Xanthine oxidase catalyzes the oxidation of retinol

In mammals, xanthine oxidase (E.C. 1.17.3.2) catalyzes the hydroxylation of a wide variety of heterocyclic substrates such as purines, pyrimidines, and pterins, in addition to aldehydes [1] as all-trans-retinaldehyde [2-5]. Here, we show that buttermilk xanthine oxidase was capable to oxidizing all-trans-retinol (t-ROL) to all-trans-retinaldehyde (t-RAL) that was successively oxidized to all-trans-retinoic acid (t-RA). A rise in the enzyme activity, when t-ROL-CRBP complex was assayed, with respect to the free t-ROL, was observed. Furthermore, treatment of the enzyme with Na2S and glutathione resulted in a significant increment in catalytic activity toward t-ROL and t-RAL, due to the reconstitution of the native structural organization of the molybdenum centre of molybdopterin cofactor of the desulfo form of xanthine oxidase.     

NAP modulates hyperglycemic–inflammatory event of diabetic retina by counteracting outer blood retinal barrier damage

Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Prolonged hyperglycemia stimulates inflammatory pathway characterized by the release of some cytokines leading to the impairment of blood retinal barrier (BRB). NAP exerts a protective effect in various eye diseases, including DR. So far, the role of NAP in the modulation of inflammatory event during early phase of this pathology has not been investigated yet. In the current study, we have studied the retinal protective effect of NAP, injected into the eye, in diabetic rats. NAP treatment exerts a dual effect downregulating interleukin (IL)-1β and its related receptors and upregulating IL-1Ra expression. We have also tested the role of this peptide in human retinal epithelial cells (ARPE19) cultured on a semipermeable support and exposed to hyperglycemic–inflammatory insult, representing a in vitro model of diabetic macular edema, a clinical manifestation of DR. The results have shown that NAP prevents outer BRB impairment by upregulating the tight junctions. In conclusion, deepened characterization of NAP action mechanism on hyperglycemic–inflammatory damage may be useful to develop a new strategy to prevent retinal damage during DR.     

Oxidative nucleophilic substitution selectively produces cambinol derivatives with antiproliferative activity on bladder cancer cell lines

Methyltrioxorhenium mediated oxidative addition/elimination nucleophilic substitution yielded alkylamino and arylamino cambinol derivatives characterized by anti-proliferative activity against wild-type and p53 mutated MGH-U1 and RT112 bladder cancer cell lines. Some of the novel compounds showed an activity higher than that of the lead compound. The reaction was highly regioselective, affording for the first time a panel of C-2 cambinol substitution products. Aliphatic primary and secondary amines, and primary aromatic amines, were used as nitrogen centered nucleophiles. Surprisingly, the antiproliferative activity of C-2 substituted cambinol derivatives was not correlated to the induction of p53 protein, as evaluated by the analysis of the cell viability on wild-type and p53 mutated cancer cell lines, and further confirmed by western blot analyses. These data suggest that they exert their antiproliferative activity by a mechanism completely different from cambinol.     

Role for non-proteolytic control of M-phase-promoting factor activity at M-phase exit

M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.     

Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture

Endothelial progenitor cells (EPCs) play a role in endogenous neovascularization of ischaemic tissues. Isolation and characterization of EPCs from circulating mononuclear cells are important for developing targeted cellular therapies and reproducibility of data are the major scientific goals. Here we compared two currently employed isolation methods, i.e. from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133(+) cells, by defining the cell morphology and functional activities. We show that EPCs from cultured PBMCs resulted in an adherent population of 23% +/- 4% merged cells positive for Dil-Ac-LDL and lectin, whereas the percentage of double positive cells in cultured CD133(+) enriched cells was 50% +/- 7% (P < 0.01). These data were obtained through a novel and a more complete method of analysis of cell calculations (specifically by dividing each microscope field into 120 subfields). When stimulated with tumour necrosis factor alpha (TNF)-alpha and glucose, cell number was reduced in EPCs from total PBMCs and, more consistently, in CD133(+) enriched cells. However, both cultured total PBMCs and CD133(+) enriched cells respond similarly to TNF-alpha or glucose-induced p38-phosphorylation. EPCs from both procedures show similar results in terms of phenotype and response to modulators of their functional activities. However, when the cell phenotype of CD133(+) enrichment-derived cells was compared with that of cells from the total PBMC, a significant increase in CD133(+) expression was observed (P < 0.01) This may have relevance during intervention studies using cultured EPCs.     

