Cristina D'Arrigo - Academia.edu (original) (raw)
Papers by Cristina D'Arrigo
We report about Transmission Electron Microscopy (TEM) of the nuclear matrix and its proteins. Us... more We report about Transmission Electron Microscopy (TEM) of the nuclear matrix and its proteins. Using immunoelectron microscopy, we have detected the presence of lamins and the nuclear mitotic apparatus protein (NuMA) in nuclear matrices isolated from rat normal hepatocytes (NH) in the presence of an RNase inhibitor and after RNA digestion. The results were compared with those of nuclear matrices isolated from persistent hepatocyte nodules (PHN). In NH, immunoelectron microscopy shows that lamins and NuMA preferentially localise within electron-dense domains of the internal nuclear matrix. After RNA digestion NuMA undergoes a sharp depletion, while lamins A and C increase in electron-transparent regions and a thin web of lamin protofibrils, which connect the electron–dense regions, are unmasked. Fourier filtering of the images shows that lamin epitopes are arrayed both in locally ordered clusters and in quasi–regular rows. In PHN, the nuclear matrix undergoes changes both in morpholo...
International journal of immunopathology and pharmacology
The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-con... more The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in alpha and beta conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53 degrees C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high beta sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native alpha-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-...
International Journal of Molecular Sciences, 2013
The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify p... more The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further OPEN ACCESS increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.
The International Journal of Biochemistry & Cell Biology, 2012
Among the different species of water-soluble -peptides (A1-42, A1-40 and N-terminal truncated ... more Among the different species of water-soluble -peptides (A1-42, A1-40 and N-terminal truncated Apeptides), Apy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Apy3-42, we separated different aggregation states of A1-42 and Apy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates.
Alzheimer's Disease Pathogenesis-Core Concepts, Shifting Paradigms and Therapeutic Targets, 2011
Nucleic Acid Therapeutics, 2013
The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions i... more The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 μM and 0.80 ± 0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.
The Journal of Physical Chemistry B, 2008
The interaction free energy of like-charged polyelectrolytes in solution is calculated in the fra... more The interaction free energy of like-charged polyelectrolytes in solution is calculated in the framework of the extended counterion condensation theory, recently given by Schurr and Fujimoto, Biophys. Chem. 2002, 101-102, 425-445. For sufficiently high linear charge density, the electrostatic free energy of two parallel identical rigid polyelectrolytes as a function of the distance between them shows a minimum at distances in the range of nanometers, increasing with the Debye screening length. This effect is due to the increasing of the counterion condensed charge and condensation volume as the two polyelectyrolytes approach.
The International Journal of Biochemistry & Cell Biology, 2011
Prion protein hPrP90-231 wt E200K and D202N mutations Neurotoxicity Familial TSE a b s t r a c t ... more Prion protein hPrP90-231 wt E200K and D202N mutations Neurotoxicity Familial TSE a b s t r a c t Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a -rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in ␣-helix, hPrP90-231 E200K is spontaneously refolded in a -structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides.
PLoS ONE, 2012
In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as i... more In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy.
Neurobiology of Aging, 2004
Macromolecular Chemistry and Physics, 2012
Vinyl functionalized multiwalled carbon nanotubes (MWCNT-vinyl) are synthesized and used as monom... more Vinyl functionalized multiwalled carbon nanotubes (MWCNT-vinyl) are synthesized and used as monomers to prepare poly(ethylene-co -norbornene)-grafted carbon nanotube composites by in situ polymerization with [Ti( η 5 -C 5 Me 5 )(NϭC t Bu 2 )Cl 2 ] activated with methylalumoxane (MAO). The glass transition temperatures ( T g ) of grafted MWCNT composites are more than 30 ° C higher than those of copolymers. This difference is rationalized by the immobilization of the copolymer chain to the MWCNT surface by a covalent bond. The Young's modulus is shown to increase by more than 200% compared to poly(ethylene-co -norbornene) by incorporating 3.4 wt% functionalized MWCNT.
