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We have localized, byin situ hybridization, two messengers, called bepl and bep4 (butanol extracted proteins), coding for cell surface proteins, involved in cell interaction, in sections of eggs of the sea urchinP. lividus. These maternal RNAs are spatially distributed in a gradient with a maximum at one pole of the egg. These results represent the first demonstration of the existence of a gradient of informational molecules in the sea urchin egg,i.e., the biological material for which the gradient theory was first proposed.

A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which-as a consequence-turns out to be non-toxic.

We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.

Amyloid beta peptide (Abeta) is the major component of amyloid plaques in the brain of individuals affected by Alzheimer's disease (AD). The formation of the plaques is due to an overproduction of Abeta by APP processing, its precursor, and to its ability to convert under specific conditions from its soluble form into highly ordered fibrillar aggregates. Although neuronal degeneration occurs near the amyloid plaques, some studies have suggested that intermediates such as protofibrils or simple oligomers are also involved in AD pathogenesis and even appear to be the more dangerous species in the onset of the pathology. Further, toxic properties of aggregates of different size have been investigated and the obtained results support the hypothesis that different aggregate sizes can induce different degeneration pathways. In the present review some of the knowledge about the biochemical routes of Abeta processing and production and the relationship among Abeta and oxidative stress, metal homeostasis, inflammatory process, and cell death are summarized. Moreover, current strategies addressing both fibrillogenesis process and different Abeta altered biochemical pathways utilized for therapies are described.

Oxidative stress and dysfunctional mitochondria are among the earliest events in AD, triggering neurodegeneration. The use of natural antioxidants could be a neuroprotective strategy for blocking cell death. Here, the antioxidant action of ferulic acid (FA) on different paths leading to degeneration of recombinant beta-amyloid peptide (rAbeta42) treated cells was investigated. Further, to improve its delivery, a novel drug delivery system (DDS) was used. Solid lipid nanoparticles (SLNs), empty or containing ferulic acid (FA-SNL), were developed as DDS. The resulting particles had small colloidal size and highly negative surface charge in water. Using neuroblastoma cells and rAbeta42 oligomers, it was demonstrated that free and SLNs-loaded FA recover cell viability. FA treatment, in particular if loaded into SLNs, decreased ROS generation, restored mitochondrial membrane potential (Deltapsi(m)) and reduced cytochrome c release and intrinsic pathway apoptosis activation. Further, FA modulated the expression of Peroxiredoxin, an anti-oxidative protein, and attenuated phosphorylation of ERK1/2 activated by Abeta oligomers.

Two cDNA clones related to Paracentrotus lividus butanol-extracted proteins, presumably belonging to cell surface proteins, were isolated by a lambda gt11 expression library of ovary poly A+ RNA. These clones, called bep1 and bep4, of 1,110 and 1,071 bp, respectively, belong to a multigene family. By sequencing analysis, a special structural organization in the coding region is detected. A single copy region is inserted between two regions different from each other but similar in the two clones, which constitute two perfectly preserved domains in the genome and are not always present together in the various members of this gene family. The bep1 and bep4 clones derive from two single genes that are polymorphic in the sea urchin genome. Expression of these clones was studied by Northern blot analysis. Both bep1 and bep4 are transcribed during oogenesis into mRNAs of 1.4 kb, which are stored in eggs and utilized during early embryogenesis. None of these RNAs is, in fact, detectable after the gastrula stage. Moreover, the transcripts of three other members of the family are present in eggs and at the 32 cell stage, but they are also synthesized in the early developmental stages.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. In this article, the suitability of cationically modified solid-lipid nanoparticles (SLN) as a nonviral vector for gene delivery was investigated, in order to obtain stable materials able to condense RNA. Cationic SLN were produced by microemulsion using Compritol ATO 888 as matrix lipid, Pluronic F68 as tenside, and dimethyldioctadecylammonium bromide (DDAB) as cationic lipid. The resulting particles were approximately 100 nm in size and showed a highly positive surface charge (+41 mV) in water. Size and shape were further characterized by scanning electron microscopy (SEM) measurements. Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex the in vitro transcribed Paracentrotus lividus bep3 RNA, we utilized both light scattering and gel mobility experiments, and protection by nuclease degradation was also investigated. By microinjection experiment, we demonstrated that the nanoparticles do not inference with the viability of the P. lividus embryo and the complex nanoparticles-bep3 permits movement of the RNA during its localization in the egg, suggesting that it could be a suitable system for gene delivery. Taken together, all these results indicate that the cationic SNL are a good RNA carrier for gene transfer system and the sea urchin a simple and versatile candidate to test biological properties of nanotechnology devices.

