Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal β-glycosidases

Sβgly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of Sβgly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure–function relationships in these enzymes. Received: May 20, 1999 / Accepted: January 10, 2000     

Cross-talk between hematopoiesis and angiogenesis signaling pathways

The relationship between hematopoietic cells and endothelial cells has been seen as an indication that a common progenitor, the hemangioblast, gives rise to both cell types in the yolk sac, the initial site of hematopoiesis and blood vessel formation during mammalian development. The existence of angioblast-like circulating endothelial precursor cells in adults humans has recently been suggested. In this review, we have summarized the principle mechanisms involved in the cross-talk signaling pathway between hematopoiesis and angiogenesis in order to further understand how the hematopoietic and vascular systems are established during the development.     

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.     

The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.     

Water quality and phytoplankton community structure in mangrove streams under different logging regimes in Cameroon

Mangrove logging is rampant, and its effects on phytoplankton dynamics have not been investigated. Phytoplankton are valuable in biomonitoring pollution in mangrove ecosystems. This study assessed the relationship between physico‐chemical characteristics of the water and observed phytoplankton community structure, in relation to anthropogenic activities. Water samples were collected from seven sites within the mangrove ecosystem that differed in wood harvesting age and proximity to human settlements. Characterization of samples followed standard methods. pH of all water samples indicated acidity. Phytoplankton species richness and soluble P were positively correlated (r = 0.822, P ≤ 0.023), but no correlations existed with logging age (r = ?0.273, P = 0553). The site with highest soluble phosphorus concentration (Site 4) had highest phytoplankton abundance, diversity and richness. A total of 127 species were identified in 58 families. Euglenaceae was the most abundant family (13 species) and Anacystis sp. the most frequent species (100%). The most common genera were Microcystis and Anacystis, bioindicators of pollution. The site at which harvesting was most recently performed had more eutrophic species, while site harvested earliest (Site 5) had the least. These results are discussed with respect to how anthropogenic activities influence water quality, phytoplankton community structure, mangrove conservation and management.