Journal of Thermal Analysis and Calorimetry, 2000
In the wool textile industry, several processes serve to improve the commercial properties of the... more In the wool textile industry, several processes serve to improve the commercial properties of the fibres such as fineness, softness, length, strength and lustrous. For example, wool is chemically treated with reductive agents then stretched and set. This leads to modifications of the original protein structure causing changes in thermal behaviour, dyeing, colouristic and wet resistance properties. A multidisciplinary approach was used to investigate treated and untreated wools, with the aim of exploiting the nature of the structural changes. SEM and TEM revealed changes on the cuticle and cortical cell morphology; structure modification were studied by FT-IR and DSC.
Journal of Polymer Science Part A: Polymer Chemistry, 2009
The in situ synthesis of ethylene-co-norbornene copolymers/multi-walled carbon nanotubes (MWNTs) ... more The in situ synthesis of ethylene-co-norbornene copolymers/multi-walled carbon nanotubes (MWNTs) nanocomposites was achieved by rare-earth half-sandwich scandium precursor [Sc(g 5 -C 5 Me 4 SiMe 3 )(g 1 -CH 2 SiMe 3 ) 2 (THF)] (1) activated by [Ph 3 C][B(C 6 F 5 ) 4 ], through a non-PFT (Polymerization Filling Technique) approach. MWNTs nanocomposites with low aluminum residue were obtained with excellent yields even though small amounts of triisobutylaluminium were needed as scavenger to prevent catalyst poisoning by MWNT impurities. MWNT bundles were disaggregated and highly coated with Poly(ethylene-co-norbornene) [P(E-co-N)] as revealed by transmission electron microscopy. Interestingly, P(E-co-N) copolymers showed T g over 130 C as well as norbornene content over 50 mol %; both values were higher than those obtained by the cationic active species in 1/[Ph 3 C][B(C 6 F 5 ) 4 ]. A series of copolymerization reactions by 1/[Ph 3 C][B(C 6 F 5 ) 4 ]/Al i Bu 3 without MWNTs produced copolymers with the same unexpected features. The NMR analysis revealed the presence of rac-ENNE and rac-ENNNE sequences. Thus, Al i Bu 3 changed the stereoirregular alternating copolymer microstructure produced by 1/[Ph 3 C][B(C 6 F 5 ) 4 ]. We conclude that Al i Bu 3 is not only a scavenger for CNT impurities, but it reacts with the THF ligand to give coordinatively unsaturated active species. Finally, P(E-co-N)/MWNT masterbatches were mixed with commercial TOPAS to produce cyclic olefin copolymer nanocomposites with excellent dispersion of filler.
Journal of Neurochemistry, 2002
... Pyroglutamate-modified amyloid β-peptides AβN3(pE) strongly affect cultured neuron and as... more ... Pyroglutamate-modified amyloid β-peptides AβN3(pE) strongly affect cultured neuron and astrocyte survival. Claudio Russo 1 ,; Elisabetta Violani 2 ,; Serena Salis 1 ,; Valentina Venezia 1 ,; Virginia Dolcini 1 ,; Gianluca Damonte 3 ,; Umberto Benatti 3 ,; ...
Journal of Neurochemistry, 2007
Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsib... more Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre-fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90-231 (hPrP90-231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild-denatured hPrP90-231 (incubated for 1 h at 53 degrees C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90-231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.
Journal of Neurochemistry, 2003
The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (Pr... more The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (PrP 106-126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106-126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, b-structured peptide. Moreover, PrP 106-126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild-type PrP 106-126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106-126.
Journal of Cellular Biochemistry, 2006
SCN- binds to the charged amino group of lysines, inducing local changes in the electrostatic fre... more SCN- binds to the charged amino group of lysines, inducing local changes in the electrostatic free energy of histones. We exploited this property to selectively perturb the histone-DNA interactions involved in the stabilization of eu and heterochromatin. Differential scanning calorimetry (DSC) was used as leading technique in combination with trypsin digestion that selectively cleaves the histone end domains. Euchromatin undergoes progressive destabilization with increasing KSCN concentration from 0 to 0.3 M. Trypsin digestion in the presence of 0.2 M KSCN show that the stability of the linker decreases as a consequence of the competitive binding of SCN- to the amino groups located in the C and N-terminal domain of H1 and H3, respectively; likewise, the release of the N-terminal domain of H4 induces an appreciable depression in both the temperature and enthalpy of melting of core particle DNA. Unfolding of heterochromatin requires, in addition to further cleavage of H4, extensive digestion of H2A and H2B, strongly suggesting that these histones stabilize the higher order structure by forming a protein network which extends throughout the heterochromatin domain.
