Cloning of the <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. FG1 dehalogenase genes and construction of hybrid pathways in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I > F, in the order.     

The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in 'trans' interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites.     

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.     

Speciation of phytate ion in aqueous solution. Protonation constants and copper(II) interactions in NaNO3aq at different ionic strengths

The acid base behavior of phytate has been studied (at t=25 degrees C by potentiometry, ISE-H+ glass electrode) in NaNO3aq at different ionic strengths (0.1 < or = I/mol L(-1) < or = 1.0). The interactions with copper(II) were investigated in the same experimental conditions in different metal to ligand (Phy) ratios (1:1 < or = Cu2+ :Phy < or = 4:1), by using both ISE-H+ and ISE-Cu2+ electrodes. Phytate acid base behavior in sodium nitrate is very similar to that in sodium chloride, previously investigated. In the experimental conditions adopted, the formation of three CuiHjPhy(12-2i-j)- species is observed: the mononuclear CuH4Phy6- and CuH5Phy5-, and the dinuclear Cu2H5Phy3-. Analysis of complex formation constants at different ionic strengths reveals that both ISE-H+ and ISE-Cu2+ electrodes gave, within the experimental error, analogous values. Dependence of complex formation constants on ionic strength was modeled by EDH (Extended Debye-Hückel) and SIT (Specific ion Interaction Theory) equations. The sequestering ability of phytate toward copper(II) has been evaluated by the calculation of pL50 (the total ligand concentration, as -log CL, able to bind 50% of metal cation), an empirical parameter already proposed for an objective "quantification" of this ability. A thorough analysis of literature data on phytate-copper(II) complexes has been performed.     

Identification of a novel potential antigen for early-phase serodiagnosis of leptospirosis

This study examined four genes encoding for predicted membrane proteins selected from the genome sequences of Leptospira interrogans. Genes were cloned and the proteins expressed in E. coli. Immunoblotting analysis of the recombinants with sera from early and convalescent phases of a leptospirosis patient showed that two proteins, namely Lp29 and Lp49, were reactive with serum from both phases of the illness. These data were further confirmed in enzyme-linked immunosorbent assay using sera from both phases of seventeen confirmed leptospirosis specimens, suggesting that these proteins are presented to the host immune system during infection. In the early phase, anti-Lp29 IgM was detected in all sera when microscopic agglutination tests (MAT), the reference method for diagnosis of leptospirosis, were negative. The gene encoding Lp49 is conserved among five tested leptospiral pathogenic serovars, while Lp29 is present in serovars that are predominant in urban settings. These recombinant antigens might be valuable for serodiagnosis of both phases of leptospirosis.     

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.     

Middle Telychian (upper Llandovery, Silurian) graptolites from boreholes of northwestern Libya: Their biostratigraphic significance and palaeogeographical implication

A low-diversity but stratigraphically and palaeobiogeographically important graptoloid association comprising Metaclimacograptus flamandi (Legrand), Parapetalolithus meridionalis (Legrand), and Torquigraptus australis Štorch was identified in the core samples of wells A1-66, F1-66 and A1-43 in the Ghadamis Basin of Libya. The former two species have been originally recovered from Algeria. All three taxa are common and widespread in the middle Telychian strata of Spain (Central Iberian Zone and western Iberian Cordillera) and France (Brittany). In European sections, the species are associated with other, moderately diverse graptoloid fauna, which enables a more precise and worldwide biostratigraphic correlation of Saharan boreholes and surface sections. The present faunal association indicates a middle Telychian age (upper crispus, griestoniensis and lowermost crenulata/tullbergi biozones) for the strata and suggests palaeobiogeographical links between North African pericratonic basins and Ibero-Armorican shelves. M. flamandi has not been found outside this NW-Gondwanan realm.     

Isolation, heterologous expression and characterization of an endo-polygalacturonase produced by the phytopathogen Burkholderia cepacia

Endo-polygalacturonases (endoPGs) belong to the glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond present in the smooth regions of pectins. Pectic substances are among the principal macromolecular components of the primary plant cell walls and are subjected to enzymatic degradation not only in the course of important physiological processes such as plant senescence and ripening, but also during infection events by plant pathogens. Here we report, for the first time, the isolation and the purification of an endoPG (PehA) from the supernatant of the plant pathogen Burkholderia cepacia strain ATCC 25416. In order to obtain adequate amounts of protein required for structural and functional studies, the gene coding for pehA was PCR-amplified and cloned in Escherichia coli cells. The recombinant protein was purified to homogeneity and characterized. PehA exhibited a pI value of 8.0 and an optimal activity at pH 3.5. Far-UV circular dichroism (CD) measurements show that PehA assumes a beta-helix fold super-secondary structural motif.     

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.