Dietary supplementation with green tea extract improves the antioxidant status and oocyte developmental competence in Italian Mediterranean buffaloes.

The aim of this work was to assess the antioxidant status and the developmental competence of oocytes recovered by ovum pick-up (OPU) in Italian Mediterranean buffaloes supplemented with green tea extracts (GTE) for 90 days. Buffalo cows (n = 16) were randomly assigned to a control group receiving no supplement and a treatment group, receiving GTE starting 90 days before OPU, carried out for five consecutive sessions. Blood samples were collected before the start of supplementation with GTE (T0) and at day 45 (T1) and day 90 (T2) of supplementation, to measure ferric reducing activity (FRAP), total antioxidant capacity (TAC), superoxide dismutase (SOD) and catalase (CAT). The antioxidant status of follicles was measured as TAC on the follicular fluid collected from the dominant follicle just prior OPU, coinciding with T2, and at the end of five repeated OPU sessions (T3). Another objective was to assess in vitro the protective effects of green tea extracts on hepatic cells exposed to methanol insult. Different concentrations of GTE (0.5 µM and 1 µM) were tested on cultured hepatic cells and viability, morphology and SOD activity were assessed at 24, 48 and 72 h. Supplementation with GTE increased (P < 0.05) the number of total follicles (8.7 ± 0.5 vs 6.9 ± 0.5), the number and the percentage of Grade A + B cumulus-oocyte complexes (COCs) compared with the control (3.7 ± 0.4 vs 2.3 ± 0.3 and 57.5 ± 4.2 vs 40.4 ± 4.9 %, respectively). Oocyte developmental competence was improved in the GTE group as indicated by the higher (P < 0.05) percentages of Grade 1,2 blastocysts (44.8 vs 29.1 %). In the GTE group, plasma TAC was higher both at T1 and T2, while FRAP increased only at T2, with no differences in SOD and CAT. The TAC of follicular fluid was higher (P < 0.05) in the GTE compared to the control both at T2 and at T3 The in vitro experiment showed that co-treatment with methanol and 1 µM GTE increased (p < 0.01) cell viability at 24 h (P < 0.01), 48 h (P < 0.05) and 72 h (P < 0.01) compared with the methanol treatment co-treatment with 1 µM GTE prevented the decrease in SOD activity observed with methanol at 24 and 48 h of culture. In conclusion, the results of in vivo and in vitro experiments suggest that supplementation with GTE increases buffalo oocyte developmental competence, by improving oxidative status and liver function.

Inhibition of long-term potentiation by CuZn superoxide dismutase injection in rat dentate gyrus: involvement of muscarinic M1 receptor.

Long-term potentiation (LTP) and long-term depression represent important processes that modulate synaptic transmission that carries out a key role in neural mechanisms of memory. Many studies give strong evidences on a role of the reactive oxygen species in the induction of LTP in CA1 region of hippocampal slices that was inhibited by adding the scavenger enzyme superoxide dismutase (SOD1). Previous data showed that SOD1 is secreted by many cellular lines, including neuroblastoma SK-N-BE cells through microvesicles by an ATP-dependent mechanism; moreover, it has been shown that SOD1 interacts with human neuroblastoma cell membranes increasing intracellular calcium levels via a phospholipase C-protein kinase C pathway activation. The aim of this study was to investigate the effect of intracerebral injection of SOD1 or the inactive form of enzyme (ApoSOD) on the modulation of synaptic transmission in dentate gyrus of the hippocampus in urethane anesthetized rats. The results of the present research showed that intracerebral injection of SOD1 and ApoSOD in the dentate gyrus of the rat hippocampal formation inhibits LTP induced by high-frequency stimulation of the perforant path. This result cannot be only explained by the dismutation of oxygen radical induced by SOD1 since also ApoSOD, that lacks the enzymatic activity, carries out the same inhibitory effect on LTP induction.

Differential p63 and p53 expression in human keloid fibroblasts and hypertrophic scar fibroblasts.

The p63 gene belongs to the p53 gene family and encodes for sequence-specific transcription factors. p63 has been characterized primarily in the context of epidermis where is implicated in the establishment of keratinocyte cell fate and in maintenance of epithelial self-renewal. DeltaNp63 isoform has been showed to be involved in several kinds of human tumors of epidermal origin, even nonmalignant, for the neoplastic and proliferative potential. Here, we report the differential expression and the cellular localization of the DeltaNp63 isoform in fibroblasts isolated from human keloids and hypertrophic scars compared to normal skin. Differently from hypertrophic scar, our results show that DeltaNp63 has a nuclear localization and is overexpressed only in keloid fibroblasts, suggesting an essential role of DeltaNp63 in vivo in human keloids. Consistent with our results, we hypothesize that DeltaNp63 overexpression may be oncogenic because of its ability to block the activity of p53 since p53 is underexpressed in fibroblasts from keloids.