In Paracentrotus lividus eggs, there are RNAs localized at the animal and vegetal poles. During the first cell division, some of these RNAs are associated with the mitotic spindle, whereas others are free in the cytoplasm. Among the RNAs bound to mitotic apparatus (MA), we have found the mitochondrial 16S rRNA. By immunohistochemistry we have also detected hsp60, a mitochondrial membrane protein, localized around the MA, suggesting that the entire mitochondria are associated with it. Western blotting of proteins prepared by cellular fractionation after detergent treatment of P. lividus eggs revealed that both hsp60 and cytochrome c are not associated with cytoskeletal elements. All the above data have been confirmed by immunoblot analyses of preparations of microtubules and MA in which the presence of hsp60 and cytochrome c were detected only in the MA fraction. Moreover, mitochondrial succinate dehydrogenase activity was determined in MA and cytoplasm fractions during the first cell division, and the localization and vitality of the organelles were also confirmed by in vivo staining with Mito red. A possible role for mitochondria in the asymmetric distribution of RNAs and in cell division is discussed.

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.

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 immunohistochemestry after isolation of the mitotic apparatus.

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.

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.

The production of heat-shock proteins in sea urchin embryos is accompanied by the appearance at the polysomal level of their relative mRNAs, as shown by their translation in a cell-free system; thus suggesting that the regulation of their production occurs at a transcriptional level. The mechanism for the inhibition of the bulk protein synthesis and for its reversal on the other hand should be looked for at a posttranscriptional level, since both these phenomena occur also in the presence of actinomycin D. The heat-shock proteins produced as early as at the mesenchyme blastula stage persist within the embryo at least till the pluteus stage.

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 lenghts 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.

Clinical and experimental biomedical studies have shown Type 2 diabetes mellitus (T2DM) to be a risk factor for the development of Alzheimer's disease (AD). This study demonstrates the effect of metformin, a therapeutic biguanide administered for T2DM therapy, on β-amyloid precursor protein (APP) metabolism in in vitro, ex vivo and in vivo models. Furthermore, the protective role of insulin against metformin is also demonstrated. In LAN5 neuroblastoma cells, metformin increases APP and presenilin levels, proteins involved in AD. Overexpression of APP and presenilin 1 (Pres 1) increases APP cleavage and intracellular accumulation of β-amyloid peptide (Aβ), which, in turn, promotes aggregation of Aβ. In the experimental conditions utilized the drug causes oxidative stress, mitochondrial damage, decrease of Hexokinase-II levels and cytochrome C release, all of which lead to cell death. Several changes in oxidative stress-related genes following metformin treatment were detected by PCR arrays specific for the oxidative stress pathway. These effects of metformin were found to be antagonized by the addition of insulin, which reduced Aβ levels, oxidative stress, mitochondrial dysfunction and cell death. Similarly, antioxidant molecules, such as ferulic acid and curcumin, are able to revert metformin's effect. Comparable results were obtained using peripheral blood mononuclear cells. Finally, the involvement of NF-κB transcription factor in regulating APP and Pres 1 expression was investigated. Upon metformin treatment, NF-κB is activated and translocates from the cytoplasm to the nucleus, where it induces increased APP and Pres 1 transcription. The use of Bay11-7085 inhibitor suppressed the effect of metformin on APP and Pres 1 expression.

