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31.
Cosseddu GM Nonno R Vaccari G Bucalossi C Fernandez-Borges N Di Bari MA Castilla J Agrimi U 《PLoS pathogens》2011,7(11):e1002370
In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrP(Sc) in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrP(Sc) in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrP(Sc) in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrP(Sc) was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrP(Sc) to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro. 相似文献
32.
Domoki F Kis B Nagy K Farkas E Busija DW Bari F 《American journal of physiology. Heart and circulatory physiology》2005,289(1):H368-H373
Diazoxide (Diaz), an activator of mitochondrial ATP-sensitive K+ (mitoKATP) channels, is neuroprotective, but the mechanism of action is unclear. We tested whether Diaz preserves endothelium-dependent (hypercapnia) or -independent [iloprost (Ilo)] cerebrovascular dilator responses after ischemia-reperfusion (I/R) in newborn pigs and whether the effect of Diaz is sensitive to 5-hydroxydecanoate (5-HD), an inhibitor of mitoKATP channels. Anesthetized, ventilated piglets (n = 48) were equipped with closed cranial windows. Changes in diameter of pial arterioles were determined with intravital microscopy in response to graded hypercapnia (5-10% CO2 - 21% O2-balance N2, n = 25) or Ilo (0.1-1 microg/ml, n = 18) before and 1 h after 10 min of global I/R. Experimental groups were pretreated with vehicle, NS-398 (a selective cyclooxygenase-2 inhibitor, 1 mg/kg), Diaz (3 mg/kg), or 5-HD (20 mg/kg) + Diaz. Potential direct effects of Diaz and 5-HD on hypercapnic vasodilation were also tested in the absence of I/R (n = 5). To confirm the direct effect of Diaz on mitochondria, mitochondrial membrane potential in cultured piglet cerebrovascular endothelial cells was monitored using Mito Tracker Red. Hypercapnia resulted in dose-dependent pial arteriolar vasodilation, which was attenuated by approximately 70% after I/R in vehicle- and NS-398-treated animals. Diaz and 5-HD did not affect the CO2 response. Diaz significantly preserved the postischemic vasodilation response to hypercapnia, but not to Ilo. Diaz depolarized mitochondria in cultured piglet cerebrovascular endothelial cells, and 5-HD completely abolished the protective effect of Diaz, both findings indicate a role for mitoKATP channels. In summary, preservation of arteriolar dilator responsiveness by Diaz may contribute to neuroprotection. 相似文献
33.
Identification and characterization of a novel, psoriasis susceptibility-related noncoding RNA gene, PRINS 总被引:2,自引:0,他引:2
34.
Atlante A de Bari L Valenti D Pizzuto R Paventi G Passarella S 《Biochimica et biophysica acta》2005,1708(1):13-22
We report here initial studies on D-lactate metabolism in Jerusalem artichoke. It was found that: 1) D-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-D-lactoyl-glutathione. 2) Externally added D-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) D-lactate was metabolized inside mitochondria by a flavoprotein, a putative D-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added D-lactate by means of a D-lactate/H(+) symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial D-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke. 相似文献
35.
Malnutrition is a common feature of gastroenterological diseases. In this study, nutritional status of the patients admitted to Department of Gastroenterology at University Hospital Center Zagreb was assessed. Anthropometric, dietetic, biochemical methods and method of Subjective Global Assessment (SGA) was used. The study group included 284 patients admitted to the Hospital. Malnutrition, as defined by SGA, was found in 61.1% of the patients, of whom 75% were moderately and 25% severely malnourished. Those patients classified as moderately and extremely malnourished by SGA were found to have statistically lower values of BMI, albumin, total proteins, calcium, iron, triglycerides, cholesterol, vitamin A and lymphocytes as compared to those who were adequately nourished. The prevalence of malnutrition in hospitalized patients treated at the Department of Gastroenterology is high. The use of nutritional screening with multiple measures would be important in the early identification and treatment of these patients and would help decrease this high prevalence. 相似文献
36.
