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1.  An Insight into the Proteome of Crithidia fasciculata Choanomastigotes as a Comparative Approach to Axenic Growth, Peanut Lectin Agglutination and Differentiation of Leishmania spp. Promastigotes 
PLoS ONE  2014;9(12):e113837.
The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.
PMCID: PMC4263474  PMID: 25503511
2.  Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte 
BMC Genomics  2014;15(1):849.
Leishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been recently reported in central Spain. Leishmania spp. parasites are transmitted to the mammalian host by the bite of sand flies. The primary vector of L. infantum in Spain is Phlebotomus perniciosus. For decades, research on these parasites has involved the axenic culture model of the promastigote stage including gene expression profiling studies performed in the post-genome era. Unlike the controversial axenic culturing of amastigotes, promastigote cultures are generally accepted and used, although with the precaution of avoiding excessive culture passage.
The primary objective of this differentiation study is to compare the gene expression profiles of promastigotes isolated from the foregut of the sand fly and amastigotes. For this purpose, P. perniciosus sand flies were infected with L. infantum and differentiated promastigotes were extracted by dissection of the foreguts. Shotgun DNA microarray hybridization analyses allowed for transcriptome comparison of these promastigotes with amastigotes obtained by infection of the U937 cell line. The results have been compared with those described in published expression analyses using axenic promastigotes.
A total of 277 up-regulated genes were found through this hybridization experiment. The comparison of these particular results with published gene expression profile analyses performed using the same experimental procedure to study cultured promastigotes in stationary phase versus amastigotes revealed considerable differences (approximately 95% of the up-regulated genes were different). We found that the up-regulation rate is lower in amastigotes than in sand fly-derived promastigotes, which is in agreement with the over-expression of genes involved in gene expression regulation and signaling in those promastigote populations.
The up-regulation rate is lower in intracellular amastigotes than in promastigotes obtained from the sand fly gut. This was also reported by us using the promastigote culture model and is an evidence for the hypothesis of promastigote preadaptation towards life in the intracellular environment. Regarding transcript abundance, the set of differentially regulated genes is notably different when using promastigotes from the sand fly foregut instead of axenic cultures.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-849) contains supplementary material, which is available to authorized users.
PMCID: PMC4203910  PMID: 25281593
Leishmania infantum; Phlebotomus perniciosus; Promastigotes; Amastigotes; Promastigote axenic culture; Gene expression profiling
3.  Myxoma of the Left Ventricle 
Texas Heart Institute Journal  2014;41(4):395-400.
This report concerns a 69-year-old woman who presented with an asymptomatic myxoma in the left ventricle. The tumor was successfully excised. We provide a very brief review of 72 other published cases of surgically treated left ventricular myxoma.
PMCID: PMC4120502  PMID: 25120392
Adult; cardiopulmonary bypass; echocardiography, transesophageal; echocardiography, transthoracic; female; heart neoplasms/surgery; heart ventricles/pathology; myxoma/surgery
4.  Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate 
Graphical abstract
•The tyrosine aminotransferase from Leishmania infantum has a cytoplasmic distribution and is able to use the oxoacid ketomethiobutyrate, as a co-substrate.•L. infantum tyrosine aminotransferase is over-expressed in infective and nitric oxide resistant parasites.•The structural differences with the mammalian TAT, together with cellular distribution, expression pattern and activity, support that LiTAT is a drug target candidate.•The structure-based model of the pharmacophore of LiTAT with specific substrate ketomethiobutyrate has been generated.
Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs.
PMCID: PMC4266777  PMID: 25516846
Leishmania infantum; Tyrosine aminotransferase; Infectivity; KMTB
6.  Dormant Intracellular Salmonella enterica Serovar Typhimurium Discriminates among Salmonella Pathogenicity Island 2 Effectors To Persist inside Fibroblasts 
Infection and Immunity  2014;82(1):221-232.
