Vibrio Trends in the Ecology of the Venice Lagoon

Vibrio is a very diverse genus that is responsible for different human and animal diseases. The accurate identification of Vibrio at the species level is important to assess the risks related to public health and diseases caused by aquatic organisms. The ecology of Vibrio spp., together with their genetic background, represents an important key for species discrimination and evolution. Thus, analyses of population structure and ecology association are necessary for reliable characterization of bacteria and to investigate whether bacterial species are going through adaptation processes. In this study, a population of Vibrionaceae was isolated from shellfish of the Venice lagoon and analyzed in depth to study its structure and distribution in the environment. A multilocus sequence analysis (MLSA) was developed on the basis of four housekeeping genes. Both molecular and biochemical approaches were used for species characterization, and the results were compared to assess the consistency of the two methods. In addition, strain ecology and the association between genetic information and environment were investigated through statistical models. The phylogenetic and population analyses achieved good species clustering, while biochemical identification was demonstrated to be imprecise. In addition, this study provided a fine-scale overview of the distribution of Vibrio spp. in the Venice lagoon, and the results highlighted a preferential association of the species toward specific ecological variables. These findings support the use of MLSA for taxonomic studies and demonstrate the need to consider environmental information to obtain broader and more accurate bacterial characterization.     

Purine salvage as metabolite and energy saving mechanism in Camelus dromedarius: the recovery of guanine

The preservation of purine ring as purine bases appears to be a common feature of camel liver. Hepatic guanine appears to be actively converted into GMP in the camel rather than further degraded. The limiting step of guanine degradation appears to be the lack of hepatic guanase activity. Higher purine bases over uric acid ratios were found in camel urine with respect to those of zebu.     

The light-harvesting antenna of brown algae: highly homologous proteins encoded by a multigene family.

Accessory light-harvesting complexes (LHCFs) were isolated from the brown alga Laminaria saccharina. Complexes specifically associated with photosystem I or II are identical with each other with respect to molecular mass, isoelectric point and behavior on anion-exchange chromatography or non-denaturing isoelectric focusing. The purified complexes also have similar pigment composition and spectroscopic properties. It is concluded that LHC antennae associated with photosystem I or II cannot be distinguished biochemically. After screening of genomic and cDNA libraries produced from L. saccharina sporophytes, six lhcf genes were isolated. Sequence analysis of these lhcf genes showed a high level of homology between the encoded polypeptides. Comparisons with coding sequences of lhcf genes from Macrocystis pyrifera and expressed sequence tags from Laminaria digitata (two other Laminariales) indicated that these proteins are probably very similar in all brown algae. Another feature common to the lhcf genes characterized was the presence of an intron in the coding region corresponding to the plastid-targeting presequence. The sequence similarity extended to the 5' and 3' UTRs of several genes. In spite of the common origin of the chloroplasts, no light-regulating elements involved in the expression of the genes encoding the higher-plant light-harvesting proteins has been found in the UTRs.     

Vacuolar apical compartment (VAC) in breast carcinoma cell lines (MCF-7 and T47D): failure of the cell-cell regulated exocytosis mechanism of apical membrane

Abstract. We have previously shown that an integral plasma membrane glycoprotein (AP2) is highly polarized to the apical domain in confluent Madin-Darby canine kidney (MDCK) epithelial cells. However, when the monolayers are prevented from forming intercellular contacts, approximately 60% of the AP2 cellular content is stored in the intracellular vacuolar apical compartment (VAC). In the current work we found that AP2 was present in the non-tumorigenic human mammary epithelial cell line MCF-10A. in the breast carcinoma cell lines MCF-7 and T47D, and in breast ductal carcinomas in vivo. By radioimmunoassay, an intracellular Compartment of AP2 was identified in the mammary cell lines in culture. In MCF-10A, this compartment behaved as in MDCK cells; namely it was observed only when the cells cannot form cell-cell contacts. However, in the carcinoma cell lines MCF-7 and T47D, a significant AP2 intracellular compartment was observed also under conditions permissive for the formation of intercellular contacts. These results were confirmed by immunofluorescence and immunoelectron microscopy experiments that showed VACs in MCF-7 and T47D, even in cells with extensive intercellular contacts. In MCF-7 cells, the addition of serum caused a partial decrease of the AP2 intracellular compartment. The exocytosis of VACs occurred towards the center of multi-cellular groups, forming intercellular lumens, similar to those transiently observed in MDCK cells and to structures described by others during embryo development. Altogether, these results suggest that VAC exocytosis is controlled by cell-cell contact signalling, which may be defective in carcinoma cells.     

