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1.
Am J Case Rep ; 21: e919745, 2020 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-32358478

RESUMO

BACKGROUND Thyroglossal duct cysts are the most common congenital neck cysts. They typically present in childhood and early adulthood, and average a size of 2-4 cm, but can also present in later adult life. CASE REPORT We present a case of a 36-year-old female patient with a very large midline neck mass, reaching the mandible superiorly. Patient history and physical examination, as well as computed tomography scan of her neck, confirmed the diagnosis of large thyroglossal duct cyst. She underwent Sistrunk procedure for thyroglossal duct cyst excision, and the specimen was sent for histopathological evaluation, which confirmed the diagnosis. CONCLUSIONS Thyroglossal duct cyst should be considered as a differential diagnosis in older patients and in patients with a relatively large neck mass.


Assuntos
Cisto Tireoglosso/patologia , Adulto , Diagnóstico Diferencial , Feminino , Humanos , Cisto Tireoglosso/diagnóstico por imagem , Cisto Tireoglosso/cirurgia , Neoplasias da Glândula Tireoide/diagnóstico , Tomografia Computadorizada por Raios X
2.
Saudi Med J ; 40(11): 1123-1128, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31707409

RESUMO

OBJECTIVES: To evaluate the feasibility and outcomes of cochlear implantation (CI) in infancy. METHODS: All infants who underwent CI from January 2011 to October 2018 at a tertiary referral center in the Kingdom of Saudi Arabia were retrospectively reviewed. Demographic data, factors associated with early detection, and any surgical difficulties or postoperative complications were extracted from the medical records. The outcome of CI was determined by a speech pathologist. RESULTS: Fifteen patients underwent CI during the study period (9 bilateral and performed simultaneously, 6 unilateral). The round window was difficult to identify in 5 cases. Incomplete electrode insertion because of cochlear ossification secondary to meningitis was documented in one patient. No major postoperative complications were encountered. The average auditory performance score was 7 and the speech intelligibility rating was 5. CONCLUSIONS: This study represents the largest national cohort of pediatric patients undergoing CI in infancy. In this series, the surgery was safe and the speech outcome was good. With implementation of the neonatal screening program in the Kingdom of Saudi Arabia, the number of infants undergoing CI is likely to increase in the near future, paving the way for more research in infant CI.


Assuntos
Implante Coclear , Surdez/fisiopatologia , Surdez/cirurgia , Avaliação de Resultados em Cuidados de Saúde , Fonoterapia , Fala , Criança , Pré-Escolar , Humanos , Lactente , Arábia Saudita
3.
J Colloid Interface Sci ; 505: 682-691, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28654883

RESUMO

A novel material composite nanofibers (PAN-CNT/TiO2-NH2) based on adsorption of Cr(VI) ions, was applied. Polyacrylonitrile (PAN) and carbon nanotube (CNTs)/titanium dioxide nanoparticles (TiO2) functionalized with amine groups (TiO2-NH2) composite nanofibers have been fabricated by electrospinning. The nanostructures and the formation process mechanism of the obtained PAN-CNT/TiO2-NH2 composite nanofibers are investigated using FTIR, XRD, XPS, SEM, and TEM. The composite nanofibers were used as a novel adsorbent for removing toxic chromium Cr(VI) in aqueous solution. The kinetic study, adsorption isotherm, pH effect, initial concentration, and thermodynamic study were investigated in batch experiments. The composite nanofibers had a positive effect on the absorption of Cr(VI) ions under neutral and acidic conditions, and the saturated adsorption reached the highest when pH was 2. The adsorption equilibrium reached within 30 and 180min with an initial solution concentration increasing from 10 to 300mg/L, and the process can be better described using nonlinear pseudo first than nonlinear pseudo second order model and Intra-particle diffusion. Isotherm data fitted well using linear and nonlinear Langmuir, Freundlich, Redlich-Peterson, and Temkin isotherm adsorption model. Thermodynamic study showed that the adsorption process is exothermic. The adsorption capacity can remain up to 80% after 5 times usage, which show good durability performance. The adsorption mechanism was also studied by UV-vis and XPS.