Salmonella enterica uses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recent in vivo studies have shown that intracellular bacteria activate the TTSS encoded by Salmonella pathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observed in vivo in bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bacteria persisting inside fibroblasts, but its induction mode and the effectors involved remain unknown. Here, we show that nongrowing dormant intracellular bacteria use the two-component system OmpR-EnvZ to induce SPI-2 expression and the PhoP-PhoQ system to regulate the time at which induction takes place, 2 h postentry. Dormant bacteria were shown to discriminate the usage of SPI-2 effectors. Among the effectors tested, SseF, SseG, and SseJ were required for survival, while others, such as SifA and SifB, were not. SifA and SifB dispensability correlated with the inability of intracellular bacteria to secrete these effectors even when overexpressed. Conversely, SseJ overproduction resulted in augmented secretion and exacerbated bacterial growth. Dormant bacteria produced other effectors, such as PipB and PipB2, that, unlike what was reported for epithelial cells, did not to traffic outside the phagosomal compartment. Therefore, permissiveness for secreting only a subset of SPI-2 effectors may be instrumental for dormancy. We propose that the S. enterica serovar Typhimurium nonproliferative intracellular lifestyle is sustained by selection of SPI-2 effectors that are produced in tightly defined amounts and delivered to phagosome-confined locations.
PMCID: PMC3911833  PMID: 24144726
8.  Aging and substitutive hormonal therapy influence in regional and subcellular distribution of ERα in female rat brain 
Age  2012;35(3):821-837.
Estrogens are not only critical for sexual differentiation it is well-known for the role of 17β-estradiol (E2) in the adult brain modulating memory, learning, mood and acts as a neuroprotector. E2 exerts its actions through two classical receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). The distribution of both receptors changes from one brain area to another, E2 being able to modulate their expression. Among the classical features of aging in humans, we find cognitive impairment, dementia, memory loss, etc. As estrogen levels change with age, especially in females, it is important to know the effects of low E2 levels on ERα distribution; results from previous studies are controversial regarding this issue. In the present work, we have studied the effects of long-term E2 depletion as well as the ones of E2 treatment on ERα brain distribution of ovariectomized rats along aging in the diencephalon and in the telencephalon. We have found that ovariectomy causes downregulation and affects subcellular localization of ERα expression during aging, meanwhile prolonged estrogen treatment produces upregulation and overexpression of the receptor levels. Our results support the idea of the region-specific neuroprotection mechanisms mediated by estradiol.
PMCID: PMC3636381  PMID: 22648398
Hormonal therapy; Aging; Estrogen receptor alpha; Brain; Estradiol; Telencephalon; Diencephalon
9.  Does film smoking promote youth smoking in middle-income countries?: A longitudinal study among Mexican adolescents 
To determine whether exposure to smoking imagery in films predicts smoking onset among never-smoking Mexican adolescents.
The analytic sample was comprised of 11- to 14-year old secondary school students who reported never having tried smoking at baseline, 83% (1741/2093) of whom were successfully followed up after one year. Exposure to 42 popular films that contained smoking was assessed at baseline, whereas smoking behavior and risk factors were assessed at baseline and follow up. Logistic regression was used to estimate bivariate and adjusted relative risks of trying smoking and current smoking at follow up.
At follow up, 36% reported having tried smoking and 8% reported having smoked in the previous month. Students who were successfully followed up were exposed to an average of 43.8 minutes of smoking in the films they reported viewing at baseline. Adjusted relative risks (ARRs) indicated that students in the two highest levels of exposure to film smoking were more than twice as likely to have smoked in the previous 30 days at follow up (ARR3v1=2.44, 95%CI 1.31, 4.55; ARR4v1=2.23, 95% CI 1.19, 4.17). The adjusted relative risk of having tried smoking by follow up reached statistical significance only when comparing the 3rd highest to the lowest exposure group (ARR3v1=1.54, 95%CI 1.01, 2.64). Having a parent or best friend who smoked at baseline were the only other variables that independently predicted both outcomes.
Exposure to movie smoking is a risk factor for smoking onset among Mexican youth.
PMCID: PMC3837702  PMID: 19959694
Media; tobacco; smoking; longitudinal; adolescents
10.  Human PAF1 inhibition of HIV-1 
Retrovirology  2013;10(Suppl 1):P39.
PMCID: PMC3848275
11.  Prevalence of medication administration errors in two medical units with automated prescription and dispensing 
To identify the frequency of medication administration errors and their potential risk factors in units using a computerized prescription order entry program and profiled automated dispensing cabinets.
Prospective observational study conducted within two clinical units of the Gastroenterology Department in a 1537-bed tertiary teaching hospital in Madrid (Spain).
