Biomedical subjects

Claudia Silva

Publications and source records attributed to Claudia Silva.

At least 19 recordsLinked to original sources

PubMed2007

Metalloproteinases are enriched in microglia compared with leukocytes and they regulate cytokine levels in activated microglia.

Microglia are resident immune cells within the central nervous system (CNS). They become activated following neurological insults and increase their expression of cytokines. Also elevated in CNS injuries are proteases, including matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs). The spectrum of metalloproteinase members expressed by microglia and by the systemic leukocytes that infiltrate the injured CNS is unknown, as are their functions. We determined the levels of transcripts encoding all 24 MMPs, nine ADAMs, and their four physiological antagonists, tissue inhibitor of metalloproteinases (TIMPs), in human microglia, B and T cells, monocytes, and neutrophils. We found a distinct pattern for each immune subset and an enrichment of metalloproteinases in microglia compared with leukocytes. When microglia were activated, there was an upregulation of transcripts for nine metalloproteinases, and reduction of TIMP3. Activation of microglia also resulted in increased levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 protein in the conditioned media of cells. The amount of secreted TNF-alpha, but not IL-1beta or IL-10, was suppressed by BB94, a broad spectrum metalloproteinase inhibitor, and by TIMP3 but not TIMP1 or TIMP2. This inhibitory profile suggests the involvement of an ADAM member in TNF-alpha secretion. We conclude that microglia bear a metalloproteinase signature distinct from systemic cells, and that following activation, microglia upregulate TNF-alpha protein levels through a combination of elevated cytokine transcripts, increased metalloproteinase level and activity, and through the decrease of TIMP3. The results have implications for the regulation of neuroinflammation and its outcomes following CNS injuries.

ADAM Proteins↗

PubMed2006

Proteolytic processing of SDF-1alpha reveals a change in receptor specificity mediating HIV-associated neurodegeneration.

Proteolytic cleavage of constitutively expressed proteins can generate peptides with novel bioactive properties. Matrix metalloproteinase (MMP)-2 cleaves the 4 amino-terminal residues of the chemokine, stromal cell-derived factor (SDF)-1alpha, yielding a highly neurotoxic molecule, SDF(5-67), which fails to bind to its cognate receptor, CXCR4. Herein, we detected SDF(5-67) in brain monocytoid cells of HIV-infected persons, particularly in those with HIV-associated dementia. SDF(5-67) activated cell type-specific expression of proinflammatory genes including IL-1beta, TNFalpha, indoleamine 2',3'-dioxygenase (IDO), and IL-10 in both astrocytic and monocytoid cells (P < 0.05). Unlike SDF-1alpha, SDF(5-67) caused neuronal membrane perturbations with ensuing neurotoxicity and apoptosis (P < 0.05) through engagement of an inducible receptor. CXCR3 antagonists and siRNA-mediated knockdown of CXCR3 inhibited SDF(5-67)-stimulated neurophysiological changes, neuronal death, and neuroimmune activation (P < 0.05). Moreover SDF(5-67) bound directly to CXCR3 in a competitive manner, mediated by its amino terminus. In vivo neuroinflammation, neuronal loss, and neurobehavioral abnormalities caused by SDF(5-67) (P < 0.05) were prevented by a CXCR3 antagonist. These studies reveal additive neuropathogenic properties exerted by a proteolytically cleaved chemokine as consequences of a change in receptor specificity, culminating in neurodegeneration.

Animals↗

PubMed2006

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress.

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H(2)O)(-1). This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na(+) dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in V(max) of the Na(+)-dependent component, with no significant change in the K(m) value of about 75 mumol l(-1). Quantitative real-time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo-inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l(-1) to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.

Adenosine Triphosphate↗

PubMed2006

Lentivirus envelope protein exerts differential neuropathogenic effects depending on the site of expression and target cell.

