Differential effects of heptanoate and hexanoate on myocardial citric acid cycle intermediates following ischemia-reperfusion.

In the normal heart, there is loss of citric acid cycle (CAC) intermediates that is matched by the entry of intermediates from outside the cycle, a process termed anaplerosis. Previous in vitro studies suggest that supplementation with anaplerotic substrates improves cardiac function during myocardial ischemia and/or reperfusion. The present investigation assessed whether treatment with the anaplerotic medium-chain fatty acid heptanoate improves contractile function during ischemia and reperfusion. The left anterior descending coronary artery of anesthetized pigs was subjected to 60 min of 60% flow reduction and 30 min of reperfusion. Three treatment groups were studied: saline control, heptanoate (0.4 mM), or hexanoate as a negative control (0.4 mM). Treatment was initiated after 30 min of ischemia and continued through reperfusion. Myocardial CAC intermediate content was not affected by ischemia-reperfusion; however, treatment with heptanoate resulted in a more than twofold increase in fumarate and malate, with no change in citrate and succinate, while treatment with hexanoate did not increase fumarate or malate but increased succinate by 1.8-fold. There were no differences among groups in lactate exchange, glucose oxidation, oxygen consumption, and contractile power. In conclusion, despite a significant increase in the content of carbon-4 CAC intermediates, treatment with heptanoate did not result in improved mechanical function of the heart in this model of reversible ischemia-reperfusion. This suggests that reduced anaplerosis and CAC dysfunction do not play a major role in contractile and metabolic derangements observed with a 60% decrease in coronary flow followed by reperfusion.     

Novel imidazoline compounds as partial or full agonists of D2-like dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI

Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a–k inspired by 2-BFI scaffold to assess imidazoline molecules as D₂-like dopamine receptor ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly full (3e and 3h) agonists, all endowed with D₂-like potency comparable to that of dopamine.     

The Sesquiterpenes Polygodial and Drimanial in vitro Affect Glutamatergic Transport in Rat Brain

Natural products including those derived from plants, have over the years greatly contributed to the development of therapeutic drugs. Polygodial and drimanial are sesquiterpenes isolated from the bark of the plant Drymis Winteri (Winteraceae) that exhibit antinociceptive properties. Since peripheral glutamate presents nociceptive actions, in this study it was investigated the effects of hydroalcooholic extracts from Drymis winteri (polygodial and drimanial) on the glutamatergic system in rat brain. Polygodial and drimanial inhibited glutamate uptake by astrocytes, as well as by cortical, hippocampal and striatal slices, and increased synaptosomal glutamate release. These concurrent effects would predispose to an increase in the extracellular glutamate concentrations, leading to possible neurotoxic effects (excitotoxicity) of these natural compounds, which would suggest the need for some caution in their therapeutic application.     

T cell selection and differentiation in AIDS disease: the model of HIV-discordant monozygotic twins

The model of monozygotic twins has been repeatedly studied to control the genetic and age-specific effects on HIV disease. Focusing on this natural model, the expression of CD27/CD45RA differentiation markers and the distribution of the Vbeta TCR repertoire was analyzed on CD4+ and CD8+ T cells. In our HIV-discordant monozygotic twins, a significant reduction of naive T cells and a parallel accumulation of effector/memory T cells was induced by HIV infection, as well as a skewing of T cell repertoire evidenced by VbetaTCR analysis. The block of HIV replication by highly active antiretroviral therapy (HAART) restored most of the T cell maturation and selection process, with some exception among CTL differentiation and repertoire. Altogether, the model of HIV-discordant monozygotic twins is a valuable tool showing that HAART is not able to completely restore the CTL profile.     

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumour cells

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca²⁺ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca²⁺ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.     

Physico-chemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid

Cationic liposomes-DNA complexes (lipoplexes) are largely used in gene delivery. Deciphering specific chemical and physical properties of lipoplexes is a necessary step to unravel the mechanisms underlying transfection and to improve transfection efficacy in each experimental model. In the present paper we investigated the physico-chemical features of lipoplexes containing a plasmid encoding for the GFP protein, in order to correlate these results with transfection efficacy. Cationic unilamellar vesicles (mean diameter 100 nm) were prepared, from the cationic DC-Chol lipid and the zwitterionic phospholipid DOPE. The two components of the liposome bilayer were used at molar ratio close to unity. ESR spectra were recorded and zeta potential zeta was measured on liposomes complexed with the plasmid. One of the main points of interest in this paper resided in the fact that both kinds of measurements were carried out in the same conditions (i.e. lipid concentration, medium composition, and pH) employed for cell transfection experiments. Transfection was performed on CHO cells; the percentage of fluorescent cells was evaluated and compared with the above physico-chemical features. It emerged that the composition and pH of the medium, the lipoplex/cell ratio, as well as the amount of lipoplex added to the cell culture were critical parameters for transfection efficacy. Finally, lipoplex surface charge played a fundamental role to achieve a high transfection level.     

The effect of body mass on physiological indicators in the performance of forestry workers

The paper presents the results of research into the effects of body mass (BM) on basic physiological indicators of work capability among forestry workers. The indicators included the maximum theoretical heart rate, the maximum heart rate in exertion tests, and the basal energy expenditure. The effects of the deviation of the actual from the ideal BM values were analysed on a sample of 8 workers. The variables included age, body height and mass. These were used to determine the maximum theoretical heart rate. The maximum heart rate was determined in an ergonomic laboratory in a programmed exertion test on a treadmill. Using standard formulae in work physiology, values of work capability indicators were calculated for the actual and ideal BM of each worker. The results, embracing individual and summary values and their absolute and relative ratios, showed that workers exceeded their ideal BM by an average of 9.9 kg. In all workers, the maximum theoretical heart rate was higher than that achieved in the exertion test. It was also found that even significant deviations of the ideal from the actual BM (the actual BM was more than 20% higher than the ideal BM) did not have any considerable effects on the maximum theoretical heart rate. The analysis of oxygen consumption showed that in relation to physical capability of the ideal BM and the maximal theoretical heart rate, physical capability of each worker was lower by an average of 11.5%. The highest reduction of physical capability was found in those workers with the least favourable ratio between the theoretical and the maximal tested heart rate. It was concluded that on average, the basal energy expenditure in each worker was higher by 7.45% due to the deviation of the actual from the ideal BM. At constant values of other factors, this means an equivalently lower capacity for daily physical performance.     

C-nucleoside analogues of furanfurin as ligands to A1 adenosine receptors

Furanfurin (2-beta-D-ribofuranosylfuran-4-carboxamide) derivatives and analogues were synthesized and their affinity for adenosine receptors was determined. The agonistic behavior of furanfurin against A1 receptors is preserved only when the furan ring is substituted with isosteric pentatomic ring systems such as oxazole, thiazole or thiophene, and the carboxamide group is unsubstituted. Replacement of the hydrogen atoms of the carboxamide group with alkyl, cycloalkyl or arylalkyl groups generates compounds endowed with moderate antagonistic activity.     

Establishment of keratinocyte colonies from small-sized cervical intraepithelial neoplasia specimens

The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue‐derived fibroblasts and keratinocytes were co‐cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate. J. Cell. Physiol. 227: 3787–3795, 2012. © 2012 Wiley Periodicals, Inc.     

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions.Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference ‘small-spored’ Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups.The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.