Loss of ecto-5'nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation

The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.     

Possible involvement of aquaporin-7 and -8 in rat testis development and spermatogenesis.

Fluid secretion and reabsorption are of central importance in male reproductive (MR) physiology. However, the related molecular mechanisms are poorly known. Here, potential roles for AQP7 and AQP8, two aquaporin water channels abundantly expressed in the MR tract, were investigated by studying their expression and distribution in the developing testis of the Wistar rat. By semiquantitative RT-PCR and immunoblotting, first expression of AQP7 was noted at postnatal day 45 (P45), with levels increasing substantially at P90 and remaining at high levels thereafter. AQP8 began to be expressed at P15, rapidly increased until P20, and remained fairly stable thereafter. Immunohistochemical analyses demonstrated AQP7 in elongated spermatids, testicular spermatozoa, and residual bodies at P45 with increased signal intensity thereafter. AQP8 was observed in primary spermatocytes from P20 to P30 and, in elongated spermatids, residual bodies and Sertoli cells at P30 and thereafter. The ontogeny and distribution of AQP7 and AQP8 in rat testis suggest involvement in major physiologic changes in testis development and spermatogenesis.     

Na+-K+-2Cl- cotransporters and Cl- channels regulate citric acid cough in guinea pigs.

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl- from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10-100 microM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl- channel inhibitors DIDS or niflumic acid (100 microM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl- due to the expression of the furosemide-sensitive Cl- transporter, NKCC1. Efflux of intracellular Cl-, in part through calcium-activated Cl- channels, may play an important role in regulating airway afferent neuron activity.     

Comparison between the allergenic airborne pollen in Trieste and at Lozzo di Cadore (Italy) in 1989

Summary A comparative study was carried out between aeroallergenic pollen spectra in Trieste and at Lozzo di Cadore. The two localities were found to be different on a five-day running mean basis as to the 8 pollen taxa monitored by the National Aerobiological Monitoring Network managed by the Italian Association of Aerobiology (AIA). In the zone of Lozzo the pollination curve of the spring flowering taxa showed a lag of about one month due to the colder climate. Lozzo di Cadore showed a poor airborne pollen content,Corylus and Graminaceae being prevailing. Trieste has a higher airborne pollen diversity and longer pollination times because of its higher floristic and vegetational complexity. In Trieste the allergenic pollen spectrum showed great quantities ofOstrya, Quercus, Oleaceae, Graminaceae and of ruderal taxa, widespread over the area, due to man's influence.     

Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.     

Design of new reaction conditions for characterization of a mutant thermophilic α-l-fucosidase

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.     

Combination of geometric morphometric and genetic approaches applied to a debated taxonomical issue: the status of Onthophagus massai (Coleoptera, Scarabaeidae) as an endemic species vicarious to Onthophagus fracticornis in Sicily

The present study deals with the phenomenon of insular speciation and discusses, as a case study, the debated taxonomical issue of the status of Onthophagus massai (Coleoptera, Sarabaeidae) as an endemic species vicarious to Onthophagus fracticornis in Sicily. The authors investigated the differentiation patterns between an insular population belonging to the supposed species O. massai (collected in its locus typicus, Piano Battaglia) and three Italian O. fracticornis populations (collected along a N–S latitudinal gradient). These patterns are described and analysed using multiple approaches: the qualitative inspection of the microsculpture of elytral surfaces, considered a diagnostic character for O. massai identification; the comparison of horn static allometries, known to be a good indicator of divergence processes between closely related species or isolated populations of the same species; the comparison of the patterns of shape and size difference of the head, epipharynx and genitalia attained with a combination of traditional and geometric (landmark and semilandmark) morphometric methods; and, finally, the estimation of the genetic relationships between Sicilian and continental populations obtained by analysing cytochrome oxidase subunit 1 mitochondrial gene sequences. The integration of the results of these approaches indicates that there is not sufficient evidence to vindicate the species status for O. massai, which should more likely be considered a small-sized version of O. fracticornis (a possible case of insular dwarfism). However, the complex pattern of shape, size and genetic variation observed between the populations analysed hinted at the possibility that a diversification process is ongoing, but not only between insular and continental populations; each population showed a tendency to evolve as an evolutionarily independent unit.     

The use of actigraphy in the monitoring of methylphenidate versus placebo in ADHD: a meta-analysis

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioral disorder of childhood. There is an increasing need to find objective measures and markers of the disorder in order to assess the efficacy of the therapy and to improve follow-up strategies. Actigraphy is an objective method for recording motor activity and sleep parameters using small, computerized, watch-like devices worn on the body, and it has been used in many clinical trials to assess methylphenidate efficacy and adverse effects in ADHD. Our article aim is to systematically review and perform a meta-analysis of the current evidence on the role of actigraphy in both the detection of changes in activity and in sleep patterns in randomized clinical trials that compared methylphenidate against placebo in the treatment of ADHD. A comprehensive literature search of PubMed/MEDLINE, Scopus, Embase, Cochrane Library, CINHAL and PsycINFO databases was carried out to find randomized clinical trials comparing methylphenidate versus placebo in children with ADHD, using actigraphic measures as an outcome. No start date limit was used and the search was updated until June 2013. The primary outcome measures were ‘total sleep time’ and daytime ‘activity mean’. As secondary outcomes, we analyzed ‘sleep onset latency’, ‘sleep efficiency’ and ‘wake after sleep onset’. Eight articles comprising 393 patients were included in the analysis. Children with ADHD using MPH compared to placebo have a significant difference of a large effect with a diminishing value in the activity mean. For the total sleep time, we found a significant and large effect in the decrease in sleep in MPH group. This study shows that MPH may effectively reduce mean activity in ADHD children, but it may negatively affect total sleep time.     

The role of oxysterols in control of endothelial stiffness

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE^−/− mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.