We investigated the neuropathogenic effects of feline immunodeficiency virus (FIV) envelope proteins in the context of both extracellular exposure and intracellular expression in feline neural cells. The envelope from the neurovirulent CSF-derived FIV V1 strain (V1-CSF) conferred infectivity to pseudotyped viruses in peripheral blood mononuclear cells (P < 0.01) in contrast to other cell types. Intracellular V1-CSF envelope expression in macrophages and microglia but not astrocytes resulted in the induction of host inflammatory genes contributing to neurotoxicity including IL-1beta, TNF-alpha, and indolamine 2',3'-dioxygenase (IDO) (P < 0.05) with concurrent neuronal death (P < 0.05). Upregulation of the endoplasmic reticulum stress genes was evident in brains from FIV-infected animals (P < 0.05) and in FIV-infected macrophages (P < 0.05) relative to controls. Intrastriatal implantation of an FIV envelope pseudotyped virus led to marked neuroinflammation and neuronal injury associated with neurobehavioral deficits (P < 0.01). Thus, lentivirus envelope proteins exert differential neuropathogenic effects through mechanisms that depend on the infected or exposed cell type.

Animals↗

PubMed2006

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose: a simple analysis by 13C NMR of urinary menthol glucuronide.

Menthol glucuronide was isolated from the urine of a healthy 70-kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U-(13)C]glucose. Glucuronide (13)C-excess enrichment levels were 4-6% and thus provided high signal-to-noise ratios (SNRs) for confident assignment of (13)C-(13)C spin-coupled multiplet components within each (13)C resonance by (13)C NMR. The [U-(13)C]glucuronide isotopomer derived via direct pathway conversion of [U-(13)C]glucose to [U-(13)C]UDP-glucose was resolved from [1,2,3-(13)C(3)]- and [1,2-(13)C(2)]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U-(13)C]glucose. In a second study, a group of four overnight-fasted patients (63 +/- 10 kg) with severe heart failure were given peppermint oil and infused with [U-(13)C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady-state plasma [U-(13)C]glucose enrichment of 4.6% +/- 0.6%. Menthol glucuronide was harvested and glucuronide (13)C-isotopomers were analyzed by (13)C NMR. [U-(13)C]glucuronide enrichment was 0.6% +/- 0.1%, and the sum of [1,2,3-(13)C(3)] and [1,2-(13)C(2)]glucuronide enrichments was 0.9% +/- 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% +/- 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% +/- 10% of GP.

Adult↗

PubMed2006

Synthesis, antioxidant activity and structure-activity relationships for a new series of 2-(N-acylaminoethyl)indoles with melatonin-like cytoprotective activity.

5-Methoxy-2-(N-acetylaminoethyl)indole (5d), a melatonin analogue derived from the transposition of the acetylaminoethyl side chain from C3 to C2 of the indole nucleus, had been previously characterized as a low affinity antagonist at MT1 and MT2 membrane receptors; this molecule is endowed with good in vitro antioxidant and cytoprotective potency in rat cerebellar cell cultures, comparable to or better than those of melatonin. In order to further investigate the role of structure-antioxidant activity relationships in cytoprotection, the structure of 5d was systematically modulated to design a new series of compounds. The 5-methoxy group was replaced by substituents with different electronic and lipophilic properties and it was moved to a different position on the indole ring. Other modifications of the lead structure involved the methylation of the indole nitrogen or its replacement by a sulfur atom. The side chain was also modified either increasing its lipophilicity or introducing an ionisable acid group. The antioxidant activity of this set of compounds was evaluated by the ABTS and conjugated dienes (CD) assays, while their cytoprotection was evaluated against kainate-induced cytotoxicity in cultured cerebellar neurons. In both antioxidant assays, the shift of the 5-methoxy group to the 4-position of the indole nucleus led to the most active radical scavenger (9), more potent than the parent compound and melatonin in the antioxidant tests, but much less effective as a cytoprotectant. Sharp structure-activity relationships were registered for cytoprotection, where the maintenance of the 5-alkoxy-2-(N-acylaminoethyl)indole scaffold appeared as the key feature to confer both antioxidant and cytoprotective activity to the structure. Some derivatives of the set, however, together with the most potent 5d, maintained a significant antioxidant and cytoprotective effect and could be employed as tools for in vivo pharmacological investigations on neuroprotective efficacy of melatonin-related indoles.