4.
Eur J Clin Microbiol Infect Dis ; 36(10): 1717-1731, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28573472

RESUMO

Point-of-care diagnosis based on nucleic acid testing aims to incorporate all the analytical steps, from sample preparation to nucleic acid amplification and detection, in a single device. This device needs to provide a low-cost, robust, sensitive, specific, and easily readable analysis. Microfluidics has great potential for handling small volumes of fluids on a single platform. Microfluidic technology has recently been applied to paper, which is already used in low-cost lateral flow tests. Nucleic acid extraction from a biological specimen usually requires cell filtration and lysis on specific membranes, while affinity matrices, such as chitosan or polydiacetylene, are well suited to concentrating nucleic acids for subsequent amplification. Access to electricity is often difficult in resource-limited areas, so the amplification step needs to be equipment-free. Consequently, the reaction has to be isothermal to alleviate the need for a thermocycler. LAMP, NASBA, HDA, and RPA are examples of the technologies available. Nucleic acid detection techniques are currently based on fluorescence, colorimetry, or chemiluminescence. For point-of-care diagnostics, the results should be readable with the naked eye. Nowadays, interpretation and communication of results to health professionals could rely on a smartphone, used as a telemedicine device. The major challenge of creating an "all-in-one" diagnostic test involves the design of an optimal solution and a sequence for each analytical step, as well as combining the execution of all these steps on a single device. This review provides an overview of available materials and technologies which seem to be adapted to point-of-care nucleic acid-based diagnosis, in low-resource areas.


Assuntos
Doenças Transmissíveis/diagnóstico , Equipamentos e Provisões , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Países em Desenvolvimento , Humanos
5.
Prog Mol Biol Transl Sci ; 142: 51-92, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27571692

RESUMO

The pectinolytic Dickeya spp. are Gram-negative bacteria causing severe disease in a wide range of plant species. Although the Dickeya genus was initially restricted to tropical and subtropical areas, two Dickeya species (D. dianthicola and D. solani) emerged recently in potato cultures in Europe. Soft-rot, the visible symptoms, is caused by plant cell wall degrading enzymes, mainly pectate lyases (Pels) that cleave the pectin polymer. However, an efficient colonization of the host requires many additional elements including early factors (eg, flagella, lipopolysaccharide, and exopolysaccharide) that allow adhesion of the bacteria and intermediate factors involved in adaptation to new growth conditions encountered in the host (eg, oxidative stress, iron starvation, and toxic compounds). To facilitate this adaptation, Dickeya have developed complex regulatory networks ensuring appropriate expression of virulence genes. This review presents recent advances in our understanding of the signals and genetic circuits impacting the expression of virulence determinants. Special attention is paid to integrated control of virulence functions by variations in the superhelical density of chromosomal DNA, and the global and specific regulators, making the regulation of Dickeya virulence an especially attractive model for those interested in relationships between the chromosomal dynamics and gene regulatory networks.


Assuntos
Enterobacteriaceae/patogenicidade , Interações Hospedeiro-Patógeno , Modelos Biológicos , Plantas/microbiologia , Adaptação Fisiológica , Virulência
6.
Cell Death Dis ; 7: e2227, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27195674

RESUMO

EEC (ectrodactily-ectodermal dysplasia and cleft lip/palate) syndrome is a rare genetic disease, autosomal dominant inherited. It is part of the ectodermal dysplasia disorders caused by heterozygous mutations in TP63 gene. EEC patients present limb malformations, orofacial clefting, skin and skin's appendages defects, ocular abnormalities. The transcription factor p63, encoded by TP63, is a master gene for the commitment of ectodermal-derived tissues, being expressed in the apical ectodermal ridge is critical for vertebrate limb formation and, at a later stage, for skin and skin's appendages development. The ΔNp63α isoform is predominantly expressed in epithelial cells and it is indispensable for preserving the self-renewal capacity of adult stem cells and to engage specific epithelial differentiation programs. Small interfering RNA (siRNA) offers a potential therapy approach for EEC patients by selectively silencing the mutant allele. Here, using a systemic screening based on a dual-luciferase reported gene assay, we have successfully identified specific siRNAs for repressing the EEC-causing p63 mutant, R304W. Upon siRNA treatment, we were able to restore ΔNp63-WT allele transcriptional function in induced pluripotent stem cells that were derived from EEC patient biopsy. This study demonstrates that siRNAs approach is promising and, may pave the way for curing/delaying major symptoms, such as cornea degeneration and skin erosions in young EEC patients.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Alelos , Sequência de Bases , Fenda Labial/metabolismo , Fenda Labial/patologia , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Displasia Ectodérmica/metabolismo , Displasia Ectodérmica/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Genes Dominantes , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Luciferases/genética , Luciferases/metabolismo , Terapia de Alvo Molecular , Mutação , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Síndrome , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo
7.
Clin Obes ; 5(1): 2-11, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25504829