Journal of Cellular Biochemistry, 2003
Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laborato... more Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laboratory [Barboro et al. [2002] Exp. Cell. Res. 279:202-218] have confirmed that lamins and the nuclear mitotic apparatus protein (NuMA) are localized inside the interphase nucleus in a polymerized form. This provided evidence of the existence of a RNA stabilized lamin/NuMA frame, consisting of a web of thin ( approximately 3 and approximately 5 nm) lamin filaments to which NuMA is anchored mainly in the form of discrete islands, which might correspond to the minilattices described by Harborth et al. [1999] (EMBO. J. 18:1689-1700). In this article we propose that this scaffold is involved in the compartmentalization of both chromatin and functional domains and further determines the higher-order nuclear organization. This hypothesis is strongly supported by the scrutiny of different structural transitions which occur inside the nucleus, such as chromatin displacement and rearrangements, the collapse of the internal nuclear matrix after RNA digestion and the disruption of chromosome territories induced by RNase A and high salt treatment. All of these destructive events directly depend on the loss of the stabilizing effect exerted on the different levels of structural organization by the interaction of RNA with lamins and/or NuMA. Therefore, the integrity of nuclear RNA must be safeguarded as far as possible to isolate the matrix in the native form. This material will allow for the first time the unambiguous ultrastructural localization inside the INM of the components of the functional domains, so opening new avenues of investigation on the mechanisms of gene expression in eukaryotes.
Experimental Cell Research, 2009
Tumor progression is characterized by definite changes in the protein composition of the nuclear ... more Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were overexpressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure. a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
We report about Transmission Electron Microscopy (TEM) of the nuclear matrix and its proteins. Us... more We report about Transmission Electron Microscopy (TEM) of the nuclear matrix and its proteins. Using immunoelectron microscopy, we have detected the presence of lamins and the nuclear mitotic apparatus protein (NuMA) in nuclear matrices isolated from rat normal hepatocytes (NH) in the presence of an RNase inhibitor and after RNA digestion. The results were compared with those of nuclear matrices isolated from persistent hepatocyte nodules (PHN). In NH, immunoelectron microscopy shows that lamins and NuMA preferentially localise within electron-dense domains of the internal nuclear matrix. After RNA digestion NuMA undergoes a sharp depletion, while lamins A and C increase in electron-transparent regions and a thin web of lamin protofibrils, which connect the electron–dense regions, are unmasked. Fourier filtering of the images shows that lamin epitopes are arrayed both in locally ordered clusters and in quasi–regular rows. In PHN, the nuclear matrix undergoes changes both in morpholo...
International journal of immunopathology and pharmacology
The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-con... more The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in alpha and beta conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53 degrees C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high beta sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native alpha-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-...
International Journal of Molecular Sciences, 2013
The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify p... more The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further OPEN ACCESS increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.
The International Journal of Biochemistry & Cell Biology, 2012
Among the different species of water-soluble -peptides (A1-42, A1-40 and N-terminal truncated ... more Among the different species of water-soluble -peptides (A1-42, A1-40 and N-terminal truncated Apeptides), Apy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Apy3-42, we separated different aggregation states of A1-42 and Apy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates.
Alzheimer's Disease Pathogenesis-Core Concepts, Shifting Paradigms and Therapeutic Targets, 2011
Nucleic Acid Therapeutics, 2013
The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions i... more The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 μM and 0.80 ± 0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.
