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41.
Magdaline Costa Koula Sourris Sue Mei Lim Qing C. Yu Claire E. Hirst Helena C. Parkington Vanta J. Jokubaitis Anthony E. Dear Hong B. Liu Suzanne J. Micallef Kathy Koutsis Andrew G. Elefanty Edouard G. Stanley 《Stem cell research》2013,10(1):103-117
The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34+KDR+ endothelial progenitor cells after 6 days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization. 相似文献
42.
Paulo Bentes de Carvalho-Neto Marcelo dos Santos Marcos Brasilino de Carvalho Ana Maria da Cunha Mercante Viviane Priscila Pina dos Santos Patrícia Severino Eloiza Helena Tajara Iuri Drumond Louro Adriana Madeira álvares da Silva-Conforti 《PloS one》2013,8(7)
FAS/FASL altered expression may cause tumor protecting immunomodulation, with a direct impact on patient prognosis. FAS expression was studied in 60 squamous cell carcinomas of the oral cavity. FAS expression did not show a significant association with tumor histopathological characteristics, but was significantly associated with lymph node positivity. FAS expression was significantly associated with disease specific death and negative FAS expression was an independent risk factor, increasing risk 4 times when compared to positive expression. When FAS and FASL expression results were combined, we were able to define high, intermediate and low risk profiles. Disease-free and disease-specific survival were significantly correlated with FAS/FASL expression profiles. The high risk category was an independent marker for earlier disease relapse and disease-specific death, with approximately 4- and 6-fold increased risk, respectively, when compared to the low risk profile. Risk profiles based on FAS/FASL expression showed that high risk was significantly associated with increased disease relapse and death, as well as shorter disease-free or disease-specific survival. This categorization, added to patient clinical data, may facilitate the choice of therapy, minimizing treatment failure and increasing disease control. 相似文献
43.
Leiz M. C. Véras Vanessa R. R. Cunha Filipe C. D. A. Lima Maria A. Guimar?es Marianne M. Vieira Yuri D. M. Campelo Vanessa Y. Sakai David F. Lima Paulo S. Carvalho Jr Javier A. Ellena Paulo R. P. Silva Luciene C. Vasconcelos Markus Godejohann Helena M. Petrilli Vera R. L. Constantino Yvonne P. Mascarenhas José Roberto de Souza de Almeida Leite 《PloS one》2013,8(6)
This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), 1H and 13C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution. 相似文献
44.
Photosynthesis Research - Light response curves (LRCs) describe how the rate of photosynthesis varies as a function of light. They provide information on the maximum photosynthetic capacity,... 相似文献
45.
Iben B. Bentsen Ida Nielsen Michael Lisby Helena B. Nielsen Souvik Sen Gupta Kamilla Mundbjerg Anni H. Andersen Lotte Bjergbaek 《Nucleic acids research》2013,41(5):3173-3189
To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein–DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein–DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling. 相似文献
46.
Martina Wallace Helena WhelanLorraine Brennan 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Chronic exposure to hyperglycaemic conditions has been shown to have detrimental effects on beta cell function. The resulting glucotoxicity is a contributing factor to the development of type 2 diabetes. The objective of this study was to combine a metabolomics approach with functional assays to gain insight into the mechanism by which glucotoxicity exerts its effects.Methods
The BRIN-BD11 and INS-1E beta cell lines were cultured in 25 mM glucose for 20 h to mimic glucotoxic effects. PDK-2 protein expression, intracellular glutathione levels and the change in mitochondrial membrane potential and intracellular calcium following glucose stimulation were determined. Metabolomic analysis of beta cell metabolite extracts was performed using GC–MS, 1H NMR and 13C NMR.Results
Conditions to mimic glucotoxicity were established and resulted in no loss of cellular viability in either cell line while causing a decrease in insulin secretion. Metabolomic analysis of beta cells following exposure to high glucose revealed a change in amino acids, an increase in glucose and a decrease in phospho-choline, n−3 and n−6 PUFAs during glucose stimulated insulin secretion relative to cells cultured under control conditions. However, no changes in calcium handling or mitochondrial membrane potential were evident.Conclusions
Results indicate that a decrease in TCA cycle metabolism in combination with an alteration in fatty acid composition and phosphocholine levels may play a role in glucotoxicity induced impairment of glucose stimulated insulin secretion.General significance
Alterations in certain metabolic pathways play a role in glucotoxicity in the pancreatic beta cell. 相似文献47.