Animals↗

PubMed2005

[Predictors of non fatal cardiovascular events in a Chilean cohort: results of the San Francisco Project].

BACKGROUND: The San Francisco Project is a prospective study aiming to determine the most important cardiovascular risk factors in the Chilean population. AIM: To report the results of the San Francisco Project after a mean follow up of five years. MATERIAL AND METHODS: Between 1997 and 1999, 920 subjects (aged 39.5 +/- 16.3 years, 382 males), free of cardiovascular events, were examined to determine their cardiovascular risk factors. The prevalence of risk factors was compared with the Chilean National Health Survey. All non fatal cardiovascular events were registered until 2003. A logistic regression model including gender, obesity, family history, educational level and classical cardiovascular risk factors, was performed to identify independent predictors of cardiovascular events. RESULTS: The prevalence of cardiovascular risk factors in the studied population was similar to that of the Chilean National Health Survey, except for serum lipid levels, that were lower in the latter. The mean follow up was 5.3 +/- 0.3 years (range 4-6 years), and 26 cardiovascular events were registered. The independent predictors of these events were age, with a relative risk (RR) of 4.3 and 95% confidence intervals (CI) of 1.9 to 9.5 (p <0.001), high blood pressure with a RR of 5.2 (95% CI 2.3-11.7, p <0.001), diabetes mellitus with a RR of 4.5 (95% CI 1.9-10.7, p <0.01) and a low socioeconomic level with a RR of 3.5 (95% CI 1.5-7.9, p <0.01). The model did not accept serum lipid levels, smoking and alcohol intake as independent predictors. CONCLUSIONS: In this cohort of Chilean people, the independent predictors of non fatal cardiovascular events were high blood pressure, diabetes mellitus and a low socioeconomic level.

Adolescent↗

PubMed2005

Aberrant cortical neurogenesis in a pediatric neuroAIDS model: neurotrophic effects of growth hormone.

OBJECTIVE: To study the effects of HIV-1 and feline immunodeficiency virus (FIV) on neural stem cell viability, together with the neurotrophic properties of growth hormone (GH) in models of pediatric neuroAIDS. DESIGN AND METHODS: Mouse neural stem cells were infected in vitro with a Sindbis virus vector (SIN-HIVenv) expressing the envelope protein from the brain-derived HIV-1 strain JR-FL using a vector expressing enhanced green fluorescent protein (SIN-EGFP) as control. Cell survival and alterations in expression of neural stem cell markers upon GH treatment was assessed. Neonatal cats were infected with a neurovirulent FIV strain and 6 weeks after infection treated with GH for 6 weeks. Twelve weeks post-infection, neural progenitor cell marker expression, neuronal loss and neuroinflammation in brain were examined using real time reverse transcription-PCR and immunohistochemical analyses. RESULTS: HIV-1 envelope expression in neural stem cells reduced nestin expression (P < 0.05) and induced cell death (P < 0.001), which was blocked by GH. In the frontal cortex of FIV-infected cats neuroinflammation, loss of differentiated neurons (P < 0.01) and aberrant neuronal progenitor cell gene expression (P < 0.05) were observed. FIV envelope expression was detected in neural progenitor and monocytoid cells. GH treatment of FIV-infected animals induced insulin-like growth factor-1 expression in neurons (P < 0.01), enhanced neuronal survival (P < 0.01) and increased nestin expression (P < 0.05). Moreover, improved neurobehavioral performance (P < 0.01) and immunological status (P < 0.001) were observed, among GH-treated animals infected with FIV. CONCLUSION: GH protects neural stem cells that are susceptible to lentivirus-mediated injury. Thus, GH may be a potential treatment for pediatric neuroAIDS because of its neurotrophic actions.

AIDS Dementia Complex↗

PubMed2005

Acquired rifamycin resistance with twice-weekly treatment of HIV-related tuberculosis.

