Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases.
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Bacteria↗
Biomedical subjects
R Blasco
Publications and source records attributed to R Blasco.
Canada goose (Branta canadensis) droppings as a potential source of pathogenic bacteria.
Canada goose droppings, collected in parks to which the public had access, were screened for a range of bacteria that could be pathogenic in man. Droppings of Canada geese, and other waterfowl, did contain such bacteria, including some that are well-known causes of illness in man. These bacteria, plus a species of Salmonella that was experimentally inoculated into droppings, were shown to survive and multiply in the droppings for up to one month after their deposition by geese. Canada geese ranged further from water than other waterfowl species and thus distributed their droppings over a larger area of park grassland. This more widespread distribution of their droppings leads Canada geese to pose a greater potential health risk than other waterfowl studied here, but variations in human responses to challenge with bacteria, and variations in human and waterfowl behaviour in public parks, renders quantification of this risk impossible.
Animals↗
Functional analysis of vaccinia virus B5R protein: role of the cytoplasmic tail.
Vaccinia extracellular enveloped virus (EEV) is important for cell-to-cell and long-range virus spread both in vitro and in vivo. Six genes have been identified that encode protein constituents of the EEV outer membrane, and some of these proteins are critical for EEV formation. The B5R gene encodes an EEV-specific type I membrane protein, and deletion of this gene markedly decreases EEV formation and results in a small plaque phenotype. Data suggest that the transmembrane domain, cytoplasmic tail, or both contain the EEV localization signals that are required for targeting of the B5R protein to EEV and for EEV formation. Here, we report the construction of mutant vaccinia viruses in which the wild-type B5R gene was replaced with a mutated one that encodes a protein with the putative cytoplasmic tail deleted. The mutated protein showed normal intracellular distribution and was properly incorporated into EEV. Vaccinia viruses expressing the B5R protein lacking the cytoplasmic tail formed plaques that were similar in type and size to those formed by wild-type viruses and produced equivalent amounts of infectious EEV. These results indicate that the B5R cytoplasmic tail is not necessary for EEV formation and points to the transmembrane domain as the major determinant for targeting the B5R protein to the outer membrane of EEV and for supporting EEV formation.
Animals↗
Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry.
We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser65 to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.
Animals↗
Recombinant swinepox virus expressing beta-galactosidase: investigation of viral host range and gene expression levels in cell culture.
Swinepox virus (SPV) has been proposed as a potential vector for generating recombinant vaccines for swine. However, little is known about important aspects of SPV biology, such as the functionality of SPV promoters or the host range of SPV. Using a transient expression assay, well-characterized vaccinia virus promoters were shown to be active in cells infected with SPV. A recombinant SPV expressing beta-galactosidase (beta-gal) was constructed and characterized. The E. coli LacZ gene was placed under the control of a strong vaccinia synthetic early/late promoter and was inserted by homologous recombination in a noncoding region of the SPV genome. The recombinant SPV expressing beta-gal was used to characterize the host range of the virus by measuring protein expression and virus production in different cell lines. In general, SPV expressed more protein and grew more efficiently than vaccinia virus in porcine cell lines. Surprisingly, the recombinant SPV was able to infect and replicate in several cell lines of nonswine origin. The virus directed regulated early and late gene expression of beta-gal in those cells and formed blue plaques in cell monolayers in the presence of X-gal. Upon infection with the recombinant SPV, there was a significant level of viral replication, and the virus can be serially passaged in some nonswine cell lines. The data presented suggest that despite the strict host tropism of SPV, the virus exhibits a relatively broad host range in cell culture.
Animals↗
Microbial photodegradation of aminoarenes. Metabolism of 2-amino-4-nitrophenol by Rhodobacter capsulatus.