Medication errors were measured using the disguised observation technique. Types of medication errors and their potential severity were described. The correlation between potential risk factors and medication errors was studied to identify potential causes.
In total, 2314 medication administrations to 73 patients were observed: 509 errors were recorded (22.0%)—68 (13.4%) in preparation and 441 (86.6%) in administration. The most frequent errors were use of wrong administration techniques (especially concerning food intake (13.9%)), wrong reconstitution/dilution (1.7%), omission (1.4%), and wrong infusion speed (1.2%). Errors were classified as no damage (95.7%), no damage but monitoring required (2.3%), and temporary damage (0.4%). Potential clinical severity could not be assessed in 1.6% of cases. The potential risk factors morning shift, evening shift, Anatomical Therapeutic Chemical medication class antacids, prokinetics, antibiotics and immunosuppressants, oral administration, and intravenous administration were associated with a higher risk of administration errors. No association was found with variables related to understaffing or nurse's experience.
Medication administration errors persist in units with automated prescription and dispensing. We identified a need to improve nurses' working procedures and to implement a Clinical Decision Support tool that generates recommendations about scheduling according to dietary restrictions, preparation of medication before parenteral administration, and adequate infusion rates.
PMCID: PMC3240760  PMID: 21890872
Farmacia; me8004me; machine learning; predictive modeling; statistical learning; privacy technology; clinical informatics; biomedical informatics; pediatrics; e-prescribing; human factors; decision-support systems; clinical; gastroenterology/organization and administration; medical order entry systems; medication errors; robotics
12.  Deep EST profiling of developing fenugreek endosperm to investigate galactomannan biosynthesis and its regulation 
Plant Molecular Biology  2012;79(3):243-258.
Galactomannans are hemicellulosic polysaccharides composed of a (1 → 4)-linked β-D-mannan backbone substituted with single-unit (1 → 6)-α-linked D-galactosyl residues. Developing fenugreek (Trigonella foenum-graecum) seeds are known to accumulate large quantities of galactomannans in the endosperm, and were thus used here as a model system to better understand galactomannan biosynthesis and its regulation. We first verified the specific deposition of galactomannans in developing endosperms and determined that active accumulation occurred from 25 to 38 days post anthesis (DPA) under our growth conditions. We then examined the expression levels during seed development of ManS and GMGT, two genes encoding backbone and side chain synthetic enzymes. Based on transcript accumulation dynamics for ManS and GMGT, cDNA libraries were constructed using RNA isolated from endosperms at four ages corresponding to before, at the beginning of, and during active galactomannan deposition. DNA from these libraries was sequenced using the 454 sequencing technology to yield a total of 1.5 million expressed sequence tags (ESTs). Through analysis of the EST profiling data, we identified genes known to be involved in galactomannan biosynthesis, as well as new genes that may be involved in this process, and proposed a model for the flow of carbon from sucrose to galactomannans. Measurement of in vitro ManS and GMGT activities and analysis of sugar phosphate and nucleotide sugar levels in the endosperms of developing fenugreek seeds provided data consistent with this model. In vitro enzymatic assays also revealed that the ManS enzyme from fenugreek endosperm preferentially used GDP-mannose as the substrate for the backbone synthesis.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-012-9909-y) contains supplementary material, which is available to authorized users.
PMCID: PMC3349874  PMID: 22527750
Fenugreek; Endosperm; Galactomannan biosynthesis; EST profiling; Hexose phosphates; Nucleotide sugars
13.  Postcesarean Thromboprophylaxis with Two Different Regimens of Bemiparin 
Objectives. To compare the effectiveness of postcesarean thromboprophylaxis with two different regimens of bemiparin. Material and Methods. The study included 646 women with cesarean delivery in our hospital within a 1-year period, randomly assigned to one of two groups for prophylaxis with 3500 IU bemiparin once daily for 5 days or 3500 IU bemiparin once daily for 10 days. Results. There was one case of pulmonary embolism (first day following cesarean). An additional risk factor was present in 98.52% of the women, most frequently emergency cesarean, anemia, or obesity. The only risk factors for thromboembolic disease significantly related to pulmonary thromboembolism were placental abruption and prematurity. There were no differences in thromboembolic events among the two thromboprophylaxis regimens. Conclusions. Cesarean-related thromboembolic events were reduced in our study population due to the thromboprophylactic measures taken. Thromboprophylaxis with 3500 IU bemiparin once daily for 5 days following cesarean was sufficient to avoid thromboembolic events.