RESUMO

Although obesity has been widely recognized for its consequences on physical health, its psychological burden in the adult populations in the Middle East remains unclear. This meta-analysis synthesized data from observational studies to investigate the association between obesity and depression among adult populations in Middle Eastern countries. Five bibliographical electronic databases were searched for studies published up to April 2014. Pooled meta-analytic estimates were derived using the random-effect models. Three case-control studies and five cross-sectional studies were identified. Meta-analysis showed significant positive associations between obesity and depression across study designs, with an overall effect of odds ratio 1.27 (95% confidence interval 1.11-1.44). The association between obesity and depression was more marked in women than men although that difference was not statistically significant. Other subgroup analysis showed that none of the potential factors including the assessment for obesity or depression, confounder control and study quality had a modification effect on the studied association. Meta-analysis of eight observational studies from five countries in the Middle East suggests an evidence of a positive association between obesity and depression among adult populations, which appeared to be more marked among women. Future research should examine the causal pathways between obesity and depression.


Assuntos
Antidepressivos/uso terapêutico , Depressão/psicologia , Obesidade/psicologia , Adulto , Estudos de Casos e Controles , Estudos Transversais , Depressão/tratamento farmacológico , Depressão/epidemiologia , Humanos , Oriente Médio/epidemiologia , Obesidade/epidemiologia , Obesidade/prevenção & controle , Padrões de Prática Médica , Distribuição por Sexo
8.
Osteoporos Int ; 22(9): 2499-506, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21069293

RESUMO

UNLABELLED: Crude incidence rates for hip fractures in individuals aged 50 and above in Lebanon were determined using data from the national hip fracture registry. For the years 2006-2008, crude rates varied between 164 and 188/100,000 for females and between 88 and 106 per 100,000 for males. Using the US 2000 white population as a reference, the calculated age-standardized rates were closest to rates derived for southern Europe. INTRODUCTION: Owing to the demographic explosion, it is projected that the rates of hip fractures would increase the most in the Middle East and Asia. Few are the population-based studies investigating the incidence of hip fractures in the region. METHODS: Using the Ministry of Health registry data, this population-based study evaluated the incidence of hip fractures in individuals aged 50 and above in Lebanon for the years 2006, 2007, and 2008. RESULTS: Hip fracture crude incidence rates varied across the years between 164 and 188 per 100,000 for females and between 88 and 106 per 100,000 for males, with a female/male ratio of 1.6-2.1. The overall mean age (SD) for hip fractures was 75.9 (9.2), 76.8 (9.0), and 77.0 (9.9) years in females in 2006, 2007, and 2008, respectively, and 74.4 (11.6), 76.3 (10.3), and 74.0 (12.1) years in males, respectively. Using the US 2000 white population as a reference, the age-standardized rates were 370.4, 335.1, and 329.0 for females and 109.7, 134.1, and 128.7 for males, for the years 2006, 2007, and 2008, respectively. CONCLUSIONS: The hip fracture age-standardized incidence rates in the Lebanese subjects receiving Ministry of Health coverage were lower than those found in northern Europe and the US and closest to rates derived for southern Europe.


Assuntos
Fraturas do Quadril/epidemiologia , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Incidência , Líbano/epidemiologia , Masculino , Pessoa de Meia-Idade , Sistema de Registros
9.
J Mol Microbiol Biotechnol ; 3(2): 309-18, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11321588

RESUMO

The pecS regulatory locus negatively modulates the expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Previous studies have shown that PecS down-regulates the expression of both pecSand pecMgenes and that PecM is required for full PecS activity. Computer-aided hydropathy analysis of PecM predicted the presence of between 8 to 10 potential transmembrane segments. We analyzed the membrane topology of PecM using the beta-lactamase gene fusion system and obtained the following unique characteristics. PecM contains 10 membrane spanning segments, with both the amino and carboxyl termini located in the cytoplasmic side of the inner membrane. The fourth periplasmic loop, which has a relatively long hydrophilic domain containing 17 amino acid residues, may play an important role in PecM function. The topological model obtained for PecM can be applied to PecM homologues in other bacteria. Measurement of the extrusion of the blue pigment indigoidine by the E. chrysanthemi derivative isogenic mutants pecS, pecM and pecS-pecM revealed that PecM is required for complete efflux of the pigment. Its relation to other efflux systems and its potential physiological role are discussed.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/patogenicidade , Proteínas de Membrana/química , Proteínas de Membrana/genética , Pigmentos Biológicos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Primers do DNA , Dickeya chrysanthemi/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
10.
Pediatr Neurosurg ; 34(1): 40-2, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11275785

