Biomedical subjects

R Blasco

Publications and source records attributed to R Blasco.

At least 19 recordsLinked to original sources

PubMed2006

Interactions between nitrate assimilation and 2,4-dinitrophenol cometabolism in Rhodobacter capsulatus E1F1.

The phototrophic, nitrate-photoassimilating bacterium Rhodobacter capsulatus E1F1 cometabolizes 2,4-dinitrophenol (DNP) by photoreducing it to 2-amino-4-nitrophenol under anaerobic conditions. DNP uptake and nitrate metabolism share some biochemical features, and in this article we show that both processes are influenced by each other. Thus, as was demonstrated for nitrate assimilation, DNP uptake requires a thermolabile periplasmic component. Nitrate assimilation is inhibited by DNP, which probably affects the nitrite reduction step because neither nitrate reductase activity nor the transport of nitrate or nitrite is inhibited. On the other hand, DNP uptake is competitively inhibited by nitrate, probably at the transport level, because the nitroreductase activity is not inhibited in vitro by nitrate, nitrite, or ammonium. In addition, the decrease in the intracellular DNP concentration in the presence of nitrate probably inactivates the nitroreductase. These results allow prediction of a negative environmental effect if nitrate and DNP are released together to natural habitats, because it may lead to a lower rate of DNP metabolism and to nitrite accumulation.

2,4-Dinitrophenol↗

PubMed2006

Cyanide metabolism of Pseudomonas pseudoalcaligenes CECT5344: role of siderophores.

Cyanide is one of the most potent and toxic chemicals produced by industry. The jewelry industry of Córdoba (Spain) generates a wastewater (residue) that contains free cyanide, as well as large amounts of cyano-metal complexes. Cyanide is highly toxic to living systems because it forms very stable complexes with transition metals that are essential for protein function. In spite of its extreme toxicity, some organisms have acquired mechanisms to avoid cyanide poisoning. The biological assimilation of cyanide needs the concurrence of three separate processes: (i) a cyanide-insensitive respiratory chain, (ii) a system for iron acquisition (siderophores) and (iii) a cyanide assimilation pathway. Siderophores are low-molecular-mass compounds (600-1500 Da) that scavenge iron (Fe(3+)) ions (usually with extremely high affinity) from the environment under iron-limiting conditions. There are two main classes of siderophores: catechol and hydroxamate types. The catechol-type siderophores chelate ferric ion via a hydroxy group, whereas the hydroxamate-type siderophores bind iron via a carbonyl group with the adjacent nitrogen. In the presence of cyanide, bacterial proliferation requires this specific metal uptake system because siderophores are able to break down cyano-metal complexes. Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide or cyano-metal complexes as nitrogen source. A proteomic approach was used for the isolation and identification, in this strain, of a protein that was induced in the presence of cyanide, namely CN0, that is involved in siderophore biosynthesis in response to cyanide. An overview of bacterial cyanide degradation pathways and the involvement of siderophores in this process are presented.

Bacterial Proteins↗

PubMed2005

Alkaline cyanide biodegradation by Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 uses cyanide, cyanate, beta-cyanoalanine, and other cyanoderivatives as nitrogen sources under alkaline conditions, which prevents volatile HCN (pK(a) 9.2) formation. The cyanide consumed by this strain is stoichiometrically converted into ammonium. In addition, this bacterium grows with the heavy metal, cyanide-containing waste water generated by the jewellery industry, and is also a cyanide-resistant strain which induces an alternative oxidase and a siderophore-based mechanism for iron acquisition in the presence of cyanide. The detection of cyanase and beta-cyanoalanine nitrilase activities in cyanide-induced cells suggests their implication in the cyanide degradation pathway.

Biodegradation, Environmental↗

PubMed2003

Flow-injection spectrophotometric determination of cyanate in bioremediation processes by use of immobilised inducible cyanase.

