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.

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.

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.

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.

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.

Sn, a light-dependent and tissue-specific gene of maize: the genetic basis of its instability.

The genetic system under investigation is defined by three major components: a gene, Sn, conferring tissue specific anthocyanin accumulation in different plant regions, light, required for color development in competent tissues, and another gene, Pl, substituting for light in its capacity to elicit pigment production. Attention is given in this paper to an Sn allele, symbolized Sn:bol3, capable of some constitutive pigmentation in seedlings and seed integuments. Sn:bol3 confers a higher pigment potential than the other alleles and is unstable. Its instability relates to its frequent changes from an original condition, indicated as Sn-s, to Sn-w, where -s and -w stand for strong and weak and refer to the two levels of seedling pigmentation. Weak derivatives arise spontaneously at a high frequency in homo- and heterozygous Sn:bol3 genotypes. In the latter, weak derivatives are also recovered on the chromosome originally devoid of Sn as if the heterozygous association had promoted "contamination" of one chromosome (recipient) with Sn coming from the other (donor). If the two chromosomes in the heterozygote are marked with contrasting alleles of R, a gene lying about two crossover units proximal to Sn, it appears that the R constitution of the recipient chromosome affects their constitution. Presence of R-r in fact leads to changes of both chromosomes in terms of Sn constitution, resulting in a majority of nonparental chromosomes, R-r Sn and r Sn-w or r sn, while replacement of R-r with R-g, a mutant derivative of R-r, leads to a drastic reduction in the yield of nonparental chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

[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.

[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).

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.

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.

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.

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.).

In excised pro(1-1) mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [(14)C]proline, [(14)C]glutamic acid, [(14)C]glutamine, and [(14)C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO(2) production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro(1-1) mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro(1-1) mutant proline catabolism prevails over proline synthesis.

A mutation causing proline requirement in Zea mays.

A conditional seedling lethal, monogenic recessive, endosperm mutant is described. Phenotypic can be accomplished when embryos are cultured in vitro on media supplemented with proline. The efficiency of the repair is proportional to the concentration of proline in the medium. Normal growth is resumed at a dose of 160 mg/l. All the data collected are most easily interpreted by assuming that the mutant, symbolized pro has a genetic block in the biosynthetic route leading to proline.This is probably the first case of a strict genetic requirement for an amino acid to be reported in Zea mays. The possible reasons for the difficulties encountered in isolating obligate auxotrophs in higher plants are briefly reviewed.

Antiparallel expression of the sense and antisense transcripts of maize alpha-tubulin genes.

In all eukaryotes alpha- and beta-tubulins are encoded by small families of closely related genes and are highly conserved. In Zea mays, at least six different alpha-tubulin coding sequences are known. We describe the isolation from scutellar nodes of the maize inbred line W22 of a clone (CTM5) coding for an alpha-tubulin. On the basis of the 3' end nucleotide sequence, this clone can be assigned to the already reported tua4 gene. Northern analysis demonstrates that CTM5 encodes a 1.5 kb transcript, which is expressed in different tissues of the seed and of the seedling. In order to define the spatial and temporal expression of alpha-tubulin genes, in situ hybridization experiments were performed on these tissues. Unexpectedly, a specific signal was detected with both antisense and sense RNA strands. Temporal and spatial distribution of the two RNAs, however, shows that high levels of the two transcripts are always discordant. In tissues where sense transcripts are highly abundant (embryos at various developmental stages, root tips, pollen grains), the antisense transcripts are expressed in relatively small amounts, while in pericarp, coleoptile, leaves, and scutellar node, where antisense transcripts accumulate, the sense transcript only reaches a very low level. Northern analysis using single-stranded DNA probes confirmed the presence of an antisense transcript of 1.5 kb, prompting speculation about the role of this transcript in the regulation of the expression of alpha-tubulin genes.

Molecular homology among members of the R gene family in maize.

The R gene family determines the timing, distribution and amount of anthocyanin pigmentation in maize. This family comprises a set of regulatory genes, consisting of a cluster of several elements at the R locus, on chromosome 10, the Lc and Sn gene lying about two units R distal and B on chromosome 2. Each gene determines a tissue-specific pigmentation of different parts of the seed and plant. The proposed duplicated function of R, Sn, Lc and B loci is reflected in cDNA sequence similarity. In this paper an extensive analysis of the predicted proteins of the R, Sn, Lc and B genes together with a search for putative sites of post-translational modification is reported. A comparison with the prosite database discloses several N-glycosylation and phosphorylation sites, as well as the basic Helix-Loop-Helix (HLH) domain of transcriptional activators. Sn, Lc, and R-S show a high conservation of these sites, while B is more divergent. Analysis of the 5' leader of mRNA sequences discloses the presence of five ATG triplets with two upstream open reading frames (uORFs) of 38 and 15 amino acids and a loop structure indicating a possible mechanism of control at the translational level. It is conceivable that possible mechanisms acting at the translational and post-translational level could modulate the expression and the activation of these transcription factors. Northern analysis of various tissues of different R alleles highlights a strict correlation between pigment accumulation in different tissues and the expression of the regulatory and structural genes suggesting that the pattern of pigmentation relies on a mechanism of differential expression of the members of the R family. Analysis of the Sn promoter discloses the presence of several sequences resembling binding sites of known transcription factors (as GAGA and GT) that might be responsible for the spatial and light-induced expression of this gene. Two regions include a short sequence homologous to the consensus binding site of the B-HLH domain suggesting a self-regulatory control of the Sn gene.