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Renata BATTINI

COLLABORATORE DI RICERCA
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Pubblicazioni

2023 - Linking endocannabinoid system, palmitoylethanolamide, and sarcopenia in view of therapeutic implications [Capitolo/Saggio]
Molinari, S.; Maretti, E.; Battini, R.; Leo, E.
abstract

Sarcopenia, a debilitating skeletal muscle disease closely connected with elderly, is becoming a major public health problem with the increasing of life expectancy. In the aim to found effective, targeted and side-effect-free therapies, understanding the endocannabinoid system (ECS) role in muscle homeostasis is of strategic importance. The skeletal muscle expresses all the ECS elements; in particular, a central role is played by the nuclear receptor PPARα and its main endogenous ligand, palmitoylethanolamide (PEA), an endocannabinoid-like molecule with an important anti-inflammatory effect. It is worth highlighting that in muscle the expression level of both PPARα receptor and its coactivator PGC1a, decreases with age, suggesting a causative relation between the lower PPARα function and sarcopenia. Therefore, the administration of PEA to the muscle can be a promising approach to counteract sarcopenia. In this regard, to promote the muscle targeting, innovative drug delivery systems, such as solid lipid nanoparticles, can be considered.

2022 - Design, Characterization, and In Vitro Assays on Muscle Cells of Endocannabinoid-like Molecule Loaded Lipid Nanoparticles for a Therapeutic Anti-Inflammatory Approach to Sarcopenia [Articolo su rivista]
Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Truzzi, Eleonora; Iannuccelli, Valentina; Leo, Eliana
abstract

Inflammatory processes play a key role in the pathogenesis of sarcopenia owing to their effects on the balance between muscle protein breakdown and synthesis. Palmitoylethanolamide (PEA), an endocannabinoid-like molecule, has been well documented for its anti-inflammatory properties, suggesting its possible beneficial use to counteract sarcopenia. The promising therapeutic effects of PEA are, however, impaired by its poor bioavailability. In order to overcome this limitation, the present study focused on the encapsulation of PEA in solid lipid nanoparticles (PEA-SLNs) in a perspective of a systemic administration. PEA-SLNs were characterized for their physico-chemical properties as well as cytotoxicity and cell internalization capacity on C2C12 myoblast cells. Their size was approximately 250 nm and the encapsulation efficiency reached 90%. Differential scanning calorimetry analyses demonstrated the amorphous state of PEA in the inner SLN matrix, which improved PEA dissolution, as observed in the in vitro assays. Despite the high internalization capacity observed with the flow cytometer (values between 85 and 94% after 14 h of incubation), the Nile Red labeled PEA-SLNs showed practically no toxicity towards myoblasts. Confocal analysis showed the presence of SLNs in the cytoplasm and not in the nucleus. These results suggest the potentiality provided by PEA-SLNs to obtain an innovative and side-effect-free tool in the medical treatment of sarcopenia

2022 - Palmitoylethanolamide-loaded Solid Lipid Nanoparticles for a therapeutic anti-inflammatory approach of Sarcopenia involving ECS [Relazione in Atti di Convegno]
Leo, Eliana Grazia; Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Iannuccelli, Valentina
abstract

2021 - Development of solid lipid nanoparticles for the delivery of an anti- inflammatory drug to muscle skeletal cells [Poster]
Maretti, Eleonora; Truzzi, Eleonora; Molinari, Susanna; Battini, Renata; Leo, Eliana Grazia
abstract

2019 - PIN1: a putative molecular target to protect skeletal muscle against age-related muscle loss [Relazione in Atti di Convegno]
Brocca, Lorenza; Grosso, Martina; Pezzini, Camilla; Semeghini, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Mucci, Adele; Righi, Valeria; Lorenzo Puri, Pier; Battini, Renata; Bottinelli, Roberto; Antonietta Pellegrino, Maria; Molinari, Susanna
abstract

Aging is associated with a progressive loss in skeletal muscle mass and strength, known as sarcopenia. Sarcopenia results in a decrease in mobility and an increased risk of developing chronic metabolic disease, thus it represents a major socio-economical problem. Age-related muscle loss cannot be consistently prevented by physical therapy and a pharmacologic therapy does not exist, probably because the molecular basis of this condition is still largely unknown. Many factors such as mitochondrial dysfunction, oxidative stress, inflammation, changes in the innervation of muscle fibers probably play an important role in age-related muscle decline. PIN1 is a widely expressed Peptydyl Prolyl cis/trans isomerase, involved in post-phosphorylation control of the function of multiple target proteins. Many evidences indicate that PIN1 controls signaling pathways involved in skeletal muscle wasting. Our results indicate that skeletal muscle of Pin1 KO mice is protected against muscle loss and weakness during aging. At the molecular level, we found 1) an increased expression of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), a transcription factor that promotes mitochondrial biogenesis, in skeletal muscle of Pin1 KO mice. Coherently with their resistance to muscle loss, we also found 2) an increase in the expression of the protein synthetic signaling proteins p70 ribosomal S6 kinase (S6K) and of its phosphorylated form in aged skeletal muscle of Pin1 KO mice compared to the wild type controls, suggesting that in these mice protein synthesis is maintained efficient. We also found 3) a significant decrease of Myostatin levels in skeletal muscle of aged Pin1 KO mice. It is well known that Myostatin is upregulated sarcopenia, it activates SMAD2/3 signaling and contributes to protein degradation and muscle atrophy. The transcriptional effects of Pin1 depletion on PGC1α and S6K genes must be mediated by a transcription factor. A putative candidate to mediate these effects is represented by the transcription factor Myocyte Enhancer Factor 2C (MEF2C), a known PIN1 target. In skeletal muscle cells a specific splice variant of MEF2C, MEF2C α1, activates the increase of skeletal muscle mass by activating the expression of IGF1 and S6K. These activities are repressed by its phosphorylation, that renders it a target for the inhibitory effect of PIN1 on its protein stability and activity. Coherently with these premises, we found 4) a decrease of MEF2C protein phosphorylation levels in aged KO mice compared to the control animals. This might at least partially contribute to the increased expression of PGC1α and of S6K. Our results indicate that PIN1 could represent a valuable pharmacological target to counteract age-related muscle loss, simultaneously modulating multiple targets in a concerted way.

2017 - Dynamic Phosphorylation of the Myocyte Enhancer Factor 2Cα1 Splice Variant Promotes Skeletal Muscle Regeneration and Hypertrophy [Articolo su rivista]
Baruffaldi, Fiorenza; Montarras, Didier; Basile, Valentina; De Feo, Luca; Badodi, Sara; Ganassi, Massimo; Battini, Renata; Nicoletti, Carmine; Imbriano, Carol; Musarò, Antonio; Molinari, Susanna
abstract

Abstract: The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases.