A new flow injection (FI) method for photometric monitoring of cyanate in bioremediation processes using immobilised native cyanase is described. The method is based on the catalytic reaction between cyanate and bicarbonate to produce ammonia and carbon dioxide in the presence of an inducible native cyanase, immobilised in a reactor packed with glass beads. Two degrees of purification of the biocatalyst were used-heated cell-free extract and purified extract of cyanase from Pseudomonas pseudoalcaligenes CECT 5344. The ammonia produced by the enzymatic reaction is finally monitored photometrically at 700 nm using a modification of the conventional Berthelot method. The method furnishes different calibration curves depending on the degree of purification of the cyanase, with linear ranges between 1.23 and 616.50 micromol L(-1) ( r(2)=0.9979, n=7) and between 1.07 and 308.25 micro mol L(-1) ( r(2)= 0.9992, n=7) for the heated cell-free extract and the purified cyanase extract, respectively. No statistically significant differences between the samples were found in the precision study evaluated at two cyanate concentration levels using one-way analysis of variance. A sampling frequency of 15 h(-1) was achieved. The method was used to monitor cyanate consumption in a cyanate bioremediation tank inoculated with Pseudomonas pseudoalcaligenes CECT 5344 strain. The correlation between cyanate degradation and ammonia production was tested using a conventional method. Finally, the method was applied to different samples collected from the bioremediation tank using the standard addition method; recoveries between 85.9 and 97.4% were obtained.

Biodegradation, Environmental↗

PubMed2003

[Animal sting and bites].

Under the heading of this subject we deal with stings by arthropods, making reference to the differences that exist between the stings of wasps and bees, commenting on the composition of the poisons and the different local and general reactions that are caused by such stings. Also discussed are the stings/bites caused by scorpions, spiders, ticks, and marine animals, with the clinical picture they provoke and the treatment that must be administered. Finally, snakebites are considered, with reference to the most frequent types of ophidia to be found in Navarra, how to differentiate between the bites of snakes and vipers, the different clinical pictures they provoke and the treatment to be applied

Humans↗

PubMed2001

Rhodococcus sp. RB1 grows in the presence of high nitrate and nitrite concentrations and assimilates nitrate in moderately saline environments.

Rhodococcus sp. RB1 was able to thrive in media with up to 0.9 M NaCl or KCl and in the presence of high concentrations of nitrate (up to 0.9 M) and nitrite (up to 60 mM), but only under oxic conditions. An adaptation period was not required for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl. Nitrate assimilation was limited by the carbon supply, but nitrite was not accumulated in the culture medium, even at nitrate concentrations as high as 0.8 M, thus suggesting that nitrite reduction does not limit nitrate assimilation. The presence of NaCl or KCl did not affect nitrate or nitrite uptake, which were completely inhibited by ammonium or glutamine. Rhodococcus sp. RB1 nitrate reductase had an apparent molecular mass of 142 kDa and used NADH and reduced bromophenol blue or viologens as electron donors, independently of the presence of salt. The enzyme was associated with an NADH-diaphorase activity and was induced by nitrate and repressed by ammonium or glutamine, thus showing typical biochemical and regulatory properties of bacterial assimilatory NADH-nitrate reductases. The enzyme was active in vitro in the presence of 3 M NaCl or KCI, but the maximal activity was observed at 0.5 M salt. Addition of 2 M NaCl increased the optimal temperature of the enzyme from 12 to 32 degrees C, but the optimal pH (10.3) was unaffected.

Cell Division↗

PubMed2001

Assimilation of D-malate by Rhodobacter capsulatus E1F1.

Rhodobacter capsulatus grew by using either L- or D-malate as carbon sources under light/anaerobic conditions. The cellular yields were the same with D- or L-malate. Both L-malate dehydrogenase and L-malic enzyme activities were detected in cell-free extracts from cells grown in both isomers. By contrast, a racemase activity converting D-malate into L-malate was induced only when D-malate was present in the culture medium. This racemase activity was Mn2+-dependent and was measured by coupling it either to the malate dehydrogenase or to the fumarase activities. The racemase activity was partially purified by anion-exchange chromatography.