The phototrophic bacterium Rhodobacter capsulatus photoreduces 2,4-dinitrophenol to 2-amino-4-nitrophenol, which is further metabolized by an aerobic pathway that is also light-dependent. The catabolism of 2-amino-4-nitrophenol requires O2 and the presence of alternative carbon (C) and nitrogen (N) sources, preferably acetate and ammonium. Rhodobacter capsulatus B10, a bacterium unable to assimilate nitrate, releases negligible amounts of nitrite when growing with 2-amino-4-nitrophenol, thus suggesting that an oxygenase, nitrite-producing activity is not involved in the metabolization of the compound. The diazotrophic growth of R. capsulatus increases in the presence of 2-amino-4-nitrophenol, but growth with ammonium is clearly inhibited by the compound. Mutant strains of R. capsulatus B10, which are affected in nifHDK, nifR1, or nifR4 genes, unable to fix dinitrogen, do not grow with 2-amino-4-nitrophenol as the sole N source. This indicates that the compound cannot be used as a N source. The nif mutants degrade 2-amino-4-nitrophenol to the same extent as the wild-type in the presence of ammonium. The compound is not used as a C source by the bacterium, either. Aromatic stable intermediates, such as 2,4-diaminophenol or 4-nitrocatechol, are not detectable in microaerobic cultures of R. capsulatus growing with 2,4-dinitrophenol or 2-amino-4-nitrophenol.
Aerobiosis↗
Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus.
The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O2 pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O2 pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1, 2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO2-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of 14CO2 from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled 14C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source.
Aerobiosis↗
Analysis of the B and T cell response in guinea pigs induced with recombinant vaccinia expressing foot-and-mouth disease virus structural proteins.
Recombinant vaccinia viruses expressing foot-and-mouth disease virus (FMDV) P1 and VP1 genes have been used to study the immune response induced by these viral polypeptides in guinea pigs. Anti-FMDV antibodies, but not neutralizing activity, were detected in the sera from immunized animals. The results indicate that both CD4+ and CD8+ FMDV-specific T cells were induced by the vaccinia recombinants. Consistently with the activation of CD4+ T cells, lymphocytes from immunized animals specifically proliferated in vitro in response to whole virus. The induction of virus-specific CD8+ T cells was determined by CTL assay of immune splenocytes restimulated in vitro with FMDV infected cells. Altogether, the results obtained indicate that both B and T cell immune responses to FMDV are elicited upon immunization of guinea pigs with vaccinia recombinants expressing FMDV structural polypeptides.
Animals↗
Specific assays for bacteria using phage mediated release of adenylate kinase.
A sensitive and rapid assay method for the specific detection of bacteria was developed using Escherichia coli and Salmonella newport as the test organisms. Bacteriophages were used to provide specific lysis of the bacteria and then the release of cell contents was measured by ATP bioluminescence. Increased sensitivity was obtained by focusing on the bacteria's adenylate kinase (AK) as the cell marker instead of ATP as conventionally used. Fewer than 10(3) E. coli cells could be readily detected in less than 1 h. Salmonella newport assays, although as sensitive, were slower and took up to 2 h. The effects of the culture medium, the phage, and the presence of non-specific bacteria were examined.
Adenylate Kinase↗
Detoxification of protoanemonin by dienelactone hydrolase.
Protoanemonin is a toxic metabolite which may be formed during the degradation of some chloroaromatic compounds, such as polychlorinated biphenyls, by natural microbial consortia. We show here that protoanemonin can be transformed by dienelactone hydrolase of Pseudomonas sp. strain B13 to cis-acetylacrylate. Although similar Km values were observed for cis-dienelactone and protoanemonin, the turnover rate of protoanemonin was only 1% that of cis-dienelactone. This indicates that at least this percentage of the enzyme is in the active state, even in the absence of activation. The trans-dienelactone hydrolase of Pseudomonas sp. strain RW10 did not detectably transform protoanemonin. Obviously, Pseudomonas sp. strain B13 possesses at least two mechanisms to avoid protoanemonin toxicity, namely a highly active chloromuconate cycloisomerase, which routes most of the 3-chloro-cis,cis-muconate to the cis-dienelactone, thereby largely preventing protoanemonin formation, and dienelactone hydrolase, which detoxifies any small amount of protoanemonin that might nevertheless be formed.