Hua Gao Tasha B. Toro Margherita Paschini Bari Braunstein-Ballew Rachel B. Cervantes Victoria Lundblad 《Genetics》2010,186(4):1147-1159
In Saccharomyces cerevisiae, association between the Est1 telomerase subunit and the telomere-binding protein Cdc13 is essential for telomerase to be recruited to its site of action. A current model proposes that Tel1 binding to telomeres marks them for elongation, as the result of phosphorylation of a proposed S/TQ cluster in the telomerase recruitment domain of Cdc13. However, three observations presented here argue against one key aspect of this model. First, the pattern of Cdc13 phosphatase-sensitive isoforms is not altered by loss of Tel1 function or by mutations introduced into two conserved serines (S249 and S255) in the Cdc13 recruitment domain. Second, an interaction between Cdc13 and Est1, as monitored by a two-hybrid assay, is dependent on S255 but Tel1-independent. Finally, a derivative of Cdc13, cdc13–(S/TQ)11→(S/TA)11, in which every potential consensus phosphorylation site for Tel1 has been eliminated, confers nearly wild-type telomere length. These results are inconsistent with a model in which the Cdc13–Est1 interaction is regulated by Tel1-mediated phosphorylation of the Cdc13 telomerase recruitment domain. We propose an alternative model for the role of Tel1 in telomere homeostasis, which is based on the assumption that Tel1 performs the same molecular task at double-strand breaks (DSBs) and chromosome termini.TELOMERE length homeostasis is a highly regulated process that must balance telomere loss (as the result of incomplete replication and/or nucleolytic degradation) with telomeric repeat addition (through the action of telomerase and/or recombination). In the budding yeast Saccharomyces cerevisiae, a key regulatory event is recruitment of telomerase to chromosome ends by the telomere end-binding protein Cdc13 (Nugent et al. 1996; Evans and Lundblad 1999; Pennock et al. 2001; Bianchi et al. 2004; Chan et al. 2008). Recruitment relies on a direct interaction between Cdc13 and the Est1 subunit of telomerase (Pennock et al. 2001), which brings the catalytic core of the enzyme to its site of action. Disruption of this interaction, due to mutations in either CDC13 (cdc13-2) or EST1 (est1-60), results in an Est (ever-shorter-telomere) phenotype, as manifested by progressive telomere shortening and an eventual senescence phenotype. The recruitment activity of Cdc13, which resides in a 15-kDa N-terminal domain (Pennock et al. 2001), is sufficient to direct telomerase even to nontelomeric sites (Bianchi et al. 2004). As predicted by the recruitment model, association of telomerase with telomeres is greatly reduced in strains expressing the recruitment-defective cdc13-2 allele (Chan et al. 2008).Telomerase action at individual telomeres is highly regulated. Using an assay that monitors telomere addition at single nucleotide resolution (single telomere extension, STEX), Lingner and colleagues showed that only ∼7% of telomeres with wild-type (i.e., 300 bp) length are elongated by telomerase during a single cell cycle (Teixeira et al. 2004). However, as telomere length declines, the extension frequency increases: ∼20% of telomeres 200 bp in length and >40% of 100-bp-long telomeres are elongated (Teixeira et al. 2004; Arneric and Lingner 2007). The mechanism by which telomerase distinguishes short from long telomeres has been the subject of intense investigation. Work from a number of laboratories has led to the proposal that Tel1-dependent phosphorylation of Cdc13 at underelongated telomeres mediates the interaction between Cdc13 and the telomerase-associated Est1 protein, thus ensuring that telomerase is directed to the shortest telomeres in a population. In support of this model, the Est1 and Est2 telomerase subunits exhibit enhanced association with telomeres that have been artificially shortened, whereas Cdc13 displays length-independent association with telomeres (Bianchi and Shore 2007; Sabourin et al. 2007). This suggests