Brucella abortus MFP: a trimeric coiled-coil protein with membrane fusogenic activity

The bacterial genus Brucella consists of a group of facultative intracellular pathogens which produces abortion and infertility in animals and a chronic debilitating febrile illness in humans. BMFP is a basic protein of Brucella abortus that belongs to a highly conserved group of homologue proteins of unknown structure and function in proteobacteria (COG2960). In this study, we report the structural and biochemical characterization of this protein. We found that BMFP has two structural domains: a carboxyl-terminal coiled-coil domain through which the protein self-associates as a trimer and a natively disordered amino-terminal domain which has propensity to adopt an amphipathic alpha-helical structure. This natively unfolded domain undergoes a structural rearrangement from unfolded to alpha-helix in the presence of high ionic strength, acidic pH, detergents, and phospholipid vesicles. Moreover, we demonstrated that the interaction of BMFP with phospholipid vesicles promotes in vitro membrane fusion. These results contribute to the elucidation of the structural and functional properties of this protein and its homologues present in most proteobacteria.     

A new role for the p85-phosphatidylinositol 3-kinase regulatory subunit linking FRAP to p70 S6 kinase activation.

The serine/threonine kinase p70 S6 kinase (p70S6K) phosphorylates the 40 S ribosomal protein S6, modulating the translation of an mRNA subset that encodes ribosomal proteins and translation elongation factors. p70S6K is activated in response to mitogenic stimuli and is required for progression through the G(1) phase of the cell cycle and for cell growth. Activation of p70S6K is regulated by phosphorylation of seven different residues distributed throughout the protein, a subset of which depends on the activity of p85/p110 phosphatidylinositol 3-kinase (PI3K); in fact, the phosphorylation status of Thr(229) and Thr(389) is intimately linked to PI3K activity. In the full-length enzyme, however, these sites are also acutely sensitive to the action of FKBP 12-rapamycin-associated protein (FRAP). The mechanism by which PI3K and FRAP cooperate to induce p70S6K activation remains unclear. Here we show that the p85 regulatory subunit of PI3K also controls p70S6K activation by mediating formation of a ternary complex with p70S6K and FRAP. The p85 C-terminal SH2 domain is responsible for p85 coupling to p70S6K and FRAP, because deletion of the C-terminal SH2 domain inhibits complex formation and impairs p70S6K activation by PI3K. Formation of this complex is not required for activation of a FRAP-independent form of p70S6K, however, underscoring the role of p85 in regulating FRAP-dependent p70S6K activation. These studies thus show that, in addition to the contribution of PI3K activity, the p85 regulatory subunit plays a critical role in p70S6K activation.     

Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3.

Heterochromatin at yeast telomeres and silent mating (HM) loci represses adjacent genes and is formed by the binding and spreading of silencing information regulators (SIR proteins) along histones. This involves the interaction between the C terminus of SIR3 and the N terminus of histone H4. Since H4 is hypoacetylated in heterochromatin we wished to determine whether acetylation is involved in regulating the contacts between SIR3 and H4. Binding of H4 peptide (residues 1-34) acetylated at lysines Lys-5, Lys-8, Lys-12, and Lys-16 to an immobilized SIR3 protein fragment (residues 510-970) was investigated using surface plasmon resonance. We find that acetylation of H4 lysines reduces binding (K(a)) of H4 to SIR3 in a cumulative manner so that the fully acetylated peptide binding is decreased approximately 50-fold relative to unacetylated peptide. Thus, by affecting SIR3-H4 binding, acetylation may regulate the formation of heterochromatin. These data help explain the hypoacetylated state of histone H4 in heterochromatin of eukaryotes.     

Two clusters of residues at the docking groove of mitogen-activated protein kinases differentially mediate their functional interaction with the tyrosine phosphatases PTP-SL and STEP.

Regulated function of mitogen-activated protein (MAP) kinases involves their selective association through docking sites with both activating MAP kinase kinases and inactivating phosphatases, including dual specificity and protein-tyrosine phosphatases (PTP). Site-directed mutagenesis on the mammalian MAP kinases ERK2 and p38alpha identified within their C-terminal docking grooves two clusters of residues important for association with their regulatory PTPs, PTP-SL and STEP. ERK2 and p38alpha mutations that resembled the sevenmaker gain-of-function mutation in the Rolled D. melanogaster ERK2 homologue failed to associate with PTP-SL, were not retained in the cytosol, and were poorly inactivated by this PTP. Additional ERK2 mutations at the docking groove showed deficient association and dephosphorylation by PTP-SL, although their cytosolic retention was unaffected. Other ERK2 mutations, resembling gain-of-function mutations in the FUS3 yeast ERK2 homologue, associated to PTP-SL and were inactivated normally by this PTP. Our results demonstrate that mutations at distinct regions of the docking groove of ERK2 and p38alpha differentially affect their association and regulation by the PTP-SL and STEP PTPs.     

