RNA binding proteins: putative regulative role in mitochondrial proteins expression.

Mitochondrial protein expression in Ratt and in human cells

In the present paper, we analyzed some aspects of the post-transcriptional regulation of two COX (cy-tochrome c oxidase) subunits, i.e. mitochondrion-encoded COXIII and nucleus-encoded COXIV. In particular, by T1 RNase protection assays, we found two proteins, present in mitochondrial extracts from adult rat brain and testis, able to bind COXIII mRNA. We also found cytoplasmic proteins present in testis, kidney and heart extracts that bind COXIV mRNA. Moreover, to study the expression of mitochondrial proteins in tumor cells, we quantitated COXIV and Hsp60 chaperonin abundance in two epithelial cell lines from human breast, one neoplastic (MDA-MB231) and the other immortalized and non-tumor…

Mitochondrial proteins regulation in Rattus norvegicus and human cells

Il trasferimento di molecole tra cellule cerebrali è mediato anche da vescicole extracellulari

Correlation between polymorphism of TYMS gene and toxicity response to treatment with 5-fluoruracil and capecitabine

Tumorigenesis is a multiphasic process in which genetic alterations guide the progressive transformation in cancer cells1. In order to evaluate the possible correlation between some gene variants and the risk of the toxicity development onset, two of the polymorphisms of the thymidylate synthase (TYMS), rs34743033 (2R/3R) and rs16430 (DEL/INS) were investigated. We enrolled in our study 47 patients from the Hospital of Sicily. Our preliminary findings suggest that there could be a linkage between the genotypes discussed and the development of the toxicity following the chemotherapy treatment. These results need to be confirmed by further studies, however this short paper offers some initial…

A 3D‑scaffold of PLLA induces the morphological differentiation and migration of primary astrocytes and promotes the production of extracellular vesicles

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly-L-lactic acid (PLLA) via thermally-induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a…

Metodo per la purificazione da sistemi di produzione batterici di proteine ricombinanti attive

Un metodo per la produzione e purificazione da sistemi di produzione batterici di proteine ricombinanti attive con pre-sequenza di sei istidine.

Possible mechanisms regulating the expression of nuclear and mitochondrial genes

Assembly of the ribonucleoprotein complex containing the mRNA of the β-subunit of the mitochondrial H+-ATP synthase requires the participation of two distal cis-acting elements and a complex set of cellular trans-acting proteins

The mRNA encoding the beta-subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) is localized in an approx. 150 nm structure of the hepatocyte of mammals. In the present study, we have investigated the cis- and trans-acting factors involved in the generation of the ribonucleoprotein complex containing beta-F1-ATPase mRNA. Two cis-acting elements (beta1.2 and 3'beta) have been identified. The beta1.2 element is placed in the open reading frame, downstream of the region encoding the mitochondrial pre-sequence of the protein. The 3'beta element is the 3' non-translated region of the mRNA. Complex sets of proteins from the soluble and non-soluble fractions of the liver interact with t…

RNA-binding proteins expressed in differentiating rat astrocytes

Replication-independent expression of H1˚ and H3.3 histone variants is probably regulated by different RNA-binding proteins

DNA in eukaryotes is wrapped around core histones to form nucleosomes, the basic units of chromatin. The linker histones H1 bind DNA where it enters and leaves the nucleosome, thus stabilizing higher order structures. Chromatin is a dynamic complex, modulated by different processes such as DNA-methylation, post-translational modifications of histones, and incorporation of specific histone variants. Throughout rat brain development, expression of H1° and H3.3 histone variants is mainly regulated at the post-transcriptional level. These proteins are of interest for their possible involvement in the replication-independent chromatin remodelling induced by extracellular stimuli. We previously c…

Aquaporin-4 distribution in control and stressed astrocytes in culture and in the cerebrospinal fluid of patients with traumatic brain injuries

Distribution of aquaporin-4 (AQP4) was studied by western analysis and immunofluorescence in rat astrocytes exposed to either hypothermic (30 °C) or hyperosmolar (0.45 M sucrose) stress, and in the cerebrospinal fluid (CSF) of patients who suffered traumatic brain injury (TBI). CSF was obtained from 5 healthy subjects and from 20 patients suffering from severe TBI. CSF samples were taken at admission and on days 3 and 5-7. Here we report that, in response to both hypothermia and hyperosmolar stress, AQP4 was markedly reduced in cultured astrocytes. We also found that AQP4 significantly increased in patients with severe brain injury in respect to healthy subjects (P < 0.002). AQP4 in CSF rem…

Physical Activity and Brain Health.