that the preferential elongation of shorter telomeres is controlled at the level of recruitment of the telomerase holoenzyme by Cdc13. Furthermore, efficient association of Est1 and Est2 with chromosome ends requires Tel1 and Mre11 (which acts in the same pathway as Tel1 for telomere length regulation; Nugent et al. 1998; Ritchie and Petes 2000) but not Mec1 (Takata et al. 2005; Goudsouzian et al. 2006). Tel1 itself is also telomere bound (Takata et al. 2004), with enhanced binding to shorter telomeres (Bianchi and Shore 2007; Hector et al. 2007; Sabourin et al. 2007; Abdallah et al. 2009), although there is considerable controversy over the degree and timing of Tel1 association with chromosome termini during the cell cycle. As expected for a key regulator of the interaction between Cdc13 and a telomerase subunit, a tel1-Δ strain has short telomeres (Lustig and Petes 1986), although telomere length is not impaired enough to confer the Est phenotype displayed by cdc13-2 and est1-60 strains.Implicit in the above proposal is that Cdc13 must be a direct substrate of Tel1. In support of this, Teng and colleagues reported several years ago that the recruitment domain of Cdc13 has a cluster of potential Tel1 (and/or Mec1) phosphorylation sites (Tseng et al. 2006). Substrates of the DNA damage kinases often contain several closely spaced phosphorylation sites, termed S/TQ cluster domains (SCDs), which are considered a structural hallmark of many DNA damage-response proteins (Traven and Heierhorst 2005). On the basis of in vitro kinase assays with GST fusions to 75- to 90-amino-acid portions of the Cdc13 recruitment domain, Tseng et al. 2006 concluded that four SQ sites in the recruitment domain of Cdc13 are overlapping substrates for both Tel1 and Mec1, leading to the proposal that telomerase recruitment in S. cerevisiae is regulated by Tel1-dependent phosphorylation of Cdc13.The above model makes a key prediction: in a tel1-Δ strain, telomerase should no longer exhibit a length-dependent pattern of elongation. However, preferential elongation of short telomeres still occurs at native chromosome ends in the absence of Tel1 (Arneric and Lingner 2007). In addition, Petes and colleagues have argued, on the basis of epistasis data, that Tel1 performs an indirect role in the telomerase pathway, rather than directly targeting a telomerase regulator (Ritchie et al. 1999; Ritchie and Petes 2000). These observations are not easily explained, if preferential recognition of short telomeres by telomerase is mediated by Tel1-dependent phosphorylation of Cdc13. In this current study, we have re-examined the evidence for phosphorylation of Cdc13 as a regulatory mechanism for telomere length homeostasis. We report on a series of observations that indicate that Tel1 contributes to telomere length control through a mechanism other than phosphorylation of the Cdc13 S/TQ cluster. 相似文献
37.
Brkić K Unić D Sutlić Z Biocina B Rudez I Barić D Lukić IK 《Collegium antropologicum》2006,30(2):395-400
Cardiac surgery (CS) with cardiopulmonary bypass (CPB) induces systemic inflammatory response by activating plasma proteins and blood cells. Activated monocytes/macrophages produce inflammatory marker neopterin (NP). The aim was to explore the NP kinetics in first 24 hours after CS according to the CPB use. Significant difference between groups was found for NP levels 12 and 24 hrs after CS, being higher in on-pump group. Strong association was found between NP levels 12 hrs after CS and the length of ICU stay for on-pump group (r=0.744, p<0.001). Strong association was found between preoperative NP levels and the length of ICU stay for those on-pump patients with elevated preoperative NP (r=0.855, p=0.001; linear regression equation y=0.50x-5.14, p<0.001). Preoperative NP levels higher than 10 nmol/L in on-pump group could predict prolonged ICU stay and outpoint patients at higher risk for developing postoperative complications and, therefore, help to determine the necessary therapeutic interventions. 相似文献
38.
