Cinzia Forni

Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the in vivo metastatic potential

Summary.The in vitro and in vivo effects of two flavonons, naringenin (NG) and hesperitin (HP) on the proliferation rate of highly metastatic murine B16-F10 melanoma cell were investigated. NG or HP treatment of melanoma cells produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, spermidine and spermine and the enhancement of transglutaminase (TGase, EC 2.3.2.13) activity. Orally administered NG or HP in C57BL6/N mice inoculated with B16-F10 cells affected the pulmonary invasion of melanoma cells in an in vivo metastatic assay. The number of lung metastases detected by a computerized image analyzer was reduced, compared to untreated animals, by about 69% in NG-treated mice and by about 36% in HP-treated mice. Survival studies showed that 50% of the NG-treated animals died 38 ± 3.1 days after tumor cell injection (control group: 18 ± 1.5 days) and HP-treated mice died 27 ± 2.3 days after cell inoculation. Taken together, these findings provide further evidences for the potential anticancer properties of dietary flavonoids as chemopreventive agents against malignant melanoma.

Antineoplastic activity of strawberry (Fragaria × ananassa Duch.) crude extracts on B16-F10 melanoma cells

The antiproliferative and differentiation potential of anthocyanin-rich strawberry fruit crude extracts (SE) were investigated on B16-F10 murine melanoma cells. Treatment of melanoma cells with SE produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, and the enhancement of tissue transglutaminase (TG2, EC 2.3.2.13) activity (used as a differentiation marker). To gain further insight into profiling altered protein expression as a potential biomarker of the SE action on melanoma cells, analysis of the proteomic profile was performed on the treated B16-F10 cells, compared to the control. Following SE treatment, 30 proteins resulted up-regulated, and 87 proteins were down-regulated. In particular proteins overexpressed in cancer cells, involved in tumor progression and metabolism, were down-regulated. The possibility that SE may affect the Warburg effect in B16-F10 melanoma cells is discussed.

Different size, shape and growth behaviour of cells in suspension cultures of strawberry (Fragaria x ananassa Duch.)

Abstract Morphology, viability, nuclear DNA contents, and anthocyanin and phenol concentrations of the cell suspension cultures of the commercial strawberry (Fragaria x ananassa Duch.) cultivar Don were analysed. The phenylalanine ammonia‐lyase activity (PAL) was also determined. The cell suspensions consisted of three different kinds of cells: small round cells (< 65 μm diameter, type A), round cells (88–100 μm, type B) and elongated cells (100–200 μm length, type C). Each cell type was single or included in cell clusters of 20 to 100 cells (type G) (200–300 μm). Growth experiments of single‐cell type and cluster cultures showed that single‐cell cultures of type A andB did not grow at all, whereas cultures of C cells had a slow growth rate. The growth rates of G cells compared with type A and B cells were significantly higher. Type C cells were always the major component of the suspension. The cell clusters had a higher percentage of viable cells in all the growth phases, and the differences in viability...

Comparative analysis of DNA nuclear content by flow cytometry on strawberry plants propagated via runners and regenerated from meristem and callus cultures.

ABSTRACT DNA variation may occur in plant species grown either in vivo or in vitro. In this study flow cytometric analyses were undertaken on Fragaria x ananassa Duch. runner plants, and on plants regenerated from callus cultures of leaf explants and from meristem cultures. Our aims were to investigate DNA variation in runner plants of different cultivars, and to compare DNA content in plants of the same cultivar obtained by different propagation procedures (i.e. from meristems or callus cultures). Plants growing in vitro and in the greenhouse were also compared. A good regeneration ability was observed in all the cultivars, with different percentages of shoot formation. No significant differences were detected in multiplication rate and rooting percentage within cultivars. This work documents the occurrence of DNA variations in strawberry plants in vivo and in vitro. Flow cytometric measurements of DNA content showed the presence of 4C nuclei, besides 2C nuclei, in runner plants of cultivar Pajaro. DNA content variations (2C/4C nuclei) were observed in plants regenerated from callus cultures. These variations were lost after transfer of the plants to the greenhouse, except for cultivar Don. The extent of such DNA variations was influenced by genotype. Our study confirms earlier reports indicating that DNA variation induced by in vitro culture could be lost or retained after transfer of the plants to the greenhouse.

Methyl jasmonate promotes anthocyanins production in Prunus salicina × Prunus persica in vitro shoot cultures.