RESUMO

Refractory gelastic seizures are often associated with hypothalamic hamartoma (HH). Presurgical evaluation in such children often points to a distinct cortical region as the source of the seizures. A case of a child with HH and refractory seizures is presented. Video-EEG monitoring revealed a well-defined epileptic focus in the left frontal region. In accordance with the current understanding of the nature of hamartoma-related seizures, the hamartoma was resected. Follow-up evaluations revealed a marked improvement in seizure frequency and global functioning.


Assuntos
Epilepsia Reflexa/cirurgia , Hamartoma/cirurgia , Doenças Hipotalâmicas/cirurgia , Riso , Pré-Escolar , Consanguinidade , Diagnóstico Diferencial , Eletroencefalografia , Epilepsia Reflexa/etiologia , Epilepsia Reflexa/genética , Hamartoma/diagnóstico , Hamartoma/genética , Humanos , Doenças Hipotalâmicas/diagnóstico , Doenças Hipotalâmicas/genética , Hipotálamo/patologia , Hipotálamo/cirurgia , Imageamento por Ressonância Magnética , Masculino , Telemetria , Gravação em Vídeo
11.
J Biol Chem ; 276(21): 17878-86, 2001 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-11279109

RESUMO

The DNA architectural proteins FIS and CRP are global regulators of transcription in Escherichia coli involved in the adjustment of cellular metabolism to varying growth conditions. We have previously demonstrated that FIS modulates the expression of the crp gene by functioning as its transcriptional repressor. Here we show that in turn, CRP is required to maintain the growth phase pattern of fis expression. We demonstrate the existence of a divergent promoter in the fis regulatory region, which reduces transcription of the fis promoter. In the absence of FIS, CRP activates fis transcription, thereby displacing the polymerase from the divergent promoter, whereas together FIS and CRP synergistically repress fis gene expression. These results provide evidence for a direct cross-talk between global regulators of cellular transcription during the growth phase. This cross-talk is manifested in alternate formation of functional nucleoprotein complexes exerting either activating or repressing effects on transcription.


Assuntos
Proteínas de Transporte/genética , Proteína Receptora de AMP Cíclico/genética , Proteínas de Escherichia coli , Sequência de Bases , Proteínas de Transporte/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli , Fator Proteico para Inversão de Estimulação , Regulação Bacteriana da Expressão Gênica , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica
12.
Mol Plant Microbe Interact ; 14(1): 10-20, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11194867

RESUMO

The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25 degrees C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dickeya chrysanthemi/metabolismo , Dickeya chrysanthemi/patogenicidade , Plantas/microbiologia , Proteínas de Bactérias/genética , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Dickeya chrysanthemi/genética , Genes Bacterianos , Magnoliopsida/microbiologia , Mutação , Doenças das Plantas/microbiologia , Polissacarídeo-Liases/biossíntese , Polissacarídeo-Liases/genética , Transcrição Gênica , Virulência/genética
13.
Folia Phoniatr Logop ; 52(4): 178-86, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10782010

RESUMO

The study was aimed at finding what factors in evoked potentials and EEG related to stuttering in subjects 6-25 years of age. Thirty-seven subjects who stuttered and 25 nonstuttering subjects, matched for age, sex and education, were evaluated employing visual evoked potentials, auditory evoked potentials, event-related potentials (P(300)), WISC-(IQ), and electroencephalography. A significant reduction of amplitude of P(100) of visual evoked potentials was found in stutterers with a significant prolongation of wave latencies I, III, V and interpeak latencies I-III and I-V in brainstem auditory evoked potentials. No significant abnormalities were recorded in P(200), N(200) and P(300) of event-related potentials in stutterers compared with the control group. The dominant EEG rhythm was slower in stutterers with a significant interhemispheric asymmetry compared with the control group. Fifty-four percent of the stutterers had pathological EEG. Epileptiform activities were recorded in 16.2% of stuttering subjects. Focal left temporal spike activity was recorded in 5.4% of stuttering subjects. The findings of this study point to a possible role of an organic etiopathogenesis of stuttering.


