Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440

Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.     

Detection of serine proteases in extracts of the domestic mite Blomia tropicalis

Previous studies have shown that the domestic mites Dermatophagoides pteronyssinus and D. farinae contain allergens with serine protease activity. These proteolytic allergens include trypsin, chymotrypsin, elastase, kallikrein, and C3/C5 convertase. However, it is not known whether the domestic mite Blomia tropicalis shares with other mite species the serine protease activities. The enzymatic activity present in extracts obtained from food-free B. tropicalis was investigated using specific substrates and inhibitors. Based upon the concentration response and inhibition profiles, and the digestion of specific substrates our data demonstrate that extracts from B. tropicalis exhibit several serine-protease-like activities. The enzyme activities detected in the B. tropicalis extracts are trypsin, elastase, chymotrypsin, kallikrein, C3/C5 convertase, and mast cell protease. Our results also demonstrate that kallikrein and C3/C5 convertase-like activities were not significantly affected by the α₁-antiprotease, a naturally occurring serine protease inhibitor which protects lung mucosa from the enzymatic action. These data strongly suggest that the Echymyopodidae mite B. tropicalis shares at least five serine proteases with members of other mite families, the Glycyphagidae and Pyroglyphidae. In addition, our data demonstrate the potential use of biochemical methods to detect serine proteases for evaluation of mite growth in vitro, or to detect environmental exposures to these enzymes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Binding of haemin by the fish pathogen Photobacterium damselae subsp. piscicida

Whole cells of virulent (DI 21 and B 51) and avirulent (ATCC 29690 and EPOY 8803-II) strains of Photobacterium damselae subsp. piscicida, grown under iron-supplemented or iron-restricted conditions, were able to bind haemin. Iron limitation resulted in an increased binding of haemin by DI 21, B 51 and ATCC 29690 cells but did not affect the haemin-binding ability of the EPOY 8803-II cells. Proteinase K treatment of whole cells markedly reduced the binding of haemin, indicating that protein receptors located at the cell surface are involved in the binding. This was confirmed by the observation that isolated total as well as outer membrane proteins from all the strains, regardless of the iron levels of the media, were able to bind haemin, with the outer membranes showing the strongest binding. Haemin binding by membrane protein extracts was not affected by heat treatment but was almost completely abolished by Proteinase K treatment, suggesting the presence of thermostable protein receptors for haemin. The capsular polysaccharide also appears to play a minor role in binding of haemin. It was concluded that constitutive as well as inducible mechanisms of haemin binding occur in P. damselae subsp. piscicida. These mechanisms would rely mainly upon the direct interaction between the haemin molecules and surface-exposed outer membrane protein receptors.     

Laminin affects polymerization,depolymerization and neurotoxicity of Abeta peptide

Amyloid deposition in Alzheimer fibrils forms neurotoxic senile plaques in a process that may be modulated by associated proteins. In this work we demonstrate the ability of laminin-1 and laminin-2 to inhibit fibril formation and toxicity on cultured rat hippocampal neurons. We confirm that the laminin-1-derived peptide YFQRYLI inhibits efficiently both fibril formation and neurotoxicity and show that the IKVAV peptide inhibits amyloid neurotoxicity despite its slight inhibition of fibril formation. On other hand, laminin-1 induces disaggregation of preformed fibrils in vitro, characterized as a progressive disassembly of fibrils into protofibrils and further clearance of these latter species, leading to a continual inhibition of amyloid neurotoxicity.     

Bovine floor plate explants secrete SCO-spondin

SCO-spondin is a large-molecular mass glycoprotein, secreted by the subcommissural organ (SCO), which has been implicated in neuronal development during ontogeny of the central nervous system. The expression of SCO-spondin is not restricted to the SCO but it also occurs in the floor plate, a key structure participating in neuronal differentiation and patterning of the neural tube. It has been postulated that SCO-spondin detected in the floor plate is released into the lumen of the neural tube, but this new route of secretion of floor plate cells needs to be further substantiated. For this purpose, we standardized long-term organ culture of bovine floor plate and performed morphological, immunological, biochemical, and gene expression analyses. The study of floor plate explants and their conditioned media allowed us to demonstrate that: (1) organ-cultured floor plate cells are actively secretory for up to 25 days; (2) SCO-spondin gene is actively transcribed and translated by the cultured floor plate cells; (3) SCO-spondin is released into the culture medium via the apical cell pole; and (4) upon release, SCO-spondin does not aggregate in the conditioned medium but remains soluble. Furthermore, in the cultured floor plate cells, SCO-spondin may be secreted through a route bypassing the Golgi apparatus.     

Calbindin and calretinin immunoreactivities in the retina of a chondrostean,Acipenser baeri

The distribution of calbindin and calretinin in the retina of the sturgeon Acipenser baeri was studied with immunocytochemistry. Western blot analysis of brain extracts, together with immunocytochemical results in the retina and brain, indicated the presence of the two calcium-binding proteins in sturgeon. Calbindin immunocytochemistry revealed only a large displaced bipolar cell type with narrowly stratified axons, similar to some mixed rod and cones bipolar cells described in teleosts. The plexus formed by the axons of these cells in the inner plexiform sublayer was similar to that formed by calbindin-immunoreactive diffuse bipolar cells of some mammals. Calretinin immunocytochemistry also stained these displaced bipolar cells, most ganglion cells including displaced ganglion cells (Dogiel cells), and some amacrine cells of the inner nuclear layer. The distribution of calbindin and calretinin immunoreactivities in the retina of a primitive bony fish indicates that these proteins are highly specific to the cell type.     

Response to oxidative stress caused by H(2)O(2) in Saccharomyces cerevisiae mutants deficient in trehalase genes

The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing. The most severe phenotype corresponded to the Delta nth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2). Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.     

Novel bacteriochlorophyll <Emphasis Type="BoldItalic">e</Emphasis> structures and species-specific variability of pigment composition in green sulfur bacteria

The relative composition of bacteriochlorophyll (BChl) homologs in five different strains of brown-colored green sulfur bacteria was investigated by HPLC-MS/MS and NMR analyses. In addition, the effect of incubation light intensities on homolog distribution was studied in one of the strains (strain Dagow III). A total of 23 different BChl e structures were detected and comprise four homologous porphyrin ring systems and eight different esterifying alcohols. Several BChl e structures are novel. These include a C-8 ethyl, C-12 methyl [E, M] BChl e(F) homolog which was identified by (1)H-NMR analyses of the isolated, main farnesyl homologs (BChl e(F)). In addition, five previously unknown homolog series with dodecanol, pentadecenol, tetradecanol, hexadecenol and phytol as the esterifying alcohols were detected. The composition of BChl e homologs from the five strains of green sulfur bacteria differed with respect to the relative abundance of the homologs (BChl e(F) : 25.6-67.0% of total BChl e content in stationary cultures). In strain Dagow III, the abundance of BChl e(F) homologs decreased upon entry into the stationary phase. In all free-living strains, the abundance of BChl e(F) was increased when the relative carotenoid content was low. The present results provide a detailed picture of pigment composition in chlorosomes and thus will help to elucidate their structure and function. Furthermore, the newly discovered BChl e molecules are valuable biomarkers for the study of the occurrence and metabolism of green sulfur bacteria in past and present ecosystems.