Kinetics of Torpedo californica acetylcholinesterase inhibition by bisnorcymserine and crystal structure of the complex with its leaving group

Natural and synthetic carbamates act as pseudo-irreversible inhibitors of AChE (acetylcholinesterase) as well as BChE (butyrylcholinesterase), two enzymes involved in neuronal function as well as in the development and progression of AD (Alzheimer's disease). The AChE mode of action is characterized by a rapid carbamoylation of the active-site Ser(200) with release of a leaving group followed by a slow regeneration of enzyme action due to subsequent decarbamoylation. The experimental AD therapeutic bisnorcymserine, a synthetic carbamate, shows an interesting activity and selectivity for BChE, and its clinical development is currently being pursued. We undertook detailed kinetic studies on the activity of the carbamate bisnorcymserine with Tc (Torpedo californica) AChE and, on the basis of the results, crystallized the complex between TcAChE and bisnorcymserine. The X-ray crystal structure showed only the leaving group, bisnoreseroline, trapped at the bottom of the aromatic enzyme gorge. Specifically, bisnoreseroline interacts in a non-covalent way with Ser(200) and His(440), disrupting the existing interactions within the catalytic triad, and it stacks with Trp(84) at the bottom of the gorge, giving rise to an unprecedented hydrogen-bonding contact. These interactions point to a dominant reversible inhibition mechanism attributable to the leaving group, bisnoreseroline, as revealed by kinetic analysis.     

Did the Mediterranean marine reflooding precede the Mio–Pliocene boundary? Paleontological and geochemical evidence from upper Messinian sequences of Tuscany,Italy

The events related to the Messinian salinity crisis are among the most deeply investigated of Earth's history. According to the current hypothesis of Neogene paleogeographic evolution, approximately 5.5 Ma ago, after evaporitic sedimentation and before the Mio–Pliocene boundary, the Mediterranean was characterized by the widespread development of non-marine environments inhabited by molluscs and ostracods of brackish affinity. The Messinian post-evaporitic deposits that testify such a dramatic environmental change are commonly referred to as ‘Lago-mare’ and have been reported from several outcrops and boreholes throughout the entire Mediterranean basin. The origin of ‘Lago-mare’ conditions is commonly interpreted as the result of the synergistic effect of both the humid climatic conditions and change of the drainage patterns at the Mediterranean scale, with the capture of Paratethyan brackish waters. A few recent studies, however, suggest that such a scenario probably represents an oversimplification of the original context, from both a paleogeographical and paleoenvironmental point of view. Unfortunately, the results of these studies have never been commonly accepted and the proposed evidences have been considered questionable. In this paper we describe the fish assemblages from the ‘Lago-mare’ deposits of two localities, Cava Serredi and Podere Torricella, located in the Neogene hinterland basins of Tuscany, central Italy. These assemblages consists of a mixture of marine euryhaline and stenohaline taxa suggesting that the depositional environments were characterized by permanently open connections with a marine basin filled with normal marine waters. In order to better define the paleontological significance of these upper Messinian fish assemblages, the oxygen, carbon and strontium isotopic composition of fish otoliths and other fossils has been measured. These isotopic compositions are strongly indicative of the presence of normal marine conditions close to the depositional environments testified by the sedimentary sequences, thereby implying that the interpretation of the geochemical results are consistent with those derived from the paleoecological analyses of the fish assemblages. Based on the integrated paleoichthyological–geochemical approach discussed in this paper it is possible to unambiguously demonstrate that normal marine rather than fresh- or brackish waters were present in the Mediterranean at least during the upper part of the ‘Lago-mare’ event, providing an unquestionable evidence that the marine refilling of the basin preceded the Mio–Pliocene boundary.     

Interferon-gamma secretion and perforin expression are impaired in CD8+ T lymphocytes from patients with undifferentiated carcinoma of nasopharyngeal type

An efficent antitumor and antiviral cellular immune response requires optimal interferon-gamma (IFN-gamma) secretion and perforin expression in CD8(+) T cells. The aim of this study was to define whether CD4(+) and CD8(+) T cells from patients with undifferentiated carcinoma of nasopharyngeal type (UCNT), a tumor regularly associated with the Epstein-Barr virus (EBV), have abnormal phenotype profiles, cytokine production, perforin and CD3-zeta expressions. Our data showed that CD4 and CD8 subset distribution was not grossly altered in the peripheral blood of UCNT patients, while tumor biopsies contained an increased proportion of CD8(+) T cells. The analysis of the CD4(+) subset showed a defect in interleukin-2 (IL-2) production and a moderate increase of IL-10 production, a situation consistent with a Th1/Th2 imbalance. We have also demonstrated that CD8(+) lymphocytes from UCNT patients had a marked impairment of IFN-gamma secretion and perforin expression. This impairment was not related to the presence of detectable EBV DNA in the plasma. In UCNT patients, the blockade of the perforin pathway and of IFN-gamma production may constitute important mechanisms for immune escape by the tumor and for impaired control of EBV replication.     

The Kiwifruit Emerging Pathogen Pseudomonas syringae pv. actinidiae Does Not Produce AHLs but Possesses Three LuxR Solos

Pseudomonas syringae pv. actinidiae (Psa) is an emerging phytopathogen causing bacterial canker disease in kiwifruit plants worldwide. Quorum sensing (QS) gene regulation plays important roles in many different bacterial plant pathogens. In this study we analyzed the presence and possible role of N-acyl homoserine lactone (AHL) quorum sensing in Psa. It was established that Psa does not produce AHLs and that a typical complete LuxI/R QS system is absent in Psa strains. Psa however possesses three putative luxR solos designated here as PsaR1, PsaR2 and PsaR3. PsaR2 belongs to the sub-family of LuxR solos present in many plant associated bacteria (PAB) that binds and responds to yet unknown plant signal molecules. PsaR1 and PsaR3 are highly similar to LuxRs which bind AHLs and are part of the canonical LuxI/R AHL QS systems. Mutation in all the three luxR solos of Psa showed reduction of in planta survival and also showed additive effect if more than one solo was inactivated in double mutants. Gene promoter analysis revealed that the three solos are not auto-regulated and investigated their possible role in several bacterial phenotypes.     

The involvement of superoxide anions in the autoxidation of various cofactors of cysteamine-oxygenase

The reduction of tetranitroblue tetrazolium with cysteamine, mediated by a number of dyes, elemental sulphur, elemental selenium and selenide, under aerobic conditions, was inhibited to various extent upon addition of superoxide dismutase. A strict parallelism between the ability to produce O2- ions and the property of those compounds to act as cofactors for cysteamine-oxygenase, to yield hypotaurine, has been observed. Based on the fact that the autoxidation of cysteamine also gives rise to O2- formation, though to a minor extent, we propose a mechanism for cysteamine-oxygenase action. This mechanism was derived from the data obtained in the model system studied.     

Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.     

Optimizing scale-up fermentation processes

There are many aims associated with the optimization of fermentation processes. Optimization is expected to increase the yield of the final product but the process must be compliant with good manufacturing practices, the available equipment and the expected final scale of operation. Dealing with genetically modified microorganisms that overproduce recombinant protein has the advantage that the vast majority of the processes use only three different species, namely Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. Standard processes for each organism are described in textbooks and serve as a basis for the development of a tailored process. This article outlines the general philosophy that we have devised to ensure an efficient approach of scaling up fermentation processes for biopharmaceutical purposes, in a multidisciplinary environment.