PMCID: PMC3253471  PMID: 22242023
14.  Acute effects of 17β-estradiol and genistein on insulin sensitivity and spatial memory in aged ovariectomized female rats 
Age  2010;32(4):421-434.
Aging is characterized by decline in metabolic function and insulin resistance, and both seem to be in the basis of neurodegenerative diseases and cognitive dysfunction. Estrogens prevent age-related changes, and phytoestrogens influence learning and memory. Our hypothesis was that estradiol and genistein, using rapid-action mechanisms, are able to modify insulin sensitivity, process of learning, and spatial memory. Young and aged ovariectomized rats received acute treatment with estradiol or genistein. Aged animals were more insulin-resistant than young. In each age, estradiol and genistein-treated animals were less insulin-resistant than the others, except in the case of young animals treated with high doses of genistein. In aged rats, no differences between groups were found in spatial memory test, showing a poor performance in the water maze task. However, young females treated with estradiol or high doses of genistein performed well in spatial memory task like the control group. Only rats treated with high doses of genistein showed an optimal spatial memory similar to the control group. Conversely, acute treatment with high doses of phytoestrogens improved spatial memory consolidation only in young rats, supporting the critical period hypothesis for the beneficial effects of estrogens on memory. Therefore, genistein treatment seems to be suitable treatment in aged rats in order to prevent insulin resistance but not memory decline associated with aging. Acute genistein treatment is not effective to restore insulin resistance associated to the early loss of ovarian function, although it can be useful to improve memory deficits in this condition.
PMCID: PMC2980599  PMID: 20467821
17β-estradiol; Genistein; Insulin sensitivity; Spatial memory; Aging; Rat
15.  Validation of the spanish version of the multiple sclerosis international quality of life (musiqol) questionnaire 
BMC Neurology  2011;11:127.
The Multiple Sclerosis International Quality Of Life (MusiQoL) questionnaire, a 31-item, multidimensional, self-administrated questionnaire that is available in 14 languages including Spanish, has been validated using a large international sample. We investigated the validity and reliability of the Spanish version of MusiQoL in Spain.
Consecutive patients with different types and severities of multiple sclerosis (MS) were recruited from 22 centres across Spain. All patients completed the MusiQoL questionnaire, the 36-Item Short Form (SF-36) health survey, and a symptoms checklist at baseline and 21 days later. External validity, internal consistency, reliability and reproducibility were tested.
A total of 224 Spanish patients were evaluated. Dimensions of MusiQoL generally demonstrated a high internal consistency (Cronbach's alpha: 0.70-0.92 for all but two MusiQoL domain scores). External validity testing revealed that the MusiQoL index score correlated significantly with all SF-36 dimension scores (Pearson's correlation: 0.46-0.76), reproducibility was satisfactory (intraclass correlation coefficient: 0.60-0.91), acceptability was high, and the time taken to complete the 31-item questionnaire was reasonable (mean [standard deviation]: 9.8 [11.8] minutes).
The Spanish version of the MusiQoL questionnaire appears to be a valid and reliable instrument for measuring quality of life in patients with MS in Spain and constitutes a useful instrument to measure health-related quality of life in the clinical setting.
PMCID: PMC3206836  PMID: 22013975
16.  Amino acid changes within the Bunyamwera virus nucleocapsid protein differentially affect the mRNA transcription and RNA replication activities of assembled ribonucleoprotein templates 
The Journal of General Virology  2011;92(Pt 1):80-84.
The genome of Bunyamwera virus (BUNV) comprises three RNA segments that are encapsidated by the virus-encoded nucleocapsid (N) protein to form ribonucleoprotein (RNP) complexes. These RNPs are the functional templates for RNA synthesis by the virus-encoded RNA-dependent RNA polymerase (RdRp). We investigated the roles of conserved positively charged N-protein amino acids in RNA binding, in oligomerization to form model RNPs and in generating RNP templates active for both RNA replication and mRNA transcription. We identified several residues that performed important roles in RNA binding, and furthermore showed that a single amino acid change can differentially affect the ability of the resulting RNP templates to regulate the transcription and replication activities of the RdRp. These results indicate that the BUNV N protein possesses functions outside of its primary role of RNA encapsidation.
