Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat

The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at < or =pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K(m) values for ABTS and SGZ at 37 degrees C were of 106 +/- 11 and 26 +/- 2 microm, respectively, with corresponding k(cat) values of 16.8 +/- 0.8 and 3.7 +/- 0.1 s(-1). Maximal enzyme activity was observed at 75 degrees C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 degrees C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable.     

Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.     

Modeling temporal and spatial colony-site dynamics in a long-lived seabird

We studied the determinants of colony site dynamics in Audouin's gull, Larus audouinii, breeding in a small archipelago of the western Mediterranean. Data on island occupation were available for a series of 25 years, since first colonization of the archipelago in 1973. Group behavior was studied in relation to the components of dispersal: permanence or abandonment (extinction) on an island previously occupied and permanence or occupation (colonization) of another island. Generalized Linear Mixed Models (GLMMs) were used to identify the relative contribution of each explanatory variable to the probability of colony abandonment. Gulls showed a low probability (3%) of abandoning one of the islands (Grossa I.), especially when the colony was increasing in numbers from time t_i-1 to t_i. However, the probability of abandoning Grossa increased up to 31% when the colony was declining. The probability of island abandonment was very high for all other islands (range 66–99%) when the colony was declining, but much lower (range 36–82%) when it was increasing. Hence, we suggest that island abandonment by Audouin's gull is at least a two-step process. The first step (dispersal of a portion of the colony) probably takes place at random, as an evolutionary load typical of a species evolved in unstable habitats. The second step, a further loss of breeding pairs, seems to feedback on the first loss of members of the colony (public information), likely perceived as a loss of colony quality. Colonization of islands by gulls abandoning Grossa I. was marginally and negatively affected by the density of breeding yellow-legged gulls, a predatory species. Results apply to conservation ecology since they highlight the need to protect not only occupied patches but also those empty at present.     

Ancistrolikokine D,a 5,8'-coupled naphthylisoquinoline alkaloid,and related natural products from Ancistrocladus likoko

A new naphthylisoquinoline alkaloid, ancistrolikokine D, and the likewise 5,8'-coupled alkaloid ancistroealaine A, as well as two further, biosynthetically related, but nitrogen-free natural products, ancistronaphthoic acid B and cis-isoshinanolone, have been isolated from Ancistrocladus likoko J. LEACUTE;ONARD (Ancistrocladaceae). The 5,8'-coupling of the new alkaloids and of the alkaloids isolated earlier hints at a close phylogenetic relationship of A. likoko to other Central African Ancistrocladus species. The compounds show moderate activities against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei rhodesiense.     

Genetic diversity of isolates of Glomus mosseae from different geographic areas detected by vegetative compatibility testing and biochemical and molecular analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34(+) cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).     

Regulation of insulin receptor signaling by the protein tyrosine phosphatase TCPTP

The human protein tyrosine phosphatase TCPTP exists as two forms: an endoplasmic reticulum-targeted 48-kDa form (TC48) and a nuclear 45-kDa form (TC45). Although targeted to the nucleus, TC45 can exit in response to specific stimuli to dephosphorylate cytoplasmic substrates. In this study, we investigated the downregulation of insulin receptor (IR) signaling by TCPTP. In response to insulin stimulation, the TC48-D182A and TC45-D182A "substrate-trapping" mutants formed stable complexes with the endogenous tyrosine-phosphorylated IR beta-subunit in 293 cells. Moreover, in response to insulin stimulation, the TC45-D182A mutant accumulated in the cytoplasm of cells overexpressing the IR and in part colocalized with the IR beta-subunit at the cell periphery. These results indicate that the IR may serve as a cellular substrate for both TC48 and TC45. In immortalized TCPTP(-/-) murine embryo fibroblasts, insulin-induced IR beta-subunit tyrosine phosphorylation and protein kinase PKB/Akt activation were enhanced relative to the values in TCPTP(+/+) cells. Importantly, the expression of TC45 or TC48 to physiological levels suppressed the enhanced insulin-induced signaling in TCPTP(-/-) cells. These results indicate that the differentially localized variants of TCPTP may dephosphorylate the IR and downregulate insulin-induced signaling in vivo.     