RATIONALE: Rifabutin was recommended in place of rifampin during treatment of HIV-related tuberculosis (TB) to facilitate concomitant potent antiretroviral therapy, but this approach has not been evaluated in a prospective study. OBJECTIVE: To evaluate the activity of intermittent rifabutin-based therapy. METHODS: Patients with culture-confirmed TB were treated under direct supervision with 2 mo of rifabutin, isoniazid, pyrazinamide, and ethambutol (given daily, thrice-weekly, or twice-weekly per the local tuberculosis control program), followed by 4 mo of twice-weekly rifabutin plus isoniazid. MEASUREMENTS: Culture-positive treatment failure or relapse. MAIN RESULTS: A total of 169 eligible patients were enrolled. Most had advanced HIV disease; the median CD4 cell count and HIV-RNA level were 90 cells/mm3 (interquartile range, 35-175) and 5.3 log10 copies/ml (interquartile range, 4.8-5.7), respectively. Nine (5.3%) patients had culture-positive treatment failure (n = 3) or relapse (n = 6). Eight of these nine (89%) cases had isolates with acquired rifamycin resistance. Treatment failure or relapse was associated with baseline CD4 lymphocyte count, being 12.3% (9/73; 95% confidence interval, 6.5-22.0%) among patients with CD4 < 100 cells/mm3 versus 0% (0/65; 95% confidence interval, 0.0-4.5%) among those with higher CD4 lymphocyte counts (p < 0.01). One hundred thirty-seven (81%) patients received antiretroviral therapy during TB treatment. Adverse events were common, but only two patients (1%) permanently discontinued study drugs. CONCLUSIONS: Intermittent rifabutin-based therapy for HIV-related TB was well tolerated, but there was a high risk of treatment failure or relapse with acquired rifamycin resistance among patients with low CD4 lymphocyte counts.

AIDS-Related Opportunistic Infections↗

PubMed2005

Lentivirus infection causes neuroinflammation and neuronal injury in dorsal root ganglia: pathogenic effects of STAT-1 and inducible nitric oxide synthase.

Distal sensory polyneuropathy (DSP) is currently the most common neurological complication of HIV infection in the developed world and is characterized by sensory neuronal injury accompanied by inflammation, which is clinically manifested as disabling pain and gait instability. We previously showed that feline immunodeficiency virus (FIV) infection of cats caused DSP together with immunosuppression in cats, similar to that observed in HIV-infected humans. In this study, we investigated the pathogenic mechanisms underlying the development of FIV-induced DSP using feline dorsal root ganglia (DRG) cultures, consisting of neurons, Schwann cells, and macrophages. FIV-infected cultures exhibited viral Ags (p24 and envelope) in macrophages accompanied by neuronal injury, indicated by neurite retraction, neuronal loss and decreased soma size, compared with mock-infected (control) cultures. FIV infection up-regulated inducible NO synthase (iNOS), STAT-1, and TNF-alpha mRNA levels in DRG cultures. Increased STAT-1 and iNOS mRNA levels were also observed in DRGs from FIV-infected animals relative to mock-infected controls. Similarly, immunolabeling studies of DRGs from FIV-infected animals showed that macrophages were the principal sources of STAT-1 and iNOS protein production. The iNOS inhibitor aminoguanidine reduced nitrotyrosine and protein carbonyl levels, together with preventing neuronal injury in FIV-infected DRG cultures. The present studies indicate that FIV infection of DRGs directly contributes to axonal and neuronal injury through a mechanism involving macrophage immune activation, which is mediated by STAT-1 and iNOS activation.

Animals↗

PubMed2005

Proteinase-activated receptor-2 induction by neuroinflammation prevents neuronal death during HIV infection.