Biodegradation, Environmental↗
Functional analysis of vaccinia virus B5R protein: essential role in virus envelopment is independent of a large portion of the extracellular domain.
Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.
Actins↗
PCR-based method for the introduction of mutations in genes cloned and expressed in vaccinia virus.
Vaccinia virus expression systems allow efficient expression of genes and facilitate functional studies of expressed proteins in cultured mammalian cells. We designed and tested a rapid method to introduce defined mutations in genes inserted and expressed in vaccinia virus. PCR mutagenesis is used to construct a recombination cassette that contains: (i) the mutated exogenous gene, (ii) recombination flanks to direct insertion into the virus genome and (iii) a selectable gene to allow easy isolation of recombinant viruses. To generate recombinant viruses, the recombination cassette is transfected into vaccinia virus-infected cells. The procedure does not require cloning, and the mutated gene versions are inserted directly into the vaccinia virus genome downstream of a vaccinia virus strong promoter. The method was tested by introducing a point mutation into Aequorea victoria green fluorescent protein (GFP), known to alter the fluorescence absorption and emission spectra of the protein. This system should facilitate and speed the isolation of virus recombinants expressing mutated versions of any given gene and can be adapted to random mutagenesis procedures, exon shuffling or other PCR-based mutagenesis protocols.
Cell Line↗
[Sjögren syndrome and subacute demyelinating polyradiculopathy: an unusual association].
INTRODUCTION: Sjögren's syndrome is a chronic inflammatory condition of unknown aetiology and autoimmune pathology. The defining feature is the dry syndrome, expressed as xerophthalmia and xerostomia. Extra-glandular involvement at many other levels may also occur. Neurological involvement is not unusual. The peripheral nervous system is most frequently involved, and a predominantly sensitive symmetrical distal polyneuropathy may be the first sign of the condition. Other patterns of peripheral involvement are also associated with the syndrome. We present a case of subacute demyelinating polyradiculopathy associated with primary Sjögren's syndrome. CLINICAL CASE: A 28 year old woman with dry syndrome presented with paraesthesia in her hands and feet, distal weakness, which had progressed proximally in the muscles of her arms and legs, and bilateral facial weakness. The condition progressed for eight weeks. When complementary tests were done, alterations typical of this condition (FR, ANA, anti-Ro and anti-La) were seen and also others typical of the dry syndrome (Schirmer's test). Therefore, in view of these findings and the clinical features, after other conditions had been ruled out, a diagnosis of primary Sjögren's syndrome was made. The type of neuropathy was determined by the clinical features, electromyography and CSF findings. Treatment with corticosteroids gave good results. CONCLUSIONS: Demyelinating polyradiculopathy is a form of peripheral nervous system involvement which is rarely seen in this disorder. In the differential diagnosis Sjögren's syndrome should be considered, an orientative history taken, autoantibodies determined and an ophthalmological examination made.
Acute Disease↗
The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein.
The assimilatory nitrate reductase from the phototrophic bacterium Rhodobacter capsulatus has been purified to electrophoretic homogeneity and its molecular and kinetic parameters determined. The native nitrate reductase is a dimer of 144 kDa composed of two subunits of 46 and 95 kDa. The purified enzyme catalyzes the electron transfer from NADH, reduced bromophenol blue or reduced viologens to nitrate. The nitrate reductase contains 1 mol FAD per mole of enzyme and also reduces cytochrome c or dichlorophenol indophenol with NADH as the electron donor. The diaphorase activity is located in the small subunit.
2,6-Dichloroindophenol↗
Evidence that Formation of Protoanemonin from Metabolites of 4-Chlorobiphenyl Degradation Negatively Affects the Survival of 4-Chlorobiphenyl-Cometabolizing Microorganisms.
