Immunohistochemical analysis of pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expression patterns in gastric cancer

Although the considerable progress against gastric cancer, it remains a complex lethal disease defined by peculiar histological and molecular features. The purpose of the present study was to investigate pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expressions, and analyze their possible correlations with clinicopathological factors. The expression patterns were examined by immunohistochemistry in 47 patients, 27 evaluated of intestinal-type, and 20 of diffuse-type, with a mean follow up of 56 months and by Western blot in AGS, N87, KATO-III, and YCC-2, -3, -16 gastric cell lines. Overall, stomach cancer showed EZH2 correlated with high levels of p53, Ki-67, and cytoplasmic pRb2/p130 (P < 0.05, and P < 0.01, respectively). Increased expression of EZH2 was found in the intestinal-type and correlated with the risk of distant metastasis (P < 0.05 and P < 0.01, respectively), demonstrating that this protein may have a prognostic value in this type of cancer. Interestingly, a strong inverse correlation was observed between p27(KIP1) expression levels and the risk of advanced disease and metastasis (P < 0.05), and a positive correlation between the expression levels of p21(WAF1) and low-grade (G1) gastric tumors (P < 0.05), confirming the traditionally accepted role for these tumor-suppressor genes in gastric cancer. Finally, a direct correlation was found between the expression levels of nuclear pRb2/p130 and low-grade (G1) gastric tumors that was statistically significant (P < 0.05). Altogether, these data may help shed some additional light on the pathogenetic mechanisms related to the two main gastric cancer histotypes and their invasive potentials.     

Controversial relationship between the expression of the RB pathway components and RB protein phosphorylation in human breast cancer

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.     

Altered anxiety-related behavior in nociceptin/orphanin FQ receptor gene knockout mice

Studies showed that nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) agonists produce anxiolytic-like actions, while little is known about the effects of blockade of NOP receptor signaling in anxiety. To this aim, we investigated the behavioral phenotype of NOP receptor gene knockout mice (NOP(-/-)) in different assays. In the elevated plus-maze and light-dark box, NOP(-/-) mice displayed increased anxiety-related behavior. In the novelty-suppressed feeding behavior and elevated T-maze, NOP(-/-) mice showed anxiolytic-like phenotype, while no differences were found in the open-field, hole-board, marble-burying, and stress-induced hyperthermia. Altogether, these findings suggest that the N/OFQ-NOP receptor system modulates anxiety-related behavior in a complex manner.     

Response of photosynthetic machinery of field-grown kiwifruit under Mediterranean conditions during drought and re-watering

Groups of Actinidia deliciosa A. Chev. C.F. Liang et A.R. Ferguson var. deliciosa kiwifruit plants were subjected to soil water shortage (D), while other groups were well irrigated (I). Variations in chlorophyll (Chl) a fluorescence indices and leaf gas exchange were determined once plants were severely stressed (25 d after the beginning of the D-cycle). Daily maximum values of photosynthetic photon flux density (PPFD) were ca. 1 650 μmol(photon) m⁻² s⁻¹, while air temperatures peaked at 34.6 °C. High irradiance per se did not greatly affect the efficiency of photosystem (PS) 2, but predisposed its synergistic reduction by D co-occurrence. Fluorescence showed transient photodamage of PS2 with a complete recovery in the afternoon in both D and I plants. Upon re-watering the efficiency of PS2 was suboptimal (95 %) at day 2 after irrigation was reinitiated. At early morning of the day 5 of re-watering, photosynthesis and stomatal conductance recovered at about 95 and 80 % of I vines, respectively, indicating some after-stress effect on stomatal aperture. Once excessive photons reached PS2, the thermal dissipation of surplus excitation energy was the main strategy to save the photosynthetic apparatus and to optimize carbon fixation. The rather prompt recovery of both Chl a fluorescence indices and net photosynthetic rate during re-watering indicated that kiwifruit photosynthetic apparatus is prepared to cope with temporary water shortage under Mediterranean-type-climates.     

The domestication of artichoke and cardoon: from Roman times to the genomic age

BACKGROUND: The history of domestication of artichoke and leafy cardoon is not yet fully understood and when and where it occurred remains unknown. Evidence supports the hypothesis that wild cardoon is the wild progenitor of both these crops. Selection for large, non-spiny heads resulted in artichoke and selection for non-spiny, large stalked tender leaves resulted in leafy cardoon. The two crops differ in their reproductive system: artichoke is mostly vegetatively propagated and perennial, while leafy cardoon is seed propagated and mostly grown as an annual plant. Here, new trends in artichoke cultivation are analysed, while the consequences of these tendencies on the conservation of artichoke genetic resources are highlighted. SCOPE: The historical and artistic records, together with recent literature on genetics and biosystematics, are examined with the aim of achieving a better understanding of the present-day knowledge on the domestication of these two crops. CONCLUSIONS: Historical, linguistic and artistic records are consistent with genetic and biosystematic data and indicate that the domestication of artichoke and cardoon diverged at different times and in different places. Apparently, artichoke was domesticated in Roman times, possibly in Sicily, and spread by the Arabs during early Middle Ages. The cardoon was probably domesticated in the western Mediterranean in a later period.     