2015 - Phosphorylation and alternative splicing of MEF2C, a dual switch function in muscle regeneration [Abstract in Rivista]
Baruffaldi, Fiorenza; Badodi, Sara; De Feo, Luca; Ganassi, Massimo; Battini, Renata; Imbriano, Carol; Nicoletti, Carmine; Musarò, Antonio; Buckingham, Margaret; Montarras, Didier; Molinari, Susanna
abstract

Muscle regeneration is a multistep process that is regulated by a restricted number of transcription factors whose activity is modulated at multiple levels. However, how different layers of regulation are coordinated to promote adult myogenesis is not yet understood. Here we show that the MEF2C transcription factor controls multiple steps of muscle regeneration, including myogenic progression of satellite cells and muscle maturation of newly generated myofibers, exhibiting multiple functions that depend on alternative splicing and post-translational modifications. Inclusion of the 1 exon in Mef2c transcripts is upregulated in proliferating mouse satellite cells and in the early phases of muscle regeneration. The encoded MEF2C1 isoform stimulates expansion of primary myoblasts ex vivo and in vivo. The pro-proliferative activity of MEF2C is mediated by phosphorylation of two phosphoserines located in exon 1. Subsequent terminal differentiation and growth of newly formed myofibers are promoted by dephosphorylated MEF2C1 and MEF2C2. Our results thus reveal an important role for regulatory interactions between alternative splicing and post translational modifications of a single transcription factor in the control of the multilayered regulatory programs required for adult myogenesis.

2015 - Phosphorylation-dependent degradation of MEF2C contributes to regulate G2/M transition [Articolo su rivista]
Badodi, Sara; Baruffaldi, Fiorenza; Ganassi, Massimo; Battini, Renata; Molinari, Susanna
abstract

The Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation.

2014 - Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb. Ganassi M, Badodi S, Polacchini A, Baruffaldi F, Battini R, Hughes SM, Hinits Y, Molinari S. Biochim Biophys Acta. 2014 Jul;1839(7):559-70. doi: 10.1016/j.bbagrm.2014.05.003. Epub 2014 May 17. [Articolo su rivista]
Ganassi, Massimo; Badodi, Sara; Polacchini, A; Baruffaldi, Fiorenza; Battini, Renata; Hughes, Sm; Hinits, Y; Molinari, Susanna
abstract

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity

2013 - IL FATTORE DI TRASCRIZIONE MEF2C AI CROCEVIA TRA PROLIFERAZIONE, SVILUPPO E DIFFERENZIAMENTO MUSCOLARE [Poster]
Ferrari, Stefano; Battini, Renata; Magli, Alessandro; Angelelli, Cecilia; Badodi, Massimo Ganassi Sara; Baruffaldi, Fiorenza; Molinari, Susanna
abstract

Il principale interesse del laboratorio è lo studio della funzione della proteina MEF2C, un fattore di trascrizione abbondante nel tessuto muscolare scheletrico dove dirige il differenziamento terminale dei precursori miogenici (mioblasti). In specifico, ci occupiamo di definire i meccanismi che regolano la funzione di MEF2C nella progressione miogenica. Abbiamo identificato un meccanismo di repressione dell'attività di MEF2C nei mioblasti proliferanti che coinvolge la fosforilazione della proteina e la sua successiva interazione fosfo-specifica con l'enzima Pin1, una peptidil-prolil cis trans isomerasi che diminuisce la stabilità di MEF2C. Questo meccanismo repressivo è importante per inibire un differenziamento prematuro dei precursori miogenici che in tal modo possono amplificarsi. La repressione Pin1-dipendente di MEF2C è attiva sia nella linea cellulare miogenica C2C12 che in cellule satelliti, vale a dire mioblasti primari di muscolo adulto, responsabili dei meccanismi di rigenerazione in caso di danno muscolare. Ci proponiamo di valutare se questo meccanismo è alterato nella distrofia di Duchenne, dove è stata osservata una diminuita capacità proliferativa delle cellule satelliti. L'attività di MEF2C è anche regolata da meccanismi di splicing alternativo del corrispondente trascritto. Nel nostro laboratorio stiamo definendo le funzioni specifiche di due isoforme di splicing di MEF2C che si differenziano per la presenza di due esoni mutualmente esclusivi: esone alpha1 e alpha 2. I nostri dati preliminari indicano che le due varianti di splicing svolgono funzioni opposte nel corso della progressione miogenica, in particolare la variante alpha1 sembra essere importante nel controllare la progressione nel ciclo delle cellule muscolari mentre la variante alpha 2 sembra essenziale per la trascrizione muscolo-specifica. Infine abbiamo osservato che in Zebrafish, Danio rerio, Mef2c svolge un ruolo importante nel regolare lo sviluppo embrionale, specificamente nella determinazione dorso-ventrale dell'embrione, inoltre la sua attività viene modulata attraverso meccanismi di splicing anche in questo organismo.

2011 - Proline Isomerase Pin1 represses terminal differentiation and Myocyte Enhancer Factor 2C function in Skeletal Muscle Cells [Poster]
Magli, Alessandro; Ganassi, Massimo; Baruffaldi, Fiorenza; Badodi, Sara; Angelelli, Cecilia; Battini, Renata; Sal, Giannino Del; Molinari, Susanna
abstract

MEF2 (myocyte enhancer factor 2) transcription factors (MEF2A-D) are highly expressed in skeletal muscle cells, they bind to a conserved AT rich DNA sequence through their N-ter MADS and MEF2 domains and activate transcription via their C-ter transcriptional activation domains (TAD), the functional domains of MEF2C are indicated in Figure 1. MEF2 proteins interact with members of the MyoD family of basic helix–loop–helix (bHLH) proteins to establish a unique transcriptional code for skeletal muscle gene activation. Recent studies have revealed multiple signaling systems that stimulate and inhibit myogenesis by altering MEF2 phosphorylation and its association with other transcriptional cofactors. We show that the Pin1 isomerase, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds, interacts with phosphorylated MEF2C in muscle cells. This interaction requires two novel phospho-Ser-Pro motifs in MEF2C: Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenesis. Furthermore Pin1 modulates the skeletal muscle differentiation program because down-regulation of Pin1 markedly promotes myogenic differentiation. We suggest that Pin1 is a novel regulator of MEF2C function and muscle differentiation, it is expressed in muscle cells and a significant proportion of Pin1 in myotubes but not in myoblasts is excluded from the nucleus. We observed a reduction of phosphorylation of the Ser(98) and Ser(110) Pin1 binding sites in differentiated myocytes. Based on these results we propose a model in which, in proliferating myoblasts, Pin1, upon binding to phosphorylated nuclear MEF2C, leads to decreased levels and transcriptional activity of MEF2C. Upon induction of terminal differentiation, to establish a full activity of MEF2 proteins, a reduced Pin1-MEF2C association is required, possibly due to the relegation of Pin1 to the cytoplasm and to a reduced level of phosphorylation of Ser98 and Ser110.

2010 - Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding. [Articolo su rivista]
Cardinale, Daniela; O. M. H., Salo Ahen; Guaitoli, Giambattista; Ferrari, Stefania; Venturelli, Alberto; Franchini, Silvia; Battini, Renata; Ponterini, Glauco; R. C., Wade; Costi, Maria Paola
abstract

Due to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, and we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.