Abstract In vitro shoot cultures of Prunus salicina × Prunus persica, “Citation®” rootstock, were treated with 50-μM methyl jasmonate (MJ) or 100-μM abscisic acid (ABA); in MJ-treated shoots, total anthocyanins increased significantly (1.88 mg/g fresh weight) relative to controls (0.43 mg/g fresh weight). Cyanidin 3-glucoside was the most abundant anthocyanin in both MJ-treated and control explants. The addition of ABA to the culture medium did not elicit anthocyanins’ accumulation.

Assessment of the antiproliferative activity on murine melanoma cells of extracts from elicited cell suspensions of strawberry, strawberry tree, blackberry and red raspberry

The antiproliferative activity of extracts from cell suspensions of strawberry (Fragaria x ananassa Duch.), blackberry (Rubus fruticosus L.), red raspberry (Rubus idaeus L.) and strawberry-tree (Arbutus unedo L.) on murine B16-F10 melanoma cells was investigated. To enhance the synthesis of the bioactive compounds, various elicitors, i.e. temperature (cold and cold/warm cycle), light (blue and red) and increased nitrogen concentration, were also applied to the cell cultures to evaluate the effects on their antiproliferative activity. The extracts from all the species reduced murine melanoma cell proliferation (between 30% and 38% relative to the control) and an improvement of antiproliferative activity on murine melanoma cells, relative to the un-elicited berry suspensions, was shown by the extracts from strawberry and strawberry-tree cells treated with red and blue light.

Proteomics and Bryophytes: a comparison between different methods of protein extraction to study protein synthesis in the aquatic moss Leptodictyum riparium (Hedw.)

Abstract Proteomics is a leading technology for the study of proteins on a genome-wide scale, the objective of which is the large-scale identification of all protein species in a cell or tissue. In plant biology the key for successful proteomic application relies on the establishment of method to obtain high-quality protein samples. In this paper we compare three different procedures, TCA precipitation in acetone,TCA and PVPP precipitation in acetone, and precipitation in Tris-HCl to be applied for the extraction and purification of proteins from the aquatic moss Leptodic-tyum riparium. Quality gels were obtained with the Tris-HCl and TCA-acetone extraction methods.

Pigments for natural dye-sensitized solar cells from in vitro grown shoot cultures

Abstract. In vitro grown shoots cultures (Prunus salicina × Prunus persica), elicited by methyl jasmonate (MJ), are reported here for the first time to prepare a natural dye for dye-sensitized solar cells (DSSC). Redox properties of the dye, its photostability, and light absorption properties suggested it as a candidate as natural photosensitizers for TiO2 photoelectrodes. Redox properties of the dye influence the DSSC production of photocurrent, thus three antioxidant assays were performed in order to characterize the antioxidant potential of this dye. The dye exhibited a high antioxidant activity in all the assays performed. Photostability assay revealed that the dye was quite stable to light. The power conversion efficiency that we obtained (0.53%) was comparable to the data by other authors with anthocyanins-based dyes from in vivo grown plants. Finally, we compared the dye with the partially purified one as photosensitizer in DSSC. The results indicated that the raw pigment from in vitro shoot cultures of P. salicina × P. persica elicited with MJ can be proposed without the needing of any other chemicals, thermal or purification process, or pH adjustments, as a dye for natural sensitized solar cells.