Fibril deposit formation of amyloid beta-protein (Abeta) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Abeta oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Abeta small oligomers and fibrillar aggregates to simulate the action of diffusible Abeta oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Abeta. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Abeta peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.

Experiments of short term and long term labeling of the nuclear and cytoplasmic RNA of sea urchin blastulae indicate that RNA capping occurs in the nucleus and are in agreement with the hypothesis that capped RNA is transferred to the cytoplasm.

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.

Alzheimer’s disease (AD) is an aging-related multi-factorial disorder to which metabolic factors contribute at what has canonically been considered a centrally mediated process. Although the exact underlying mechanisms are still unknown, obesity is recognized as a risk factor for AD and the condition of insulin resistance seems to be the link between the two pathologies. Using mice with high fat diet (HFD) obesity we dissected the molecular mechanisms shared by the two disorders. Brains of HFD fed mice showed elevated levels of APP and Aβ 40 /Aβ 42 together with BACE, GSK3β and Tau proteins involved in APP processing and Aβ accumulation. Immunofluorescence, Thioflavin T staining experiments, confirmed increased Aβ generation, deposition in insoluble fraction and plaques formation in both the hippocampus and the cerebral cortex of HFD mice. Presence of Aβ 40 /Aβ 42 in the insoluble fraction was also shown by ELISA assay. Brain insulin resistance was demonstrated by reduced presence of insulin receptor (IRs) and defects in Akt-Foxo3a insulin signaling. We found reduced levels of phospho-Akt and increased levels of Foxo3a in the nuclei of neurons where proapototic genes were activated. Dysregulation of different genes related to insulin resistance, especially those involved in inflammation and adipocytokines synthesis were analyzed by Profiler PCR array. Further, HFD induced oxidative stress, mitochondrial dysfunction and dynamics as demonstrated by expression of biomarkers involved in these processes. Here, we provide evidence that obesity and AD markers besides insulin resistance are associated with inflammation, adipokine dyshomeostasis, oxidative stress and mitochondrial dysfunction, all mechanisms leading to neurodegeneration.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder, characterized by loss of memory and impairment of multiple cognitive functions. Amyloid beta peptide (Aβ) is the main component of amyloid plaques observed in the brain of individuals affected by AD. Oxidative stress and mitochondrial dysfunction, induced by Aβ, are among the earliest events in AD, triggering neuronal degeneration and cell death. Use of natural molecules with antioxidant properties could be a suitable strategy for inhibiting the cell death cascade. Here, by employing the sea urchin Paracentrotus lividus as a model system, and Aβ oligomers, we tested the effectiveness of ferulic acid (FA), a natural antioxidant, as a putative AD neuroprotective compound. By microscopic inspection we observed that FA is able to reverse morphological defects induced by Aβ oligomers in P. lividus embryos. In addition, FA is able to neutralize reactive oxygen species (ROS), recover mitochondrial membrane potential, and block apoptotic pathways. Moreover, this model system has allowed us to obtain information about down- or up-regulation of some key molecules--Foxo3a, ERK, and p53--involved in the antioxidant mechanism.

The success of biomedical researches has led to improvement in human health and increased life expectancy. An unexpected consequence has been an increase of age-related diseases and, in particular, neurodegenerative diseases. These disorders are generally late onset and exhibit complex pathologies including memory loss, cognitive defects, movement disorders and death. Here, it is described as the use of simple animal models such as worms, fishes, flies, Ascidians and sea urchins, have facilitated the understanding of several biochemical mechanisms underlying Alzheimer's disease (AD), one of the most diffuse neurodegenerative pathologies. The discovery of specific genes and proteins associated with AD, and the development of new technologies for the production of transgenic animals, has helped researchers to overcome the lack of natural models. Moreover, simple model systems of AD have been utilized to obtain key information for evaluating potential therapeutic interventions and for testing efficacy of putative neuroprotective compounds.