PMCID: PMC3052533  PMID: 20943890
17.  Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum 
BMC Genomics  2010;11:31.
The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.
Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.
The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.
PMCID: PMC2845110  PMID: 20074347
18.  Association of Monoclonal Expansion of Epstein-Barr Virus-Negative CD158a+ NK Cells Secreting Large Amounts of Gamma Interferon with Hemophagocytic Lymphohistiocytosis▿  
We report the first case of hemophagocytic lymphohistiocytosis (HLH) induced by the monoclonal expansion of Epstein-Barr virus (EBV)-negative NK cells. Consanguinity of the patient's parents made it necessary to discard familial HLH in the patient and her sister with identical HLA markers and demonstrate that no cause other than the expansion of NK cells, which secrete high levels of gamma interferon, was inducing HLH in this patient.
PMCID: PMC2620662  PMID: 19020108
19.  Genetic association study and meta-analysis of the HTR2C Cys23Ser polymorphism and migraine 
The Journal of Headache and Pain  2007;8(4):231-235.
The objectives of this study were to determine if the HTR2C Cys23Ser polymorphism is associated with migraine in a case-control study, and to perform a meta-analysis with present and previous available studies. The HTR2C gene is located at the Xq24-q28 chromosomal band. This band was linked to migraine with aura (MA) in two Australian families. Using the HTR2C Cys23Ser allelic variant, this gene has been ruled out as a migraine gene in 3 out of 4 studies. Only the Japanese study reported a higher risk for MA (OR=6.11; 95% CI=1.70−21.97, p trend<0.01). We performed a case-control study with 335 migraine subjects and 335 sex- and age-matched controls, and a meta-analysis pooling the results of the available data from MA subsets of patients. In the association study we found no significant differences among migraine and MA patients for this polymorphism. In the meta-analysis, under the fixed-effect model, the Ser allele did not confer higher risk for suffering MA (pooled OR=1.1; 99% CI=0.8−1.5, p=0.499). Our study did not confirm the HTR2C Cys23Ser polymorphism as a risk factor for migraine and MA.
PMCID: PMC3451673  PMID: 17901921
HTR2C gene; Migraine; Migraine with aura; Case-control study; Meta-analysis
20.  Increased Excision of the Salmonella Prophage ST64B Caused by a Deficiency in Dam Methylase 
Journal of Bacteriology  2005;187(23):7901-7911.
Salmonella enterica mutants defective in Dam methylase are strongly attenuated in virulence and release a large amount of proteins to the extracellular medium. The extent to which these two phenotypes are linked is unknown. Using a proteomic approach, we identified Sb6, Sb13, and Sb36 as proteins present in larger amounts in culture supernatants of an S. enterica serovar Typhimurium dam mutant than in those of the wild-type strain. These three proteins are encoded in the Salmonella prophage ST64B. Higher amounts of ST64B phage DNA and tailless viral capsids were also detected in supernatant extracts of the dam mutant, suggesting that Dam methylation negatively regulates the excision of ST64B. Reverse transcription-PCR analysis revealed that the expression of two ST64B genes encoding a putative antirepressor and a phage replication protein increases in the dam mutant. The SOS response also augments the excision of ST64B. Infection assays performed with phage-cured strains demonstrated that ST64B does not carry genes required for virulence in the mouse model. Evidence was also obtained discarding a relationship between the high excision of ST64B and the envelope instability or virulence attenuation phenotype. Taken together, these data indicate that ST64B excises at a high rate in dam mutants due to the loss of repression exerted by Dam on phage genes and induction of the SOS response characteristic of these mutants. The exacerbated excision of ST64B does not however contribute to the incapacity of dam mutants to cause disease.
PMCID: PMC1291290  PMID: 16291663
21.  Regulatory Regions of smeDEF in Stenotrophomonas maltophilia Strains Expressing Different Amounts of the Multidrug Efflux Pump SmeDEF 
The smeT-smeDEF region and the smeT gene, which encodes the smeDEF repressor, are highly polymorphic. Few changes in smeT might be associated with smeDEF overexpression. The results obtained with cellular extracts suggest that mutant SmeT proteins cannot bind to the operator and that other transcription factors besides SmeT are involved in the regulation of smeDEF expression.