Physical activity (PA) has been central in the life of our species for most of its history, and thus shaped our physiology during evolution. However, only recently the health consequences of a sedentary lifestyle, and of highly energetic diets, are becoming clear. It has been also acknowledged that lifestyle and diet can induce epigenetic modifications which modify chromatin structure and gene expression, thus causing even heritable metabolic outcomes. Many studies have shown that PA can reverse at least some of the unwanted effects of sedentary lifestyle, and can also contribute in delaying brain aging and degenerative pathologies such as Alzheimer&#8217;s Disease, diabetes, and multiple s…

Toxic effects on astrocytes of extracellular vesicles from CSF of multiple sclerosis patients: a pilot in vitro study.

Multiple sclerosis (MS) is an autoimmune and degenerative disorder of the central nervous system (CNS) that causes a progressive loss of motor and cognitive perfor-mances. Moreover, since the earlier phases, axonal loss as well as neuronal degener-ation and a failure of oligodendrocytes to promote myelin repair have been demon-strated. In previous studies, it has been shown that the treatment of rat neuronal primary cultures with serum from MS patients can be toxic for neurons. Here we report a pilot investigation showing that CSF from patients contains extracellular vesicles (EVs) able to induce cell death in rat cultured astrocytes. Although these data are still preliminary, they suggest …

Regolazione dell’espressione delle subunità III e IV della citocromo c ossidasi

Extracellular Membrane Vesicles as Vehicles for Brain Cell-to-Cell Interactions in Physiological as well as Pathological Conditions.

Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping…

PEP-19 and LPI camstatins are RNA-binding proteins

Oligodendroglioma cells shed microvesicles which contain TRAIL as well as molecular chaperones and induce cell death in astrocytes.

Microvesicles (MVs) shed from G26/24 oligodendroglioma cells were previously reported to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and eventually neuronal apoptosis, when added to primary cultures of rat cortical neurons. These effects were reduced but not abolished by functional monoclonal antibodies against Fas-L. In order to investigate whether MVs contain other factors able to induce cell death, we tested them for TRAIL and found clear evidence of its presence in the vesicles. This finding suggests the possibility that Fas-L and TRAIL cooperate in inducing brain cell death. Aimed at understanding the route through which the vesicles deliver their mess…

Pippin protein expression changes during cell differentiation

PIPPin is a CSD-containing protein with the ability to interact both with mRNAs encoding histone variants and chromatin. A major fraction of chromatin-bound PIPPin is sumoylated and sumoylation seems to be controlled by thyroid hormones, both in vivo and in vitro. We studied its expression in different tissues and cell lines and even in tumor cells and found that, even if more expressed in the brain respect to other tissues of the adult rat, it is also expressed in brain tumors and in cell lines as different as kidney NRK cells and PC12. The expression of the protein is strongly increased by treatments that induce differentiation, such as treatment of PC12 with NGF. We also found an increas…

EXPRESSION OF PIPPIN PROTEIN AND CELL DIFFERENTIATION.

We previously described a CSD-containing protein that seemed to bind mRNAs encoding histone variants and was present both in the nucleus and in the cytoplasm of specific populations of brain cells. Since other CSD-containing proteins have the ability to interact both with RNA and chromatin, we investigated the possibility that PIPPin binds to chromatin and indeed found that about 50% of nuclear PIPPin cannot be extracted from nuclei with salt and is instead extracted with acid, together with histones. Interestingly, a major fraction of chromatin-bound PIPPin is sumoylated and sumoylation seems to be controlled by thyroid hormones, both in vivo and in vitro. In order to study the functions o…