Proteinase-activated receptors (PARs), a newly discovered subgroup of G-protein coupled receptors, are widely expressed by neural cells, but their roles in the nervous system remain uncertain. In this study, we report that PAR-2 was up-regulated on neurons in conjunction with neuroinflammation in brain tissue from patients with HIV-1-associated dementia. The inflammatory cytokines TNF-alpha and IL-1beta were also increased in HIV-1-associated dementia brains compared with patients without dementia (p < 0.05), but these same cytokines induced PAR-2 expression on neurons. Enhanced PAR-2 expression and subsequent activation prevented neuronal cell death and induction of the tumor suppressor, p53, caused by the HIV-encoded protein, Tat (p < 0.01). Intrastriatal implantation of a PAR-2 peptide agonist also inhibited Tat-induced neurotoxicity in a mouse model of HIV neuropathogenesis (p < 0.05). Moreover, PAR-2 null animals showed more severe neuroinflammation and neuronal loss caused by Tat neurotoxicity (p < 0.05). TNF-alpha protected wild-type neurons from Tat-related neurotoxicity, but in PAR-2-deficient neurons, the same concentrations of TNF-alpha were cytotoxic (p < 0.001). Thus, neuroinflammation can exert protective effects by which it induces PAR-2 expression with the ensuing abrogation of neuronal death.

AIDS Dementia Complex↗

PubMed2005

Peripheral nerve-derived HIV-1 is predominantly CCR5-dependent and causes neuronal degeneration and neuroinflammation.

HIV-related peripheral neuropathy is a major neurological complication of HIV infection, although little is known about its pathogenesis. We amplified HIV-1 C2V3 envelope sequences from peroneal nerves obtained from HIV/AIDS patients. Sequence analysis and infectious recombinant viruses containing peripheral nerve-derived C2V3 sequences indicated a predominance of CCR5-dependent and macrophage-tropic HIV-1, although dual tropic viruses using both CCR5 and CXCR4 were identified. The neuropathogenic effects of recombinant HIV-1 clones were investigated using a novel dorsal root ganglion culture system that was comprised of sensory neurons, macrophages and Schwann cells from transgenic rats expressing human CD4 and CCR5 on monocytoid cells. Despite restricted viral replication, HIV-1 infection caused a reduction in the percentage of neurons with neuritic processes together with significant neurite retraction, which was accompanied by induction of IL-1beta and TNF-alpha expression, depending on the individual virus. Our results suggest that HIV-1 infection of the peripheral nervous system causes axonal degeneration, possibly through the induction of pro-inflammatory cytokines.

Adult↗

PubMed2005

Molecular systematics of rhizobia based on maximum likelihood and Bayesian phylogenies inferred from rrs, atpD, recA and nifH sequences, and their use in the classification of Sesbania microsymbionts from Venezuelan wetlands.

A well-resolved rhizobial species phylogeny with 51 haplotypes was inferred from a combined atpD + recA data set using Bayesian inference with best-fit, gene-specific substitution models. Relatively dense taxon sampling for the genera Rhizobium and Mesorhizobium was achieved by generating atpD and recA sequences for six type and 24 reference strains not previously available in GenBank. This phylogeny was used to classify nine nodule isolates from Sesbania exasperata, S. punicea and S. sericea plants native to seasonally flooded areas of Venezuela, and compared with a PCR-RFLP analysis of rrs plus rrl genes and large maximum likelihood rrs and nifH phylogenies. We show that rrs phylogenies are particularly sensitive to strain choice due to the high levels of sequence mosaicism found at this locus. All analyses consistently identified the Sesbania isolates as Mesorhizobium plurifarium or Rhizobium huautlense. Host range experiments on ten legume species coupled with plasmid profiling uncovered potential novel biovarieties of both species. This study demonstrates the wide geographic and environmental distribution of M. plurifarium, that R. galegae and R. huautlense are sister lineages, and the synonymy of R. gallicum, R. mongolense and R. yanglingense. Complex and diverse phylogeographic, inheritance and host-association patterns were found for the symbiotic nifH locus. The results and the analytical approaches used herein are discussed in the context of rhizobial taxonomy and molecular systematics.

Base Sequence↗

PubMed2005

Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation.