Helena Rudnicka Tadeusz Debniak Cezary Cybulski Tomasz Huzarski Jacek Gronwald Jan Lubinski Bohdan Gorski 《Molecular biology reports》2013,40(12):6619-6623
BRCA1 and BRCA2 are two major genes associated with familial breast and ovarian cancer susceptibility. In Poland standard BRCA gene test is usually limited to Polish founder BRCA1 mutations: 5382insC, C61G and 4153delA. To date, just a few single large genomic rearrangements (LGRs) of BRCA1 gene have been reported in Poland. Here we report the first comprehensive analysis of large mutations in BRCA1 and BRCA2 genes in this country. We screened LGRs in BRCA1 and BRCA2 genes by multiplex ligation-dependent probe amplification in 200 unrelated patients with strong family history of breast/ovarian cancers and negative for BRCA1 Polish founder mutations. We identified three different LGRs in BRCA1 gene: exons 13-19 deletion, exon 17 deletion and exon 22 deletion. No LGR was detected in BRCA2 genes. Overall, large rearrangements accounted for 3.7 % of all BRCA1 mutation positive families in our population and 1.5 % in high-risk families negative for Polish founder mutation. 相似文献
48.
Leandro Rodrigues da Cunha Teresa Helena Macedo da Costa Eloisa Dutra Caldas 《Biological trace element research》2013,151(1):30-37
Breast milk samples collected from 18 nursing mothers between the 15th and 90th day of lactation were digested in nitric acid in a microwave, and total mercury (THg) levels were quantified by atomic fluorescence spectrometry. Participants responded to a 24-h dietary recall questionnaire on the 74th and 76th day of lactation and to a Food Frequency Questionnaire querying the frequency of fish intake over the last 90 days. Usual intake was estimated using the PC-SIDE software package. A meal of fish was offered on the 75th day of lactation. Mothers’ individual mean THg levels ranged from <0.76 to 22.7 ng/mL during the period, and the mean level for all samples (n?=?142) was 6.47?±6.04 ng/mL. The multilevel mixed linear model used showed high heterogeneity of the mercury levels among the mothers, and THg levels did not change significantly over the period under study. However, a significant increase in THg levels was observed after the intervention with the fish meal. Exposure increased for most infants on the 90th day of lactation, with intakes exceeding the THg provisional tolerable weekly intake (PTWI) at least once during the period for 77.8 % of samples. Mothers consumed mostly food from the fat and grain groups, and a significant correlation was detected between consumption of food of these groups and breast milk THg levels (p?=?0.006 and 0.007). A significant correlation was also found between vegetable consumption and carbohydrate intake and THg levels in the samples (p?=?0.015 and 0.045, respectively). No correlation was found between mothers’ daily fish consumption frequency and THg levels. Although this study showed that mercury intake by infants during lactation may exceed the toxicologically safe exposure level (PTWI), we nevertheless believe that the benefits of lactation for both the mother and the infant outweigh the eventual risks that this exposure may represent. 相似文献
49.
SUMOylation (small ubiquitin‐like modifier conjugation) is an important post‐translational modification which is becoming increasingly implicated in the altered protein dynamics associated with brain ischemia. The function of SUMOylation in cells undergoing ischemic stress and the identity of small ubiquitin‐like modifier (SUMO) targets remain in most cases unknown. However, the emerging consensus is that SUMOylation of certain proteins might be part of an endogenous neuroprotective response. This review brings together the current understanding of the underlying mechanisms and downstream effects of SUMOylation in brain ischemia, including processes such as autophagy, mitophagy and oxidative stress. We focus on recent advances and controversies regarding key central nervous system proteins, including those associated with the nucleus, cytoplasm and plasma membrane, such as glucose transporters (GLUT1, GLUT4), excitatory amino acid transporter 2 glutamate transporters, K+ channels (K2P1, Kv1.5, Kv2.1), GluK2 kainate receptors, mGluR8 glutamate receptors and CB1 cannabinoid receptors, which are reported to be SUMO‐modified. A discussion of the roles of these molecular targets for SUMOylation could play following an ischemic event, particularly with respect to their potential neuroprotective impact in brain ischemia, is proposed. 相似文献