Chromatography, Ion Exchange↗

PubMed2001

Bioremediation of an area contaminated by a fuel spill.

In order to decontaminate a large area of restricted access contaminated by a fuel spill, laboratory and field studies were developed in two steps: (a) monitoring of the laboratory experiment on bacterial growth under aerobic and anaerobic conditions with and without addition of nutrients; and (b) use of the best conditions obtained in (a) for the decontamination of the soil. A hydraulic barrier was installed both to clean the aquifer and to avoid migration of hydrocarbons as a consequence of their solution in the groundwater and subsequent displacement. The objective was to create an ideal environment for the treatment of the affected area that favoured the growth of the indigenous bacteria (Pseudomonas and Arthrobacter) that biodegrade the hydrocarbons. Monitoring of the changes in the total concentration of petroleum hydrocarbons in the soil subjected to bacterial action was performed by gas chromatography. In a field study, the progress of biodegradation of hydrocarbons was evaluated in situ by changes in subsurface CO2/O2 levels by means of an analyser equipped with an infrared detector. Biostimulation and oxygen were the most influential factors for the biodegradation of the hydrocarbons. The use of bioventing of the soil was shown as an excellent technology to promote in situ bioremediation of the polluted area.

Air Movements↗

PubMed2001

Role for draTG and rnf genes in reduction of 2,4-dinitrophenol by Rhodobacter capsulatus.

The phototrophic bacterium Rhodobacter capsulatus is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a draTG deletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants of R. capsulatus affected in ntrC or rpoN exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in rnfA or rnfC, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in R. capsulatus, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction.

2,4-Dinitrophenol↗

PubMed2001

Set of vectors for the expression of histidine-tagged proteins in vaccinia virus recombinants.

Vaccinia virus expression vectors are widely used to direct the expression of proteins in eukaryotic cells. Here, we describe a new set of plasmid vectors designed for the expression of histidine-tagged proteins in the vaccinia system. To facilitate the rapid isolation of virus recombinants, the plasmids contain a viral gene (F13L) that serves as an efficient selection marker based on virus plaque phenotype. Histidine codons and restriction sites derived from pET-16b bacterial expression plasmid were included, thus facilitating the transfer of genes between E. coli and vaccinia expression plasmids. Plasmids in which the gene is placed downstream of either a strong vaccinia virus or a T7 promoter were constructed, allowing for constitutive or conditional expression, respectively, of the foreign protein.

Amino Acid Sequence↗

PubMed2000

Puromycin resistance (pac) gene as a selectable marker in vaccinia virus.

The antibiotic puromycin, an inhibitor of protein synthesis, was shown to inhibit vaccinia virus (VV) replication. We evaluated the use of puromycin-resistance (pac) gene as a selectable marker in VV. A recombinant vaccinia virus expressing pac (VV-pac) under the control of a viral early/late promoter was constructed and characterized. VV-pac grew in the presence of puromycin at concentrations that were inhibitory for the parental VV and toxic for the cells. Isolation of recombinant VV usually relies on plaque purification under selective conditions. Because virus plaquing was not feasible under inhibitory puromycin concentration, a protocol based on serial passage of virus was devised. The usefulness of this procedure in selecting pac expressing viruses was tested by isolating a recombinant VV.

Animals↗

PubMed2000

Role of mast cells in the pathogenesis of postburn inflammatory response: reactive oxygen species as mast cell stimulators.