Evidence for the presence of photorespiration in desiccation-sensitive leaves of the C4 'resurrection' plant Sporobolus stapfianus during dehydration stress

The possible role of photorespiration as a general stress protectionmechanism, and in C₄ plant metabolism, is controversial. Inparticular, the potential involvement of photorespiration inthe acquisition of desiccation tolerance in ‘resurrection’plants is unknown. An investigation was carried out into whetherphotorespiration is present in leaves of the C₄ resurrectionplant Sporobolus stapfianus Gandoger (Poaceae) and whether itfunctions as a mechanism of stress resistance in the desiccation-tolerantyounger leaves (YL) of this plant. It is shown that the enzymesinvolved in the photorespiratory pathway maintain their activityuntil 88% relative water content (RWC) in both YL and desiccation-sensitiveolder leaves (OL). In subsequent stages of dehydration stress,the enzymatic activity declined similarly in both YL and OL.The content of the phorespiratory metabolite, serine, and ethanolamine,a direct product of serine decarboxylation, is higher in theearly stages of dehydration (88% RWC) in OL, suggesting a transientlyenhanced photorespiratory activity in these leaves. This wasconfirmed by simultaneous gas exchange and fluorescence measurements,showing suppression of the electron transport rate in OL exposedto non-photorespiratory conditions (2% O₂) at 85% RWC. It isconcluded that a higher photorespiratory electron transportoccurs in desiccation-sensitive OL, and it is therefore proposedthat the capacity to scavenge excess electrons through photorespirationdoes not contribute to protect leaves of the desiccation-tolerantYL of S. stapfianus during the stress. Key words: Ethanolamine, glycine, photorespiratory enzymes, photosynthesis, poikilohydric plant, serine Received 5 June 2007; Revised 3 September 2007 Accepted 17 September 2007     

Targeting the miRNA-155/TNFSF10 network restrains inflammatory response in the retina in a mouse model of Alzheimer’s disease

Age-related disorders, such as Alzheimer’s disease (AD) and age-related macular degeneration (AMD) share common features such as amyloid-β (Aβ) protein accumulation. Retinal deposition of Aβ aggregates in AMD patients has suggested a potential link between AMD and AD. In the present study, we analyzed the expression pattern of a focused set of miRNAs, previously found to be involved in both AD and AMD, in the retina of a triple transgenic mouse model of AD (3xTg-AD) at different time-points. Several miRNAs were differentially expressed in the retina of 3xTg-AD mice, compared to the retina of age-matched wild-type (WT) mice. In particular, bioinformatic analysis revealed that miR-155 had a central role in miRNA-gene network stability, regulating several pathways, including apoptotic and inflammatory signaling pathways modulated by TNF-related apoptosis-inducing ligand (TNFSF10). We showed that chronic treatment of 3xTg-AD mice with an anti-TNFSF10 monoclonal antibody was able to inhibit the retinal expression of miR-155, which inversely correlated with the expression of its molecular target SOCS-1. Moreover, the fine-tuned mechanism related to TNFSF10 immunoneutralization was tightly linked to modulation of TNFSF10 itself and its death receptor TNFRSF10B, along with cytokine production by microglia, reactive gliosis, and specific AD-related neuropathological hallmarks (i.e., Aβ deposition and Tau phosphorylation) in the retina of 3xTg-AD mice. In conclusion, immunoneutralization of TNFSF10 significantly preserved the retinal tissue in 3xTg-AD mice, suggesting its potential therapeutic application in retinal degenerative disorders.Subject terms: Alzheimer''s disease, Neurodegeneration     

Unusual composition of thylakoid membranes of the resurrection plant Boea hygroscopica: Changes in lipids upon dehydration and rehydration

Boea hygroscopica is a resurrection plant that is able to pass from biosis to anabiosis and vice versa following slow dehydration, but loses this ability following a rapid water loss. Fresh leaves were detached from plants grown in well-watered conditions and subjected to either rapid or slow dehydration and rehydration. Upon rehydration only slowly dried leaves revived. Analysis of thylakoid membranes revealed a rather small amount of total lipids (1,4–2 μmol g^?1 dry weight) in comparison with other flowering plants. The main glycolipid was digalactosyldiacylglycerol (DGDG) rather than monogalactosyldiacylglycerol (MGDG) as is common in higher plants. Linoleic acid was the main fatty acid (30–40 mol% of total fatty acids), while linolenic acid was present from 14 to 26 mol%. In both the fresh and rehydrated leaves nearly all lipid components were present in similar amounts. Following dehydration the DGDG/MGDG molar ratio, which was 1.1 in control and rehydrated leaves, doubled by the end of the rapid drying period and was three times as high in slowly dried leaves. The total polar lipid/free sterol molar ratio as well as the free fatty acid level assumed the highest values in the rapidly dehydrated leaves. A shift towards the more unsaturated fatty acids was observed in all lipid classes upon dehydration irrespective of whether it was slow or rapid. Our data show only small differences between rapidly and slowly dehydrated leaves which can be correlated to the capacity of slowly dehydrated leaves to revive.     

Long-chain fatty acid transport in bacteria andyeast. Paradigms for defining the mechanism underlying this protein-mediated process

Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems. Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both [1] exhibit saturable long-chain fatty acid transport at low ligand concentration; [2] have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and [3] can be easily manipulated using the tools of molecular genetics. In E. coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS). FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional. This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane. In S. cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p. Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E. coli, there is not a tight linkage between transport and activation. Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1 strain support the hypothesis that Fat1p is required for optimal levels of long-chain fatty acid transport.