Milvia Chicca

The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application

The random amplified polymorphic DNA (RAPD) technique was applied to settle a lawsuit involving unauthorized commercialization of a patented strawberry variety of high economical relevance (‘Marmolada’®). Because of economical involvements, the molecular approach was added to the more traditional morphological examination in a double‐blind test. All plants belonging to the patented variety were unambiguously identified (13 plants among a total of 31 plants examined). The results were accepted as evidence in the court. This study confirms that the RAPD technique is especially suitable for identification of asexually reproduced plant varieties for forensic or agricultural purposes.

Xanthine oxidase activity in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a serious respiratory pathology characterized by irreversible limitation of expiratory flow and includes chronic obstructive bronchitis, chronic airflow limitation, and emphysema. To determine whether xanthine oxidase activity increased in the airspaces of COPD patients, we examined bronchoalveolar lavage fluid (BAL) from COPD patients recruited during a 2-year clinical study. Filtered BAL supernatant from COPD patients and healthy nonsmoking controls was examined by fluorometric analysis of DNA unwinding (FADU) and spectrophotometric assays (cytochrome c reduction kinetics and uric acid kinetics). Compared to controls, filtered BAL supernatant of subjects with COPD exhibited a detectable clastogenic activity probably related to superoxide production. The method of BAL preparation as an acellular system strongly suggests that superoxide production may be due to xanthine oxidase activity.

Detection of xanthine oxidase activity products by EPR and HPLC in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease

Xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22), a molybdenum-containing hydroxylase that produces superoxide and uric acid from purine substrates and molecular oxygen, is involved in the oxidative stress underlying several human pathologies including lung diseases. An enzymatic activity similar to xanthine oxidase was previously reported in bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease (COPD-BAL), by fluorometric analysis of DNA unwinding and cytochrome c reduction kinetics. Here we report the detection of xanthine oxidase activity products by electron paramagnetic resonance (EPR) in presence of the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and reversed-phase high-performance liquid chromatography (RP-HPLC) in COPD-BAL (n = 14, average age of patients 65 years, range 38-81) and BAL from healthy nonsmoker controls (n = 6, average age 64 years, range 44-73). Superoxide DMPO adducts were detected in COPD-BAL and in an in vitro system containing xanthine and xanthine oxidase (XA/XO), but not in BAL controls and when superoxide dismutase (SOD, 1000 I.U./ml) was added to COPD-BAL. The HPLC analyses after addition of xanthine showed production of uric acid in COPD-BAL and in the XA/XO system but not in BAL controls. These results support the involvement of xanthine oxidase in the mechanisms of superoxide production by BAL supernatant, which increases oxidative stress in chronic obstructive pulmonary disease.

Molecular cytogenetic analysis of the karyotype of the European Atlantic sturgeon, Acipenser sturio

A karyotype analysis was carried out on the European Atlantic sturgeon, Acipenser sturio (2n=121 ± 3). The telomeric sequence repeat (TTAGGG)n detected by fluorescent in situ hybridization (FISH) was mostly localized at the telomeres of all chromosomes. Ribosomal DNA (rDNA) genes were detected by silver staining techniques and by FISH with digoxigenin-labelled probe for 28S rDNA. Silver staining detected active NORs in the telomeric regions of six chromosomes, and by FISH one or two additional minor sites were detected. The 5S rDNA was found in the interstitial region of a small metacentric pair. The 5S rRNA gene was completely sequenced for the first time in a sturgeon species. The A. sturio karyotype organization is discussed in relation to phylogenesis of the species within the Acipenseridae and to polyploidization events characterizing sturgeon evolution.

Role of Xanthine Oxidase Activation and Reduced Glutathione Depletion in Rhinovirus Induction of Inflammation in Respiratory Epithelial Cells

Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-κB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential.

Histological and ultrastructural investigation of early gonad development and sex differentiation in Adriatic sturgeon (Acipenser naccarii, Acipenseriformes, Chondrostei)

Gonad development and sex differentiation from embryos to 594‐day‐old individuals were investigated in farmed Acipenser naccarii using light and transmission electron microscopy. The migrating primordial germ cells first appear along the dorsal wall of the body cavity in embryos 1.5 days before hatching. The gonadal ridge, containing a few primary primordial germ cells (PGC‐1) surrounded by enveloping cells, appears in 16‐day‐old larvae. At 60 days, the undifferentiated gonad is lamellar and PGC‐1 multiply, producing PGC‐2. In 105‐day‐old juveniles, a distinct germinal area with advanced PGC‐2 appears on the lateral side near the mesogonium and the first blood vessels are visible. At 180 days, putative ovaries with a notched gonadal epithelium and putative testes with a smooth one appear, together with adipose tissue on the distal side. In 210‐day‐old juveniles, active proliferation of germ cells begins in the putative ovaries, whereas putative testes still contain only a few germ cells. The onset of meiosis and reorganization of stromal tissue occurs in ovaries of 292‐day‐old individuals. Ovaries with developed lamellae enclosing early oocyte clusters and follicles with perinucleolar oocytes occur at 594 days. Meiotic stages are never found, even in anastomozing tubular testes of 594‐day‐old individuals. Steroid producing cells are detected in the undifferentiated gonad and in the differentiated ones of both sexes. Anatomical differentiation of the gonad precedes cytological differentiation and female differentiation largely precedes that of the male. Gonad development and differentiation are also associated with structural changes of connective tissue, viz. collagen‐rich areas are massive in developing testes and reduced in ovaries. J. Morphol., 2008.

