Stereospecific synthesis and bio-activity of novel beta(3)-adrenoceptor agonists and inverse agonists

Since it is widely distributed into the body, beta(3)-adrenoceptor is becoming an attractive target for the treatment of several pathologies such as obesity, type 2 diabetes, metabolic syndrome, cachexia, overactive bladder, ulcero-inflammatory disorder of the gut, preterm labour, anxiety and depressive disorders, and heart failure. New compounds belonging to the class of arylethanolamines bearing one or two stereogenic centres were prepared in good yields as racemates and optically active forms. They were, then, evaluated for their intrinsic activity towards beta(3)-adrenoceptor and their affinity for beta(1)- and beta(2)-adrenergic receptors. Stereochemical features were found to play a crucial role in determining the behaviour of such compounds. In particular, alpha-racemic, (alphaR)- and (alphaS)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}-2- methylpropanoic acid, (alpha-rac, beta-rac)-, (alphaR, betaS)- and (alphaR, betaR)- 2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid were found to be endowed with beta(3)-adrenoceptor agonistic activity. Whereas, (alphaS, betaS)- and (alphaS, betaR)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid behaved as beta(3)-adrenoceptor inverse agonists. Such compounds showed no affinity for beta(1)- and beta(2)-adrenergic receptor, respectively. Thus, resulting highly selective beta(3)-adrenoceptor ligands.     

Oxytocin stimulates in vitro angiogenesis via a Pyk-2/Src-dependent mechanism

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.     

Potential allelopathic effect of neo-clerodane diterpenes from Teucrium chamaedrys (L.) on stenomediterranean and weed cosmopolitan species

The allelopathic effects of neo-clerodane diterpenes, isolated from Teucrium chamaedrys (L.), have been evaluated on the seed germination and seedling growth of four coexisting Mediterranean species (Dactylis hispanica, Petrorhagia velutina, Phleum subulatum and Petrorhagia saxifraga) and two cosmopolitan weeds (Amaranthus retroflexus and Avena fatua). All of the structures have been elucidated on the basis of their spectroscopic features. The bioassays data, analyzed by principal component analysis, showed more negative effects on weeds respect to coexisting species. Moreover D. hispanica, P. velutina, P. subulatum showed both stimulating or inhibiting effects depending on the type of metabolite and the concentration used in the test.     

Oligomeric Yeast Frataxin Drives Assembly of Core Machinery for Mitochondrial Iron-Sulfur Cluster Synthesis

Mitochondrial biosynthesis of iron-sulfur clusters (ISCs) is a vital process involving the delivery of elemental iron and sulfur to a scaffold protein via molecular interactions that are still poorly defined. Analysis of highly conserved components of the yeast ISC assembly machinery shows that the iron-chaperone, Yfh1, and the sulfur-donor complex, Nfs1-Isd11, directly bind to each other. This interaction is mediated by direct Yfh1-Isd11 contacts. Moreover, both Yfh1 and Nfs1-Isd11 can directly bind to the scaffold, Isu1. Binding of Yfh1 to Nfs1-Isd11 or Isu1 requires oligomerization of Yfh1 and can occur in an iron-independent manner. However, more stable contacts are formed when Yfh1 oligomerization is normally coupled with the binding and oxidation of Fe²⁺. Our observations challenge the view that iron delivery for ISC synthesis is mediated by Fe²⁺-loaded monomeric Yfh1. Rather, we find that the iron oxidation-driven oligomerization of Yfh1 promotes the assembly of stable multicomponent complexes in which the iron donor and the sulfur donor simultaneously interact with each other as well as with the scaffold. Moreover, the ability to store ferric iron enables oligomeric Yfh1 to adjust iron release depending on the presence of Isu1 and the availability of elemental sulfur and reducing equivalents. In contrast, the use of anaerobic conditions that prevent Yfh1 oligomerization results in inhibition of ISC assembly on Isu1. These findings suggest that iron-dependent oligomerization is a mechanism by which the iron donor promotes assembly of the core machinery for mitochondrial ISC synthesis.ISC³ biosynthesis is an essential function that eukaryotic cells initiate in mitochondria and probably other cellular compartments using three core components: a sulfur donor, an iron donor, and an ISC assembly scaffold (1, 2). In yeast mitochondria, the cysteine-desulfurase, Nfs1, and the iron-chaperone, Yfh1, are believed to provide sulfur and iron, respectively, for ISC assembly on the Isu1 scaffold (1), whereas the Nfs1-binding protein, Isd11, has been shown to stabilize Nfs1 (3). These components are highly conserved and the human orthologues of Yfh1 (frataxin), Isu1 (ISCU), and Isd11 (ISD11) are implicated in the etiology of severe disorders including Friedreich ataxia and mitochondrial myopathy (4).Previous studies have underscored the complexity of the interactions among eukaryotic ISC assembly components as well as their metal dependence. Supplementation of mitochondrial lysates with Fe²⁺ under aerobic conditions led to co-isolation of Yfh1 and Isu1 along with Nfs1 and Isd11 by pulldown or immunoprecipitation assays (5–7). Furthermore, aerobic preincubation of histidine-tagged Yfh1 monomer with Fe²⁺ enabled Isu1 to be pulled down by Yfh1 in the absence of other proteins (5). These studies have led to the current view that iron delivery for yeast ISC synthesis involves direct contacts between iron-loaded monomeric Yfh1 and Isu1 (5–7). Although Yfh1 oligomerization is normally coupled with iron binding, oxidation, and storage (5, 8), the possibility that Isu1 might also interact with oligomeric Yfh1 has remained largely unexplored.Similar to Yfh1, human frataxin was found to interact with multiple ISC assembly components in human cells; however, in this case immunoprecipitation data suggested that frataxin binds to ISCU indirectly, via nickel-dependent contacts with ISD11 (9). Whether direct interactions occur between Yfh1 and Isd11 has not yet been examined.While previous studies focused primarily on Yfh1-Isu1 and frataxin-ISD11 interactions, it is likely that the coordinate delivery of potentially toxic sulfur and iron to Isu1/ISCU involves multiple close interactions whereby the sulfur donor and the iron donor simultaneously interact with each other and with the ISC scaffold, as proposed for prokaryotic ISC assembly (10). However, it is currently unknown whether monomeric Yfh1/frataxin may form direct contacts with more than one partner, and the structure of the eukaryotic ISC assembly machinery is completely undefined. We show that iron oxidation-dependent oligomerization enables Yfh1 to have simultaneous direct interactions with Nfs1-Isd11 and Isu1. Our data provide insights about the sequence of events and the molecular architecture required for the initial step in mitochondrial ISC assembly.     

Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer's disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre-adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes.     

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

Tamoxifen-independent recombination in the RIP-CreER mouse

Background

The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas.

Principal Findings

Here, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains.

Significance

Evidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic β-cells.