Nuclear morphometry in canine acanthomatous ameloblastomas and squamous cell carcinomas.

The aim of this study was to evaluate whether morphometrical analysis can be of diagnostic value for canine acanthomatous ameloblastoma. We calculated, by means of an automated image analyser, some morphometric nuclear parameters, in particular: mean nuclear area (MNA), mean nuclear perimeter (MNP), maximum and minimum diameters (MDx and MDm) coefficient of variation of the nuclear area (NACV), largest to smallest dimension ratio (LS ratio), and form factor (FF), in 8 canine acanthomatous ameloblastomas, and we compared these morphometric data to those of 13 squamous cell carcinomas of canine gingiva. The results indicated a progressive increase of the MNA, NACV, MNP and MDm proceeding from acanthomatous ameloblastomas (MNA: 42.11+/-8.74; NACV: 28,36+/-7,23; MNP: 24.18+/- 2.68; MDm: 5.69+/-0.49) to squamous cell carcinomas (MNA:49,69+/-9,10; NACV: 30,89+/-7,75; MNP: 25.63+/-2.54; MDm: 6.64+/-0.73). On the contrary, the LS ratio and the FF resulted greater in acanthomatous ameloblastomas (LS ratio: 1,63+/-0,12; FF: 1,13+/-0,002) than in SCCs (LS ratio: 1,40+/-0,12; FF:0.91+/-0.38). Moreover, the MNA, MNP,MDx and MDm resulted similar (MNA: p=0.89; MNP: p=0,65; MDm: p=0,16; MDx: p=0,13) in a subset of four acanthomatous ameloblastomas with cellular atypia (MNA:49,01+/-6,88; MNP: 26,28+/-1,99; MDm: 6.08+/-0.41; MDx: 10.18+/-0.88) and in squamous cell carcinomas (MNA:49.69+/-9,10; MNP: 25.63+/-2.54; MDm: 6.64+/-0.73; MDx: 9.26+/-1.05). While the NACV values resulted higher in typical acanthomatous ameloblastoma (29,99+/-6,06) than in atypical acanthomatous ameloblastoma (26,74+/-8,84) and similar to those of the SCCs (30,89+/-7,75). These results seem to confirm that acanthomatous ameloblastoma is a malignant or potentially malignant lesion and emphasizes that nuclear morphometry analysis can be an useful diagnostic and prognostic method in canine oral pathology.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Dietary and hypothyroid hypercholesterolemia induces hepatic apolipoprotein E expression in the rat: direct role of cholesterol.

Apolipoprotein E (apo E) exerts a protective effect against atherosclerosis, related to its role in intracellular cholesterol removal and remnants clearance. In this study we investigated the effect of dietary and hypothyroid hypercholesterolemia, induced respectively by a high cholesterol diet and by propylthiouracil, on hepatic apo E expression in Wistar male rats. The Northern and Western blot analysis of hepatic mRNA and protein levels showed a 2-3-fold increase of apo E in hypercholesterolemic rats compared to controls. The incubation of FAO rat hepatoma cells with 25-OH cholesterol and mevalonate led to a three-fold increase of apo E mRNA, demonstrating a direct role of cholesterol on apo E expression. This effect was completely abolished by elevating intracellular cAMP levels with forskolin. Immunoblot and immunofluorescence analysis revealed that 25-OH cholesterol/mevalonate strongly increased also apo E protein synthesis and secretion in FAO cells. Our data demonstrate that hypercholesterolemia, apart of the cause (diet or hypothyroidism) induces liver apo E expression in the rat and that this effect can be directly related, via cAMP, to cholesterol.

Morphometric study of nucleolar organiser regions (AgNOR) in HPV-associated precancerous lesions and microinvasive carcinoma of the oral cavity.

A morphometric study of silver-stained nucleolar organiser regions (NOR) was performed on histological sections from routinely paraffin-embedded blocks of 26 oral biopsy specimens (21 cases of leukoplakia consisting of 13 low, 4 moderate, 4 severe degree of dysplasia, and 5 cases of microinvasive carcinomas). In situ hybridisation (ISH) for HPV-DNA was performed on serial sections of the same samples. The following parameters were studied: V NOR (single AgNOR volume per nucleus), TV NOR (total AgNOR volume per nucleus), and R.I. (AgNOR's roundness index). The results highlight that not all the morphometric features of AgNORs allow the discrimination between lesions with dysplasia of low, moderate and severe degree and microinvasive carcinoma. TV NOR appeared useful, while the other morphometric parameters appeared statistically not significant i differentiating between the different lesions. These findings suggest that high values of TV NOR in oral dysplasia could represent a risk marker, identifying a subgroup of lesions with a worse prognosis, constituting then a possible indication for rigorous clinical management and/or for complex treatment of these HPV-associated preneoplastic lesions.