2010 - pDNA condensation capacity and in vitro gene delivery properties of cationic solid lipid nanoparticles [Articolo su rivista]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical–chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.

2010 - PROLINE ISOMERASE PIN1 REPRESSES TERMINAL DIFFERENTIATION AND MYOCYTE ENHANCER FACTOR 2C FUNCTION IN SKELETAL MUSCLE CELLS [Articolo su rivista]
Magli, Alessandro; Angelelli, Cecilia; Ganassi, Massimo; Baruffaldi, Fiorenza; V., Matafora; Battini, Renata; A., Bachi; G., Messina; A., Rustighi; G., Del Sal; S., Ferrari; Molinari, Susanna
abstract

Reversible proline-directedphosphorylation at Ser/Thr-Pro motifs has anessential role in myogenesis, a multistep processstrictly regulated by several signalling pathwaysthat impinge on two families of myogeniceffectors, the basic Helix Loop Helix (bHLH)myogenic transcription factors and the MyocyteEnhancer Factor 2 (MEF2) proteins. Thequestion of how these signals are deciphered bythe myogenic effectors remains largelyundefined. In this study we show that thepeptidyl-prolyl isomerase Pin1, which catalyzesthe isomerization of phosphorylated Ser/Thr-Propeptide bonds to induce conformational changesof its target proteins, acts as an inhibitor ofmuscle differentiation as its knock-down inmyoblasts promotes myotube formation. Withthe aim of clarifying the mechanism of Pin1function in skeletal myogenesis, we investigatedwhether MEF2C, a critical regulator of themyogenic program that is the endpoint of severalsignalling pathways, might serve as a/the targetfor the inhibitory effects of Pin1 on muscledifferentiation. We show that Pin1 interactsselectively with phosphorylated MEF2C inskeletal muscle cells, both in vitro and in vivo.The interaction with Pin1 requires two novelcritical pSer/Thr-Pro motifs in MEF2C, Ser98and Ser110, which are phosphorylated in vivo.Overexpression of Pin1 decreases MEF2Cstability and activity, and its ability to cooperatewith MyoD to activate myogenic conversion.Collectively these findings reveal a novel role forPin1 as a regulator of muscle terminaldifferentiation and suggest that Pin1 mediatedrepression of MEF2C function could contributeto this function.

2010 - The role of protamine in the gene delivery by cationic SLN on different cell lines [Abstract in Atti di Convegno]
Vighi, Eleonora; Montanari, Monica; Ruozi, Barbara; Battini, Renata; Leo, Eliana Grazia
abstract

In this study we produced cationic SLN using stearicacid, stearylamine and protamine sulfate astransfection promoter, in order to improve SLN transfection capacity without the use of adjuvantsubstances. In fact, protamine is a cationic smallprotein with high arginine content that is FDAapproved for the parenteral administration. Since protamine is a nuclear proteinthat helps DNA packaging in sperm cells, it is also used as transfection acceleratorin the gene delivery.Therefore, in this work we optimized the protamineamount in SLN formulation in order to evaluate therole of this protein in the transfection activity ofcationic SLN.

2009 - Cationic solid lipid nanoparticles containing Protamine as transfection vector on neuroblastoma cell line [Relazione in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Tosi, Giovanni; Battini, Renata; Montanari, Monica; Leo, Eliana Grazia
abstract

Sono stati presentati i risultati di studi effettuati in vitro sull'efficienza di transfezione di SLN allestite con diversi componenti tra i quali protamina.

2009 - Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells [Articolo su rivista]
Ruozi, Barbara; Montanari, Monica; Vighi, Eleonora; Tosi, Giovanni; Tombesi, Andrea; Battini, Renata; Restani, Cinzia; Leo, Eliana Grazia; Forni, Flavio; Vandelli, Maria Angela
abstract

In this study the mechanism of the internalisation and the cellular distribution of 5’ fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amount of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalisation mechanism. The intracellular distribution of the oligo was analysed by confocal laser scanning microscopy (CLSM) treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the co-localization studies, fluorescent labelled markers, specific for the different cellular compartments, were co-incubated with FITC-ODN.The different lipidic vesicles affect the internalisation mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1h from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 h, oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite amount of oligo with the cell membranes.

2008 - Veicolazione intracellualre di DNA plasmidico mediante solid lipid nanoparticles (SLN) cationiche [Relazione in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Tosi, Giovanni; Montanari, Manuela; Battini, Renata; Leo, Eliana Grazia
abstract

Veicolazione intracellualre di DNA plasmidico mediante solid lipid nanoparticles (SLN) cationiche

2007 - Dotap/Udca vesicles: novel approach in oligonucleotide delivery [Articolo su rivista]
Ruozi, Barbara; Battini, Renata; Montanari, Monica; Mucci, Adele; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

The relatively hydrophilic bile acid, ursodeoxycholic acid (UDCA), was used as additive to DOTAP cationic liposomes to evaluate the effect on the cellular uptake of an oligonucleotide. Nuclear magnetic resonance studies were applied to estimate the relative amount of incorporated UDCA into the lipidic bilayers. The interaction between the new formulations and 5’fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN) was studied using gel electrophoresis experiments. Besides, DOTAP or DOTAP/UDCA vesicles (MixVes; DOTAP/UDCA 1:0.25, 1:0.5, 1:1 and 1:2 molar ratio) were complexed with PS-ODN and tested after transfections the cellular uptake and the localization of the oligo in HaCaT cell line by the use of cytofluorimetric and confocal microscopic analysis. DOTAP lipid formulated in presence of defined amount of UDCA forms more stable, flexible and active MixVes. In particular, the MixVes at 1:0.25 and 1:0.5 molar ratio increase and modify the cellular uptake of PS-ODN if compared with DOTAP liposomes 3 h after the transfection studies. Besides, the in vitro data suggest that these new formulations are not toxic.

2007 - Nuove formulazioni liposomiali per la veicolazione di materiale genico in cellule cheratinocito simili [Abstract in Atti di Convegno]
Ruozi, Barbara; Montanari, Monica; Tosi, Giovanni; Mucci, Adele; Vighi, Eleonora; Battini, Renata; Leo, Eliana Grazia; Forni, Flavio; Vandelli, Maria Angela
abstract

Nuove formulazioni liposomiali per la veicolazione di materiale genico in cellule cheratinocito simili

2007 - Re-dispersible cationic solid lipid nanoparticles (SLNs) freeze-dried without cryoprotectors: Characterization and ability to bind the pEGFP-plasmid [Articolo su rivista]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.

2006 - Alternative strategies in medicinal chemistry to face drug resistance in anticancer therapy. [Abstract in Atti di Convegno]
Cardinale, Daniela; Ferrari, Stefania; Franchini, Silvia; Corsini, Enrico; Battini, Renata; Costi, Maria Paola
abstract

...