RNA-binding CSD-C2 protein and its interactors in nerve cell differentiation

RNA-Binding Proteins which interact with mRNAs for H1° and H3.3 histone variants

Oligodendroglioma cells synthesize the differentiation-specific linker histone H1˚ and release it into the extracellular environment through shed vesicles

Chromatin remodelling can be involved in some of the epigenetic modifications found in tumor cells. One of the mechanisms at the basis of chromatin dynamics is likely to be synthesis and incorporation of replacement histone variants, such as the H1° linker histone. Regulation of the expression of this protein can thus be critical in tumorigenesis. In developing brain, H1° expression is mainly regulated at the post-transcriptional level and RNA-binding proteins (RBPs) are involved. In the past, attention mainly focused on the whole brain or isolated neurons and little information is available on H1° expression in other brain cells. Even less is known relating to tumor glial cells. In this st…

H1.0 Linker Histone as an Epigenetic Regulator of Cell Proliferation and Differentiation

H1 linker histones are a class of DNA-binding proteins involved in the formation of supra-nucleosomal chromatin higher order structures. Eleven non-allelic subtypes of H1 are known in mammals, seven of which are expressed in somatic cells, while four are germ cell-specific. Besides having a general structural role, H1 histones also have additional epigenetic functions related to DNA replication and repair, genome stability, and gene-specific expression regulation. Synthesis of the H1 subtypes is differentially regulated both in development and adult cells, thus suggesting that each protein has a more or less specific function. The somatic variant H1.0 is a linker histone that was recognized…

RNA binding proteins in brain cells differentiation

Extracellular Vesicles Shed by Melanoma Cells Contain a Modified Form of H1.0 Linker Histone and H1.0 mRNA-binding Proteins

Extracellular vesicles (EVs) are shed in the extracellular environment by both prokaryotes and eukaryotes. Although produced from both normal and cancer cells, malignant cells release a much higher amount of EVs, which also contain tumor-specific proteins and RNAs. We previously found that G26/24 oligodendroglioma cells shed EVs that contain the pro-apoptotic factors FasL and TRAIL1-2. Interestingly, G26/24 release, via EVs, extracellular matrix remodelling proteases3, and H1° histone protein4, and mRNA. To shed further light on the role of EVs in discarding proteins and mRNAs otherwise able to counteract proliferative signals, we studied a melanoma cell line (A375). We found that also thes…

Cell-to-cell communications among brain cells by extracellular vesicles

Rat PIPPin is probably part of a large complex of RNA-binding proteins

Throughout rat brain development, expression of histones variants is mainly regulated at the post-transcriptional level. We previously cloned two cDNAs encoding, respectively, PIPPin (or CSD-C2), a brain-enriched protein able to bind the 3’end of H1° and H3.3 mRNAs, and LPI (longer isoform of PEP-19). Both PEP-19 and LPI are brain-specific. By western blot, we found that PIPPin expression in PC12 cells is enhanced by NGF-induced differentiation. We investigated the RNA-binding properties of the three proteins using their 6 histidine-tagged recombinant fusions and found that they all bind H1° and H3.3 RNAs. Since PEP-19 and LPI are camstatins, we also analyzed whether calmodulin could interf…

RNAbinding proteins involved in nerve cell differentiation.

Proteine di fusione, metodo di preparazione, e relativi anticorpi per la diagnosi di patologie mitocondriali

ATTIVITÀ RNA LEGANTE DELLA PROTEINA CSD-C2 RICOMBINANTE PRODOTTA IN ESCHERICHIA COLI RNA BINDING ACTIVITY OF RECOMBINANT CSD-C2 PROTEIN EXPRESSED IN ESCHERICHIA COLI

Identification of nuclear proteins which interact with H1° mRNA.

In developing rat brain the synthesis of H1° histone is mainly regulated at posttranscriptional level and probably depends on RNA-binding proteins (RBPs) (1). We previously identified RBPs apparently specific for this messenger (2) and cloned two novel proteins by screening an expression cDNA library by binding to radiolabeled RNA (3-10). Here we report the use of biotinylated H1° RNA as bait to isolate by chromatography nuclear proteins which interact with H1° mRNA. We identified by mass spectrometry abundant RBPs and molecular chaperones. By western blot we also evidenced, among the RNA-bound proteins, the cold shock domain-containing protein 2 (CSD-C2, also know as PIPPin), a brain-enric…

STUDIO DELL’ESPRESSIONE DI GENI CODIFICANTI PROTEINE MITOCONDRIALI IN RATTUS NORVEGICUS.