An EPG Study of the Probing Behavior of Adult Bemisia tabaci Biotype Q (Hemiptera: Aleyrodidae) Following Exposure to Cyantraniliprole

ABSTRACT Cyantraniliprole is a novel insecticide for control of multiple chewing and sucking insect pest species including the sweetpotato whitefly Bemisia tabaci (Gennadius), which is one of the most important polyphagous pests in tropical, subtropical, and Mediterranean regions. This study aims to evaluate the effects of cyantraniliprole on the probing behavior of B. tabaci on tomato. Electrical penetration graph data indicated that on plants treated with cyantraniliprole (foliar application), adult whiteflies of the genetic variant Q2 were not able to reach the phloem and consequently did not perform the activities represented by E1 and E2 waveforms, i.e., phloem salivation (during which inoculation of geminiviruses occurs) and phloem sap ingestion (during which geminiviruses are acquired by the whiteflies), respectively. The complete failure of B. tabaci biotype Q adults to feed from the phloem of tomato plants treated with cyantraniliprole could be explained by rapid cessation of ingestion because of the mode of action of this insecticide. Overall, these findings indicated that cyantraniliprole might represent a useful new tool for producers to protect tomato plants from damage by B. tabaci.

Correlation of adverse effects of cisplatin administration in patients affected by solid tumours: A retrospective evaluation

Cisplatin is the most common antineoplastic drug used for the therapy of solid tumours. To date, researchers have focused on the dosage to be administered for each specific tumour, mainly considering the local adverse effects. The aim of this study was to correlate the severity of the adverse effects with: i) the dosage of cisplatin; ii) the specific site of the tumour; iii) the association with other drugs; and iv) the symptoms. We analysed data from 123 patients with 11 different tumour classes undergoing therapy from 2007 to 2008 at St. Anna Hospital (Ferrara, Italy), using the Spearman non-parametric correlation index. Even though significant correlations were found among the variables, the overall results showed that the main factor influencing the severity of the adverse effects was the dosage of cisplatin administered.

Immunocytochemical studies on the pituitary gland of Anguilla anguilla L., in relation to early growth stages and diet-induced sex differentiation

Mammalian and teleost antisera against pituitary hormones were used to identify and localize pituitary cell types in the European eel (Anguilla anguilla L.). The investigation was conducted on unpigmented glass eels of 5.6-6.2 of total body length (L(T)) caught in river mouths, then on yellow eels reared from the pigmented glass eel (or elver) stage up to 12-14 cm of L(T), in an eel farm in warm freshwater. Treated elvers were fed with commercial paste food supplemented with mature carp ovaries, containing oestradiol, that induced an early ovarian differentiation and a higher growth rate. The antisera detected seven types of immunoreactive (ir) cells, six of which were already found in glass eel adenohypophysis, suggesting differentiation of these cell types during the leptocephalus stage. In 12-14 cm treated yellow eels with small ovaries, a seventh type (ir-GtH) was detected in the proximal pars distalis; in the same animals the ir-TSH cells increased in number and size. From unpigmented glass eels to 12-14 cm yellow eels, the whole pituitary volume of controls increased nearly four times, while that of treated ones increased nearly six times. The larger volume of pituitary in treated eels was mainly due to volume increase of proximal pars distalis and rostral pars distalis. The %GH, that is the potential index of GH production, was significantly higher in treated yellow eels with gonads differentiating into ovaries than in controls; no difference was detected in %PRL between treated and control eels. The above results strongly suggest that in eels the feminizing effects of oestrogen is first exerted on the pituitary, probably through the hypothalamus, and later on the gonads.

Correlation between age and DNA damage detected by FADU in human peripheral blood lymphocytes.

Fluorometric analysis of DNA unwinding (FADU) is a fast and reliable method for detecting single strand DNA breaks as an index of DNA damage induced by clastogenic agents. A study of damage detected by FADU was conducted on DNA extracted from peripheral blood lymphocytes of 128 healthy nonsmoking regular donors (ranging in age from 19 to 67 years) and from 5 umbilical cord blood samples. DNA damage was measured as percentage of unwound DNA after alkalinization. Statistical analyses, both parametric (Pearson r correlation coefficient, b regression coefficient, ANOVA) and nonparametric (Kruskal-Wallis H test, Spearman rs rank correlation coefficient), support a significant correlation between age of donors and amount of DNA damage. The same results are found when adult donors are divided in four age classes and the ANOVA test performed among the mean percentages of unwound DNA of each class. Furthermore, donors of the same age belonging to different blood groups (A, B, AB and O) do not show any difference in DNA damage detected by FADU.