A combination of population genetics and phylogenetic inference methods was used to delineate Bradyrhizobium species and to uncover the evolutionary forces acting at the population-species interface of this bacterial genus. Maximum-likelihood gene trees for atpD, glnII, recA, and nifH loci were estimated for diverse strains from all but one of the named Bradyrhizobium species, and three unnamed "genospecies," including photosynthetic isolates. Topological congruence and split decomposition analyses of the three housekeeping loci are consistent with a model of frequent homologous recombination within but not across lineages, whereas strong evidence was found for the consistent lateral gene transfer across lineages of the symbiotic (auxiliary) nifH locus, which grouped strains according to their hosts and not by their species assignation. A well resolved Bayesian species phylogeny was estimated from partially congruent glnII+recA sequences, which is highly consistent with the actual taxonomic scheme of the genus. Population-level analyses of isolates from endemic Canarian genistoid legumes based on REP-PCR genomic fingerprints, allozyme and DNA polymorphism analyses revealed a non-clonal and slightly epidemic population structure for B. canariense isolates of Canarian and Moroccan origin, uncovered recombination and migration as significant evolutionary forces providing the species with internal cohesiveness, and demonstrated its significant genetic differentiation from B. japonicum, its sister species, despite their sympatry and partially overlapped ecological niches. This finding provides strong evidence for the existence of well delineated species in the bacterial world. The results and approaches used herein are discussed in the context of bacterial species concepts and the evolutionary ecology of (brady)rhizobia.

Base Sequence↗

PubMed2005

Comparison of morphine, ketorolac, and their combination for postoperative pain: results from a large, randomized, double-blind trial.

BACKGROUND: Meta-analyses report similar numbers needed to treat for nonsteroidal antiinflammatory drugs (NSAIDs) and opioids. Differences in baseline pain intensity among the studies from which these numbers needed to treat were derived may have confounded the results. NSAIDs have an opioid-sparing effect, but the importance of this effect is unclear. Therefore, the authors sought to compare the proportions of subjects who obtain pain relief with ketorolac versus morphine after surgery and to determine whether the opioid-sparing effect of an NSAID reduces the magnitude of opioid side effects. METHODS: The study was a double-blind, randomized controlled trial. The authors randomly assigned 1,003 adult patients to receive 30 mg ketorolac or 0.1 mg/kg morphine intravenously. They calculated the proportion of subjects who achieved at least 50% reduction in pain intensity 30 min after analgesic administration. Further, so long as pain intensity 30 min after analgesic administration was 5 or more out of 10, patients received 2.5 mg morphine every 10 min until pain intensity was 4 or less out of 10. The authors assessed the presence of opioid-related side effects. RESULTS: Five hundred patients received morphine and 503 received ketorolac. Fifty percent of patients in the morphine group achieved pain relief, compared with 31% in the ketorolac group (difference, 19%; 95% confidence interval, 13-25%). The ketorolac-morphine group required less morphine (difference, 6.5 mg; 95% confidence interval, -5.8 to -7.2) and had a lower incidence of side effects (difference, 11%; 95% confidence interval, 5-16%) than the morphine group. CONCLUSIONS: Opioids are more efficacious analgesics than NSAIDs, although historic data for these two drugs yield similar numbers needed to treat. Adding NSAIDs to the opioid treatment reduces morphine requirements and opioid-related side effects in the early postoperative period.

Adult↗

PubMed2005

Bradyrhizobium canariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicum bv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta.

