Isolation and characterization of <Emphasis Type="Italic">Clostridium difficile</Emphasis> from shellfish and marine environments

This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater, sediment, and zooplankton were collected at five sampling stations in the Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marine sediment. Majority of the C. difficile isolates were toxin A/B positive. Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.     

Rubiginoside, a farnesyl glycoside from Lepisanthes rubiginosa.

Chemical investigation of the methanolic fraction of Lepisanthes rubiginosa bark has led to the isolation and characterisation of a new tetrasaccharide derivative of farnesol named rubiginoside along with known triterpenoid saponins.     

LC-MS and GC-MS metabolite profiling of nickel(II) complexes in the latex of the nickel-hyperaccumulating tree Sebertia acuminata and identification of methylated aldaric acid as a new nickel(II) ligand

Targeted liquid chromatography-mass spectrometry (LC-MS) technology using size exclusion chromatography and metabolite profiling based on gas chromatography-mass spectrometry (GC-MS) were used to study the nickel-rich latex of the hyperaccumulating tree Sebertia acuminata. More than 120 compounds were detected, 57 of these were subsequently identified. A methylated aldaric acid (2,4,5-trihydroxy-3-methoxy-1,6-hexan-dioic acid) was identified for the first time in biological extracts and its structure was confirmed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. After citric acid, it appears to be one of the most abundant small organic molecules present in the latex studied. Nickel(II) complexes of stoichiometry NiII:acid=1:2 were detected for these two acids as well as for malic, itaconic, erythronic, galacturonic, tartaric, aconitic and saccharic acids. These results provide further evidence that organic acids may play an important role in the transport and possibly in the storage of metal ions in hyperaccumulating plants.     

Cytotoxic turrianes of Kermadecia elliptica from the New Caledonian rainforest

In the course of an automated screening for small molecules presenting cytotoxic activity, eight new cyclophanes named kermadecins A-H (1-8), have been isolated from the bark of a New Caledonian plant, Kermadecia elliptica, Proteaceae. A LC/APCI-MS study performed on kermadecins A, B and C, provided a reliable method for the detection of other analogues existing in small quantities in the extract. This led to the isolation of five other members of this chemical series. The structures were elucidated by extensive mono- and bi-dimensional spectroscopy and mass spectrometry. The cytotoxic activity of four of them was evaluated on various cell lines.     

Regulation and activity of cytosolic 5'-nucleotidase II. A bifunctional allosteric enzyme of the Haloacid Dehalogenase superfamily involved in cellular metabolism

In many vertebrate tissues, cytosolic 5'-nucleotidase II (cN-II) either hydrolyses or phosphorylates a number of purine (monophosphorylated) nucleosides through a scheme common to the Haloacid Dehalogenase superfamily members. It possesses a pivotal role in purine cellular metabolism and it acts on anti-tumoural and antiviral nucleoside analogues, thus being of potential therapeutic importance. cN-II is Mg2+-dependent, regulated and stabilised by several factors such as allosteric effectors ATP and 2,3-DPG, although these are not directly involved in the reaction stoichiometry. We review herein the experimental knowledge currently available about this remarkable enzymatic activity.     

Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: A review

Endogenous ribonucleotides and deoxyribonucleotides play a crucial role in cell function. The determination of their levels is of fundamental interest in numerous applications such as energy metabolism, biochemical processes, or in understanding the mechanism of nucleoside analog compounds. Nucleoside analogs are widely used in anticancer therapy. Their mechanisms of action are related to their structural similarity with natural nucleotides. Numerous assays have been described for the determination of endogenous nucleotides or anticancer nucleotide analogs in different matrices such as cellular cultures, tissue or peripheral blood mononuclear cells. The determination of these compounds is challenging due to the large difference of concentrations between ribonucleotides and deoxyribonucleotides, the presence of numerous endogenous interferences in complex matrices and the high polarity of the molecules due to the phosphate moiety. The extraction was generally performed at low temperature and was based on protein precipitation using acid or solvent mixture. This first phase could be coupled with extraction or cleaning step of the supernatant. Liquid chromatography coupled with UV detection and based on ion-exchange chromatography using non-volatile high salt concentrations was largely described for the quantification of nucleotides. However, the development of LC–MS and LC–MS/MS during the last ten years has constituted a sensitive and specific tool. In this case, analytical column was mostly constituted by graphite or C18 stationary phase. Mobile phase was usually based on a mixture of ammonium buffer and acetonitrile and in several assays included a volatile ion-pairing agent. Mass spectrometry detection was performed either with positive or negative electrospray mode according to compounds and mobile phase components. The purpose of the current review is to provide an overview of the most recent chromatographic assays (over the past ten years) developed for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy. We focused on sample preparation, chromatographic separation and quantitative considerations.     

Inhibition of IGF-1 signalling enhances the apoptotic effect of AS602868, an IKK2 inhibitor, in multiple myeloma cell lines

Multiple myeloma (MM) is a B cell neoplasm characterized by bone marrow infiltration with malignant plasma cells. IGF-1 signalling has been explored as a therapeutic target in this disease. We analyzed the effect of the IKK2 inhibitor AS602868, in combination with a monoclonal antibody targeting IGF-1 receptor (anti-IGF-1R) in human MM cell lines. We found that anti-IGF-1R potentiated the apoptotic effect of AS602868 in LP1 and RPMI8226 MM cell lines which express high levels of IGF-1R. Anti-IGF-1R enhanced the inhibitory effect of AS602868 on NF-κB pathway signalling and potentiated the disruption of mitochondrial membrane potential caused by AS602868. These results support the role of IGF-1 signalling in MM and suggest that inhibition of this pathway could sensitize MM cells to NF-κB inhibitors.