2006 - Solid lipid nanoparticles: a new cationic lipid matrix composition for gene transfer, [Abstract in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Forni, Flavio; Leo, Eliana Grazia
abstract

Solid lipid nanoparticles: a new cationic lipid matrix composition for gene transfer

2005 - Acidi colici nella preparazione di liposomi cationici per la transfezione in cellule HaCaT [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Mucci, Adele; Tosi, Giovanni; Forni, Flavio
abstract

Acidi colici nella preparazione di liposomi cationici per la transfezione in cellule HaCaT

2005 - Liposome evolution: novel approach in gene transfer [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Mucci, Adele; Schenetti, Luisa; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

Liposome evolution: novel approach in gene transfer

2005 - Liposome-oligonucleotides interaction for in vitro uptake by COSI and HaCaT cells [Articolo su rivista]
Ruozi, Barbara; Battini, Renata; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

Liposomes are considered very promising delivery systems for antisense therapeutic approach, offering drug protection and facilitating oligonucleotide cell internalization. The present study was aimed to investigate the influence of phospholipid composition of the liposomal systems both on the encapsulation and on the oligonucleotide carrier capacity in vitro. Liposomes composed of neutral ( phosphatidylcholine, cholesterol and dioleoylphosphatidylethanolamine) and/or cationic lipids (N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride salt, DOTAP) with different molar ratios were complexed with 50 fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN). The interaction was evaluated using atomic force microscopy (AFM), gel electrophoresis and HPLC analysis. Cytofluorimetric analysis and fluorescence microscopy were applied to evaluate the uptake and intracellular distribution of fluorescently labelled PS-ODN after transfection in two cell lines, COS I ( fibroblast cell) and HaCaT ( immortalized keratinocyte cell). The AFM studies reveal that the liposome/PS-ODN interaction leads the formation of a new irregular structure that completely hides the PS-ODN. Gel electrophoresis experiments and HPLC analysis have clearly demonstrated that also neutral liposomes are able to keep a little amount of PS-ODN but without strain to the complexation; the interaction was weak and rapidly destabilized when the complex was added to the cells. Transfection experiments performed with different incubation times show that DOTAP liposomes increase the rate of cellular uptake of PS-ODN and seem to influence its intracellular distribution in COS I cells where the oligonucleotide looks localized in nucleoli. Similar behaviour, at a lesser extent, is exhibited in HaCaT cells.

2004 - Liposome-oligonucleotides interaction; in vitro studies of cellular uptake [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Forni, Flavio; Vandelli, Maria Angela
abstract

Liposome-oligonucleotides interaction; in vitro studies of cellular uptake

2004 - Low-density lipoprotein (LDL) receptor/transferrin fusion protein: in vivoproduction and functional evaluation as a potential therapeutic tool forlowering plasma LDL cholesterol. [Articolo su rivista]
Razzini, Giorgia; Parise, Flavia; Calebiro, D; Battini, Renata; Bagni, Bruno; Corazzari, Tolmino; Tarugi, Patrizia Maria; Angelelli, Cecilia; Molinari, Susanna; Falqui, L; Ferrari, Stefano
abstract

A soluble form of human low-density lipoprotein receptor (LDL-R) fused in frame with rabbit transferrin (LDL-Rs(hu)/Tf(rab)) is assessed in vivo as a therapeutic tool for lowering plasma LDL cholesterol. The cDNA encoding LDL-Rs(hu)/Tf(rab) is expressed in mice, using a hydrodynamics-based gene transfer procedure. The transgene is transcribed in the liver of transduced animals and the corresponding protein is secreted into the bloodstream. Circulating LDL-Rs(hu)/Tf(rab) binds LDL specifically, thus indicating that it is correctly processed through the cellular compartments in vivo. More importantly, the expression of LDL-Rs(hu)/Tf(rab) allows the removal of injected human (125)I-labeled LDL ((123)I-LDL) from the bloodstream of transduced CD1 mice, which show faster LDL plasma clearance, anticipating by approximately 90 min the same clearance value observed in control animals. A similar effect is observed in transduced LDL-R(-/-) mice, in which the clearance of injected human LDL depends solely on the presence of circulating LDL-Rs(hu) /Tf(rab). In these animals the extent of plasma LDL clearance is directly related to the concentration of LDL-Rs(hu)/Tf(rab) in the blood. Finally, LDL-Rs(hu)/Tf(rab) does not alter the pattern of LDL organ distribution: in transduced animals, as well as in control animals, liver and bladder are the predominantly labeled organs after (123)I-LDL injection. However, LDL-Rs(hu)/Tf(rab) has a quantitative effect on LDL tissue deposition: in treated animals LDL-Rs(hu)/Tf(rab) determines an increase in radioactivity in the liver at early times after (123)I-LDL injection and a progressive labeling of the bladder, starting 20 min after injection.

2004 - Sviluppo di nuovi sistemi liposomiali per la veicolazione di oligonucleotidi in cellule cheratinocito simili [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Forni, Flavio; Mucci, Adele; Vandelli, Maria Angela
abstract

Sviluppo di nuovi sistemi liposomiali per la veicolazione di oligonucleotidi in cellule cheratinocito simili

2004 - TGFβ/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts [Articolo su rivista]
Tagliafico, Enrico; S., Brunelli; A., Bergamaschi; L., De Angelis; R., Scardigli; D., Galli; Battini, Renata; P., Bianco; Ferrari, Sergio; G., Cossu; Ferrari, Stefano
abstract

Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. The gene expression profile of four clonal derived lines of mesoangioblasts was determined by DNA micro-array analysis: it was similar in the four lines but different from 10T1/2 embryonic fibroblasts, used as comparison. Many known genes expressed by mesoangioblasts belong to response pathways to developmental signalling molecules, such as Writ or TGFbeta/BMP Interestingly, mesoangioblasts express receptors of the TGFbeta/BMP family and several Smads and, accordingly, differentiate very efficiently into smooth muscle cells in response to TGFbeta and into osteoblasts in response to BMP In addition, insulin signalling promotes adipogenic differentiation, possibly through the activation of IGF-R. Several Writs and Frizzled, Dishevelled and Tcfs are expressed, suggesting the existence of an autocrine loop for proliferation and indeed, forced expression of Frzb-1 inhibits cell division. Mesoangioblasts also express many neuro-ectodermal genes and yet undergo only abortive neurogenesis, evens after forced expression of neurogenin 1 or 2, MASH or NeuroD. Finally, mesoangioblasts express several pro-inflammatory genes, cytokines and cytokine receptors, which may explain their ability to be recruited by tissue inflammation. Our data define a unique phenotype for mesoangioblasts, explain several of their biological features and set the basis for future functional studies on the role of these cells in tissue histogenesis and repair.

