Postjunctional Origin of the indirect-like sympathomimetic effect of metanephrine and normetanephrine on blood pressure

The effects of metanephrine and normetanephrine have been compared with those from equiactive doses of the origin compounds, adrenaline and noradrenaline, on the pressor responses in rat, in order to determine whether their effects are owed, at least partially, to a releasing presynaptic action of the catecholamines in normal animals as well as those pretreated with reserpine, guanethidine and 6-OH-dopamine. Their effects have likewise been studied in isolated perfused renal arteries both in normal and reserpinized rats. None of the adrenolytic agent used were able either to reduce the duration of the hypertensive response or to accelerate tachyphylaxis. Identical results were obtained in renal artery preparations. It is thus concluded that the catecholamines stored in presynaptic endings are not involved in the observed phenomena and it is suggested that they might depend on the high doses required to produce effects equiactive to those of the origin substances.     

Placental tissue metabolome analysis by GC-MS: Oven-drying is a viable sample preparation method

Abstract

Analysis of the human placenta metabolome has great potential to advance the understanding of complicated pregnancies and deleterious fetal outcomes in remote populations, but samples preparation can present unique challenges. Herein, we introduce oven-drying as a simple and widely available method of sample preparation that will facilitate investigations of the placental metabolome from remote and under-studied populations. Placentae from complicated and uncomplicated pregnancies were prepared in three ways (oven-dried at 60?°C, fresh, lyophilized) for metabolome analysis via gas chromatography-mass spectrometry (GC-MS). Multiple computer models (e.g. PLS-DA, ANN) were employed to classify and determine if there was a difference in placentae metabolome and a group of metabolites with high variable importance in projection scores across the three preparations and by complicated vs. control groups. The analyses used herein were shown to be thorough and sensitive. Indeed, significant differences were detected in metabolomes of complicated vs. uncomplicated pregnancies; however, there were no statistical differences in the metabolome of placentae prepared by oven-drying vs. lyophilization vs. fresh placentae. Oven-drying is a viable sample preparation method for placentae intended for use in metabolite analysis via GC-MS. These results open many possibilities for researching metabolome patterns associated with fetal outcomes in remote and resource-poor communities worldwide.     

2-(1H-Pyrazol-4-yl)acetic acids as CRTh2 antagonists

High throughput screening identified the pyrazole-4-acetic acid substructure as CRTh2 receptor antagonists. Optimisation of the compounds uncovered a tight SAR but also identified some low nanomolar inhibitors.     

Tor1, Sch9 and PKA downregulation in quiescence rely on Mtl1 to preserve mitochondrial integrity and cell survival

Here we show that Mtl1, member of the cell wall integrity pathway of Saccharomyces cerevisiae, plays a positive role in chronological life span (CLS). The absence of Mtl1 shortens CLS and causes impairment in the mitochondrial function. This is reflected in a descent in oxygen consumption during the postdiauxic state, an increase in the uncoupled respiration and mitochondrial membrane potential and also a descent in aconitase activity. We demonstrate that all these effects are a consequence of signalling defects suppressed by TOR1 (target of rapamycin) and SCH9 deletion and less efficiently by Protein kinase A (PKA) inactivation. Mtl1 also plays a role in the regulation of both Bcy1 stability and phosphorylation, mainly in response to glucose depletion. In postdiauxic phase and in conditions of glucose depletion, Mtl1 negatively regulates TOR1 function leading to Sch9 inactivation and Bcy1 phosphorylation converging in PKA inhibition. Slt2/Mpk1 kinase partially contributes to Bcy1 phosphorylation, although additional targets are not excluded. Mtl1 links mitochondrial dysfunction with TOR and PKA pathways in quiescence, glucose being the main signalling molecule.     

Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer''s disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.     

Matrix Metalloproteinase 2 (MMP-2) Degrades Soluble Vasculotropic Amyloid-β E22Q and L34V Mutants,Delaying Their Toxicity for Human Brain Microvascular Endothelial Cells

Patients carrying mutations within the amyloid-β (Aβ) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Aβ synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AβE22Q and AβL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Aβ peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Aβ-(1–16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Aβ degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Aβ peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AβE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Aβ species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.