The late Messinian Lago-Mare episode in the Mediterranean Basin: Preliminary report on the occurrence of Paratethyan ostracod fauna from central Crete (Greece)

In the Crete Island, late Messinian Lago-Mare facies are not well known. At present, the occurrence in Crete of the uppermost Messinian post evaporitic deposits is a matter of debate. According to several authors, the well-known late Messinian Lago-Mare facies does not occur in Crete. In this paper the preliminary results obtained from the biostratigraphical analysis of some sections sampled in the Messarà Plain will be shown. Nearby Faneromeni and Ano Akria villages, the Miocene/Pliocene boundary is well exposed. There, gypsum-bearing clay, laminated microcrystalline gypsum and gypsum-rudites characterize the evaporitic deposits of the Messinian stage. In these areas, above the Messinian evaporite, post-evaporitic fine-laminated polychrome clays, with intercalations of sandstones and conglomerates, have been found. In both the Faneromeni and Ano Akria area, the Pliocene grey clays and conglomerates rest unconformably on the uppermost Messinian post-evaporitic deposits. A 20 cm-spaced sampling has been performed in both the sections, for more than 100 samples collected. The results of the micropaleontological analysis performed on the Faneromeni and Ano Akria sections point to the occurrence of ostracod assemblages containing: Loxocauda limata (Schneider in Agalarova et al.), Loxocauda sp., Cytherura pyrama Schneider, Cyprideis anlavauxensis Carbonnel, Cyprideis agrigentina Decima, Amnicythere palimpsesta (Livental), Amnicythere propinqua (Livental), Amnicythere accicularia (Olteanu), Amnicythere costata (Olteanu), Amnicythere multituberculata (Livental), Amnicythere sp. D (Miculan in Bassetti et al.), Amnicythere sp. 2 Gliozzi and Grossi, Amnicythere sp., Euxinocythere (Maeotocythere) praebaquana (Livental in Agalarova et al.), Mediocytherideini indet., Pontoniella pontica (Agalarova), Camptocypria sp. 1 Gliozzi and Grossi, Caspiocypris sp., Zalanyiella venusta (Zalanyi), Tyrrhenocythere sp., Loxoconcha rhombovalis Pokorny, Loxoconcha eichwaldi Livental, Loxoconcha sp. A (Miculan in Bassetti et al.), Loxocorniculina djafarovi (Schneider in Suzin). In the analysed samples, reworked planktonic foraminifers and well-preserved charophyte gyrogonites have been also found. The ostracod assemblages found in the Messarà Plain belong to the Loxocorniculina djafarovi Zone (sensu Carbonnel, 1978), which characterizes the uppermost Messinian deposits of the whole Mediterranean Basin. At that time, the well-known Lago-Mare biofacies was also widespread on the Crete Island. The presence of Paratethyan ostracods in the post-evaporitic Messinian deposits of both Faneromeni and Ano Akria sections suggests that in the Crete Island the latest Messinian sedimentation took place in brackish water palaeoenvironments.     

beta1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.     

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

BACKGROUND: Ocular neovascular disorders, such as diabetic retinopathy and age-related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper-dependent adenovirus (HD-Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization. METHODS: We first analyzed efficiency and stability of intraretinal gene transfer following intravitreous injection in mice. A HD-Ad vector expressing green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (HD-Ad/GFP) was compared with a first-generation (E1/E3-deleted) Ad vector carrying an identical GFP expression cassette (FG-Ad/GFP). We also constructed HD-Ad vectors expressing a soluble form of the VEGF receptor (sFlt-1) in a constitutive (HD-Ad/sFlt-1) or doxycycline (dox)-inducible (HD-Ad/S-M2/sFlt-1) manner and tested their therapeutic efficacy upon intravitreous delivery in a rat model of oxygen-induced retinopathy (OIR). RESULTS: HD-Ad/GFP promoted long-lasting (up to 1 year) transgene expression in retinal Müller cells, in marked contrast with the short-term expression observed with FG-Ad/GFP. Intravitreous injection of HD-Ad vectors expressing sFlt-1 resulted in detectable levels of sFlt-1 and inhibited retinal neovascularization by more than 60% in a rat model of OIR. Notably, the therapeutic efficacy of the inducible vector HD-Ad/S-M2/sFlt-1 was strictly dox-dependent. CONCLUSIONS: HD-Ad vectors enable stable gene transfer and regulated expression of angiostatic factors following intravitreous injection and thus are attractive vehicles for the gene therapy of neovascular diseases of the retina.