2003 - Cationic liposomes for gene transfection [Articolo su rivista]
Ruozi, Barbara; Forni, Flavio; Battini, Renata; Vandelli, Maria Angela
abstract

Cationic liposomes spontaneously interact with the negatively charged DNA to form a stable complex that promotes the gene transfer to cells. The mode of formation and the size of cationic liposomes/DNA complexes were investigated using the atomic force microscopy (AFM). Also the most important physical-chemical factors involved in cationic liposome-mediated gene transfection, e.g. size and lipidic composition, were evaluated through the transfection of complexes with different liposomes/DNA molar ratio into three types of cultured cells. Cationic liposomes, composed of a neutral lipid (phosphatidilcoline), a cationic lipid dimethyldioctadecylammonium bromide (DDAB), a co-lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and a phospholipid derivative of polyethylene glycol (DSPE-mPEG) at different molar ratio, were mixed with a plasmid pCMVbeta to form liposomes/DNA complexes. We have demonstrated that the complexes were made by complicated structures in which the liposomes tend to aggregate and the DNA is surrounded by lipidic material. In vitro transfection efficiency by liposomes/plasmid pCMVbeta complexes was found to depend on the kind of lipid associated in the liposomes and the liposomes/DNA mixing ratio. The importance of associating DOPE in cationic liposomes was confirmed; this co-lipid is able to improve the ability of cationic liposomes to transfect cells but in addition, the AFM images and the EtBr fluorescence experiments have suggested that this lipid can also play an important role to facilitate the formation of stable liposomes, which efficaciously protect the DNA by nuclease digestion.

2001 - Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain [Articolo su rivista]
Messi, Patrizia; Bondi, Moreno; Sabia, Carla; Battini, Renata; Manicardi, Giuliano
abstract

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus. L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degreesC for 120 min and storage at 4 degreesC for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa. (C) 2001 Elsevier Science B.V. All rights reserved.

2001 - Interazione tra liposomi cationici e DNA plasmidico: studi di transfezione genica in diverse linee cellulari [Relazione in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Forni, Flavio; Vandelli, Maria Angela
abstract

Interazione tra liposomi cationici e DNA plasmidico: studi di transfezione genica in diverse linee cellulari

2001 - The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4 [Articolo su rivista]
Borghi, S.; Molinari, Susanna; Razzini, G.; Parise, F.; Battini, Renata; Ferrari, Stefano
abstract

Targeting of myocyte enhancer binding factor 2 (MEF2) proteins to the nucleus depends on a C-terminal bipartite nuclear localization signal (NLS). By expression of green fluorescent protein (GFP)/MEF2 fusion proteins in transfected myoblasts, we show that MEF2C contains an additional 13 amino acids domain, located immediately upstream of the NLS, which contributes to its nuclear retention. We also show that the NLS present in MEF2 proteins is required for efficient nuclear localization of histone deacetylase 4 (HDAC4). In muscle cells, transfected HDAC4 is largely cytoplasmic or, to a lesser extent, pancellular. Co-transfection of either MEF2A or MEF2C causes HDAC4 to accumulate in the nucleus in association with MEF2. This effect strongly depends on MEF2 NLS; it also requires the specific interaction of HDAC4 with MEF2, since the isolated NLS is not sufficient for targeting HDAC4 to the nucleus and other nuclear proteins, such as NF-Y, cannot substitute MEF2. Therefore, we demonstrate that HDAC4, different from HDAC5, is mainly a cytoplasmic resident protein, requiring a trans-acting NLS for nuclear localization. The physiological implications of MEF2 carrying its own inhibitor to the nucleus are discussed.

2000 - Veicolazione di DNA plasmidico in cellule COS attraverso vettori liposomiali [Abstract in Atti di Convegno]
Ruozi, Barbara; Battini, Renata; Guerra, Paolo; Forni, Flavio
abstract

Veicolazione di DNA plasmidico in cellule COS attraverso vettori liposomiali

1999 - Cellular calcium handling in brain slices from calbindin D(28K)- deficient mice [Articolo su rivista]
L., Pasti; G., Carmignoto; T., Pozzan; Battini, Renata; Ferrari, Stefano; G., Lally; Pc, Emson
abstract

CELLULAR calcium handling was examined in brain slices from transgenic antisense mice with a regional deficiency in the neuronal calcium binding protein calbindin D-28k and from their non transgenic wild type litter mate controls. Depolarization of brain slices with NMDA or potassium produced a prolonged elevation of neuronal calcium signal in neurons in brain slices from calbindin D-28k-deficient transgenic mice. This effect was selective and was seen only in brain areas where the antisense construct produced a significant depletion of calbindin D-28k protein. In other regions where calbindin D28k protein was not modified by the construct and in all glial cells whether from wild type or transgenic mice, cellular calcium handling was normal. NeuroReport 10:2367-2372

1999 - Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia [Articolo su rivista]
F., Parise; L., Simone; M. A., Croce; Ghisellini, Margherita; Battini, Renata; S., Borghi; Tiozzo, Roberta; Ferrari, Sergio; CALANDRA BUONAURA, Sebastiano; Ferrari, Stefano
abstract

A cDNA sequence encoding a soluble form of the human low-density lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated. In-frame fusion of the two cDNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-terminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through the pathway of the transferrin receptor. Cells transfected with the LDL-R/transferrin cDNA translate, glycosylate, and secrete the corresponding protein in the culture medium. The secreted protein binds LDL in a ligand-blotting experiment. Finally, the chimeric protein mediates the binding and internalization of LDL in mutant cells lacking the LDL receptor. In fact, Watanabe rabbit fibroblasts, incubated with the chimeric protein show a fourfold increase in LDL binding, a fivefold increase in LDL internalization, and a sixfold increase in LDL degradation, with respect to unincubated fibroblasts.

1999 - Glutamatergic synaptic responses and long-term potentiation are impaired in the CA1 hippocampal area of calbindin D(28K)-deficient mice [Articolo su rivista]
Jouvenceau, A.; Potier, B.; Battini, Renata; Ferrari, Stefano; Dutar, P.; Billard, Jm
abstract

The contribution of the cytosolic calcium binding protein calbindin D-28K (CaBP) to glutamatergic neurotransmission and synaptic plasticity was investigated in hippocampal CA1 area of wild-type and antisense transgenic CaBP-deficient mice, with the use of extracellular recordings in the ex vivo slice preparation. The amplitude of non-N-methyl-D-aspartate receptor (non-NMDAr)-mediated extracellular field excitatory postsynaptic potentials (fEPSPs) recorded in control medium was significantly greater in CaBP-deficient mice, whereas the afferent fiber volley was not affected. In contrast, the amplitude of NMDAr-mediated fEPSPs isolated in a magnesium-free medium after blockade of non-NMDAr and GABAergic receptors was significantly depressed in these animals. No alteration in the magnitude of paired-pulse facilitation was found, indicating that the presynaptic calcium mechanisms controlling glutamate release were not altered in CaBP-deficient mice. The magnitude and time course of the short-term potentiation (STP) of fEPSPs induced by a 30 Hz conditioning stimulation, which was blocked by the NMDAr antagonist 2-amino-5-phosphonovalerate acid (2-APV), was not impaired in the transgenic mice, whereas long-term potentiation (LTP) induced by a 100 Hz tetanus was not maintained. The long-term. depression (LTD) induced by low-frequency stimulation (1 Hz, 15 min) in the presence of the GABA antagonist bicuculline was not altered. These results argue for a contribution of CaBP to the mechanisms responsible for the maintenance of long-term synaptic potentiation, at least in part by modulating the activation of NMDA receptors.