Extracellular membrane vesicle shedding and the blood-brain barrier

Extracellular Vesicle‐Associated RNA as a Carrier of Epigenetic Information

Post-transcriptional regulation of messenger RNA (mRNA) metabolism and subcellular localization is of the utmost importance both during development and in cell differentiation. Besides carrying genetic information, mRNAs contain cis-acting signals (zip codes), usually present in their 5'- and 3'-untranslated regions (UTRs). By binding to these signals, trans-acting factors, such as RNA-binding proteins (RBPs), and/or non-coding RNAs (ncRNAs), control mRNA localization, translation and stability. RBPs can also form complexes with non-coding RNAs of different sizes. The release of extracellular vesicles (EVs) is a conserved process that allows both normal and cancer cells to horizontally tran…

Hsp 56 mRNA in Paracentrotus lividus embryos binds to a mitochondrial protein

We previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is constitutively expressed during development, that it has a specific territorial distribution, both in normal and heat-shocked embryos, and that its amount increases after heat shock [Roccheri MC, Patti M, Agnello M, Gianguzza F, Carra E, Rinaldi AM. Localization of mitochondrial Hsp56 chaperonin during sea urchin development. Biochem Biophys Res Commun 2001;287:1093-98] and cadmium treatment [Roccheri MC, Agnello M, Boneventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004;321:80-7]. In this study, we looked at Hsp56 mRNA …

Mitochondria and Development

DEVELOPMENT OF A HOME-BASED TRAINING PROGRAM FOR PATIENTS WITH PARKINSON DISEASE: NEUROBIOLOGICAL AND MOTOR SKILLS EFFECT

Neurodegenerative diseases are inherited diseases of the central nervous system, which cause progressive damage to specific populations of neurons and lead to a deterioration in the quality of life (1,2). Parkinson disease (PD) is a progressive neurodegenerative disease and is the second most common after AD, and is characterized by postural instability, tremor and rigidity. Moreover, physical activity can reduces risk of other geriatric diseases such as diabetes, hypertension, osteoporosis and cardiovascular disease, which may also contribute to PD pathogenesis (3). We enrolled 12 subjects (age: 62.74 ± 4.94; height: 175,5cm ± 7,41 cm; weight: 75,5 ± 17,95 kg) affected by PD. An home-based…

Analysis of interaction between messenger RNA encoding H3.3 histone variant and pippin protein by biolayer interferometry

Analysis of cytochrome oxidase subunits III and IV expression in debveloping rat brain.

Genomic and non-genomic mechanisms of action of thyroid hormones and their catabolite 3,5-diiodo-l-thyronine in Mammals

Since the realization that the cellular homologs of a gene found in the retrovirus that contributes to erythroblastosis in birds (v-erbA), i.e. the proto-oncogene c-erbA encodes the nuclear receptors for thyroid hormones (THs), most of the interest for THs focalized on their ability to control gene transcription. It was found, indeed, that, by regulating gene expression in many tissues, these hormones could mediate critical events both in development and in adult organisms. Among their effects, much attention was given to their ability to increase energy expenditure, and they were early proposed as anti-obesity drugs. However, their clinical use has been strongly challenged by the concomita…

Two immortalized rat astrocyte cell lines as in vitro model for specific cell proliferation studies: cytogenetic and epigenomic characterization and diversification

Here we report differences between: 1) a heterogeneous population of primary rat brain astrocytes (Primary), in culture since several years ago, and 2) a cloned cell line (Clone), obtained from the Primary cells. Both populations maintain astrocyte morphology but, according to cytogenetic and epigenomic characterization, differ for the chromosomal asset from rat normal cells (42 chromosomes): Primary cells show mostly a bimodal karyotype with 41 or 43 chromosomes, and Clone has a unique-modal karyotype of 43 chromosomes. Interestingly, we also found that both cell lines show genome-wide DNA hypomethylation, with Clone showing even more pronounced demethylation respect to Primary cells. Thes…

Hsp56 expression in Paracentrotus lividus embryos

Histone H1° and H3.3 RNA-binding proteins identified in the developing rat brain

Histone H1° RNA-binding proteins in developing rat brain.

