| Name | Maria Luisa |
| Surname | Balestrieri |
| Institution | Università degli Studi della Campania Luigi Vanvitelli |
| marialuisa.balestrieri@unicampania.it | |
| Address | Department of Biochemistry, Biophysics and General Pathology, University of Campania "L. Vanvitelli", Via L. De Crecchio 7, 80138 Naples, Italy |
| Resume | Download |
Downregulation of levels of endothelial progenitor cells (EPCs) during in-vitro short-term exposure to high glucose concentrations relates to reduced activity of silent information regulator 1 (SIRT1) and increased synthesis of platelet-activating factor (PAF). We investigated the possible relationship between PAF and SIRT1 pathways in EPCs during altered glucose homeostasis.
The occurrence of N-methylated tryptamine derivatives in bergamot plant (Citrus bergamia Risso et Poit) is reported for the first time. Interestingly, the most abundant of these substances is N,N,N-trimethyltryptamine, which has not been previously identified in any citrus plant. The N-methylated tryptamine derivatives were identified and quantitated in leaves, peel, juice, and seeds by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. N,N,N-Trimethyltryptamine was confirmed by MS(3) and comparison with the synthesized authentic standard. In addition, the study of the distribution of tryptophan, tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, and N,N,N-trimethyltryptamine indicated that these compounds are differently expressed in the various tissues of the bergamot plant. Intriguingly, chemically synthesized N,N,N-trimethyltryptamine was reported to possess nicotine-like activity being a stimulant of parasympathetic ganglia by exerting its action on acetylcholine receptors. On this basis, the identification of N,N,N-trimethyltryptamine at a relatively high level in leaves suggests a possible role in a physiological mechanism of plant defense.
Breast cancer, a leading cause of cancer related deaths worldwide, is one of the most common neoplasms in women. The increased generation of reactive oxygen species (ROS) in breast lesion is critically involved in the mutagenic processes that drive to breast carcinoma initiation and progression. To date, the molecular events occurring in the tissue adjoin the cancer lesion have not been elucidated. Here, we investigated the role of excess ROS generation during human breast carcinogenesis by evaluating oxidative stress biomarkers, tissue transglutaminase (t-TGase) activity, and expression levels of ubiquitin and cyclooxygenase-2 (COX-2) in the normal tissue adjoin to fibroadenoma (nFA), atypical ductal hyperplasia (nADH), and invasive ductal carcinoma (nIDC) from 45 breast cancer patients. We found that lipid peroxidation and nitric oxide production significantly increased in nIDC respect to nFA and nADH (P < 0.005) whereas the 4-hydroxy-2-nonenal (HNE) protein-adducts increased only in nADH (P < 0.005). The increased lipid damage observed in nIDC correlates with estrogen receptor exposure in IDC (R(2) = 0.89). Moreover, nIDC and invasive ductal carcinoma (IDC) showed a 10-fold higher t-TGase activity compared to nFA and nADH. Contrary, COX-2 expression levels significantly decreased nIDC and IDC respect to the nFA and nADH (P < 0.001). The analysis of the free ubiquitin expression revealed equal levels in nADH and nIDC samples whereas high molecular weight-ubiquitin conjugate increased about fivefold only in nIDC (P < 0.01 vs. nADH). These novel findings reveal an interplay between membrane lipid peroxidation, t-TGase activity, and COX-2 expression levels in the tissue adjoining to neoplastic lesion during breast cancer progression.
We analyzed the effects of tight glycemic control on regenerative potential of myocardium during acute myocardial infarction.
The presence of pipecolic acid and pipecolic acid betaine, also known as homostachydrine, is herein reported for the first time in Citrus genus plants. Homostachydrine was found in fruits, seeds, and leaves of orange, lemon, and bergamot (Citrus bergamia Risso et Poit). As homostachydrine was not commercially available, as a comparative source, extracts of alfalfa leaves ( Medicago sativa L.) were used, in which homostachydrine is present at high concentration. Then, the results where confirmed by comparison with an authentic standard synthesized and purified starting from pipecolic acid. The synthesized standard was characterized by a ESI-MS/MS study using a 3D ion-trap mass spectrometer. When subjected to MS/MS fragmentation in positive ion mode, homostachydrine, unlike its lower homologue proline betaine (also known as stachydrine), showed a pattern of numerous ionic fragments that allowed unambiguous identification of the compound. For the quantitation in the plant sources, high sensitivity and specificity were achieved by monitoring the transition (158 → 72), which is absent in the fragmentation patterns of other major osmolytes commonly used as markers for studies of abiotic stress. As for the metabolic origin of homostachydrine, the occurrence in citrus plants of pipecolic acid leads to the hypothesis that it could act as a homostachydrine precursor through direct methylation.