In the present study, we studied epithelial-mesenchymal transition (EMT) with fetal and postnatal serial skin sections. E-cadherin, occludin and zonula occludens 1 (ZO-1)-expressing cells appear in the dermal area from E18.5 to postnatal day 9 (P9), with highest expression from P2 to P5. The co-expression of mesenchymal marker alpha-smooth muscle (alpha-SMA), fibronectin and vimentin with E-cadherin in these dermal cells was further examined. Almost no dermal cells express alpha-SMA before P0. From P2 to P6, cells expressing both E-cadherin and alpha-SMA appear in the dermis. In contrast, fibronectin-releasing cells were detected in the dermis as early as on E15.5, although on P5, some dermal cells was found weakly expressing both fibronectin and E-cadherin, most cells strongly expressing fibronectin did not express E-cadherin. Vimentin was mainly expressed in both endothelial and blood-derived cells and did not show co-expression with E-cadherin. Confocal microscopy studies further found that during EMT, E-cadherin appears intracellularly, while the expression of alpha-SMA starts from the membrane area and moves to the cytosol of the cells. Our data are the first in vivo evidence that EMT occurs during mouse skin development. Dermal cells are derived from EMT and other origins, including blood, during skin development. 相似文献
39.
One popular way of measuring visual attentional processes in the rat is using 5-choice serial reaction time task (5-CSRTT). This paradigm requires subjects to detect brief flashes of light presented in a pseudorandom order in one of five spatial locations over a large number of trials. For this task, the animals are trained for approximately 30-40 daily sessions during which they gradually learn to respond in the appropriate aperture within a certain amount of time. If they fail to respond, respond in the wrong hole or at an inappropriate time, a short period of darkness (time-out) is presented as punishment and no reward is delivered. The 5-CSRTT provides the possibility to test the effects of various neural, pharmacological and behavioral manipulations on discrete and somewhat independent measures of behavioral control, including accuracy of discrimination, impulsivity, perseverative responses and response latencies. 相似文献
40.
Beluzić R Cuk M Pavkov T Barić I Vugrek O 《Biochemical and biophysical research communications》2008,368(1):30-36
Recently, S-adenosylhomocysteine hydrolase deficiency was confirmed for the first time in an adult. Two missense mutations in codons 89 (A>V) and 143 (Y>C) in the AdoHcyase gene were identified [N.R.M. Buist, B. Glenn, O. Vugrek, C. Wagner, S. Stabler, R.H. Allen, I. Pogribny, A. Schulze, S.H. Zeisel, I. Bari?, S.H. Mudd, S-Adenosylhomocysteine hydrolase deficiency in a 26-year-old man, J. Inh. Metab. Dis. 29 (2006) 538-545]. Accordingly, we have proven the Y143C mutation to be highly inactivating [R. Beluzi?, M. Cuk, T. Pavkov, K. Fumi?, I. Bari?, S.H. Mudd, I. Jurak, O. Vugrek, A single mutation at tyrosine 143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound co-factor NAD, Biochem. J. 400 (2006) 245-253]. Now we report that the A89V exchange leads to a 70% loss of enzymatic activity, respectively. Circular dichroism analysis of recombinant p.A89V protein shows a significantly reduced unfolding temperature by 5.5 degrees C compared to wild-type. Gel filtration of mutant protein is almost identical to wild-type indicating assembly of subunits into the tetrameric complex. However, electrophoretic mobility of p.A89V is notably faster as shown by native polyacrylamide gel electrophoresis implicating changes to the overall charge of the mutant complex. 'Bioinformatics' analysis indicates that Val(89) collides with Thr(84) causing sterical incompatibility. Performing site-directed mutagenesis changing Thr(84) to 'smaller' Ser(84) but preserving similar physico-chemical properties restores most of the catalytic capabilities of the mutant p.A89V enzyme. On the other hand, substitution of Thr(84) with Lys(84) or Gln(84), thereby introducing residues with higher volume in proximity to Ala(89) results in inactivation of wild-type protein. In view of our mutational analysis, we consider changes in charge and the sterical incompatibility in mutant p.A89V protein as main reason for enzyme malfunction with AdoHcyase deficiency as consequence. 相似文献