Lactate Threshold Training Program on Patients with Multiple Sclerosis: A Multidisciplinary Approach

Physical activity could play a key role in improving the quality of life, particularly in patients with nervous system diseases such as multiple sclerosis (MS). Through lactacid anaerobic training, this study aims to investigate the effects at a bio-psycho-physical level to counteract the chronic fatigue associated with the pathology, and to improve mental health at a psychological and neurotrophic level. Eight subjects (age: 34.88 ± 4.45 years) affected by multiple sclerosis were involved. A lactate threshold training program was administered biweekly for 12 weeks at the beginning of the study (T0), at the end of the study (T1) and at 9 months after the end of the study (T2), with physical…

Hsp56 protein and mRNA distribution in normal and stressed P. lividus embryos

It was previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is con- stitutively expressed during development, that it increases after heat-shock and cadmium treatment, and that it has a specific territorial distribution, both in normal and heat-shocked embryos, as shown by immunolocalization experiments. In this work, we analyzed by Western blot the territorial distribution of the protein in plutei exposed to heat-shock or sublethal cadmium concentrations, and we found that Hsp56 increases in both ectodermal and en- dodermal cells. Moreover, by “in situ” hybridization, we looked at Hsp56 mRNA during normal development and under stress conditions. We found that th…

RNA-Binding Proteins as Epigenetic Regulators of Brain Functions and Their Involvement in Neurodegeneration.

A central aspect of nervous system development and function is the post-transcriptional regulation of mRNA fate, which implies time- and site-dependent translation, in response to cues originating from cell-to-cell crosstalk. Such events are fundamental for the establishment of brain cell asymmetry, as well as of long-lasting modifications of synapses (long-term potentiation: LTP), responsible for learning, memory, and higher cognitive functions. Post-transcriptional regulation is in turn dependent on RNA-binding proteins that, by recognizing and binding brief RNA sequences, base modifications, or secondary/tertiary structures, are able to control maturation, localization, stability, and tr…

Analysis of cytochrome c oxidase subunits III and IV in developing rat brain

Analysis of cytochrome oxidase subunits III and IV expression in developing rat brain.

Brain-specific RNA-binding protein

3D cultures of primary astrocytes on Poly-L-lactic acid scaffolds

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro tissues with morphological and functional features similar to the biological tissue of the human body. Polymeric materials can be used in contact with biological systems in replacing destroyed tissue by transplantation [1]. Several biopolymers, including poly L (lactic acid) (PLLA), have been used in biomedical applications to set scaffolds with ductile proprieties and biodegradation kinetics [2]. In particular, the PLLA scaffold topography mimics the natural extracellular matrix and makes it a good candidate for neural tissue engineering. We report about of 3D system the PLLA porous scaffolds prepar…

Co-culture of rat brain cells as a tool for studying cell-cell interactions

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) in response to interaction with other brain cells (astrocytes, pericytes and neurons). BCECs are characterized by tight junctions (TJ), maturation and stabilization of which require different proteins, such as occludin. When co-cultured with astrocytes and neurons, BCECs were found to form a monolayer resembling the natural BBB: paracellular flux of dopamine and sucrose (i.e. compounds which are unable to cross the BBB in vivo) significantly decreased (1), while the transendothelial electrical resistance (TEER) increased. In these conditions, BCECs produced a larger amount of occludin and tended to localize it at t…

An In Vitro Model of Glioma Development

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are…

3'-Untranslated regions of oxidative phosphorylation mRNAs function in vivo, as enhancers of translation

Recent findings have indicated that the 3´-untranslated region (3´-UTR) of the mRNA encoding the β-catalytic subunit of the mitochondrial H+-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255–5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849–855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3´-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3´-UTR of the mRNAs encoding the α- and β-subunits of the rat H+-ATP syn…