Pollutants toxicity towards aquatic macrophytes

In India there is no present government policy to survey and evaluate adverse drug events (ADEs) / Pharmacovigilance programme in veterinary medicine. Therefore, essential information such as frequency, severity of treated animal ADRs and reliable data about frequent ADR-producing drugs remains unknown. The objective of the study is to assess and communicate risks and benefits in the market. Ultimately to educate the veterinarians and the stake holders on the safety and efficacy of veterinar y drugs and biologicals. To this purpose, a 12 month pilot study based on WHO r ecommendations was conducted to monitor ADRs in the livestock treated by field veterinarian in the state of T amil Nadu in India for frequently used drugs and common labeled signs. The present study warrants for the need for sustained veterinary pharmacovigilance programmers in livestock for timely ADR presenting drug reactions and drug safety improvement.O of the ways adopted by person for suicide is drowning. Criminals also hide their crime by throwing person in water after murder. To identify antemortem drowning or postmortem drowning, many postmortem findings are considered along with diatom test from bone marrow but many fallacies are there and false results are obtained. To overcome all these difficulties, the new method is proposed which is easy, less cumbersome, economical and confirmatory test; drowning by diatom test from tracheal fluid obtained by distilled water washed tracheal fluid.H susceptibility to xenobiotics, such as pharmaceutical drugs and genotoxicants, is highly variable. Much variability is conferred by polymorphisms in P450 genes and in housekeeping genes involved in basic DNA repair and metabolism. Since humans express multiple P450 genes, it is often difficult to discern which variant P450 gene confers xenobiotic susceptibility. In addition, epidemiological studies are often limited due to small patient populations. As a model xenobiotic, the potent liver carcinogen aflatoxin B1 (AFB1) was used, which is metabolically activated by CYP1A2 and CYP3A4. Since (budding yeast) do not express P450 genes that can metabolically activate AFB1, it was possible to phenotype CYP1A2 variants and 2) profile the yeast genome for resistance to AFB1. AFB1 activation based on DNA adducts, Rad51 polymorphisms, cell survival, and genome instability was characterized. Considering that 31% of (Saccharomyces cerevisiae) budding yeast genes are very similar to human genes, it was hypothesized that genes that confer AFB1 resistance in budding yeast will also confer resistance in humans. CYP1A2 was introduced into the diploid yeast collection containing ~5000 single gene deletions. Using state-of-the-art next generation DNA sequencing, ~500 genes were identified that confer AFB1 resistance, including genes that function in DNA repair, checkpoint response and adaptation, DNA damage tolerance, and oxidative stress. Future studies will be focused on identifying whether down-regulation of these human orthologs also confer AFB1 sensitivity. The author and his co-workers are now profiling resistance to additional P450-activated xenobiotics and phenotyping CYP1A1 polymorphisms.C kidney disease (CKD) is associated with a decreased expression and activity of several cytochrome P450 enzymes. This may result in drug-associated toxicity in CKD patients taking drugs that are metabolized by affected isozymes. The objective of this study was to determine the mechanism of hepatic drug metabolizing enzyme down-regulation in CKD. Hepatic CYP3A1, CYP3A2 and CYP2C11 mRNA expression were determined in rats with surgically induced CKD. Chromatin Immunoprecipitation (ChIP) was performed to determine nuclear receptor and epigenetic mediated differences in the promoter region of these enzymes. Hepatic CYP3A and CYP2C11 mRNA expression was significantly decreased in CKD rats compared to controls (P<0.05). RNA polymerase II binding to the CYP3A and CYP2C11 promoter regions was decreased in CKD rats (P<0.05). ChIP also revealed a decreased PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). HNF4α binding to the CYP3A and CYP2C11 promoter regions was also decreased compared to controls (P<0.05). The decrease in PXR and HNF4α binding was concurrent with diminished histone 4 acetylation in the CYP3A2 promoter locus for nuclear receptor