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Biomedical subjects

R Negri

Publications and source records attributed to R Negri.

At least 19 recordsLinked to original sources
PubMed2004

Hippocampal gene expression is modulated by hypergravity.

We used the cDNA microarray technique to monitor simultaneously possible changes induced by hypergravity in the expression level of thousands of hippocampal genes. We tested the mRNA level of about 5000 genes in the hippocampus of mice subjected to 1.09 g (1g) or to 1.85 g (2g) for five repeated 1-h daily rotations in a centrifuge (g = 9.81 m/s2). Data were compared with those obtained for mice kept stationary (C). The ratios 1g/C and 2g/C identified genes affected by rotation and rotation + hypergravity, respectively, whereas 2g/1g ratio identified those affected by hypergravity. We found that about 200 genes were affected by rotation and/or rotation + hypergravity. Almost all the genes affected by rotation + hypergravity were up-regulated, only five being down-regulated. The modulated genes code for proteins involved in a wide range of cellular functions (DNA/RNA metabolism, protein processing, intermediate metabolism, cytoskeleton and motility, cell cycle and apoptosis, signal transduction, neuronal structure/function), suggesting that rotation + hypergravity may affect several aspects of the hippocampal function in order to compensate for environmental changes. Six genes directly or indirectly involved in synaptic transmission and plasticity (proSAAS, neuroblastoma ras oncogene, ESTs moderately similar to thymosin beta-10, syndet, inhibin beta E and Ngfi-A binding protein 2) were found to be significantly modulated by hypergravity and unaffected or only slightly affected by rotation. The modulation by hypergravity of these genes suggests that this stimulus might induce plastic remodelling of the hippocampal circuits, possibly both at structural and functional level.

Animals↗
PubMed2002

Preliminary study of gene expression levels in human T-cells exposed to cosmic radiations.

Several experiments demonstrated the influence of microgravity on mitogenic activation of T cells at molecular level. To discriminate between effects of microgravity and cosmic radiations, in this work we studied the effects of high cosmic radiations on the genetic expression in human T cells boarded in a stratospheric balloon (BIRBA-1 mission, 22 hours of flight). The genetic expression was analyzed by the cDNA microarray hybridization technology, which allows the comparative and simultaneous estimate of hundreds of mRNAs Activated cells react to the ionizing stress by activating genes involved in cell cycle check-point, oxidative stress response, heat shock proteins production or by repressing genes involved in antigen recognition.

Journal Article↗
PubMed2001

Sequence dependence of translational positioning of core nucleosomes.

The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modifications of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is specified by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wild-type and a mutant sequence differ by +2 bp, corresponding to an overall helical periodicity of approximately 10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The DNA immediately flanking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.

Animals↗
PubMed2001

A possible prebiotic synthesis of purine, adenine, cytosine, and 4(3H)-pyrimidinone from formamide: implications for the origin of life.

The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO(3) and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution.

Catalysis↗
PubMed2001

RNA polymerase III transcription complexes on chromosomal 5S rRNA genes in vivo: TFIIIB occupancy and promoter opening.

Quantitative analysis of multiple-hit potassium permanganate (KMnO(4)) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established.

Base Sequence↗
PubMed2001

Cell cycle arrest determines the intensity of the global transcriptional response of Saccharomyces cerevisiae to ionizing radiation.

Whole-genome analysis was performed using DNA microarrays to define the changes in the gene expression patterns occurring in Saccharomyces cerevisiae cells exposed to ionizing radiation. The effects of sublethal dose on wild-type, rad53 (enhanced sensitivity to radiation and impaired in a cell cycle damage checkpoint), and rad6 (enhanced sensitivity to radiation and functional cell cycle block by radiation) mutant backgrounds and of a higher dose on the wild-type and G(2)-phase-arrested cells were analyzed. Several gene pathways were identified as being implicated in the response to radiation. In particular, the cell cycle blockage that occurred in the wild-type strain after a high radiation dose and in the rad6 mutant after a lower dose entailed modifications of defined gene expression patterns, which are described here and are compared with the gene modulation patterns observed in the rad53 strain in the absence of efficient blockage. Loss of the RAD53 function caused a major increase in the number of genes modulated by radiation. Given that Rad53-Sad1p, the protein encoded by RAD53, has functions other than those directly connected to cell cycle arrest, we determined the gene patterns that were modulated upon irradiation of rad53 cells that had been forced to arrest in G(2) phase by nocodazole treatment. These differential whole-genome analyses shed light on the multiplicity of functions of the pivotal Rad53-Sad1p protein. The results obtained describe how the cells respond to different irradiation conditions by modulating important gene classes, including those associated with stress defense, ribosomal proteins, histones, ergosterol and GCR1-controlled sugar metabolism.

Cell Cycle↗
PubMed2000

Helical repeat of DNA in the nucleosome core particle.