1997 - Absence of mef2 binding to the A/T-rich element in the muscle creatine kinase (MCK) enhancer correlates with lack of early expression of the MCK gene in embryonic mammalian muscle [Articolo su rivista]
Ferrari, S.; Molinari, S.; Melchionna, R.; Angelis, M. G. C.; Battini, R.; De Angelis, L.; Kelly, R.; Cossu, G.
abstract

During skeletal muscle development, different types of muscle fibers are generated, which express different combinations of muscle-specific gene products, For example, the muscle creatine kinase gene (MCK) is highly expressed in fetal but not embryonic myotubes, We performed transient transfections of CAT reporter constructs, driven by the MCK promoter with variable lengths of 5'-flanking sequence, into primary cultures of embryonic and fetal muscle cells, Reporter activity was observed in fetal but not embryonic muscle cells, We assayed the ability of nuclear extracts prepared from embryonic and fetal muscle and C2C12 myotubes to bind specific regulatory elements in the MCK enhancer, The profile of DNA/protein complexes resulting from electrophoretic mobility shift assays was qualitatively the same with all extracts used when the oligonucleotide probes represented the MCK E-box, MHox site, CArG-box, and AP2 site, In contrast, no binding activity to the MEF2 site was observed with embryonic nuclear extract, Interestingly, MEF2 mRNAs and proteins were detected in both fetal and embryonic muscle, with the exception of the MEF2D1b isoform, which is restricted to fetal muscle, Furthermore, we found that protein phosphatase inhibitors included in the preparation of embryonic nuclear extracts or added to the medium of transfected embryonic myotubes can restore MEF2 DNA binding activity, as well as reporter activity driven by the MCK promoter and partial transcriptional activation of the endogenous MCK gene, We propose that phosphorylation of MEF2 regulates its activity and represents an important aspect of the mechanism controlling stage-specific transcription during skeletal myogenesis.

1997 - Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation [Articolo su rivista]
Grande, Alexis; Manfredini, Rossella; M., Pizzanelli; Tagliafico, Enrico; R., Balestri; F., Trevisan; D., Barbieri; C., Franceschi; Battini, Renata; Ferrari, Stefano; Ferrari, Sergio
abstract

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function,we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.

1996 - Deficits in memory and hippocampal long-term potentiation in mice with reduced calbindin D28K expression [Articolo su rivista]
Molinari, Susanna; Battini, Renata; Ferrari, Stefano; L., Pozzi; As, Killcross; Tw, Robbins; A., Jouvenceau; Jm, Billard; P., Dutar; Y., Lamour; Wa, Baker; H., Cox; Pc, Emson
abstract

The influx of calcium into the postsynaptic neuron is likely to be an important event in memory formation. Among the mechanisms that nerve cells may use to alter the Lime course or size of a spike of intracellular calcium are cytosolic calcium binding or ''buffering'' proteins. To consider the role in memory formation of one of these proteins, calbindin D-28K, which is abundant in many neurons, including the CA1 pyramidal tells of the hippocampus, transgenic mice deficient in calbindin D-28K have been created. These mice show selective impairments in spatial learning paradigms and fail to maintain long-term potentiation. These results suggest a role for calbindin D28K protein in temporally extending a neuronal calcium signal, allowing the activation of calcium-dependent intracellular signaling pathways underlying memory function.

1996 - Phosphorylation-dependent binding of MEF2 to the A/T rich element in the muscle creatine kinase enhancer correlates with lack of expression in embryonic mammalian muscle [Articolo su rivista]
Ferrari, S.; Molinari, S.; Melchionna, R.; Cusella-De Angelis, M. G.; Battini, R.; De Angelis, L.; Kelly, R.; Cossu, G.
abstract

1994 - Specific binding to vitamin D response elements of chicken intestinal DNA-binding activity is not related to the vitamin D receptor [Articolo su rivista]
Ferrari, Stefano; Battini, Renata; Molinari, Susanna
abstract

In this report we confirm that the putative vitamin D response element (VDRE), located between -320 and -360 in the chicken calbindin-D-28k gene, is not a binding site for the vitamin D-3 receptor (VDR). In examining the ability of chicken intestinal nuclear extracts (CINE) to bind known VDREs, we observed a specific VDRE-binding activity, which is distinct from VDR. In fact, VDR-depleted CINE retains the ability to bind the rat osteocalcin VDRE. The VDRE-binding activity binds DNA with high affinity and contacts it as the same guanine residues as VDR. Its specificity in binding structural variants of the AGGTCA repeat is broader than that of VDR, as direct repeats spaced by 3, 4, and 5 base pairs are almost equally effective competitors when added to the probe in molar excess. Palindromic arrangements of the same motif are lower affinity competitors. The retinoid-X-receptors is involved in the binding complex, as incubation of CINE with antibody to retinoid-X receptor results in a quantitative supershift. Antibodies to retinoic acid receptors (RAR alpha and -beta), T-3 receptor, or chicken ovalbumin up-stream promoter-transcription factor has no apparent effect. These data suggest that species specificty is a relevant aspect of VDR/DVRE recognition, and that a novel factor(s), different from VDR, might be involved in the effect of vitamin D on gene expression.

1992 - Induction of Calbindin-D28K by 1,25-dihydroxyvitamin D3 in cultured chicken intestinal cells [Articolo su rivista]
Ferrari, Stefano; Molinari, Susanna; Battini, Renata; Cossu, G; Lamonfava, S.
abstract

Intestinal cells from chicken embryos were grown in chemically defined, serum-free medium. The majority of cultured cells exhibits an epithelial-like morphology. As demonstrated by indirect immunofluorescence, the epithelial cells, and not the contaminating fibroblasts, express Calbindin-D28K only after 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, is added to the culture medium. The highly sensitive reverse transcriptase-polymerase chain reaction shows that both Calbindin-D28K mRNA and the corresponding primary unprocessed transcripts (pre-mRNA) are dramatically increased in cultured intestinal cells treated with 1,25-dihydroxyvitamin D3, thus indicating that Calbindin-D28K is induced by the increased rate of transcription of the corresponding gene. © 1992.