Immunohistochemical Distribution of Cytochrome P4501A in Larvae and Fingerlings of the Siberian Sturgeon, Acipenser baeri

In this paper we investigate by means of immunohistochemistry, the tissue distribution of constitutive cytochrome P4501A (CYP1A), from hatching until 30 days posthatching in developing Siberian sturgeon, Acipenser baeri. For this purpose, a polyclonal (BN-1) antiserum developed against a conservative sequence of piscine CYP1A and a monoclonal (C10-7) antiserum directed against cod CYP1A were used on paraffin-embedded samples. From hatching onwards, distinct CYP1A immunoreactivity was distinctly observed in the following tissues and cells: envelope of oil droplets, matrix and syncytium of the yolk-sac, sinusoids, biliary epithelial cells and hepatocytes. In the digestive tract, buccopharyngeal, oesophageal, gastric and intestinal epithelia, as well as the cytoplasm and brush border of enterocytes were CYP1A-positive. Interestingly, gastric glands and melanin-plug present within lumen of the digestive system were strongly immunoreactive. Kidney (epithelia of renal tubules), gills (pillar and endothelial cells), skin (epithelial cells), muscle fibres of heart and eye (retina) were positive. In brain, we observed a strong CYP1A staining in the developing telencephalon and especially in olfactory system, as well as in those nerve fibres running ventrally toward the posterior brain. A strong CYP1A staining was observed in vascular endothelia of all organs/tissues, especially in the liver. In general, the intensity of CYP1A immunostaining increased during larval development, suggesting besides its known metabolic function (endogenous and/or exogenous), a possible participation of this heme-protein in control of cell division, regulation of growth and differentiation.     

Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K_m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca²⁺ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.     

On the possibility for generic modeling of submarine groundwater discharge

We simulate large-scale dynamics of submarine groundwater discharge (SGD) in three different coastal aquifers on the Mediterranean Sea. We subject these aquifers to a wide range of different groundwater management conditions, leading to widely different net groundwater drainage from land to sea. The resulting SGD at steady-state is quantifiable and predictable by simple linearity in the net land-determined groundwater drainage, defined as total fresh water drainage minus groundwater extraction in the coastal aquifer system. This linearity appears to be general and independent of site-specific, variable and complex details of hydrogeology, aquifer hydraulics, streamlines and salinity transition zones in different coastal systems. Also independently of site-specifics, low SGD implies high seawater content due to seawater intruding into the aquifer and mixing with fresh groundwater within a wide salinity transition zone in the aquifer. Increasing SGD implies decreasing seawater content, decreased mixing between seawater and fresh groundwater and narrowing of the salinity transition zone of brackish groundwater in the aquifer.     

Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements.     

Alternative pathway respiration is associated with ammonium ion sensitivity in spinach and pea plants

Spinach and pea plants were grown in hydroponic culture with nitrate orammonium salts as the nitrogen source. Dry matter accumulation andphotosynthetic rate declined in spinach plants fed with ammonium salts, whereasthey did not change in pea plants compared with nitrate-fed plants. Measurementof organic nitrogen and free amino acid content showed that ammonium ions wereassimilated in shoots in spinach plants and in roots in pea plants. Ammoniumionnutrition led to a decline in starch content in both species. Organic acidsincreased in roots of pea plants fed with ammonium ions whereas they declinedinspinach plants. In both species ammonium ions increased root respiration ratebut the contribution of both routes (cytochromic and alternative pathway) tothis increase was different depending on the species. In spinach plants,ammonium ions increased the cytochromic path and decreased the alternativepathway, whereas in pea plants both routes were stimulated mainly through thealternative pathway. The differences in the sensitivity to ammonium ionsbetweenboth species are discussed in terms of differences in the availability of Cskeletons and energy, which could be due in part to differences in the capacityto stimulate the alternative pathway.