Highly diverse Bradyrhizobium strains nodulate genistoid legumes (brooms) in the Canary Islands, Morocco, Spain and the Americas. Phylogenetic analyses of ITS, atpD, glnII and recA sequences revealed that these isolates represent at least four distinct evolutionary lineages within the genus, namely Bradyrhizobium japonicum and three unnamed genospecies. DNA-DNA hybridization experiments confirmed that one of the latter represents a new taxonomic species for which the name Bradyrhizobium canariense is proposed. B. canariense populations experience homologous recombination at housekeeping loci, but are sexually isolated from sympatric B. japonicum bv. genistearum strains in soils of the Canary Islands. B. canariense strains are highly acid-tolerant, nodulate diverse legumes in the tribes Genisteae and Loteae, but not Glycine species, whereas acid-sensitive B. japonicum soybean isolates such as USDA 6(T) and USDA 110 do not nodulate genistoid legumes. Based on host-range experiments and phylogenetic analyses of symbiotic nifH and nodC sequences, the biovarieties genistearum and glycinearum for the genistoid legume and soybean isolates, respectively, were proposed. B. canariense bv. genistearum strains display an overlapped host range with B. japonicum bv. genistearum isolates, both sharing monophyletic nifH and nodC alleles, possibly due to the lateral transfer of a conjugative chromosomal symbiotic island across species. B. canariense is the sister species of B. japonicum, as inferred from a maximum-likelihood Bradyrhizobium species phylogeny estimated from congruent glnII+recA sequence partitions, which resolves eight species clades. In addition to the currently described species, this phylogeny uncovered the novel Bradyrhizobium genospecies alpha and beta and the photosynthetic strains as independent evolutionary lineages. The type strain for B. canariense is BTA-1(T) (=ATCC BAA-1002(T)=LMG 22265(T)=CFNE 1008(T)).

Bacterial Proteins↗

PubMed2005

Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes.

We used phylogenetic and population genetics approaches to evaluate the importance of the evolutionary forces on shaping the genetic structure of Rhizobium gallicum and related species. We analysed 54 strains from several populations distributed in the Northern Hemisphere, using nucleotide sequences of three 'core' chromosomal genes (rrs, glnII and atpD) and two 'auxiliary' symbiotic genes (nifH and nodB) to elucidate the biogeographic history of the species and symbiotic ecotypes (biovarieties) within species. The analyses revealed that strains classified as Rhizobium mongolense and Rhizobium yanglingense belong to the chromosomal evolutionary lineage of R. gallicum and harbour symbiotic genes corresponding to a new biovar; we propose their reclassification as R. gallicum bv. orientale. The comparison of the chromosomal and symbiotic genes revealed evidence of lateral transfer of symbiotic information within and across species. Genetic differentiation analyses based on the chromosomal protein-coding genes revealed a biogeographic pattern with three main populations, whereas the 16S rDNA sequences did not resolve that biogeographic pattern. Both the phylogenetic and population genetic analyses showed evidence of recombination at the rrs locus. We discuss our results in the light of the contrasting views of bacterial species expressed by microbial taxonomist and evolutionary biologists.

Base Sequence↗

PubMed2005

[Identification of point mutations in the 21-hydroxylase gene in patients affected with congenital adrenal hyperplasia].

INTRODUCTION: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder due to impaired cortisol secretion. Approximately 95% of CAH cases are caused by defects in the 21-hydrodylase2 (CYPA2) gene. The spectrum of clinical manifestations includes a severe and mild form of expression. The frequency of the following point mutations was determined: P30L, IVS2-12A/C-G splice, Del 8pb, I172N, cluster Ex6, V281L, Q318X, R356W and P453S. MATERIALS AND METHODS: The 58 patients consisted of 48 with the severe form of CAH and 10 with the mild form. Point mutations in the hydroxylase gene were isolated by allele-specific PCR and PCR-ACRS (amplification created restriction site), and their frequency was determined. RESULTS: Alternate alleles were identified in 82.8% of the samples. The most frequent mutations were IVS2-12A/C-G splice (26.7%), Q318X (21.5%), V281L (12.1%) and I172N (12.1%). DISCUSSION: The most frequent mutations were similar to those observed in other countries, except for Q318X. Although its frequency was higher but similar to that observed in Latin American countries, it contrasted with those of other continents and indicated the possible influence of genetic background in its expression. Several of the mutations were associated with specific clinical forms related to the enzyme activity. In the milder forms of CAH, several alleles were detected. These were important because these patients can have children with the virilizing and salt wasting forms. Recognition of the allelic forms of CAH will permit more specific genetic counseling and prenatal diagnosis.

Adrenal Hyperplasia, Congenital↗