Although the crystal structure of nucleosome core particle is essentially symmetrical in the vicinity of the dyad, the linker histone binds asymmetrically in this region to select a single high-affinity site from potentially two equivalent sites. To try to resolve this apparent paradox we mapped to base-pair resolution the dyads and rotational settings of nucleosome core particles reassembled on synthetic tandemly repeating 20 bp DNA sequences. In agreement with previous observations, we observed (1) that the helical repeat on each side of the dyad cluster is 10 bp maintaining register with the sequence repeat and (2) that this register changes by 2 bp in the vicinity of the dyad. The additional 2 bp required to effect the change in the rotational settings is accommodated by an adjustment immediately adjacent to the dyad. At the dyad the hydroxyl radical cleavage is asymmetric and we suggest that the inferred structural asymmetry could direct the binding of the linker histone to a single preferred site.

Animals↗
PubMed1999

RhoA activity is required for fibronectin assembly and counteracts beta1B integrin inhibitory effect in FRT epithelial cells.

FRT thyroid epithelial cells synthesize fibronectin and organize a network of fibronectin fibrils at the basal surface of the cells. Fibronectin fibril formation is enhanced by the overexpression of the ubiquitous beta1A integrin and is inhibited by the expression of the dominant-negative beta1B subunit. We tested the hypotheses that RhoA activity might mediate the integrin-dependent fibronectin fibrillogenesis and might counteract beta1B integrin inhibitory effect. FRT-beta1A cells were transfected with a vector carrying a dominant negative form of RhoA (RhoAN19) or treated with the C3 transferase exoenzyme. Both treatments inhibited fibronectin assembly and caused loss of actin microfilaments and adhesion plaques. On the other hand, FRT-beta1B cells were transfected with the constitutively activated form of RhoA (RhoAV14) or treated with the E. coli cytotoxic necrotizing factor 1, which directly activates RhoA. Either treatment restored microfilament and adhesion plaque assembly and promoted fibronectin fibril organization. A great increase in fibronectin fibril assembly was also obtained by treatment of FRT-beta1B cells with TGF-beta. Our data indicate that RhoA is required to promote fibronectin matrix assembly in FRT cells and that the activation of the signal transduction pathway downstream of RhoA can overcome the inhibitory effect of beta1B integrin.

Actin Cytoskeleton↗
PubMed1998

Manganese water-soluble porphyrin senses DNA conformation.

We have determined the sites that are preferentially cleaved by Mn(T4MPyP) (where T4MPyP is the dianion of 5, 10, 15, 20, tetrakis (4-N-methylpyridine)porphyrin) on synthetic DNAs and on both intrinsically curved and average-shaped natural DNA sequences. On the basis of cleavage selectivity and of DNase I footprinting we show that the recognition specificity by this compound is based on steric properties: the preferred conformation is a DNA minor groove narrower than average and dimensionally defined. This conclusion is reached on the basis of: (i) the localization of the preferential cleavage sites at the 3' extremity of short A-tracts, known to undergo minor groove directional narrowing; (ii) the effects of temperature on cleavage specificity on curved sequences; (iii) the localization of cleavage sites in synthetic constructs whose crystal and solution structure was previously defined, and in programmed sequence variants thereoff; (iv) the effects of base substitutions on cleavage efficiency; (v) DNase I footprinting analysis. Several of these evidences argue against the possibility that Mn(T4MPyP)/DNA site selection occurs on the basis of electrostatic potential effects.Mn(T4MPyP) provides a tool for the analysis of DNA conformation whose selectivity is complementary to that of DNase I and hydroxyl radicals.

Animals↗
PubMed1998

Changing nucleosome positions in vivo through modification of the DNA rotational information.

The effects of the rotational information of DNA in determining the in vivo localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5 S rRNA repeat gene. The distribution of the phased series of flexibility signals present in this DNA has been altered by inserting in its centre a 25 bp tract. The effects of such alteration on the in vivo distribution of the helically phased, alternatively located ncps have been determined relative to a reference 21 bp insertion mutant. The results show that the answers provided in vitro and in vivo by the yeast 5 S rRNA gene sequence to specific modifications of the DNA rotational frame are similar, thus pointing to the relevance of DNA rotational information in vivo.

Chromosomes, Fungal↗
PubMed1998

Beta1B integrin interferes with matrix assembly but not with confluent monolayer polarity, and alters some morphogenetic properties of FRT epithelial cells.

Beta1B is a beta1 integrin splice variant that differs from the ubiquitous beta1A in the terminal portion of the cytosolic tail. The expression of this variant in CHO cells results in reduced fibroblast adhesion and motility (Balzac, E et al., J. Cell Biol. 127, 557-565 (1994)). We have evaluated the phenotypic changes induced by the expression of beta1B in the FRT epithelial cell line. Stable transfectants of FRT cells expressing beta1B or beta1A human integrins were obtained. The transfected integrins associated with the endogenous alpha subunits and were delivered to the plasma membrane. Beta1B expressing cells attached less efficiently and spread less on fibronectin, laminin or type IV collagen coated dishes. A great reduction of fibronectin fibrils associated to the basal membrane of non-confluent beta1B transfected cells was observed. This was paralleled by the disappearance of microfilament bundles and loss of basally located focal adhesions. On the contrary, upon beta1A transfection, a higher amount of fibronectin fibrils, together with microfilament bundles and focal adhesions, was observed. Expression of beta1B did not significantly modify the ability to manifest the polarized phenotype when cells were grown to confluence on filters in two-chamber-systems. Beta1B-transfected cells showed reduced motile properties when embedded as aggregates in type I collagen gels. Moreover, formation of polarized cysts in suspension culture was impaired. The results show that beta1B, by interfering with focal adhesion organization, microfilament and fibronectin assembly, cell spreading and migration, affects some morphogenetic properties of FRT epithelial cells.

Actin Cytoskeleton↗
PubMed1997

Mechanism of degradation of 2'-deoxycytidine by formamide: implications for chemical DNA sequencing procedures.

We describe the reaction of formamide with 2'-deoxycytidine to give pyrimidine ring opening by nucleophilic addition on the electrophilic C(6) and C(4) positions. This information is confirmed by the analysis of the products of formamide attack on 2'-deoxycytidine, 5-methyl-2'-deoxycytidine, and 5-bromo-2'-deoxycytidine, residues when the latter are incorporated into oligonucleotides by DNA polymerase-driven polymerization and solid-phase phosphoramidite procedure. The increased sensitivity of 5-bromo-2'-deoxycytidine relative to that of 2'-deoxycytidine is pivotal for the improvement of the one-lane chemical DNA sequencing procedure based on the base-selective reaction of formamide with DNA. In many DNA sequencing cases it will in fact be possible to incorporate this base analogue into the DNA to be sequenced, thus providing a complete discrimination between its UV absorption signal and that of the thymidine residues. The wide spectrum of different sensitivities to formamide displayed by the 2'-deoxycytidine analogues solves, in the DNA single-lane chemical sequencing procedure, the possible source of errors due to low discrimination between C and T residues.

Bromodeoxycytidine↗
PubMed1997

Nucleosomes as topological rheostats.

Induction of transcription in eukaryotic promoters is accompanied by removal or remodeling of nucleosomes. Given that this process causes release of torsional stress, the question is asked relative to its fate and to its effects on local DNA conformation. Is it dispersed by free rotation through surrounding nucleosomes or does it stay locally to be used in the modulation or activation of the transcription machinery? The results of the calculations relative to the onset of writhing suggest that the free energy made available by removal of nucleosomes is in the range of values that corresponds to the transition linking difference, thus pointing to a possible regulatory mechanism for the local use of free energy in promoters.

Animals↗
PubMed1996

One-step, one-lane chemical DNA sequencing by N-methylformamide in the presence of metal ions.

We report on a chemical method that allows DNA sequencing by a single reaction. It is based on treatment of 5'-end-labeled DNA with N-methylformamide in the presence of manganese. This method allows the manipulation of samples to be kept to a minimum and consists of a single chemical step that requires about 30 minutes to complete base degradation, phosphodiester bond cleavage and denaturation. Examples of one-treatment, one-lane DNA sequencing of both radioactively and fluorescently 5'-end-labeled DNAs are reported.

Formamides↗
PubMed1996

Collagen interaction with apically expressed beta 1 integrins: loss of transepithelial resistance and reorganization of cultured thyroid cell monolayer.

We have cultured FRT rat thyroid cells to confluency on filters in bicameral systems and allowed a type I collagen solution to gel on their apical compartment. A dramatic drop in transepithelial resistance occurred within 2 h after collagen addition. This drop in transepithelial resistance was dependent upon collagen concentration. Cells interacting with the collagen lost their apical microvilli, formed pseudopods and displayed rearrangements in the distribution of actin and uvomorulin. After 24 h the cells had reorganized in two layers, one facing the other, with opposite orientations. We found that in approximately 15 to 20% of the cells within a confluent monolayer alpha 1 and beta 1 integrin subunits were localized in a subdomain of the apical plasma membrane, other than on the basolateral surface, as they normally are. By double immunofluorescence, after addition of diluted collagen solutions, we were able to detect collagen fibers that were bound to these integrin-containing apical subdomains. The addition of anti-beta 1 antibodies to the apical domain significantly delayed the drop in transepithelial resistance induced by the collagen gel. These data suggest that members of the beta 1 integrin family that are expressed on the apical domain may act as receptors for collagen fibers and may play a role in promoting changes in cell orientation.

Actins↗
PubMed1995

Changing nucleosome positions through modification of the DNA rotational information.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.

Base Sequence↗
PubMed1995

Multiple overlapping positions of nucleosomes with single in vivo rotational setting in the Hansenula polymorpha RNA polymerase II MOX promoter.

In vivo nucleotide-level mapping of nucleosomes in the promoter of the methanol oxidase (MOX) gene in the yeast Hansenula polymorpha is reported. The 4 nucleosomes analyzed are organized in families; they localize in alternative positions along a unique rotational phase, and the linker regions can be occupied by alternative nucleosomes. This organization underscores a substantial freedom of choice by histone octamers when nucleating on a promoter region.

Alcohol Oxidoreductases↗