PMCID: PMC415575  PMID: 15155232
22.  Cloning and Characterization of SmeT, a Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF 
Antimicrobial Agents and Chemotherapy  2002;46(11):3386-3393.
We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF. SmeT belongs to the TetR and AcrR family of transcriptional regulators. The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes. Experiments with S. maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor. S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5′ end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PsmeT. The level of expression of smeT is higher in smeDEF-overproducing S. maltophilia strain D457R, which suggests that SmeT represses its own expression. Band-shifting assays have shown that wild-type strain S. maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region. That cellular factor(s) was absent from smeDEF-overproducing S. maltophilia strain D457R. The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein. This change allowed overexpression of both smeDEF and smeT in D457R. It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R. This suggests that the wild-type protein is not dominant over the mutant SmeT.
PMCID: PMC128709  PMID: 12384340
23.  Expression of Multidrug Efflux Pump SmeDEF by Clinical Isolates of Stenotrophomonas maltophilia 
The presence of the multidrug efflux pump SmeDEF was assessed in a collection of clinical isolates of Stenotrophomonas maltophilia. All isolates encoded this pump, as demonstrated by PCR. Forty-seven percent of the strains overproduced a protein of the same size that was immunoreactive against an anti-SmeF antibody, and 33% overexpressed the gene semD when they were tested by reverse transcription-PCR. A correlation between smeDEF overexpression and antibiotic resistance was observed.
PMCID: PMC90562  PMID: 11353642
24.  Cloning and Characterization of SmeDEF, a Novel Multidrug Efflux Pump from Stenotrophomonas maltophilia 
Antimicrobial Agents and Chemotherapy  2000;44(11):3079-3086.
Stenotrophomonas maltophilia is a nosocomial bacterial pathogen intrinsically resistant to several antibiotics. The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood. A library of chromosomal DNA from a spontaneous multidrug-resistant S. maltophilia D457R mutant (A. Alonso and J. L. Martinez, Antimicrob. Agents Chemother. 41:1140–1142, 1997) was screened for complementation of erythromycin susceptibility on an antibiotic-hypersusceptible Escherichia coli ΔacrAB strain. Cloning and further analysis revealed that a 6-kbp region constituting a transcriptional unit was capable of complementing the antibiotic-susceptible phenotype of an E. coli ΔacrAB strain. We identified three open reading frames, smeD, smeE and smeF, which code for members of the membrane fusion protein, resistance nodulation division, and outer membrane factor families, respectively. Drug susceptibility assays indicated that the SmeDEF system cloned in E. coli mediates resistance to a wide range of antibiotics. Ethidium bromide and norfloxacin accumulation experiments in the presence and in the absence of carbonyl cyanide m-chlorophenylhydrazone showed that this system constitutes a drug efflux pump dependent on the membrane proton motive force. The presence of high levels of smeDEF mRNA in the multiresistant D457R mutant was consistent with the high levels of SmeF (formerly Omp54) observed in the same strain. In contrast, transcription levels of smeDEF in the D457 strain were tiny, which correlates with the low levels of SmeF observed for this strain. Also, for both the D457 and D457R strains, we observed growth phase-dependent regulation in which the highest level of transcription corresponded to early exponential phase, with transcription decreasing throughout the growth curve to undetectable levels at 24 h.
PMCID: PMC101606  PMID: 11036026
25.  Stenotrophomonas maltophilia D457R Contains a Cluster of Genes from Gram-Positive Bacteria Involved in Antibiotic and Heavy Metal Resistance 
A cluster of genes involved in antibiotic and heavy metal resistance has been characterized from a clinical isolate of the gram-negative bacterium Stenotrophomonas maltophilia. These genes include a macrolide phosphotransferase (mphBM) and a cadmium efflux determinant (cadA), together with the gene cadC coding for its transcriptional regulator. The cadC cadA region is flanked by a truncated IS257 sequence and a region coding for a bin3 invertase. Despite their presence in a gram-negative bacterium, these genetic elements share a common gram-positive origin. The possible origin of these determinants as a remnant composite transposon as well as the role of gene transfer between gram-positive and gram-negative bacteria for the acquisition of antibiotic resistance determinants in chronic, mixed infections is discussed.
PMCID: PMC89961  PMID: 10858330

Results 1-25 (26)