Numerous compounds, many of them osmolytes, were quantified in natural juices and in frozen concentrate juices from fruits of plants of the Citrus genus. L-proline, N-methyl-L-proline (hygric acid), N,N-dimethyl-L-proline (stachydrine), 4-hydroxy-L-prolinebetaine (betonicine), 4-hydroxy-L-proline, γ-aminobutyric acid (Gaba), 3-carboxypropyltrimethylammonium (GabaBet), N-methylnicotinic acid (trigonelline), and choline in the fruit juices of yellow orange, blood orange, lemon, mandarin, bitter orange (Citrus aurantium), chinotto (Citrus myrtifolia), and grapefruit were analyzed by sensitive HPLC-ESI-tandem mass spectrometry procedure. It was found that the most represented osmolytes in the juices, that is, L-proline, stachydrine, and betonicine, can be quantified with minimal sample preparation and short analysis time (about 1 min) also by flow injection analysis (FIA) ESI-MS/MS with the same results as obtained by HPLC ESI-MS/MS. In all of the juices, discrete amounts of choline and trigonelline were present. Conversely, GabaBet was always below detection limits. Notably, N-methyl-L-proline and 4-hydroxy-L-prolinebetaine, which were discovered for the first time in the juice of bergamot (Citrus bergamia Risso et Poit), are also present in all of the citrus juices examined.
The content of proline and various compounds deriving from its metabolism (4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine) was determined in fruits and seeds of Bergamot (Citrus bergamia Risso et Poit), growing in the Calabria region (South Italy). A HPLC-ESI-tandem mass spectrometry method, which allowed rapid determination of L-proline, 4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine in juice and extracts of bergamot fruit with minimum sample preparation and short analysis time (about 10 min), is presented. Proline and 4-hydroxy-L-proline levels in the samples were also determined by HPLC analysis with fluorescence detection and the results compared to those obtained with HPLC-ESI-tandem mass spectrometry. For the first time, the presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine in the fruits of a plant of the Citrus genus is reported.
Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor-α (TNF-α) reduced their number and biological activity. Yet, signal transduction events linked to TNF-α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in- flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose-dependent improvement of EPC number during exposure to TNF-α (P < 0.05 vs. TNF-α alone). Analysis of the signal transduction pathway activated by TNF-α revealed that the increased expression of p-p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF-α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF-α) as well as the inhibitor of Erk1/2-specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF-α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D-DIGE and MALDI-TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin-related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway.
Endothelial progenitor cell (EPC) therapy is a promising approach to promote angiogenesis and endothelial repair in patients with cardiovascular diseases (CVD). However, their release of proinflammatory mediators may compromise the therapeutic efficacy. Little is known about the role of Platelet-Activating Factor (PAF) in EPC functional response. Here, we investigated the expression of PAF receptor (PAF-R) in early EPC and the release of PAF under stimulation with factors involved in endothelial dysfunction. Results indicated that early EPC express the PAF-R and respond to PAF signaling via a transient increase of cytoplasmic Ca(2+) concentration. EPC release PAF in a time dependent manner upon stimulation with tumor necrosis factor-alpha (TNF-alpha) or high-glucose concentration with a peak at 30 min and 10 min (p<0.01 vs. control), respectively. PAF, starting at concentration of 50 ng/ml, exerted a detrimental effect on EPC number with a concomitant increase of p38 activity. Furthermore, both the reduction of early EPC number and the enhanced p38 activity induced by PAF were abolished by CV3988, a PAF receptor antagonist. These novel findings, revealing that early EPC respond to PAF signaling, unveil an inflammatory pathway that may play a crucial role in the outcome of cardiovascular cell therapy with EPC.
The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre-ovulatory, ovulatory, post-ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post-ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre-ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (μm/24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post-ovulatory phases (63.7 ± 11.2), with intermediate values in the pre-ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre-ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post-ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (μmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre-ovulatory and post-ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species-specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.