Extracellular membrane vesicles can mediate intercellular transfer of molecules

Many eukaryotic cell types, including neural cells, release into the extracellular environment vesicles of different sizes and composition. Neurons and astrocytes shed extracellular vesicles which contain FGF2 and VEGF and could be involved in interaction with endothelial cells, to form the blood-brain barrier. Also brain tumor cells, such as glioblastomas, release vesicles in the extracellular space. Microvesicles (MVs) shed from G26/24 oligodendro¬glioma cells were previously reported to contain FAS-L and to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and neuronal apoptosis, when added to primary cultures of rat cortical neurons. More recently, they were …

Cell-to-Cell Communication in Learning and Memory: From Neuro- and Glio-Transmission to Information Exchange Mediated by Extracellular Vesicles

Most aspects of nervous system development and function rely on the continuous crosstalk between neurons and the variegated universe of non-neuronal cells surrounding them. The most extraordinary property of this cellular community is its ability to undergo adaptive modifications in response to environmental cues originating from inside or outside the body. Such ability, known as neuronal plasticity, allows long-lasting modifications of the strength, composition and efficacy of the connections between neurons, which constitutes the biochemical base for learning and memory. Nerve cells communicate with each other through both wiring (synaptic) and volume transmission of signals. It is by now…

Extracellulare membrane vesicles as vehicles for cell-to-cell and cell-to-extracellular matrix functional interactions

Extracellular microvesicles as brain cell-to-cell communication carriers

Analysis of cytochrome c oxidase subunits III and IV expression in developing rat brain

Espressione del messaggero per l’Hsp56 in embrioni di Paracentrotus lividus

Mitochondrial protein expression in rattus norvegicus and human cells

Molecular Determinants of Malignant Brain Cancers: From Intracellular Alterations to Invasion Mediated by Extracellular Vesicles

Malignant glioma cells invade the surrounding brain parenchyma, by migrating along the blood vessels, thus promoting cancer growth. The biological bases of these activities are grounded in profound alterations of the metabolism and the structural organization of the cells, which consequently acquire the ability to modify the surrounding microenvironment, by altering the extracellular matrix and affecting the properties of the other cells present in the brain, such as normal glial-, endothelial- and immune-cells. Most of the effects on the surrounding environment are probably exerted through the release of a variety of extracellular vesicles (EVs), which contain many different classes of mol…

Confocal microscopy study of the distribution, content and activity of mitochondria during Paracentrotus lividus development

Summary In the present paper we applied confocal microscopy andfluorescence technologies for studying the distribution andthe oxidative activity of sea urchin ( Paracentrotus lividus )mitochondria during development, by in vivo incubating eggsand embryos with cell-permeant MitoTracker probes. Wecalculated, by a mathematical model, the intensity values, the variations of intensity, and the variation index of incorporatedfluorochromes. Data demonstrate that mitochondrial massdoes not change during development, whereas mitochondrialrespirationincreases.Inaddition,startingfrom16blastomeresstage, some regions of the embryo contain organelles moreactive in oxygen consumption. Introduction The con…

CHARACTERIZATION OF TRANSFORMED CELL LINES OBTAINED FROM PRIMARY RAT CORTICAL ASTROCYTES

Brain cancers are complex and heterogeneous; most of them derive from glial cells[1], and are called gliomas, further subdivided into astrocytomas, oligodendrogliomas, ependymomas and glioastrocytomas[2]. The malignant cells undergo modifications of their metabolism and behaviour, and acquire the ability to migrate along the blood vessels in small groups (model of the guerrilla war)[3], thus invading the surrounding brain parenchyma. Most important, they have the capacity to affect the surrounding microenvironment, by altering both the extracellular matrix and the properties of the normal cells present in the brain, including glial-, endothelial-, and immune-cells, further promoting cancer …

Mitochondrial protein expression in rat and human cells.

The dynamic properties of neuronal chromatin are modulated by triiodothyronine.