Thermal trauma has a direct effect on mast cells, triggering the secretion of histamine. This secretion leads to an enhanced xanthine oxidase activity and an increased production of reactive oxygen species (ROS), the latter being produced after burns through differing mechanisms. As ROS have been shown to have deleterious effects on cellular membranes, a lesion of the mast cell membrane could close the circle of autoinjury due to the vasoactive actions of mast cell mediators. Our studies were designed to assess the potentiality of ROS as stimulators of mast cell degranulation after burns by comparing two groups of rats treated, respectively, with SOD and saline solution after a scald injury. Plasma levels of tryptase and histamine were analyzed as markers of mast cell activity. A comparison of the mean increases of tryptase between baseline and 3-h postburn levels in the two groups shows significant differences (p < 0.001) (control: 0.13+/-0.04, SOD: 0.03+/-0.01). When comparing the mean increases between the baseline and 3 h postburn levels of histamine in the two groups, significant differences were also found (p < 0.001) (control group: 2.70+/-0.57. SOD group: 1.22+/-0.32). The lower levels of histamine and tryptase induced by SOD provides indirect evidence that ROS are involved in the process, causing the release of such mediators by mast cells, which may in turn suggest that ROS can act as stimulators of mast cell degranulation in burns.

Animals↗

PubMed2000

Sequence and analysis of a swinepox virus homologue of the vaccinia virus major envelope protein P37 (F13L).

P37 (F13L gene product), the most abundant protein in the envelope of the extracellular virus form of the prototype poxvirus, vaccinia virus (VV), is a crucial player in the process leading to acquisition of the envelope, virus egress and transmission. We have cloned and sequenced a swinepox virus (SPV) gene homologous to VV F13L. The SPV gene product, termed P42, was 54% identical to P37, the VV F13L gene product, and, among the poxviruses, was most similar (73% identity) to the myxoma virus homologue. The SPV P42 gene contained late transcription signals and was expressed only at late times during infection. The protein was palmitylated, and showed an intracellular distribution similar to that of VV P37, both by immunofluorescence and by subcellular fractionation. As with VV P37, SPV P42 was incorporated in extracellular enveloped SPV particles, but was absent from the intracellular mature virus form. To check the ability of SPV P42 to function in the context of VV infection, we inserted the SPV gene into a VV deficient in P37, which is severely blocked in virus envelopment and cell-to-cell transmission. Despite correct expression of SPV P42, the resulting recombinant VV showed no rescue of extracellular virus formation or cell-to-cell virus spread. The lack of function of SPV P42 in the VV genetic background suggests that specific interactions between SPV P42 or VV P37 and other viral proteins is required to drive the envelopment process.

Amino Acid Sequence↗

PubMed2000

Intracellular localization of vaccinia virus extracellular enveloped virus envelope proteins individually expressed using a Semliki Forest virus replicon.

The extracellular enveloped virus (EEV) form of vaccinia virus is bound by an envelope which is acquired by wrapping of intracellular virus particles with cytoplasmic vesicles containing trans-Golgi network markers. Six virus-encoded proteins have been reported as components of the EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a nonglycosylated transmembrane protein, and one (F13L) is a palmitylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. We have investigated the fates of these proteins after expressing them individually in the absence of vaccinia infection, using a Semliki Forest virus expression system. Significant amounts of proteins A33R and A56R efficiently reached the cell surface, suggesting that they do not contain retention signals for intracellular compartments. In contrast, proteins A34R and F13L were retained intracellularly but showed distributions different from that of the normal infection. Protein A36R was partially retained intracellularly, decorating both the Golgi complex and structures associated with actin fibers. A36R was also transported to the plasma membrane, where it accumulated at the tips of cell projections. Protein B5R was efficiently targeted to the Golgi region. A green fluorescent protein fusion with the last 42 C-terminal amino acids of B5R was sufficient to target the chimeric protein to the Golgi region. However, B5R-deficient vaccinia virus showed a normal localization pattern for other EEV envelope proteins. These results point to the transmembrane or cytosolic domain of B5R protein as one, but not the only, determinant of the retention of EEV proteins in the wrapping compartment.

Animals↗

PubMed2000

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus.

We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.

