contrast, the microfilament inhibitor cytochalasin B had no effect on localization of BEP1 and BEP4 antigens localization, which appears to be due only to microtubules. Moreover, the presence of bep mRNAs and BEP proteins in a microtubule preparation has been demonstrated. Maintenance of the asymmetric distribution of BEP proteins during cellular division of eggs and embryos, by association with mitotic spindle, is also shown.

Temporal-spatial expression of two Paracentrotus lividus cell surface proteins.

The temporal expression of two cell surface proteins, called BEP1 and BEP4, during Paracentrosus lividus embryonic development was studied. These proteins are found in both monomeric and dimeric forms in egg and embryos and we have established that their specific form is related to their being in the cytoplasm or on the cell surface. The spatial distribution of BEP1 and BEP4 proteins in eggs and embryos was established by whole mount immunohistochemistry. These proteins are located in the animal part of unfertilized and fertilized eggs; thereafter they are much less represented in structures derived from the vegetal cells of the embryo such as the micromeres of the 16 cell stage, the primary mesenchyme of blastula and the gut of gastrula. At the prism stage BEP1 and BEP4 proteins are present to some ectodermal parts and thereafter, at the pluteus stage, to the oral region.

Spatial distribution of collagen type I mRNA in Paracentrotus lividus eggs and embryos.

We have identified the presence of type I collagen (COLL1alpha) mRNA in Paracentrotus lividus unfertilized egg, indicating a maternal origin of this mRNA. By in situ whole mount hybridization the spatial distribution of COLL1alpha mRNA in egg and embryo at different developmental stages was established. Moreover, the presence of COLL1alpha gene in Paracentrotus lividus genome was analyzed by Southern blot experiments. The localization pattern indicates that the maternal mRNA is placed in the fertilized egg in a fixed position, relative to the embryonic axes. Furthermore, the embryonic expression is spatially restricted during development, suggesting involvement in sea urchin embryo cell specification events. The presence of two bands in Southern blot hybridization may indicate that two genes specific for COLL1alpha are present in the sea urchin genome.

A 54-kDa protein specifically associates the 3' untranslated region of three maternal mRNAs with the cytoskeleton of the animal part of the Paracertrotus lividus egg.

Bep mRNAs, i.e., maternal messengers coding for cell surface proteins, are localized in the animal part of Paracertrotus lividus egg and embryos. Here we have examined the involvement of the cytoskeleton in asymmetric distribution of bep3 mRNA. Moreover, in order to understand whether and how cis- and trans-acting factors are necessary for bep3 mRNA localization, we have looked for in vitro-specific interactions between egg proteins and bep3 mRNA. By northwestern assay we have identified a 54-kDa protein that binds to the 3'UTR of bep3 mRNA. This 54-kDa protein also permits association of 3'UTR of bep3 with cytoskeleton elements, indicating its involvement in the localization process. Binding of 54-kDa protein to 3'UTR of bep1 and bep4 has also been demonstrated, suggesting that a binding motif is shared with these other two mRNAs of the same gene family. Northwestern analyses carried out utilizing proteins extracted from different developmental stages indicate that the 54-kDa protein is the only protein able to bind to the 3'UTR of bep3.

Centrifugation does not alter spatial distribution of 'BEP4' mRNA in paracentrotus lividus EGG.

Paracentrotus lividus unfertilized eggs were centrifuged in a sucrose gradient, so to split each into two parts: a nucleated light fragment and an anucleated heavy fragment. Northern blot analyses utilizing a bep4 probe as animal marker and H2A histone gene and 12S-mit RNA as controls indicate that the eggs are elongated along the animal-vegetal axis during centrifugation and thereafter split into an animal and a vegetal half. Treatment of the eggs with colchicine before centrifugation abolishes the animal localization of bep4 mRNA.

"BEP" RNAs and proteins are situated in the animal side of sea urchin unfertilized egg, which can be recognized by female pronuclear localization.

Microsurgery experiments demonstrate that the animal side of the unfertilized sea urchin Paracentrotus lividus egg coincides with the side of the egg pronucleus location. It is demonstrated by means of in situ hybridization and immunostaining of whole mounts of animal or vegetal halves that the previously identified bep 1 and bep4 RNAs and their proteins are located in the animal part of the unfertilized egg and much less in the vegetal part. The addition of Fabs against BEP1 and BEP4 causes exogastrulation.

Cloning, expression, and localization of a new member of a Paracentrotus lividus cell surface multigene family.

