Differential requirement of ATP and extra-ribosomal proteins for ribosome inactivation by eight RNA N-glycosidases.

The requirement of ATP and extra-ribosomal proteins for the inactivation of ribosomes by eight plant RNA N-glycosidases [ribosome-inactivating proteins (RIPs)] was investigated. Tritin, pokeweed antiviral protein and barley RIP depend, as gelonin [Sperti, S., Brigotti, M., Zamboni, M., Carnicelli, D. and Montanaro, L. (1991) Biochem. J., 277, 281-284], on the presence of ATP and extra-ribosomal proteins for full inactivation of ribosomes, while bryodin, lychnin, momordin, momorcochin and saporin inactivate isolated Artemia salina ribosomes suspended in buffer saline.     

Enzymatic synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid in a membrane reactor

Summary Dehydrocholic acid (3,7,12-trioxo-5-cholanic acid) (0.5% concentration) was completely and selectively reduced to 12-ketoursodeoxycholic acid (3, 7-dihydroxy-12-oxo- 5-cholanic acid) in a membrane reactor by means of 3-hydroxysteroid dehydrogenase and 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with the glucose-glucose dehydrogenase system.     

Purification and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity

Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of Citrullus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antiviral protein from roots, PAP-R) and from the leaves of Bryonia dioica (bryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an Mr at approx, 30,000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912).     

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Molecular Biology Reports - Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this,...     

Molecular Mechanism of Inhibition of the Mitochondrial Carnitine/Acylcarnitine Transporter by Omeprazole Revealed by Proteoliposome Assay,Mutagenesis and Bioinformatics

The effect of omeprazole on the mitochondrial carnitine/acylcarnitine transporter has been studied in proteoliposomes. Externally added omeprazole inhibited the carnitine/carnitine antiport catalysed by the transporter. The inhibition was partially reversed by DTE indicating that it was caused by the covalent reaction of omeprazole with Cys residue(s). Inhibition of the C-less mutant transporter indicated also the occurrence of an alternative non-covalent mechanism. The IC₅₀ of the inhibition of the WT and the C-less CACT by omeprazole were 5.4 µM and 29 µM, respectively. Inhibition kinetics showed non competitive inhibition of the WT and competitive inhibition of the C-less. The presence of carnitine or acylcarnitines during the incubation of the proteoliposomes with omeprazole increased the inhibition. Using site-directed Cys mutants it was demonstrated that C283 and C136 were essential for covalent inhibition. Molecular docking of omeprazole with CACT indicated the formation of both covalent interactions with C136 and C283 and non-covalent interactions in agreement with the experimental data.     

Neuroprotective activities of bacopa,lycopene, astaxanthin, and vitamin B12 combination on oxidative stress-dependent neuronal death

Oxidative stress is considered the common effector of the cascade of degenerative events in many neurological conditions. Thus, in this paper we tested different nutraceuticals in H₂O₂ in vitro model to understand if could represent an adjuvant treatment for neurological diseases. In this study, nutraceuticals bacopa, lycopene, astaxanthin, and vitamin B12 were used alone or in combination in human neuronal differentiated SH-SY5Y cells upon hydrogen peroxide-induced injury and neuroprotective, neuronal death pathways were analyzed. The nutraceuticals analyzed were able to protect H₂O₂ cytotoxic effects, through increasing cell viability and proteins involved in neuroprotection pathways and restoring proteins involved in cell death pathways. On this basis, it is possible to propose the use of these compounds as dietary supplement for the prevention or as adjuvant to the only symptomatic treatments so far available for neurodegenerative diseases.     

Localization of Mitochondrial Carnitine/Acylcarnitine Translocase in Sensory Neurons from Rat Dorsal Root Ganglia

The carnitine/acylcarnitine transporter is a transport system whose function is essential for the mitochondrial β-oxidation of fatty acids. Here, the presence of carnitine/acylcarnitine carrier (CACT) in nervous tissue and its sub-cellular localization in dorsal root ganglia (DRG) neurons have been investigated. Western blot analysis using a polyclonal anti-CACT antibody produced in our laboratory revealed the presence of CACT in all the nervous tissue extracts analyzed. Confocal microscopy experiments performed on fixed and permeabilized DRG neurons co-stained with the anti-CACT antibody and the mitochondrial marker MitoTracker Red clearly showed a mitochondrial localization for the carnitine/acylcarnitine transporter. The transport activity of CACT from DRG extracts reconstituted into liposomes was about 50 % in respect to liver extracts. The experimental data here reported represent the first direct evidence of the expression of the carnitine/acylcarnitine transporter in sensory neurons, thus supporting the existence of the β-oxidation pathway in these cells.     

Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53‐dependent pathway

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53‐wild type U2OS cells (and not of p53‐null Saos and p53‐mutant MG63 cells) by slowing‐down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin‐induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub‐G1 population, Bcl‐2 downregulation, caspase‐3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination‐induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine‐alpha. Moreover, the doxorubicin‐induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53‐dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy. J. Cell. Physiol. 228: 198–206, 2013. © 2012 Wiley Periodicals, Inc.