1991 - Effect of ELF pulsed electromagnetic fields on protein kinase c activation process in HL-60 leukemia cells [Articolo su rivista]
Monti, M. G.; Pernecco, L.; Moruzzi, M. S.; Battini, R.; Zaniol, P.; Barbiroli, B.
abstract

The activation of protein kinase C in HL-60 cells has been used as a model to investigate the molecular effects of the interaction of ELF pulsed electromagnetic fields (PEMFs) with the living systems. The shape of the pulsed magnetic field used in our experiments was a positive triangle (50 Hz, 8 mT peak). Protein kinase C is activated by association with plasma membranes; the membrane-associated enzyme binds phorbol esters. In the present study the process of protin kinase C activation was studied by measuring the binding of [3H]-phorbol-12,13-dibutyrate. The extent of labelled PDBu binding to HL-60 cells was increased by exposing the cells to the ELF electromagnetic field. Scatchard analysis of PDBu binding data showed an increased number of binding sites for the PDBu in the cells exposed to the electromagnetic field for 10, 15 or 20 min. Addition of EGTA to the culture medium resulted in a smaller stimulation of protein kinase C activation in the cells exposed to PEMF. © 1991 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1991 - Elf electromagnetic fields affect gene expression of hegenerating rat liver following partial hepatectomy [Articolo su rivista]
Battini, Renata; Monti, Maria Giuseppina; Moruzzi, Maria Stella; Ferrari, Stefano; Zaniol, P; Barbiroli, B.
abstract

Pulsed extremely-low-frequency magnetic fields (ELF-PEMFs) influence the expression of oncogenes c-myc and c-ras and of ornithine decarboxylase (ODC) in the regenerating rat liver following partial hepatectomy. In fact, while the mRNA's encoding both oncogenes are present in very low amounts in the normal liver, their concentration is dramatically increased during regeneration. Ornithine decarboxylase and c-myc mRNA's reach a maximum during the early phases of regeneration (3 hours after surgery) and decrease thereafter. c-ras mRNA reaches a maximum 40 hours after the operation. Treatment with ELF-PEMFs delivered to the animals immediately after the operation and every 12 hours thereafter increases the concentration of both oncogenes and of ornithine decarboxylase mRNA's at 3 hours (c-myc and ODC) and at 40 hours (c-ras) respectively.

1990 - Differentiation-dependent expression of apolipoprotein A-I in chicken myogenic cells in culture [Articolo su rivista]
Ferrari, Stefano; Battini, Renata; Cossu, G.
abstract

Northern blot hybridization experiments showed that Apolipoprotein A-I (Apo A-I) mRNA is present at high concentration in chicken myotubes cultured in vitro, while it is virtually absent in fibroblasts and myoblasts. Myotubes are also capable of translating and secreting in the culture medium a protein which is specifically immunoprecipitated by anti-Apo A-I antibodies and has the same electrophoretic mobility as Apo A-I purified from circulating high-density lipoproteins. The appearance of Apo A-I mRNA in myotubes depends on the transcriptional activation of the corresponding gene, as it was shown by hybridizing 32P-labeled RNA synthesized in isolated nuclei to Apo A-I cDNA. The activation of the Apo A-I gene is regulated by the muscle cell coordinately with muscle-specific genes. In fact, treatment with TPA, a powerful inhibitor of differentiation, efficiently prevents myoblasts from producing Apo A-I mRNA, as well as muscle actin mRNA, and causes myotubes to quickly cease Apo A-I mRNA synthesis. The existence of a strict relationship between Apo A-I mRNA concentration and myogenic cell differentiation was also confirmed by experiments with quail myoblasts transformed with a temperature-sensitive mutant of the Rous Sarcoma Virus. Cells raised at the permissive temperature (undifferentiated phenotype) do not contain Apo A-I as well as α-actin mRNAs, while shifting to the nonpermissive temperature (differentiated phenotype) causes a rapid increase in Apo A-I and α-actin mRNA concentration.

1990 - Functional analysis of the promoter region of the gene encoding chicken calbindin D28K [Relazione in Atti di Convegno]
Ferrari, S.; Battini, R.; Pike, W. J.
abstract

1990 - Identification of chicken calbindin D28K pre-messenger RNA sequences by polymerase chain reaction [Articolo su rivista]
Ferrari, Stefano; Battini, Renata
abstract

A transcribed RNA sequence encompassing the junction between the first intron and the second exon of the chicken calbindin D28K gene was copied in a cDNA fragment and subsequently amplified by polymerase chain reaction. When intestinal RNA is used as template, the appearance of the 161 bp amplified fragment is strictly dependent on the vitamin D status of the animal. In fact no amplified fragment is obtained when the RNA is extracted from the intestine of vitamin D-deficient chickens, while it is easily detected when the RNA is extracted only 30 min after injection with 1,25-dihydroxycholecalciferol. Conversely, the amplified fragment is obtained, irrespectively of the vitamin D status of the animal, when the RNA template is extracted from the brain. The appearance of unspliced RNA sequences upon vitamin D induction is followed, after a 30 min lag, by the appearance of the corresponding mature mRNA sequences. © 1990.

1989 - Expression and secretion of chicken apolipoprotein AI in transfected COS cells [Articolo su rivista]
Dixon, J. L.; Battini, R.; Ferrari, S.; Redman, C. M.; Banerjee, D.
abstract

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein-lipid assembly. apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10°C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion. © 1989.

1989 - Tissue-specific regulation of the concentration of Calbindin D28K mRNA in the developing chicken [Articolo su rivista]
Ferrari, S.; Battini, R.; Drusiani, E.
abstract

A BamHI-HindIII restriction fragment containing the 5′-terminal portion of the gene encoding chicken Calbindin D28K was sequenced and used as a probe in Northern-blot hydridizations to RNA extracted from the brain and intestine of chickens at various stages of development. In both tissues Calbindin D28K mRNA consists of a family of three species, which differ by size. In the intestine Calbindin D28K mRNAs appear at hatching and reach a peak at day 7. In the brain the same RNA species are easily detected at least 7 days before hatching, show a moderate increase at hatching and remain essentially constant during the first 10 days of adult life. The concentration of Calbindin D28K mRNAs in the intestine is strictly dependent on Vitamin D, while it is not in the cerebellum. © 1989.

1988 - Expression of c-myc and induction of DNA synthesis by platelet-poor plasma in human diploid fibroblasts. [Articolo su rivista]
Ferrari, Sergio; Calabretta, Bruno; Battini, Renata; Cosenza, Sc; Owen, Ta; Soprano, Kj; Baserga, R.
abstract

When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence, WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.

1988 - Nucleotide sequence of the promoter region of the gene encoding chicken calbindin D28K [Articolo su rivista]
Ferrari, S.; Drusiani, E.; Battini, R.; Fregni, M.
abstract

1987 - Molecular cloning of a cDNA for a human ADP/ATP carrier which is growth-regulated. [Articolo su rivista]
Battini, Renata; Ferrari, Sergio; Kaczmarek, L; Calabretta, Bruno; Chen, St; Baserga, R.
abstract

We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated. The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae. The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria. We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier. The steady-state RNA levels of the ADP/ATP carrier are growth-regulated. They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin. RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid.

1987 - Structural and functional analysis of a growth-regulated gene, the human calcyclin [Articolo su rivista]
Ferrari, Sergio; Calabretta, Bruno; Deriel, Jk; Battini, Renata; Ghezzo, F; Lauret, E; Griffin, C; Emanuel, Bs; Gurrieri, F; Baserga, R.
abstract

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5' flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5' and 3' flanking regions. The proximate 5' flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.

