Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation

Six glycine residues of human muscle acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.     

Involvement of PI3K and MAPK signaling in bcl-2-induced vascular endothelial growth factor expression in melanoma cells

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.     

Apples increase nitric oxide production by human saliva at the acidic pH of the stomach: a new biological function for polyphenols with a catechol group?

Dietary inorganic nitrate is secreted in saliva and reduced to nitrite by bacterial flora. At the acidic pH of the stomach nitrite is present as nitrous acid in equilibrium with nitric oxide (*NO), and other nitrogen oxides with nitrating and nitrosating activity. *NO in the stomach exerts several beneficial effects, but nitrosating/nitrating species have been implicated as a possible cause of epithelial neoplasia at the gastroesophageal junction. We investigated the effects of apple extracts on *NO release by human saliva at pH 2. A water extract obtained from apple homogenate increased *NO release caused by acidification of saliva. Data show that polyphenols were responsible for this activity, with chlorogenic acid and (+)-catechin the most active and concentrated species. However, ferulic acid, a hydroxycinnamic acid with only one aromatic hydroxyl group, did not increase *NO release. Fructose, the most representative sugar in apples, was also inactive. Interestingly, ascorbic acid in saliva induced a SCN(-)-enhanced burst of *NO but, unlike apple, the release was transient. The simultaneous addition of ascorbic acid and apple extract caused a burst of *NO followed by the increased steady-state level characteristic of saliva containing apple extract. Chlorogenic acid and (+)-catechin, but not ferulic acid, formed o-semiquinone radicals and nitrated polyphenols, suggesting the scavenging of *NO(2) by o-semiquinones. Our results propose that some apple polyphenols not only inhibit nitrosation/nitration but also promote *NO bio-availabilty at the gastric level, a previously unappreciated function.     

Fra1 activity in the frog, Rana esculenta, testis: a new potential role in sperm transport

Using an anti-Fos family member antibody, we have previously described in Rana esculenta testis the presence of a nuclear, 43 kDa protein that we hypothesized to be Fra1. With the assistance of an antibody against Fra1 that does not cross-react with other Fos family members, here we report data on Fra1 expression, localization, and putative activity in Rana esculenta testis during its annual reproductive cycle. Western blot analysis confirms that the nuclear, 43 kDa protein is Fra1. Immunocytochemistry validates the Western blot results and shows cytoplasmic and nuclear immunostaining of Fra1 in peritubular myoid cells, efferent ducts, and blood vessels. We report for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells during sperm transport from the tubular compartment to efferent ducts.     

Potential physiological role of plant glycosidase inhibitors

Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological applications.     

Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus

The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations. The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results. The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased. The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i). It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).     

Synthesis,antimalarial activity,and cellular toxicity of new arylpyrrolylaminoquinolines

A set of nine new arylpyrrolyl derivatives of 7-chloro-4-aminoquinoline, characterized by different substituents on the phenyl ring or different distance between the pyrrolic nitrogen and the 4-aminoquinoline, has been synthesized and tested for their activity against D-10 (CQ-S) and W-2 (CQ-R) strains of Plasmodium falciparum. All compounds exhibited activity against the CQ-S strain in the low nM range, comparable to that of chloroquine. Some of them were also highly active against the CQ-R strain and not toxic against normal cells. The antimalarial activity of this new class of compounds seems to be related to the inhibition of heme detoxification process of parasites, as in the case of chloroquine.     

TNFalpha up‐regulates SLUG via the NF‐kappaB/HIF1alpha axis,which imparts breast cancer cells with a stem cell‐like phenotype

Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44⁺/CD24^? tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010. © 2010 Wiley‐Liss, Inc.