Animals↗

PubMed1999

Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans.

We have studied the concerted degradation of two monochlorodibenzofurans by a bacterial consortium, consisting of the chlorodibenzofurans-cometabolizing and chlorosalicylates-excreting strain Sphingomonas sp RW16, and Pseudomonas sp RW10, which mineralized the released chlorosalicylates. Neither of the organisms was able to grow with chlorodibenzofurans alone. Degradation of 2-chloro- and 3-chlorodibenzofuran proceeded to the end products 5-chloro- and 4-chlorosalicylate, respectively, when the initial dioxygenase of Sphingomonas sp RW 16 attacked the unchlorinated aromatic ring of the heterocyclic dibenzofuran molecule. 2-Hydroxypenta-2,4-dienoate, formed upon meta-cleavage of the intermediary chlorotrihydroxybiphenyls, served as a growth substrate for the sphingomonad. Presumably, most of the chlorosalicylates were excreted and degraded further by Pseudomonas sp RW10. Mineralization of both chlorosalicylates proceeded through a converging pathway, via 4-chlorocatechol, and protoanemonin. Chlorosalicylates were mineralized by the pseudomonad only when their concentration in the culture medium was below 1.5 mM. In the case of initial dioxygenation taking place on the chlorinated aromatic ring, salicylate and chlorinated hydroxypentadienoates should be formed. The metabolic fate of putative chlorohydroxypentadienoates is not clear; ie, they may be channeled into unproductive catabolism and, thus, represent the critical point in the breakdown of the carbon of these two chlorodibenzofurans by Sphingomonas sp RW16.

Journal Article↗

PubMed1999

Complementation of P37 (F13L gene) knock-out in vaccinia virus by a cell line expressing the gene constitutively.

Vaccinia virus produces two different infectious forms, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Acquisition of the EEV envelope occurs by wrapping of IMV with vesicles of the trans-Golgi network (TGN). The most abundant protein in the envelope of EEV, P37, is a 37 kDa palmitylated protein encoded by the F13L gene. P37 is located in the inner side of the EEV envelope and accumulates in the TGN during infection. Deletion of gene F13L results in a severe defect in the wrapping process, although normal levels of IMV are produced. A cell line, derived from RK-13 cells, was obtained that stably expressed P37 (RK(P37)), and the properties of the protein were studied in the absence of other viral polypeptides. P37 produced in RK(P37) cells differed from P37 produced in vaccinia-infected cells in terms of hydrophobicity and intracellular distribution. Despite these differences, RK(P37) cells partially complemented the phenotypic defect of vaccinia virus P37- mutants. EEV production and cell-to-cell virus spread by mutant viruses were increased significantly in RK(P37) cells when compared to normal RK-13 cell cultures. Infection of RK(P37) cells with P37- virus substantially altered the hydrophobicity and the intracellular distribution of P37 in those cells. These results indicate the requirement of the infection context for determination of the normal palmitylation and intracellular localization of P37.

Animals↗

PubMed1999

3-nitroadipate, a metabolic intermediate for mineralization of 2, 4-dinitrophenol by a new strain of a Rhodococcus species.

The bacterial strain RB1 has been isolated by enrichment cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and energy source and characterized, on the basis of 16S rRNA gene sequence comparison, as a Rhodococcus species closely related to Rhodococcus opacus. Rhodococcus sp. strain RB1 degrades 2,4-dinitrophenol, releasing the two nitro groups from the compound as nitrite. The release of nitro groups from 2,4-dinitrophenol occurs in two steps. First, the 2-nitro group is removed as nitrite, with the production of an aliphatic nitro compound identified by 1H nuclear magnetic resonance and mass spectrometry as 3-nitroadipate. Then, this metabolic derivative is further metabolized, releasing its nitro group as nitrite. Full nitrite assimilation upon reduction to ammonia requires that an additional carbon source be supplied to the medium.

2,4-Dinitrophenol↗