Proteolytically Inactive Insulin-Degrading Enzyme Inhibits Amyloid Formation Yielding Non-Neurotoxic Aβ Peptide Aggregates

Insulin-degrading enzyme (IDE) is a neutral Zn²⁺ peptidase that degrades short peptides based on substrate conformation, size and charge. Some of these substrates, including amyloid β (Aβ) are capable of self-assembling into cytotoxic oligomers. Based on IDE recognition mechanism and our previous report of the formation of a stable complex between IDE and intact Aβ in vitro and in vivo, we analyzed the possibility of a chaperone-like function of IDE. A proteolytically inactive recombinant IDE with Glu111 replaced by Gln (IDEQ) was used. IDEQ blocked the amyloidogenic pathway of Aβ yielding non-fibrillar structures as assessed by electron microscopy. Measurements of the kinetics of Aβ aggregation by light scattering showed that 1) IDEQ effect was promoted by ATP independent of its hydrolysis, 2) end products of Aβ-IDEQ co-incubation were incapable of “seeding” the assembly of monomeric Aβ and 3) IDEQ was ineffective in reversing Aβ aggregation. Moreover, Aβ aggregates formed in the presence of IDEQ were non-neurotoxic. IDEQ had no conformational effects upon insulin (a non-amyloidogenic protein under physiological conditions) and did not disturb insulin receptor activation in cultured cells. Our results suggest that IDE has a chaperone-like activity upon amyloid-forming peptides. It remains to be explored whether other highly conserved metallopeptidases have a dual protease-chaperone function to prevent the formation of toxic peptide oligomers from bacteria to mammals.     

Thermodynamic Modeling of Internal Equilibria Involved in the Activation of Trypsinogen

Abstract

The effect of activating dipeptides, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine β-trypsin (β-trypsin), on equilibria involved in the binding of strong ligands (i.e., n-butylamine, the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI) and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen (trypsinogen) was investigated at pH 5.5 (I = 0.1 M) and T = 21.0 ± 0.5°C; under the same experimental conditions, thermodynamics for the binding of strong ligands to β-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to β-trypsin and to its zymogen are described according to an induced-fit formalism, taking into account ligand-linked interaction(s) between different functional and structural domains of the (pro)enzyme possibly involved in the trypsinogen-to-β-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17/V-terminus of β-trypsin or activating dipeptides bind) and the primary and/or secondary recognition subsite(s) (where strong ligands associate) present in the (pro)enzyme. Such an analysis allows to dissect the contributions due to the primary recognition subsite, where small mono-functional ligands (e.g., n-butylamine) bind, from those of the secondary subsite(s), which are additional recognition clefts for macromolecular inhibitors (e.g., BPTI and PSTI).     

In vitro effect of morphine on oxidative phosphorylation in mitochondria on non-neural cells

In vitro assays carried on rat liver mitochondria show that morphine is a strong inhibitor of oxidative phosphorylations. Thus the neurotropic drug has also a more general effect on non-nervous cells, effect masked till now by the more impressive effects on the nervous system.     

Interaction between cystatin-derived peptides and papain

The interaction between papain and synthetic peptides which tentatively mimic cystatin surfaces was investigated both enzymatically and structurally. Measurements of dissociation equilibrium constants for the interaction of papain with these peptides modified by successive deletions or substitutions demonstrated that the QVVAG segment, which is highly conserved throughout members of the cystatin superfamily, is essential for the interaction. The glycylcontaining (N-terminal) fragments and PW-containing (C-terminal) fragments were found to be of lesser importance, since each could be deleted without significantly modifying the interaction. These fragments improved the stability of the interacting QVVAG region, which appeared to be substrate-like in all peptides tested, as it was cleaved at the A-G bond upon peptide-papain interaction. Replacement of the A residue at the scissile bond of the QVVAG by a blocked cysteinyl residue reduced the rate of cleavage of the susceptible bond and therefore shifted the resulting peptide from a substrate to an inhibitor. Derivatization of this substituted peptide at its N- and C-terminal ends by fluoresceinyl groups resulted in a dramatic decrease in theK _i to 0.5 µM. This improvement in the inhibitory properties of the substituted and derivatized peptides was correlated with structural changes as analyzed by molecular dynamic calculations. The results were compared to those proposed for the mechanism of inhibition by natural inhibitors of the cystatin superfamily.