activation. The uremic toxin indoxyl sulfate also mediates a decrease in CYP3A expression. A novel mechanism of drug metabolizing enzyme regulation in CKD was demonstrated. The results show that decreased CYP3A and CYP2C11 mRNA expression is secondary to decreased PXR and HNF4α binding as a result of histone modulation in CKD. These data may partially explain why patients with CKD have a higher incidence of adverse medication events than patients with normal kidney function.oxic Epidermal Necrolysis (TEN) is a rare, life threatening skin reaction that is usually drug-induced. Anti-convulsants such as phenytoin, carbamazepine and phenobarbital and some antibiotics such as co-trimoxazole, quinolones and cephalosporins have been identified as common causes of drug-induced TEN. TEN has many serious complications including dehydration, increased energy expenditure and local or systemic infections. Many studies and case reports were published in the literature supporting the association between phenytoin and the development of TEN. Also many other factors can put the patient at higher risk for developing TEN while on phenytoin those include advanced age, malignancy and radiation exposure. More than one mechanism has been proposed to explain TEN pathophysiology. Hypersensitivity due to toxic metabolites of involved drugs is one theory. Genetic basis for drug-induced TEN has been proposed where there is inherited or acquired deficiency in phase 2 detoxification enzymes. Few studies have also indicated an association between HLA*1502 and phenytoin induced TEN. Family history of hypersensitivity reactions to medications should be documented and discussed with the patients. TEN’s treatment requires multidisciplinary approach to identify and withdraw the causative agent, controlling fluid and temperature homeostasis, preventing multi-organ damage, and treating systemic complications. Supportive therapy is the main strategy of treatment. Phenytoin-induced TEN carries a high mortality and morbidity rate, so accurate diagnosis and rapid treatment is essential to treat and prevent complications. It’s vital to make sure that the “right” patient is taking the “right” dose of the “right” medication. Medications history documentation with special drug allergy card indicating any history of drug reaction is recommended.T indiscriminate discharge of pollutants, such as heavy metals, surfactants and drugs, generated by anthropogenic activities is causing a tremendous hazard to both aquatic and terrestrial habitats and to human health. Therefore in recent years, the interest towards the toxic effects of these molecules on living organisms has increased. Plants have to cope with the detrimental effects of pollutants, e.g., typical symptoms of their toxicity are decrease of growth rate and chlorophylls, root detachment and leaf chlorosis and necrosis. The ability of plants to survive depends on the metabolic responsiveness of detoxification mechanisms. In fact, consequence to the toxicity is the elicitation of stress response that involved changes in the activity of enzymes, such as peroxidases, as well as enzymes of phenylpropanoid pathway, which is responsible for the synthesis of a diverse array of phenolic metabolites. These compounds are often induced by stress and serve specific roles in plant protection as well as structural components of the cell wall. The ability of plants to withstand the toxicity and accumulate pollutants is the base of environmental phytotechnologies, since numerous species can be an interesting tool for remediation of contaminated soils and waters. The mechanisms of plant defence against pollutant toxicity have been studied in floating macrophyte-based model systems. The results obtained will be discussed together with the future perspective of phytoremediation of polluted waters.Results: The total of 609 battery manufacturing male workers were consisting of 285(46.8%) workers with blood lead level (BLL) <40 μg/dl and 324 (53.2%) workers with BLL ≥40 μg/dl. High frequency (3, 4, 6, 8 Khz) hearing loss at hearing threshold above 25 dB in either ear was significantly more prevalent in workers with blood lead level ≥40 μg/dl (Adjusted Odds ratio=2.66, 95%CI: 1.86 3.80, P<0.001 and Adjusted Odds ratio=1.60, 95%CI: 1.13-2.27, P<0.008 for age and work duration respectively). Mean noise exposure level was 84.0 dBALeq.