Assuntos
Eletroencefalografia , Gagueira/fisiopatologia , Adolescente , Adulto , Encéfalo/fisiopatologia , Mapeamento Encefálico , Criança , Dominância Cerebral/fisiologia , Potenciais Evocados , Feminino , Humanos , Masculino , Tempo de Reação/fisiologia , Gagueira/diagnóstico
14.
Mol Microbiol ; 34(3): 562-75, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10564497

RESUMO

Activation of the two divergent Escherichia coli cai and fix operons involved in anaerobic carnitine metabolism is co-dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the specific carnitine-sensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, purified on a heparin column and ran as a 15 kDa protein on SDS-PAGE. DNase I footprinting and interference experiments identified two sites, F1 and F2, with apparently comparable affinities for the binding of CaiF in the cai-fix regulatory region. These sites share a common perfect inverted repeat comprising two 11 bp half-sites separated by 13 bp, and centred at -70 and -127 from the fix transcription start site. They were found to overlap the two low-affinity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co-operatively to the F1/CRP2 and F2/CRP3 sites of the intergenic cai-fix region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP-CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the fix promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N-terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.


Assuntos
Carnitina/metabolismo , Colicinas , Proteína Receptora de AMP Cíclico/genética , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Transativadores/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carnitina/genética , Proteínas de Transporte , Proteína Receptora de AMP Cíclico/metabolismo , Pegada de DNA , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Immunoblotting , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Transativadores/isolamento & purificação , Transativadores/metabolismo , Transcrição Gênica
15.
Mol Microbiol ; 34(4): 641-50, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10564505

RESUMO

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes including several pectate lyases encoded by the pel genes. We characterized a novel cluster of pectinolytic genes consisting of the three adjacent genes pehV, pehW and pehX, whose products have polygalacturonase activity. The high similarity between the three genes suggests that they result from duplication of an ancestral gene. The transcription of pehV, pehW and pehX is dependent on several environmental conditions. They are induced by pectin catabolic products and this induction results from inactivation of the KdgR repressor which controls almost all the steps of pectin catabolism. The presence of calcium ions strongly reduced the transcription of the three peh genes. Their expression was also affected by growth phase, osmolarity, oxygen limitation and nitrogen starvation. In addition, the pehX transcription is affected by catabolite repression and controlled by the activator protein CRP. PecS, which was initially isolated as a repressor of virulence factors, acts as an activator of the peh transcription. We showed that the three regulators KdgR, PecS and CRP act by direct interaction with the promoter regions of the peh genes. Analysis of simultaneous binding of KdgR, PecS, CRP and RNA polymerase indicated that the activator effect of PecS results from a competition between PecS and KdgR for the occupation of overlapping binding sites. Thus, to activate peh transcription, PecS behaves as an anti-repressor against KdgR.


Assuntos
Dickeya chrysanthemi/genética , Genes Bacterianos , Poligalacturonase/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte , Proteína Receptora de AMP Cíclico/metabolismo , DNA Bacteriano/análise , Dickeya chrysanthemi/enzimologia , Dickeya chrysanthemi/crescimento & desenvolvimento , Dickeya chrysanthemi/patogenicidade , Glicosídeo Hidrolases/genética , Dados de Sequência Molecular , Família Multigênica , Poligalacturonase/biossíntese , Poligalacturonase/fisiologia , Polissacarídeo-Liases/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico
16.
J Bacteriol ; 181(19): 5948-57, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10498706

RESUMO

The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases which attack pectin, the major constituent of the plant cell wall. Of these enzymes, the alkaline isoenzyme named PelD in strain 3937 and PelE in strain EC16 has been described as being particularly important, based on virulence studies of plants. Expression of the pelD and pelE genes is tightly modulated by various regulators, including the KdgR repressor and the cyclic AMP-cyclic AMP receptor protein (CRP) activator complex. The use of a lacZ reporter gene allowed us to quantify the repression of E. chrysanthemi 3937 pelD expression exerted by PecS, another repressor of pectinase synthesis. In vitro DNA-protein interaction experiments, centered on the pelD and pelE wild-type or pelE mutated promoter regions, allowed us to define precisely the sequences involved in the binding of these three regulators and of RNA polymerase (RNAP). These studies revealed an unusual binding of the KdgR repressor and suggested the presence of a UP (upstream) element in the pelD and pelE genes. Investigation of the simultaneous binding of CRP, KdgR, PecS, and the RNAP to the regulatory region of the pelD and pelE genes showed that (i) CRP and RNAP bind cooperatively, (ii) PecS partially inhibits binding of the CRP activator and of the CRP-RNAP complex, and (iii) KdgR stabilizes the binding of PecS and prevents transcriptional initiation by RNAP. Taken together, our data suggest that PecS attenuates pelD and pelE expression rather than acting as a true repressor like KdgR. Overall, control of the pelD and pelE genes of E. chrysanthemi appears to be both complex and novel.


