Changes of the hepatic proteome in hepatitis B-infected mouse model at early stages of fibrosis

Liver fibrosis (LF) is the accumulation of extracellular matrix (ECM) proteins due to chronic liver injury. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to perform a comparative analysis of cytosolic and nuclear protein patterns of nontransgenic (NTg) and HBV transgenic (Tg) mice livers at early stages of fibrosis. We identified several candidate proteins, involved in a variety of pathways, which could be used as putative biomarkers for LF early detection.     

NAADP+ is an agonist of the human P2Y11 purinergic receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an intracellular second messenger releasing Ca2+ from intracellular stores in different cell types. In addition, it is also active in triggering [Ca2+](i) increase when applied extracellularly and various underlying mechanisms have been proposed. Here, we used hP2Y(11)-transfected 1321N1 astrocytoma cells to unequivocally establish whether extracellular NAADP+ is an agonist of the P2Y(11) receptor, as previously reported for beta-NAD+ [I. Moreschi, S. Bruzzone, R.A. Nicholas, et al., Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes, J. Biol. Chem. 281 (2006) 31419-31429]. Extracellular NAADP+ triggered a concentration-dependent two-step elevation of [Ca2+](i) in 1321N1-hP2Y(11) cells, but not in wild-type 1321N1 cells, secondary to the intracellular production of IP(3), cAMP and cyclic ADP-ribose (cADPR). Specifically, the transient [Ca2+](i) rise proved to be related to IP(3) overproduction and to consequent Ca2+ mobilization, while the sustained [Ca2+](i) elevation was caused by the cAMP/ADP-ribosyl cyclase (ADPRC)/cADPR signalling cascade and by influx of extracellular Ca2+. In human granulocytes, endogenous P2Y(11) proved to be responsible for the NAADP+-induced cell activation (as demonstrated by the use of NF157, a selective and potent inhibitor of P2Y(11)), unveiling a role of NAADP+ as a pro-inflammatory cytokine. In conclusion, we provide unequivocal evidence for the activation of a member of the P2Y receptor subfamily by NAADP+.     

Antagonism of metabotropic glutamate receptor type 5 attenuates l-DOPA-induced dyskinesia and its molecular and neurochemical correlates in a rat model of Parkinson's disease

Metabotropic glutamate receptor type 5 (mGluR5) modulates dopamine and glutamate neurotransmission at central synapses. In this study, we addressed the role of mGluR5 in l-DOPA-induced dyskinesia, a movement disorder that is due to abnormal activation of both dopamine and glutamate receptors in the basal ganglia. A selective and potent mGluR5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl] pyridine, was tested for its ability to modulate molecular, behavioural and neurochemical correlates of dyskinesia in 6-hydroxydopamine-lesioned rats treated with l-DOPA. The compound significantly attenuated the induction of abnormal involuntary movements (AIMs) by chronic l-DOPA treatment at doses that did not interfere with the rat physiological motor activities. These effects were paralleled by an attenuation of molecular changes that are strongly associated with the dyskinesiogenic action of l-DOPA (i.e. up-regulation of prodynorphin mRNA in striatal neurons). Using in vivo microdialysis, we found a temporal correlation between the expression of l-DOPA-induced AIMs and an increased GABA outflow within the substantia nigra pars reticulata. When co-administered with l-DOPA, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl] pyridine greatly attenuated both the increase in nigral GABA levels and the expression of AIMs. These data demonstrate that mGluR5 antagonism produces strong anti-dyskinetic effects in an animal model of Parkinson's disease through central inhibition of the molecular and neurochemical underpinnings of l-DOPA-induced dyskinesia.     

Differential, age-dependent MEK-ERK and PI3K-Akt activation by insulin acting as a survival factor during embryonic retinal development

Programmed cell death is a genuine developmental process of the nervous system, affecting not only projecting neurons but also proliferative neuroepithelial cells and young neuroblasts. The embryonic chick retina has been employed to correlate in vivo and in vitro studies on cell death regulation. We characterize here the role of two major signaling pathways, PI3K-Akt and MEK-ERK, in controlled retinal organotypic cultures from embryonic day 5 (E5) and E9, when cell death preferentially affects proliferating neuroepithelial cells and ganglion cell neurons, respectively. The relative density of programmed cell death in vivo was much higher in the proliferative and early neurogenic stages of retinal development (E3-E5) than during neuronal maturation and synaptogenesis (E8-E19). In organotypic cultures from E5 and E9 retinas, insulin, as the only growth factor added, was able to completely prevent cell death induced by growth factor deprivation. Insulin activated both the PI3K-Akt and the MEK-ERK pathways. Insulin survival effect, however, was differentially blocked at the two stages. At E5, the effect was blocked by MEK inhibitors, whereas at E9 it was blocked by PI3K inhibitors. The cells which were found to be dependent on insulin activation of the MEK-ERK pathway at E5 were mostly proliferative neuroepithelial cells. These observations support a remarkable specificity in the regulation of early neural cell death.     