Evaluation in rabbits of different anti-SHIV vaccine strategies based on DNA/fowlpox priming and virus-like particle boosting

Two different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of defining a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic simian/human immunodeficiency virus, SHIV(89.6P). The two regimens were based on three priming immunizations with either an expression plasmid plus a fowlpox (FP) recombinant vector or with two FP recombinant vectors, each one expressing either the SIV(mac239) gag/pol or the HIV-1env(89.6P) genes. In both protocols, priming immunizations were followed by two boosts with SHIV-mimicking virus-like particles (VLP). A complete SHIV-specific response was observed in all animals. Interestingly, the DNA vaccine was three to 10 times more efficient than the FP recombinant in inducing an anti-gag humoral response. Real-time PCR confirmed the memory effect on T-cell subsets secreting interleukin-4 and interferon-gamma, as a consequence of stimulation of both arms of the immune system. Although both protocols were almost equally effective in eliciting homologous neutralizing antibodies and highlighted the efficacy of VLP administration for boosting, protocol A seemed to be more effective in promoting a balanced T-cell memory immune response and appears more promising for vaccine purposes.     

Type of mycorrhizal associations in two coastal nature reserves of the Mediterranean basin

We analysed the mycorrhizal types of two coastal Mediterranean plant communities. Plants belonging to 82 species of 46 families growing in two Mediterranean study sites located within a Regional Natural Park in Italy (Macchia Lucchese) and in a National Park in Croatia (Brijuni) were assessed for the type of their mycorrhizal associations. Mycorrhizas occurred in 83% and 90% of the plant species surveyed in Macchia Lucchese and in Brijuni, respectively. On the basis of macroscopic and microscopic characteristics, six different mycorrhizal types were found, and their co-occurrence in the same plant community showed the large mycorrhizal diversity in Mediterranean vegetation. Different occurrence patterns of mycorrhizal types have been recognised in diverse habitats within Macchia Lucchese, from sand dune plant communities to sclerophyllous woodland. Mycorrhizal type abundance calculated on a floristic basis was very different from that obtained using a vegetation cover index.     

4-Aminopyrazolo[3,4-d]pyrimidine (4-APP) as a novel inhibitor of the RNA and DNA depurination induced by Shiga toxin 1

Shiga toxin 1 (Stx1) catalyses the removal of a unique and specific adenine from 28S RNA in ribosomes (RNA-N-glycosidase activity) and the release of multiple adenines from DNA (DNA glycosylase activity). Added adenine behaves as an uncompetitive inhibitor of the RNA-N-glycosidase reaction binding more tightly to the Stx1–ribosome complex than to the free enzyme. Several purine derivatives and analogues have now been assayed as inhibitors of Stx1. Most of the compounds showed only minor differences in the rank order of activity on the two enzymatic reactions catalysed by Stx1. The survey highlights the importance of the amino group in the 6-position of the pyrimidine ring of adenine. Shifting (2-aminopurine) or substituting (hypoxanthine, 6-mercaptopurine, 6-methylpurine) the group greatly decreases the inhibitory power. The presence of a second ring, besides the pyrimidine one, is strictly required. Substitution, by introducing an additional nitrogen, of the imidazole ring of adenine with triazole leads to loss of inhibitory power, while rearrangement of the nitrogen atoms of the ring from the imidazole to the pyrazole configuration greatly enhances the inhibitory power. Thus 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), the isomer of adenine with the five-membered ring in the pyrazole configuration, is by far the most potent inhibitor of both enzymatic reactions catalysed by Stx1. This finding opens perspectives on therapeutic strategies to protect endothelial renal cells once endocytosis of Stx1 has occurred (haemolytic uraemic syndrome). In the RNA-N-glycosidase reaction 4-APP binds, as adenine, predominantly to the Stx1–ribosome complex (uncompetitive inhibition), while inhibition of the DNA glycosylase activity by both inhibitors is of the mixed type.