1987 - The gene encoding human vimentin is located on the short arm of chromosome 10 [Articolo su rivista]
Ferrari, S.; Cannizzaro, L. A.; Battini, R.; Huebner, K.; Baserga, R.
abstract

The gene for vimentin, an intermediate-filament protein, is growth regulated. We used Southern blot analysis and in situ chromosome hybridization to determine the location of the human vimentin gene. Our results show that there is only one copy of the vimentin gene and that it is located on the short arm of chromosome 10 (10pter-10q23) close to the interleukin-2 receptor gene, which is also growth regulated. In situ hybridization studies suggest that the most likely location of the vimentin gene is 10p13. Sequence similarities and homologies of human vimentin to other genes are presented.

1986 - Coding sequence and growth regulation of the human vimentin gene. [Articolo su rivista]
Ferrari, Sergio; Battini, Renata; Kaczmarek, L; Rittling, S; Calabretta, Bruno; de Riel, Jk; Philiponis, V; Wei, Jf; Baserga, R.
abstract

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.

1986 - Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein. [Articolo su rivista]
Calabretta, Bruno; Battini, Renata; Kaczmarek, L; de Riel, Jk; Baserga, R.
abstract

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.

1984 - Isolation of a cDNA clone containing a sequence complementary to the intestinal calcium-binding protein of the chick [Articolo su rivista]
Ferrari, S.; Battini, R.; Leone, A.; Ferrari, S.; Torelli, G.; Barbiroli, B.
abstract

The present work describes the construction of a cDNA library in pBR322 plasmid from an mRNA population enriched for the intestinal calcium-binding protein (CaBP) mRNA of the chick. We report the isolation of one recombinant clone containing a vitamin D-regulated sequence, which is complementary to part of the CaBP mRNA. Northern blot hybridization experiments allowed us to identify a 1900 nucleotide RNA species as the CaBP mRNA. © 1984.

1983 - Peptide chain initiation and analysis of in vitro translation products in rat heart undergoing hypertrophic growth [Articolo su rivista]
Mezzetti, G.; Ferrari, S.; Davalli, P.; Battini, R.; Corti, A.
abstract

The cytosol fraction of rat heart contains an initiation factor-like protein component that behaves like the eukaryotic factor (eIF-2) in binding [35S]-Met-tRNAf in the presence of GTP. The ternary initiator complex thus formed is able to bind to heart ribosomes. In the left ventricle of rat heart undergoing hypertrophic growth upon constriction of the descending aorta, the [35S]-Met-tRNAf binding activity of the cytosol protein(s) gradually increases after the operation from 40%, at 48 h, to 90%, at 10 days; slightly lower activation is seen in the [35S]-Met-tRNAf binding to ribosomes. Polysomal RNA is extracted from sham operated and hypertrophic rat hearts and translated in a reticulocyte cell free system. The translation products are analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis: no clearcut qualitative difference is observed in the pattern obtained from sham operated and hypertrophic animals. © 1983.

1981 - Adsorption of p-aminobenzenesulfonamide and its N1-phenyl and N1-pyridyl derivatives at the electrode-Solution interface [Articolo su rivista]
Battini, R.; Gavioli, G. B.; Grandi, G.; Benedetti, L.; Andreoli, R.
abstract

The adsorption of some sulfa-drugs, p-NH2ØSO2NH2, p-NH2ØSO2NHO, p-NH2ØSO2NHPy, was studied by a differential capacitance method at pH=12 at the dropping mercury electrode. The area occupied by each molecule of the N1-derivative compounds adsorbed at the mercury. electrolyte solution interface is {reversed tilde equals}80 Å2 which corresponds closely to the area expected for a molecule adsorbed flat on the electrode surface with its p-NH2Ø-S moiety. The ΔGads0(θ→0) values obtained at different potentials suggest that the adsorption process is less favoured when the potential decreases; however, the N1-pyridine derivative is always less adsorbed than the other sulfanilamides. © 1981 Elsevier Sequoia S.A.

1981 - Co-ordination properties of N-protected amino acids. Solution and solid state behavior of bis(benzyloxycarbonyl-L-tryptophanato)nickel(II) and its mixed complexes [Articolo su rivista]
Battini, Renata; Gavioli, Giovanna; Grandi, Giulia; Menabue, Ledi; Pellacani, Gian Carlo; Saladini, Monica; Corradi, Anna
abstract

Polarographic measurements show that nickel(II) ion and benzyloxycarbonyl-L-tryptophan (Z-L-tryptophan) form complexes in methanol with 1 : 2 and 1 : 4 metal-to-ligand ratios, depending on the ligand concentration. A complete scheme of the reactions which take place in solution and at the electrode is proposed. The 1 : 2 complex [NiL2]·2H2O and some amine adducts have been obtained in the solid state. In the solids the amino-acid is co-ordinated only through the carboxylate group.

1981 - Interfacial behaviour of N1-substituted sulfanilamides at the mercury-solution interface. Adsorption of N1-2-pyrimidinyl and N1-2(4,6-dimethyl) pyrimidinyl derivatives and structural parameters [Articolo su rivista]
Battistuzzi Gavioli, G.; Grandi, G.; Benedetti, L.; Andreoli, R.; Battini, R.
abstract

The adsorption of two N1-sulfanilamide derivatives, N1-2-pyrimidinyl and N1-2(4,6-dimethyl)pyrimidinyl, was studied by a differential capacitance method at pH=12 at the dropping mercury electrode. The area occupied by each molecule of these compounds, adsorbed at the mercury-electrolyte solution interface, corresponds well to the area expected for a molecule adsorbed flat on the electrode with its pNH2Ø-S moiety. This is confirmed by the correlation between the adsorption free energy (at constant potential) and the polarographic "substituent constant" which reflects the effect of N1-substitution on the electronic structure on the pNH2Ø-S moiety. Finally, the good linear correlation between ΔGads0 values and the activity parameters related to the biological activity of sulfanilamides suggests the presence of an adsorption step in the mechanism of action able to account for the different bacteriostatic potencies of the compounds. © 1981 Elsevier Sequoia S.A.

1981 - 409 - Adsorption of p-aminobenzenesulfonamide and its N1-phenyl and N1-pyridyl derivatives at the electrode-solution interface [Articolo su rivista]
Battini, R.; Battistuzzi Gavioli, G.; Grandi, G.; Benedetti, L.; Andreoli, R.
abstract

The adsorption of some sulfa-drugs, p-NH2ØSO2NH2,p-NH2ØSO2NHO,p-NH2ØSO2NHPy, was studied by a differential capacitance method at pH = 12 at the dropping mercury electrode. The area occupied by each molecules of the N1-derivative compounds adsorbed at the mercury{curly logical or}electrolyte solution interface is {reversed tilde equals} 80 Å2 which corresponds closely to the area expected for a molecule adsorbed flat on the electrode surface with its p-NH2ØO moiety. The ΔGoods (θ → 0) values obtained at different potentials suggest that the adsorption process is less favoured when the potential decreases; however, the N1-pyridine derivative is always less adsorbed than the other sulfanilamides. © 1981.