Rapid detection of sex chromosomal aneuploidies by QF-PCR: application in 200 men with severe oligozoospermia or azoospermia

Klinefelter syndrome is the most common genetic cause of severe male factor infertility. Cytogenetic evaluation of metaphase chromosomes generally has a long turnaround time. We describe a reliable molecular genetic method that can be completed in 2 working days to identify the presence of any extra X chromosomes. The quantitative fluorescent (QF) 5-plex PCR includes the amplification of amelogenin, which is present on both sex chromosomes in a biallelic form, a polymorphic short tandem repeat (STR) on the pseudoautosomal region of X and Y (X22), two polymorphic X-specific STRs (DXS6803, DXS6809), and a Y-specific marker (SY134), in a single tube. The presence of an extra X chromosome is recognized either by a supernumerary peak or an increased peak area based on criteria we have developed. The application of the method on 200 patients resulted in the identification of 14 patients (7%) with Klinefelter syndrome or a variant form (2 SRY-positive 46,XX men), as well as an additional patient with 47,XYY karyotype. The QF-PCR method, along with Y chromosome microdeletion testing, can be used as a first-step genetic analysis in azoospermic or severely oligozoospermic patients for the rapid identification of sex chromosome aneuploidies.     

EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR

Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, may augment metabolic and vascular actions of insulin. Therefore, we investigated effects of EGCG treatment to simultaneously improve cardiovascular and metabolic function in spontaneously hypertensive rats (SHR; model of metabolic syndrome with hypertension, insulin resistance, and overweight). In acute studies, EGCG (1-100 microM) elicited dose-dependent vasodilation in mesenteric vascular beds (MVB) isolated from SHR ex vivo that was inhibitable by N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase antagonist) or wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor]. In chronic studies, 9-wk-old SHR were treated by gavage for 3 wk with EGCG (200 mg.kg(-1).day(-1)), enalapril (30 mg.kg(-1).day(-1)), or vehicle. A separate group of SHR receiving L-NAME (80 mg/l in drinking water) was treated for 3 wk with either EGCG or vehicle. Vasodilator actions of insulin were significantly improved in MVB from EGCG- or enalapril-treated SHR (when compared with vehicle-treated SHR). Both EGCG and enalapril therapy significantly lowered systolic blood pressure (SBP) in SHR. EGCG therapy of SHR significantly reduced infarct size and improved cardiac function in Langendorff-perfused hearts exposed to ischemia-reperfusion (I/R) injury. In SHR given L-NAME, beneficial effects of EGCG on SBP and I/R were not observed. Both enalapril and EGCG treatment of SHR improved insulin sensitivity and raised plasma adiponectin levels. We conclude that acute actions of EGCG to stimulate production of nitric oxide from endothelium using PI 3-kinase-dependent pathways may explain, in part, beneficial effects of EGCG therapy to simultaneously improve metabolic and cardiovascular pathophysiology in SHR. These findings may be relevant to understanding potential benefits of green tea consumption in patients with the metabolic syndrome.     

Factors affecting laboratory bioassays with diatomaceous earth on stored wheat: effect of insect density, grain quantity, and cracked kernel containment

Laboratory bioassays were carried out to evaluate the effect of insect density (10, 30, 60, and 100 adults), wheat quantity (10, 30, 60, and 100 g), and cracked kernel containment (5, 15, 30, and 50%) on the efficacy of diatomaceous earth (DE). Three beetle species, Sitophilus oryzae (L.), Rhyzopertha dominica (F.), and Tribolium confusum Jacquelin du Val, as well as two DE formulations, Insecto and SilicoSec, and one DE enhanced with pyrethrum, PyriSec (all commercially available) were tested. In the first two series of bioassays, the three DE formulations were applied at three dose rates, 500, 1000 and 1,500 ppm. In the third series, the dose rates used were 500 and 1,000 ppm. Dead adults were counted 14 d later. For insect density, wheat quantity, and cracked kernel containment, significant differences were noted in mortality levels of the tested species among the three DE formulations and among doses. No significant differences were noted in the mortality levels among the four adult densities of any of the insects tested. The increase of wheat quantity used in the bioassays increased significantly adult mortality of T. confusum. The increase of cracked wheat containment decreased significantly adult mortality of S. oryzae.     

The E3-ubiquitin ligase TRIM50 interacts with HDAC6 and p62, and promotes the sequestration and clearance of ubiquitinated proteins into the aggresome

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.     

<Emphasis Type="Italic">Cabruca</Emphasis> agroforests in southern Bahia,Brazil: tree component,management practices and tree species conservation

In southern Bahia, Brazil, cabrucas are the traditional agroforests in which cacao trees are planted under thinned-out native forests. To analyze the role of cabrucas in tree species conservation, we inventoried the non-cocoa trees in 1.0 ha plots of cabruca in 16 cocoa farms and compared our results with a similar survey undertaken in the early 1960s in the same region to analyze the long term changes. We also interviewed 160 cocoa farmers to investigate their preferences for species and the main practices used in managing shade trees. The cabrucas showed high levels of tree diversity for an agroforestry system (Shannon index ranging from 2.21 to 3.52) and also high variation in structure and composition among the different farms. Forest specialist trees accounted for most species (63.9%) in the survey and were among the species most preferred by the farmers, although we found evidence that some of these trees are gradually being replaced by other species. Our results indicate that cabrucas are poor substitutes for undisturbed forests in terms of tree species richness, but their presence in human-altered landscapes is of utmost importance to the conservation of forest tree species as they increase overall heterogeneity and may serve as ecological corridors, additional habitats, and buffer zones.     

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP₂. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP₂ binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP₂ binding. The study provides the first structural view of the activated ezrin bound to PIP₂ and to F-actin.