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cultured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the…

Aquaporins and Brain Tumors

Brain primary tumors are among the most diverse and complex human cancers, and they are normally classified on the basis of the cell-type and/or the grade of malignancy (the most malignant being glioblastoma multiforme (GBM), grade IV). Glioma cells are able to migrate throughout the brain and to stimulate angiogenesis, by inducing brain capillary endothelial cell proliferation. This in turn causes loss of tight junctions and fragility of the blood–brain barrier, which becomes leaky. As a consequence, the most serious clinical complication of glioblastoma is the vasogenic brain edema. Both glioma cell migration and edema have been correlated with modification of the expression/localization …

Lactate as a Metabolite and a Regulator in the Central Nervous System

More than two hundred years after its discovery, lactate still remains an intriguing molecule. Considered for a long time as a waste product of metabolism and the culprit behind muscular fatigue, it was then recognized as an important fuel for many cells. In particular, in the nervous system, it has been proposed that lactate, released by astrocytes in response to neuronal activation, is taken up by neurons, oxidized to pyruvate and used for synthesizing acetyl-CoA to be used for the tricarboxylic acid cycle. More recently, in addition to this metabolic role, the discovery of a specific receptor prompted a reconsideration of its role, and lactate is now seen as a sort of hormone, even invol…

Histone RNA-binding proteins in the rat brain

Microscopic evidence of the primary astrocytes' morphological differentiation and migration inside porous Poly-L-lactic acid 3D‑scaffolds

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro and/or in vivo tissues with morphological and functional features similar to the biological tissue of the human body [1]. In the attempt to construct suitable tissue models, a critical step is the setting of 3D scaffolds that mimic the supportive structures of a natural extracellular matrix microenvironment into which cells are normally embedded. In this context, the generation of 3D cultures of brain cells is of particular interest. For instance, the poly L‐lactic acid (PLLA) polymer is wildly used because of its biocompatible and biodegradable potential; the PLLA scaffold topography simulates the n…

Involvement of Thyroid Hormones in Brain Development and Cancer

Simple Summary Development and function of the mammalian brain clearly require precise regulation of gene expression at both the transcriptional and post-transcriptional level. Thyroid hormones have been recognized to play a fundamental role in these processes, by acting at multiple levels and in different brain cell types, through direct effects on transcription, mediated by nuclear receptors, and also by triggering transduction pathways at the plasma membrane. At the same time, due to their effects on proliferation, differentiation, and cell metabolism, thyroid hormones may have a critical role in different kinds of cancer, including brain cancer. Abstract The development and maturation o…

Identificazione di proteine che interagiscono con il messaggero dell’istone H1° nel cervello di ratto in sviluppo

Regulation of mRNA transport, localization and translation in the nervous system of mammals (Review).

Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5' or 3' to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3'-untranslated (3'-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on…

Shedding of extracellular membrane vesicles from both normal and tumor cells in culture

Tumor cells of different origins shed extracellular membrane vesicles (MVs), that contain angiogenetic- and pro-apoptotic-factors as well as matrix metalloproteases (MMPs). In addition, also neurons and astrocytes in culture produce VEGF- and FGF2- containing MVs, while oligodendroglioma (G26/24) cells release FasL-containing MVs that inhibit neurite sprouting and cause neuronal apoptosis. Starting from these observations, we have been analyzing composition of MVs produced by both normal and tumor cells in culture. We found that MVs from G26/24 cells contain TRAIL, Hsp70, and VEGF. We also traced the route of shed MVs, by adding vesicles that contain 35S-labeled proteins to unlabeled neuron…

RNA-binding activity of the rat calmodulin-binding PEP-19 protein and of the long PEP-19 isoform

Synthesis of H1˚ histone protein, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins, we have been searching for RNA-binding proteins involved in the post-transcriptional regulation of the H1˚ gene. We recently reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). Herein we show that LPI, as well as PEP-19, can bind H1˚ RNA. Moreover, in order to improve production…

Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenet…

Role of RNA-binding proteins in the replication-independent expression of H1° and H3.3 histone variants

Enzymatic Spermine Metabolites Induce Apoptosis Associated with Increase of p53, caspase-3 and miR-34a in Both Neuroblastoma Cells, SJNKP and the N-Myc-Amplified Form IMR5

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptot…