Dietary cyanidin 3-glucoside from purple corn ameliorates doxorubicin-induced cardiotoxicity in mice.

BACKGROUND AND AIMS: Anthracyclines are effective anticancer drugs that have improved prognosis of hundred thousand cancer patients worldwide and are currently the most common chemotherapeutic agents used for the treatment of blood, breast, ovarian and lung cancers. However, their use is limited because of a cumulative dose-dependent and irreversible cardiotoxicity that can cause progressive cardiomyopathy and congestive heart failure. Aim of the present study was to determine the cardioprotective activity of a dietary source of cyanidin 3-glucoside (C3G), such as purple corn, against doxorubicin (DOX)-induced cardiotoxicity in mice. METHODS AND RESULTS: In vitro studies on murine HL-1 cardiomyocytes showed that pretreatment with both pure C3G and purple corn extract improved survival upon DOX treatment. However, C3G and purple corn extract did not affect the cytotoxic effect of DOX on human cancer cell lines. We then validated in vivo the protective role of a C3G-enriched diet against DOX-induced cardiotoxicity by comparing the effect of dietary consumption of corn isogenic lines with high levels of anthocyanins (purple corn - Red diet - RD) or without anthocyanins (yellow corn - Yellow diet - YD) incorporated in standard rodent diets. Results showed that mice fed RD survived longer than mice fed YD upon injection of a toxic amount of DOX. In addition, ultrastructural analysis of hearts from mice fed RD showed reduced histopathological alterations. CONCLUSION: Dietary intake of C3G from purple corn protects mice against DOX-induced cardiotoxicity.

Genome-wide analysis of p53-regulated transcription in Myc-driven lymphomas.

The tumour suppressor p53 is a transcription factor that controls cellular stress responses. Here, we dissected the transcriptional programmes triggered upon restoration of p53 in Myc-driven lymphomas, based on the integrated analysis of p53 genomic occupancy and gene regulation. p53 binding sites were identified at promoters and enhancers, both characterized by the pre-existence of active chromatin marks. Only a small fraction of these sites showed the 20 base-pair p53 consensus motif, suggesting that p53 recruitment to genomic DNA was primarily mediated through protein-protein interactions in a chromatin context. p53 also targeted distal sites devoid of activation marks, at which binding was prevalently driven by sequence recognition. In all instances, the relevant motif was the canonical unsplit consensus element, with no clear evidence for p53 recruitment by split motifs. At promoters, p53 binding to the consensus motif was associated with gene induction, but not repression, indicating that the latter was most likely indirect. Altogether, our data highlight key features of genome recognition by p53 and provide unprecedented insight into the pathways associated with p53 reactivation and tumour regression, paving the way for their therapeutic application.

The influence of music on exercise in a group of sedentary elderly women: an important tool to help the elderly to stay active.

AIM: The aim of this study was to verify how listening to instrumental asynchronous music, with tempo of 90 bpm, can affect the aerobic physical performance in elderly women engaged in a continuous and constant exercising, predominantly aerobic, consisting of walking routines. METHODS: Twenty women (N.=20, age=75.8±4.2 years) volunteered to the study and underwent a six-week period of physical exercising. All women were previously sedentary, as they had not trained systematically within the last 5 years. The experimental group (Eg=10) performed all the exercise sessions and tests listening to music. The control group (Cg=10) performed the same program without listening to music. Total distances covered, heart rates before and after the tests and the rates of perceived exertion (RPE) were measured. RESULTS: Significant differences between groups (P<0.01) were found in RPE. No statistically significant differences were observed in total distances covered and heart rates, although there was an increase of 9.83% in the total distance covered by the Eg compared to the Cg, in accordance with other previous researches. CONCLUSION: The results are in line with those reported by other authors in different populations and ages, confirming that music may be considered an important tool in supporting elderly people involved in physical exercising.

Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis.

The tumor-suppressor p53 can induce various biological responses. Yet, it is not clear whether it is p53 in vivo promoter selectivity that triggers different transcription programs leading to different outcomes. Our analysis of genome-wide chromatin occupancy by p53 using chromatin immunoprecipitation (ChIP)-seq revealed 'p53 default program', that is, the pattern of major p53-bound sites that is similar upon p53 activation by nutlin3a, reactivation of p53 and induction of tumor cell apoptosis (RITA) or 5-fluorouracil in breast cancer cells, despite different biological outcomes. Parallel analysis of gene expression allowed identification of 280 novel p53 target genes, including p53-repressed AURKA. We identified Sp1 as one of the p53 modulators, which confer specificity to p53-mediated transcriptional response upon RITA. Further, we found that STAT3 antagonizes p53-mediated repression of a subset of genes, including AURKA.

Blood orange juice inhibits fat accumulation in mice.

OBJECTIVE: To analyze the effect of the juice obtained from two varieties of sweet orange (Citrus sinensis L. Osbeck), Moro (a blood orange) and Navelina (a blond orange), on fat accumulation in mice fed a standard or a high-fat diet (HFD). METHODS: Obesity was induced in male C57/Bl6 mice by feeding a HFD. Moro and Navelina juices were provided instead of water. The effect of an anthocyanin-enriched extract from Moro oranges or purified cyanidin-3-glucoside (C3G) was also analyzed. Body weight and food intake were measured regularly over a 12-week period. The adipose pads were weighted and analyzed histologically; total RNA was also isolated for microarray analysis. RESULTS: Dietary supplementation of Moro juice, but not Navelina juice significantly reduced body weight gain and fat accumulation regardless of the increased energy intake because of sugar content. Furthermore, mice drinking Moro juice were resistant to HFD-induced obesity with no alterations in food intake. Only the anthocyanin extract, but not the purified C3G, slightly affected fat accumulation. High-throughput gene expression analysis of fat tissues confirmed that Moro juice could entirely rescue the high fat-induced transcriptional reprogramming. CONCLUSION: Moro juice anti-obesity effect on fat accumulation cannot be explained only by its anthocyanin content. Our findings suggest that multiple components present in the Moro orange juice might act synergistically to inhibit fat accumulation.

KINKY POLLEN encodes a SABRE-like protein required for tip growth in Arabidopsis and conserved among eukaryotes.

In higher plants, pollen tubes and root hairs share an ancient growth process named tip growth. We have isolated three allelic Arabidopsis mutant lines showing kinky-shaped pollen tubes and, when homozygous, showing shorter and thicker root hairs. The ultrastructure of pollen tubes in these kinky pollen (kip) mutants is similar to that of the wild type; however, time-lapse studies suggest that aberrant pollen tube shape is caused by periodic growth arrests alternated with phases of tube axis reorientation. The KIP gene encodes a protein of 2587 amino acids that is predicted to be targeted to the secretory pathway. KIP mRNA was detected in all organs investigated but was most abundant in pollen and roots. KIP has putative homologues in many eukaryotes, including mammals and yeast, and is similar to the Arabidopsis SABRE gene, whose mutation causes a dwarf phenotype. The phenotype of the kip/sab double mutant suggests related functions for both genes, however, the KIP protein is mostly required for tip-growth.

A maize r1 gene is regulated post-transcriptionally by differential splicing of its leader.

Anthocyanin biosynthesis in Zea mays is controlled by regulatory genes of the r1/b1 family that encode bHLH transcription factors. Analysis of the 381 nucleotide leader sequence of a member of this family, Sn, discloses the presence of five ATG triplets upstream of the coding region and three upstream open reading frames (uORFs) of 38, 15 and 13 amino acids respectively. RT-PCR studies revealed that a splicing event occurs in the leader region in the different tissues tested. Splicing deletes 146 nucleotides which include uORF2 and uORF3. By trans-activation experiments in maize protoplasts we find that the spliced leader, compared to the non-spliced one, reduces the number of pigmented protoplasts by four-fold. We suggest a multilevel regulation of the Sn transcription factor acting not only at the transcriptional but also at the post-transcriptional level.

Regulation of the CCAAT-Binding NF-Y subunits in Arabidopsis thaliana.

NF-Y is a CCAAT-specific binding factor composed of three distinct subunits. In vertebrates and fungi all three subunits are encoded by evolutionary conserved single copy genes. In this report we have cloned twenty-three NF-Y genes in A. thaliana, assessed their mRNA expression levels in a large number of tissues and confirmed that indeed multiple CCAAT-binding activities are present. Alignments of the genes coding for the three NF-Y subunits yield a considerable amount of information concerning the divergence/conservation of protein subdomains and of single residues within the conserved parts. Careful evaluation of mRNA expression levels by sensitive RT-PCR assays provide evidence that all three subunits have members that are ubiquitous and others that are tissue-specific and induced only after the switch to reproductive growth phase, in flowers and siliques.

Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis.

An Arabidopsis thaliana line that is mutant for the R2R3 MYB gene, AtMYB4, shows enhanced levels of sinapate esters in its leaves. The mutant line is more tolerant of UV-B irradiation than wild type. The increase in sinapate ester accumulation in the mutant is associated with an enhanced expression of the gene encoding cinnamate 4-hydroxylase, which appears to be the principal target of AtMYB4 and an effective rate limiting step in the synthesis of sinapate ester sunscreens. AtMYB4 expression is downregulated by exposure to UV-B light, indicating that derepression is an important mechanism for acclimation to UV-B in A.thaliana. The response of target genes to AtMYB4 repression is dose dependent, a feature that operates under physiological conditions to reinforce the silencing effect of AtMYB4 at high activity. AtMYB4 works as a repressor of target gene expression and includes a repression domain. It belongs to a novel group of plant R2R3 MYB proteins involved in transcriptional silencing. The balance between MYB activators and repressors on common target promoters may provide extra flexibility in transcriptional control.

The developmental expression of the maize regulatory gene Hopi determines germination-dependent anthocyanin accumulation.

The Hopi gene is a member of the maize r1 gene family. By genetic and molecular analyses we report that Hopi consists of a single gene residing on chromosome 10 approximately 4.5 cM distal to r1. Hopi conditions anthocyanin deposition in aleurone, scutellum, pericarp, root, mesocotyl, leaves, and anthers, thus representing one of the broadest specifications of pigmentation pattern reported to date of all the r1 genes. A unique feature of the Hopi gene is that seeds are completely devoid of pigment at maturity but show a photoinducible germination-dependent anthocyanin accumulation in aleurone and scutellum. Our analysis has shown that the Hopi transcript is not present in scutellum of developing seeds but is induced only upon germination and that the simultaneous presence of both C1 and Hopi mRNAs is necessary to achieve A1 activation in scutella. We conclude that the expression pattern of the Hopi gene accounts for the germination-dependent anthocyanin synthesis in scutella, whereas the developmental competence of germinating seeds to induce anthocyanin production in scutella results from the combination of the light-inducible expression of C1 and the developmentally regulated expression of the Hopi gene.

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana.

Transcription factors containing a conserved DNA-binding domain similar to that of the proto-oncogene c-myb have been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turn-helix motifs, the R2 and R3 repeats. It was estimated that Arabidopsis thaliana contains more than 100 R2R3-MYB genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated. Sequence data for more than 90 different A. thaliana R2R3-MYB genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the A. thaliana genome.

A maize anthocyanin transactivator induces pigmentation in hairy roots of dicotyledonous species.

Several dicotyledonous species were infected with an Agrobacterium rhizogenes binary vector harbouring the plasmid 121.Sn which contains the maize gene Sn under the constitutive promoter CaMV35S. In maize, Sn transactivates the anthocyanin pathway in different tissues. The aim of this work was to test the efficiency of this gene to regulate the anthocyanin pathway in heterologous systems and verify its suitability as a reporter gene. The pigmentation of the hairy roots was compared with hairy roots stained for ß-glucuronidase activity, which were used as a control. In two polymorphic genotypes of Lotus angustissimus, DNA integration and expression were assayed. The maize gene is competent to induce anthocyanin pigmentation in different species, but the complexity of the regulatory mechanisms of anthocyanin synthesis restricts the use of Sn as a reporter gene.

Ectopic anthocyanin pigmentation in maize as a tool for defining interactions between homologous regulatory factors.

The duplicated R and Sn genes are involved in the regulation of the maize anthocyanin biosynthetic pathway, encoding tissue-specific products that are homologous to the helix-loop-helix transcriptional activators. Sn determines the pigmentation of the mesocotyl, leaf basis and pericarp, while R determines pigmentation in various tissues, but not in the mesocotyl. In the progeny derived from test-crosses of R/Sn heterozygous plants, a high frequency of R plants exhibiting mesocotyl pigmentation was observed; these derivatives were defined as R*. In R* plants, the presence of this novel trait was not accompanied by the acquisition of Sn or by gross DNA rearrangements in the R profile. Accordingly, RT-PCR analysis showed that mesocotyl pigmentation in R* was attributable to the resident R gene. The occurrence of R* was observed with all R alleles tested, and was enhanced when a P component was present. The heritability of R* was shown only in the case of the standard R-r allele, which carries a functional P component. In addition, we observed that R* can influence other R alleles, transferring the ability to pigment the mesocotyl. R* is unstable, showing a tendency to return to its original state after a few generations. In R* plants there was a correlation between observed ectopic pigmentation and an increase in the level of A1 transcript but, surprisingly, not in the accumulation of R transcript. The results obtained from the analysis of test crosses of rSn/r delta plants suggest that an unlinked genetic factor accounts for the ectopic pigmentation. Concomitant occurrence of epigenetic events might explain the observed instability and reversibility noted above. Further study of this phenomenon might help to elucidate the basis of the interaction between homologous and non-homologous regulators.

Light-Dependent Spatial and Temporal Expression of Pigment Regulatory Genes in Developing Maize Seeds.

Both light and developmental stimuli are directly involved in the regulation of plant gene expression. In maize, activation of the anthocyanin pathway represents an excellent model system for studying the interactions between an external factor, such as light, and internal factors that regulate plant and seed development. By analyzing in detail the aleurone and pericarp seed layers, different developmental windows for light have been found in the two tissues[mdash]the former in the advanced stages of development and the latter in the early stages of seed development. Transcriptional control of the structural genes involved in anthocyanin deposition within the pericarp is known to be exerted by the Sn and pl genes, whereas the aleurone is controlled by the R and C1 regulatory genes. By using in situ hybridization analysis, we detected tissue-specific expression of Sn and R in the seed layers, revealing a correlation between structural gene activation and anthocyanin accumulation. In addition, RNA gel blot analysis revealed that Sn expression is enhanced by light, whereas the R gene expression is not. However, the light-induced expression of the myb-type genes C1 and pl, detected by reverse transcriptase-polymerase chain reaction, was found to be the limiting factor for conferring the developmental competence of the pericarp and the aleurone layers to light responsiveness.

[Parma Stroke Data Bank: embolic stroke]. / Parma Stroke Data Bank: ictus embolico.

Cerebral infarction is one of the three main causes of death in most countries. It is very frequent and, since it is more often disabiliting rather than fatal, it is of high social impact. The correct classification of patients and the accurate diagnostic definition of the various subtypes of stroke is of great prognostic and therapeutic importance since cerebral infarction is not a single entity. In this study we report our findings concerning 244 patients with embolic infarction recorded in the Parma Stroke Data Bank hospital register. Clinical features were studied (risk factors, symptomatology of the onset, degree of severity within 3 days of the onset, post-stroke complications) as were instruments readings (TAC) and evolution (outcome, mortality, personal performance and environmental integration, both 4 weeks after the clinical onset and after one year).

The reduced expression of endogenous duplications (REED) in the maize R gene family is mediated by DNA methylation.

The duplicated R and Sn genes regulate the maize anthocyanin biosynthetic pathway and encode tissue-specific products that are homologous to helix-loop-helix transcriptional activators. As a consequence of their coupling in the genome, Sn is partially silenced. Genomic restriction analysis failed to reveal gross structural DNA alterations between the strong original phenotype and the weak derivatives. However, the differences in pigmentation were inversely correlated with differences in the methylation of the Sn promoter. Accordingly, treatment with 5-azacytidine (AZA), a demethylating agent, restored a strong pigmentation pattern that was transmitted to the progeny and that was correlated with differential expression of the Sn transcript. Genomic sequencing confirmed that methylation of the Sn promoter was more apparent in the less pigmented seedlings and was greatly reduced in the AZA revertants. In addition, some methylcytosines were located in non-symmetrical C sequences. These findings provide an insight into Sn and R interaction, a process that we have termed Reduced Expression of Endogenous Duplications (REED). We propose that increasing the copy number of regulatory genes by endogenous duplication leads to such epigenetic mechanisms of silencing. Further understanding of the REED process may have broader implications for gene regulation and may identify new levels of regulation within eukaryotic genomes.

[Parma stroke data bank: atherothrombotic and lacunar stroke]. / Parma stroke data bank: ictus aterotrombotico ed ictus lacunare.

In an epidemiological research about stroke, we studied 235 patients with atherothrombotic brain infarctions and 81 patients with lacunes. It was a longitudinal study concerning patients admitted to our Medical Division during the acute phase and followed up for one year after the onset. We report some anamnestic data, the frequency of positive brain CT scan, main risk factors, symptoms at the onset, severity degree of the stroke within the first 72 hours, complications during, and outcome after, the first four weeks, including personal performances and environmental fitness, mortality rate and frequency of relapses. We also report some of these data after one year from the acute episode.

Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.).

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.

Prevalence and prognostic value of peripheral arterial disease in stroke patients.

As atherosclerosis is a multi-systemic disease, each patient presenting clinical manifestation of atherosclerosis such as a stroke or RIND should be, from a vascular point of view, globally evaluated. The availability of Doppler ultrasound technique enables us to discover the presence of Peripheral Artery Disease (PAD) which is not always overt in the patient's history. Furthermore the presence of PAD is associated with a poorer prognosis in stroke patients.