The Journal of Physical Chemistry B, 2008
The interaction free energy of like-charged polyelectrolytes in solution is calculated in the fra... more The interaction free energy of like-charged polyelectrolytes in solution is calculated in the framework of the extended counterion condensation theory, recently given by Schurr and Fujimoto, Biophys. Chem. 2002, 101-102, 425-445. For sufficiently high linear charge density, the electrostatic free energy of two parallel identical rigid polyelectrolytes as a function of the distance between them shows a minimum at distances in the range of nanometers, increasing with the Debye screening length. This effect is due to the increasing of the counterion condensed charge and condensation volume as the two polyelectyrolytes approach.
The International Journal of Biochemistry & Cell Biology, 2011
Prion protein hPrP90-231 wt E200K and D202N mutations Neurotoxicity Familial TSE a b s t r a c t ... more Prion protein hPrP90-231 wt E200K and D202N mutations Neurotoxicity Familial TSE a b s t r a c t Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a -rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in ␣-helix, hPrP90-231 E200K is spontaneously refolded in a -structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides.
PLoS ONE, 2012
In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as i... more In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy.
Neurobiology of Aging, 2004
Macromolecular Chemistry and Physics, 2012
Vinyl functionalized multiwalled carbon nanotubes (MWCNT-vinyl) are synthesized and used as monom... more Vinyl functionalized multiwalled carbon nanotubes (MWCNT-vinyl) are synthesized and used as monomers to prepare poly(ethylene-co -norbornene)-grafted carbon nanotube composites by in situ polymerization with [Ti( η 5 -C 5 Me 5 )(NϭC t Bu 2 )Cl 2 ] activated with methylalumoxane (MAO). The glass transition temperatures ( T g ) of grafted MWCNT composites are more than 30 ° C higher than those of copolymers. This difference is rationalized by the immobilization of the copolymer chain to the MWCNT surface by a covalent bond. The Young's modulus is shown to increase by more than 200% compared to poly(ethylene-co -norbornene) by incorporating 3.4 wt% functionalized MWCNT.
Journal of Thermal Analysis and Calorimetry, 2000
In the wool textile industry, several processes serve to improve the commercial properties of the... more In the wool textile industry, several processes serve to improve the commercial properties of the fibres such as fineness, softness, length, strength and lustrous. For example, wool is chemically treated with reductive agents then stretched and set. This leads to modifications of the original protein structure causing changes in thermal behaviour, dyeing, colouristic and wet resistance properties. A multidisciplinary approach was used to investigate treated and untreated wools, with the aim of exploiting the nature of the structural changes. SEM and TEM revealed changes on the cuticle and cortical cell morphology; structure modification were studied by FT-IR and DSC.
Journal of Polymer Science Part A: Polymer Chemistry, 2009
The in situ synthesis of ethylene-co-norbornene copolymers/multi-walled carbon nanotubes (MWNTs) ... more The in situ synthesis of ethylene-co-norbornene copolymers/multi-walled carbon nanotubes (MWNTs) nanocomposites was achieved by rare-earth half-sandwich scandium precursor [Sc(g 5 -C 5 Me 4 SiMe 3 )(g 1 -CH 2 SiMe 3 ) 2 (THF)] (1) activated by [Ph 3 C][B(C 6 F 5 ) 4 ], through a non-PFT (Polymerization Filling Technique) approach. MWNTs nanocomposites with low aluminum residue were obtained with excellent yields even though small amounts of triisobutylaluminium were needed as scavenger to prevent catalyst poisoning by MWNT impurities. MWNT bundles were disaggregated and highly coated with Poly(ethylene-co-norbornene) [P(E-co-N)] as revealed by transmission electron microscopy. Interestingly, P(E-co-N) copolymers showed T g over 130 C as well as norbornene content over 50 mol %; both values were higher than those obtained by the cationic active species in 1/[Ph 3 C][B(C 6 F 5 ) 4 ]. A series of copolymerization reactions by 1/[Ph 3 C][B(C 6 F 5 ) 4 ]/Al i Bu 3 without MWNTs produced copolymers with the same unexpected features. The NMR analysis revealed the presence of rac-ENNE and rac-ENNNE sequences. Thus, Al i Bu 3 changed the stereoirregular alternating copolymer microstructure produced by 1/[Ph 3 C][B(C 6 F 5 ) 4 ]. We conclude that Al i Bu 3 is not only a scavenger for CNT impurities, but it reacts with the THF ligand to give coordinatively unsaturated active species. Finally, P(E-co-N)/MWNT masterbatches were mixed with commercial TOPAS to produce cyclic olefin copolymer nanocomposites with excellent dispersion of filler.
