Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury

Many older patients, because of their high prevalence of coronary artery disease, are candidates for percutaneous coronary interventions (PCI), but the effects of vascular aging on restenosis after PCI are not yet well understood. Balloon injury to the right carotid artery was performed in adult and old rats. Vascular smooth muscle cell (VSMC) proliferation, apoptotic cell death, together with Akt induction, telomerase activity, p27kip1, and endothelial nitric oxide synthase (eNOS) expression was assessed in isolated arteries. Neointima hyperplasia and vascular remodeling along with endothelial cell regeneration were also measured after balloon injury. Arteries isolated from old rats exhibited a significant reduction of VSMC proliferation and an increase in apoptotic death after balloon injury when compared with adult rats. In the vascular wall of adult rats, balloon dilation induced Akt phosphorylation, and this was barely present in old rats. In arteries from old rats, Akt-modulated cell cycle check points like telomerase activity and p27kip1 expression were decreased and increased, respectively, compared with adults. After balloon injury, old rats showed a significant reduction of neointima formation and an increased vascular negative remodeling compared with adults. These results were coupled by a marked delay in endothelial regeneration in aged rats, partially mediated by a decreased eNOS expression and phosphorylation. Interestingly, chronic administration of L-arginine prevented negative remodeling and improved reendothelialization after balloon injury in aged animals. A decreased neointimal proliferation, an impaired endothelial regeneration, and an increase in vascular remodeling after balloon injury were observed in aged animals. The molecular mechanisms underlying these responses seem to be a reduced Akt and eNOS activity.     

Arrhythmias after tetralogy of fallot repair

Tetralogy of Fallot is the most common cyanotic congenital heart disease, with a good outcome after total surgical correction. In spite of a low perioperative mortality and a good quality of life, late sudden death remains a significant clinical problem, mainly related to episodes of sustained ventricular tachycardia and ventricular fibrillation. Fibro-fatty substitution around infundibular resection, intraventricular septal scar, and patchy myocardial fibrosis, may provide anatomical substrates of abnormal depolarization and repolarization causing reentrant ventricular arrhythmias. Several non-invasive indices based on classical examination such as ECG, signal-averaging ECG, and echocardiography have been proposed to identify patients at high risk of sudden death, with hopeful results. In the last years other more sophisticated invasive and non-invasive tools, such as heart rate variability, electroanatomic mapping and cardiac magnetic resonance added a relevant contribution to risk stratification. Even if each method per se is affected by some limitations, a comprehensive multifactorial clinical and investigative examination can provide an accurate risk evaluation for every patient.     

New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib,dasatinib, and nilotinib in human plasma

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.     

ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.     

Improving the spatial orientation of human teeth using a virtual 3D approach

Since teeth are resistant to decomposition processes, they provide important and at times unique sources of information about fossil humans. Fortunately, dental remains reflect significant evolutionary changes. These changes make a very important and often exclusive contribution to the definition of new taxa or the attribution of fossil specimens to existing taxa.The traditional approach to dental morphometric analyses usually focuses on the recording of several measures of the tooth with calipers, especially the two basic crown diameters (buccolingual and mesiodistal). However, since these measures do not adequately represent the complex morphology of the tooth, 2D images and 3D digital models of dental morphology have been used. For both types of analysis, the possibility of correctly comparing homologous teeth depends on the adoption of a common orientation system. The lack of such a system makes it difficult to compare the results of different studies.Here we describe a new method for orienting teeth specifically devised for the upper and lower first molar (M1). Samples of unworn maxillary (n = 15) and mandibular (n = 15) first molars of modern humans were scanned with a Roland Picza 3D digitizer. The 3D virtual models were used to compare our new orientation method with those proposed in the literature. The new orientation system, which meets a geometric criterion, is based on three points identified on the cervical line and ensures acceptable repeatability of the spatial positioning and orientation independent of the shape and wear of the first molar under investigation. This orientation system is a first step toward the creation of a virtual set of hominid and fossil human first molars, which will allow us to make comparisons via a sophisticated and noninvasive approach. This pilot study also provides guidelines to extend the new methodology to the other types of teeth.     

Formation and hydrolysis of triacylglycerol and sterols epoxides: role of unsaturated triacylglycerol peroxyl radicals

Epoxidation of unsaturated pure triacylglycerols (TAGs), cholesterol, and phytosterols was investigated using air and 18O2 oxidation experiments. Oxidized lipids were analyzed using both triple quadrupole mass spectrometry (MS), ion-trap MS in the direct infusion mode, and triple quadrupole MS in tandem with a liquid chromatograph (LC-MS/MS). Pure 1,2-distearoyl-3-oleoyl-glycerol (SSO) samples were heated in sealed vials under air or 18O2 atmosphere at 160 degrees C for 1 h. LC-MS/MS analysis of 18O-labeled oxidized TAGs revealed that hydroperoxides and epoxide TAGs are formed mainly during this first step. Then, oxidized TAGs were incubated under an inert atmosphere, separately with 1,2-dipalmitoyl-3-oleoyl-glycerol (PPO) at 160 degrees C for 90 min, and with cholesterol and stigmasterol at 100 degrees C for 10 min. Subsequent LC-MS/MS analysis revealed the occurrence of epoxidation products of PPO, cholesterol, and sitosterol. Therefore, we showed the epoxidation of unsaturated lipids proceeds readily in contact with hydroperoxide TAGs, in the absence of molecular oxygen. Dual oxidation experiments using both air and 18O2 allowed investigation of oxygen atom transfer during epoxidation of lipids. Moreover, the experimental oxidation design presented can be used to study fragmentation pathways, as illustrated for 5,6-epoxycholesterol (CE) on both triple quadrupole and ion-trap MS. We report for the first time the occurrence of 5,6;22,23-diepoxystigmasterol (StDE) and 5,6;22,23-diepoxybrassicasterol (BDE) in autoxidized vegetable oils. Additionally, acid-catalyzed hydrolysis of epoxidized lipids, with emphasis on phytosterol polyol formation, was investigated using a model gastric medium. For confirmation, almost all identified products were synthesized and characterized by MS.     

Isoform specific function of calpain 2 in regulating membrane protrusion

Previous studies have demonstrated a role for calpains in cell migration through their capacity to regulate focal adhesion dynamics and rear retraction. In this study, we provide evidence that calpains also modulate membrane protrusion activity in fibroblasts. We find that an immortalized Capn4(-/-) fibroblast line displays an altered morphology, characterized by numerous thin membrane projections and increased transient membrane activity. Furthermore, we show that protrusion kinetics of lamellipodia at the leading edge are improperly regulated in Capn4(-/-) cells, leading to impaired net forward lamellipodial extension. To address the isoform specific functions of calpain 1 and calpain 2 during cell protrusion, we stably introduced small interfering RNAs (siRNAs) targeting each isoform into a fibroblast cell line. Despite a loss in calpain 1 activity, calpain 1 knockdown cells show normal morphology and membrane protrusion dynamics. However, cells in which calpain 2 is knocked down are characterized by a protrusive morphology, increased transient membrane activity and altered protrusion kinetics, similar to the Capn4(-/-) fibroblasts. Additionally, we find that calpain 2, but not calpain 1, is required for proteolysis of the cytoskeletal and focal adhesion proteins FAK, paxillin, spectrin, and talin. Together, our findings support a novel role for calpain 2 in limiting membrane protrusions and in regulating lamellipodial dynamics at the leading edge of migrating cells.