A rapid decline in cell viability of different PCB-metabolizing organisms was observed in soil microcosms amended with 4-chlorobiphenyl. The toxic effect could not be attributed to 4-chlorobiphenyl but was due to a compound formed from the transformation of 4-chlorobiphenyl by the natural microflora. Potential metabolites of 4-chlorobiphenyl, 4-chlorobenzoate and 4-chlorocatechol, caused similar toxic effects. We tested the hypothesis that the toxic effects are due to the formation of protoanemonin, a plant-derived antibiotic, which is toxic to microorganisms and which has been shown to be formed from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway. Consistent with our hypothesis, addition to soil microcosms of strains able to reroute intermediary 4-chlorocatechol from the 3-oxoadipate pathway and into the meta-cleavage pathway or able to mineralize 4-chlorocatechol by a modified ortho-cleavage pathway resulted in reversal of this toxic effect. Surprisingly, while direct addition of protoanemonin influenced both the viability of fungi and the microbial activity of the soil microcosm, there was little effect on bacterial viability due to its rapid degradation. This rapid degradation accounts for our inability to detect this compound in soils amended with 4-chlorocatechol. However, significant accumulation of protoanemonin was observed by a mixed bacterial community enriched with benzoate or a mixture of benzoate and 4-methylbenzoate, providing the metabolic potential of the soil to form protoanemonin. The effects of soil heterogeneity and microcosm interactions are discussed in relation to the different effects of protoanemonin when applied as a shock load and when it is produced in small amounts from precursors over long periods.
Journal Article↗
[Primary intraventricular hemorrhage: an analysis of eight cases].
OBJECTIVE: Primary intraventricular haemorrhage (HIVP) is a rare condition, of diverse aetiology and variable course. We present a series of eight cases of HIVP diagnosed by CT and analyze the different aetiologies. MATERIALS AND METHODS: A retrospective series of 8 cases diagnosed by CT, the risk factors, clinical behaviour, complications and prognosis are evaluated. Neuroimaging findings (CT, MR and/or DIVAS) are described. RESULTS: The most commonly associated factor is arterial hypertension (75%). Clinically, presentation was uniform as evidence of sudden intracranial hypertension with signs of meningeal irritation with little or no neurological focal signs. The commonest complication was hydrocephalus (25%) and the chances of survival in most cases were good. Three causes of bleeding were found: an arteriovenous malformation, an aneurysm and a blood coagulation disorder. Three hypertensive patients with no obvious cause of bleeding had periependimary laguna infarcts. CONCLUSIONS: HIVP is an unusual condition of diverse aetiology, uniform clinical presentation and has a good short term prognosis. We suggest that pathology of the small vessels, induced by hypertension, may play a part in the aetiology of a subgroup of patients with no obvious cause of bleeding.
Adult↗
[Partial simple vegetative crisis: importance of electroencephalographic findings].
INTRODUCTION: 50% of the patients with cerebral tumours present with epileptic crises, which may be partial or generalized. The commonest partial crises have motor symptoms. These make up 30% of the simple partial crises. Partial simple crises with purely vegetative-type symptoms are very uncommon (less than 5%). They are considered to be caused by discharges in the internal regions of the temporal lobes, mainly in the limbic system. This means that it is very difficult to identify them using techniques of surface EEG. CLINICAL CASE: We describe the case of a patient with a cerebral tumour. The initial clinical features were short episodes of generalized coldness and sweating which had been present for the previous two weeks, without any other symptoms. During a routine EEG, focal critical paroxystic activity was recorded in the right temporal region. This coincided with a clinical episode similar to those described. CONCLUSIONS: The episodes were labelled partial simple vegetative crises. In this case the EEG was crucial for diagnosis and subsequently to recommend suitable treatment. However, difficulty in recording this type of crisis with a surface EEG makes correct diagnosis of these patients very difficult, since the epileptogenic focus is deeply situated.
Anticonvulsants↗
A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia virus.
We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air-water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia virus, the formation of the spherical immature virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped virus, and confirm that this form possesses an extra membrane overlying the IMV.
Chemical Phenomena↗