Journal of Neurochemistry, 2002
... Pyroglutamate-modified amyloid β-peptides AβN3(pE) strongly affect cultured neuron and as... more ... Pyroglutamate-modified amyloid β-peptides AβN3(pE) strongly affect cultured neuron and astrocyte survival. Claudio Russo 1 ,; Elisabetta Violani 2 ,; Serena Salis 1 ,; Valentina Venezia 1 ,; Virginia Dolcini 1 ,; Gianluca Damonte 3 ,; Umberto Benatti 3 ,; ...
Journal of Neurochemistry, 2007
Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsib... more Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre-fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90-231 (hPrP90-231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild-denatured hPrP90-231 (incubated for 1 h at 53 degrees C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90-231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.
Journal of Neurochemistry, 2003
The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (Pr... more The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (PrP 106-126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106-126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, b-structured peptide. Moreover, PrP 106-126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild-type PrP 106-126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106-126.
Journal of Cellular Biochemistry, 2006
SCN- binds to the charged amino group of lysines, inducing local changes in the electrostatic fre... more SCN- binds to the charged amino group of lysines, inducing local changes in the electrostatic free energy of histones. We exploited this property to selectively perturb the histone-DNA interactions involved in the stabilization of eu and heterochromatin. Differential scanning calorimetry (DSC) was used as leading technique in combination with trypsin digestion that selectively cleaves the histone end domains. Euchromatin undergoes progressive destabilization with increasing KSCN concentration from 0 to 0.3 M. Trypsin digestion in the presence of 0.2 M KSCN show that the stability of the linker decreases as a consequence of the competitive binding of SCN- to the amino groups located in the C and N-terminal domain of H1 and H3, respectively; likewise, the release of the N-terminal domain of H4 induces an appreciable depression in both the temperature and enthalpy of melting of core particle DNA. Unfolding of heterochromatin requires, in addition to further cleavage of H4, extensive digestion of H2A and H2B, strongly suggesting that these histones stabilize the higher order structure by forming a protein network which extends throughout the heterochromatin domain.
Journal of Cellular Biochemistry, 2003
Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laborato... more Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laboratory [Barboro et al. [2002] Exp. Cell. Res. 279:202-218] have confirmed that lamins and the nuclear mitotic apparatus protein (NuMA) are localized inside the interphase nucleus in a polymerized form. This provided evidence of the existence of a RNA stabilized lamin/NuMA frame, consisting of a web of thin ( approximately 3 and approximately 5 nm) lamin filaments to which NuMA is anchored mainly in the form of discrete islands, which might correspond to the minilattices described by Harborth et al. [1999] (EMBO. J. 18:1689-1700). In this article we propose that this scaffold is involved in the compartmentalization of both chromatin and functional domains and further determines the higher-order nuclear organization. This hypothesis is strongly supported by the scrutiny of different structural transitions which occur inside the nucleus, such as chromatin displacement and rearrangements, the collapse of the internal nuclear matrix after RNA digestion and the disruption of chromosome territories induced by RNase A and high salt treatment. All of these destructive events directly depend on the loss of the stabilizing effect exerted on the different levels of structural organization by the interaction of RNA with lamins and/or NuMA. Therefore, the integrity of nuclear RNA must be safeguarded as far as possible to isolate the matrix in the native form. This material will allow for the first time the unambiguous ultrastructural localization inside the INM of the components of the functional domains, so opening new avenues of investigation on the mechanisms of gene expression in eukaryotes.
Experimental Cell Research, 2009
Tumor progression is characterized by definite changes in the protein composition of the nuclear ... more Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were overexpressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure. a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s e v i e r. c o m / l o c a t e / y e x c r