How plants cope with abiotic stress

In India there is no present government policy to survey and evaluate adverse drug events (ADEs) / Pharmacovigilance programme in veterinary medicine. Therefore, essential information such as frequency, severity of treated animal ADRs and reliable data about frequent ADR-producing drugs remains unknown. The objective of the study is to assess and communicate risks and benefits in the market. Ultimately to educate the veterinarians and the stake holders on the safety and efficacy of veterinar y drugs and biologicals. To this purpose, a 12 month pilot study based on WHO r ecommendations was conducted to monitor ADRs in the livestock treated by field veterinarian in the state of T amil Nadu in India for frequently used drugs and common labeled signs. The present study warrants for the need for sustained veterinary pharmacovigilance programmers in livestock for timely ADR presenting drug reactions and drug safety improvement.O of the ways adopted by person for suicide is drowning. Criminals also hide their crime by throwing person in water after murder. To identify antemortem drowning or postmortem drowning, many postmortem findings are considered along with diatom test from bone marrow but many fallacies are there and false results are obtained. To overcome all these difficulties, the new method is proposed which is easy, less cumbersome, economical and confirmatory test; drowning by diatom test from tracheal fluid obtained by distilled water washed tracheal fluid.H susceptibility to xenobiotics, such as pharmaceutical drugs and genotoxicants, is highly variable. Much variability is conferred by polymorphisms in P450 genes and in housekeeping genes involved in basic DNA repair and metabolism. Since humans express multiple P450 genes, it is often difficult to discern which variant P450 gene confers xenobiotic susceptibility. In addition, epidemiological studies are often limited due to small patient populations. As a model xenobiotic, the potent liver carcinogen aflatoxin B1 (AFB1) was used, which is metabolically activated by CYP1A2 and CYP3A4. Since (budding yeast) do not express P450 genes that can metabolically activate AFB1, it was possible to phenotype CYP1A2 variants and 2) profile the yeast genome for resistance to AFB1. AFB1 activation based on DNA adducts, Rad51 polymorphisms, cell survival, and genome instability was characterized. Considering that 31% of (Saccharomyces cerevisiae) budding yeast genes are very similar to human genes, it was hypothesized that genes that confer AFB1 resistance in budding yeast will also confer resistance in humans. CYP1A2 was introduced into the diploid yeast collection containing ~5000 single gene deletions. Using state-of-the-art next generation DNA sequencing, ~500 genes were identified that confer AFB1 resistance, including genes that function in DNA repair, checkpoint response and adaptation, DNA damage tolerance, and oxidative stress. Future studies will be focused on identifying whether down-regulation of these human orthologs also confer AFB1 sensitivity. The author and his co-workers are now profiling resistance to additional P450-activated xenobiotics and phenotyping CYP1A1 polymorphisms.C kidney disease (CKD) is associated with a decreased expression and activity of several cytochrome P450 enzymes. This may result in drug-associated toxicity in CKD patients taking drugs that are metabolized by affected isozymes. The objective of this study was to determine the mechanism of hepatic drug metabolizing enzyme down-regulation in CKD. Hepatic CYP3A1, CYP3A2 and CYP2C11 mRNA expression were determined in rats with surgically induced CKD. Chromatin Immunoprecipitation (ChIP) was performed to determine nuclear receptor and epigenetic mediated differences in the promoter region of these enzymes. Hepatic CYP3A and CYP2C11 mRNA expression was significantly decreased in CKD rats compared to controls (P<0.05). RNA polymerase II binding to the CYP3A and CYP2C11 promoter regions was decreased in CKD rats (P<0.05). ChIP also revealed a decreased PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). HNF4α binding to the CYP3A and CYP2C11 promoter regions was also decreased compared to controls (P<0.05). The decrease in PXR and HNF4α binding was concurrent with diminished histone 4 acetylation in the CYP3A2 promoter locus for nuclear receptor activation. The uremic toxin indoxyl sulfate also mediates a decrease in CYP3A expression. A novel mechanism of drug metabolizing enzyme regulation in CKD was demonstrated. The results show that decreased CYP3A and CYP2C11 mRNA expression is secondary to decreased PXR and HNF4α binding as a result of histone modulation in CKD. These data may partially explain why patients with CKD have a higher incidence of adverse medication events than patients with normal kidney function.oxic Epidermal Necrolysis (TEN) is a rare, life threatening skin reaction that is usually drug-induced. Anti-convulsants such as phenytoin, carbamazepine and phenobarbital and some antibiotics such as co-trimoxazole, quinolones and cephalosporins have been identified as common causes of drug-induced TEN. TEN has many serious complications including dehydration, increased energy expenditure and local or systemic infections. Many studies and case reports were published in the literature supporting the association between phenytoin and the development of TEN. Also many other factors can put the patient at higher risk for developing TEN while on phenytoin those include advanced age, malignancy and radiation exposure. More than one mechanism has been proposed to explain TEN pathophysiology. Hypersensitivity due to toxic metabolites of involved drugs is one theory. Genetic basis for drug-induced TEN has been proposed where there is inherited or acquired deficiency in phase 2 detoxification enzymes. Few studies have also indicated an association between HLA*1502 and phenytoin induced TEN. Family history of hypersensitivity reactions to medications should be documented and discussed with the patients. TEN’s treatment requires multidisciplinary approach to identify and withdraw the causative agent, controlling fluid and temperature homeostasis, preventing multi-organ damage, and treating systemic complications. Supportive therapy is the main strategy of treatment. Phenytoin-induced TEN carries a high mortality and morbidity rate, so accurate diagnosis and rapid treatment is essential to treat and prevent complications. It’s vital to make sure that the “right” patient is taking the “right” dose of the “right” medication. Medications history documentation with special drug allergy card indicating any history of drug reaction is recommended.T indiscriminate discharge of pollutants, such as heavy metals, surfactants and drugs, generated by anthropogenic activities is causing a tremendous hazard to both aquatic and terrestrial habitats and to human health. Therefore in recent years, the interest towards the toxic effects of these molecules on living organisms has increased. Plants have to cope with the detrimental effects of pollutants, e.g., typical symptoms of their toxicity are decrease of growth rate and chlorophylls, root detachment and leaf chlorosis and necrosis. The ability of plants to survive depends on the metabolic responsiveness of detoxification mechanisms. In fact, consequence to the toxicity is the elicitation of stress response that involved changes in the activity of enzymes, such as peroxidases, as well as enzymes of phenylpropanoid pathway, which is responsible for the synthesis of a diverse array of phenolic metabolites. These compounds are often induced by stress and serve specific roles in plant protection as well as structural components of the cell wall. The ability of plants to withstand the toxicity and accumulate pollutants is the base of environmental phytotechnologies, since numerous species can be an interesting tool for remediation of contaminated soils and waters. The mechanisms of plant defence against pollutant toxicity have been studied in floating macrophyte-based model systems. The results obtained will be discussed together with the future perspective of phytoremediation of polluted waters.Results: The total of 609 battery manufacturing male workers were consisting of 285(46.8%) workers with blood lead level (BLL) <40 μg/dl and 324 (53.2%) workers with BLL ≥40 μg/dl. High frequency (3, 4, 6, 8 Khz) hearing loss at hearing threshold above 25 dB in either ear was significantly more prevalent in workers with blood lead level ≥40 μg/dl (Adjusted Odds ratio=2.66, 95%CI: 1.86 3.80, P<0.001 and Adjusted Odds ratio=1.60, 95%CI: 1.13-2.27, P<0.008 for age and work duration respectively). Mean noise exposure level was 84.0 dBALeq.