We have isolated and characterized a cDNA clone corresponding to a new member of bep (butanol, extracted, proteins) Paracentrotus lividus multigene family coding for cell surface proteins. The cDNA, called bep3, encodes a 370 amino acid protein and shares the same structural organization in the coding region with other members of the same gene family already characterized. Expression of this clone studied by Northern blot and by whole mount hybridization shows that the bep3 messenger is transcribed during oogenesis and utilized till the gastrula stage, whereas at the prism stage, unlike other members of the same gene family, new synthesis of messenger occurs. By whole mount hybridization spatial distribution of bep3 messenger in egg and embryos is established. This messenger appears located in the animal half of the unfertilized egg and moves to the cortical zone after fertilization; it is not present in the structures derived by the vegetal part of the embryo, such as the micromeres of the 16-cell stage, the primary mesenchyme cells of the blastula, and the primary intestine of the gastrula. At the prism stage instead, hybridization of bep3 messenger is restricted to the part of the embryo that will give origin to the oral region as successively confirmed by hybridization at the pluteus stage. The result of whole mount hybridization was confirmed by Northern blot hybridization of separated meso-macromere and micromere RNAs. A Southern blot experiment demonstrates that bep3 is codified by a single copy gene. Conservation of the bep multigene family in several Mediterranean and Japanese sea urchin species has also been analyzed.

Effect of retinoic acid and valproate on sea urchin development.

The effect of valproate and retinoic acid on Paracentrotus lividus development and fertilization is reported. Retinoic acid at concentrations between 5 microM and 7 microM causes severe disturbance of development and at 10 microM complete embryo degeneration if added in the period from fertilization on. Concentrations up to 7 microM are uneffective if retinoic acid addition is started 16 h after fertilization. Valproate at concentrations between 1 mM and 5 mM causes severe disturbances of development if added from 5 min after fertilization on. Concentrations of 20 mM are lethal to all embryos. If the drug is added 16 h after fertilization no effect is observed up to the concentration of 10 mM. The effect of valproate is specific, because its isomere 2-en at 10 mM has very little effect on development. Both drugs affect fertilization only at higher concentrations: more than 1 microM for retinoic acid and more than 10 mM for valproate.

Effect of doxorubicin and phenytoin on sea urchin development.

The effect of doxorubicin and phenytoin on Paracentrotus lividus development and fertilization is reported. Doxorubicin at concentrations between 1.4 and 1.8 microM causes severe disturbances of development and at 2.0 microM, complete embryonic degeneration, if added in the period from fertilization on. Concentrations up to 20 microM are ineffective on development if the treatment is started at 12 h after fertilization. Phenytoin at concentrations between 4.0 and 7.0 microM causes severe disturbances of development, if added from 5 min after fertilization on. Concentrations of 10 microM are lethal to all embryos. Again, if the drug is added at 12 h after fertilization, no effect is observed up to the concentration of 20 microM. Both drugs are ineffective on the fertilization process up to the concentration of 20 microM for doxorubicin and of 200 microM for phenytoin.

Spatial distribution of two maternal messengers in Paracentrotus lividus during oogenesis and embryogenesis.

We demonstrated that two mRNAs that are synthesized during the vitellogenic period of oogenesis and that code for cell surface proteins are asymmetrically distributed in the unfertilized egg of Paracentrotus lividus. At fertilization, these RNAs rapidly localize in the cortical zone at the animal pole of the egg. They are then detected in the mesomeres and the macromeres, but not in the micromeres, and thereafter are found in the ectoderm but not in the vegetal plate, mesenchyme cells, or early intestine. They disappear in late gastrula. The proteins synthesized by these mRNAs show the same territorial location during the period examined here, which included the unfertilized egg and the 16-blastomere stage. These conclusions were reached on the basis of in situ hybridization and immunostaining experiments, as well as Northern and Western blot analyses of isolated blastomeres. The possible significance of this asymmetric distribution of these two mRNAs and proteins in the establishment of the animal/vegetal axis is discussed.

Identification of a protein encoded by a mouse simple repeated sequence.

In order to study the possibility that a mouse repeated 'simple sequence', containing an ORF, could encode a protein, we inserted a fragment of a cDNA clone into the expression vector pEX31C. The fragment containing the short sequence 'CAGAGAGG' was expressed as MS2 polymerase fusion protein in Escherichia coli. This fusion protein (H5fp) was injected subcutaneously into rabbit and the corresponding polyclonal antibodies generated. Western blot analysis of proteins extracted from different mouse tissues established that anti H5fp antibodies recognized, in vivo, an antigen of 28 KDa. Immunolocalization with anti H5fp antibodies showed the presence of the related antigen in the cytoplasm of the examined sections. An hypothetical secondary structure for the protein was predicted by the Chou and Fasman method.