Assuntos
Dickeya chrysanthemi/genética , Regulação Bacteriana da Expressão Gênica , Polissacarídeo-Liases/biossíntese , Fatores de Transcrição/metabolismo , Álcalis , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína Receptora de AMP Cíclico/metabolismo , Dickeya chrysanthemi/enzimologia , Genes Bacterianos , Isoenzimas/biossíntese , Modelos Genéticos , Dados de Sequência Molecular , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/metabolismo , Transcrição Gênica
17.
Mol Plant Microbe Interact ; 12(5): 385-90, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10226371

RESUMO

Previously, we have cloned and characterized the pir (plant inducible regulator) gene, which is responsible for hyperinduction of the synthesis of an isozyme of pectate lyase (PLe) in Erwinia chrysanthemi EC16 in the presence of potato extract and sodium polypectate (NaPP). The Pir protein purified from Escherichia coli overexpressing pir is able to bind to the promoter region of pir as a dimer. Self-regulation of pir by its own translational product (Pir) was suggested from the findings that Pir binds at the promoter region of pir and that the hyperinduction of the pirlux construct in response to plant extract was observed only in pir+ but not in pir mutant EC16. Thus, hyperinduction of PLe was thought to be mainly due to overproduction of Pir. On the other hand, KdgR and PecS, which have been reported to be the major regulatory proteins for the synthesis of pectic enzymes, did not bind to the promoter region of pir. Thus, the regulation of Pir synthesis seems to be independent of KdgR and PecS. Also, its expression was insensitive to catabolite repression as predicted from failure of cyclic AMP (cAMP)-CRP (cAMP recognizing protein) to bind at the pir promoter region.


Assuntos
DNA Helicases/biossíntese , DNA Helicases/genética , Proteínas de Ligação a DNA , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Polissacarídeo-Liases/biossíntese , Transativadores/biossíntese , Transativadores/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , DNA Helicases/metabolismo , Primers do DNA/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Dickeya chrysanthemi/enzimologia , Indução Enzimática , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/metabolismo
18.
Biochim Biophys Acta ; 1442(2-3): 148-60, 1998 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-9804934

RESUMO

Erwinia chrysanthemi is a broad host range phytopathogenic enterobacterium responsible for soft-rot disease of many plant species. The pecT gene encodes a repressor that negatively regulates the expression of virulence factors, such as pectinases, motility or exopolysaccharide synthesis. The cloned pecT gene was overexpressed using a phage T7 system. The purification of PecT involved the use of a TSK-heparin column and delivered the PecT protein that was purified to near homogeneity. The purified repressor displayed a 34 kDa apparent molecular mass. Gel-filtration experiments revealed that the PecT protein is a dimer. Band-shift assays demonstrated that the tetramer of the PecT protein could specifically bind in vitro to the regulatory regions of the pectate lyase genes with variable affinities. In addition, we demonstrated that PecT represses its own synthesis by interacting independently with two 200 bp regions, R1 and R2, located from -382 to -632 and -17 to -234, respectively, from the distal P1 promoter and from -465 to -715 and -100 to -317 from the P2 proximal promoter. We propose a model that explains the regulation exerted by PecT on its target genes and that integrates the phenotype obtained with a PecT overproducing pec-1 mutant or a pecT mutant.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dickeya chrysanthemi/enzimologia , Dickeya chrysanthemi/genética , Polissacarídeo-Liases/genética , Sequências Reguladoras de Ácido Nucleico , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição , Transcrição Gênica , Proteínas de Bactérias/biossíntese , Sequência de Bases , Clonagem Molecular , Escherichia coli , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Plantas/microbiologia , Plasmídeos , Reação em Cadeia da Polimerase , Polissacarídeo-Liases/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Proteínas Repressoras/metabolismo , Transativadores/biossíntese
19.
Proc Natl Acad Sci U S A ; 95(24): 14034-9, 1998 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-9826648

RESUMO

The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein.


Assuntos
Proteínas de Bactérias , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Dickeya chrysanthemi/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Polissacarídeo-Liases/genética , Transativadores/genética , Transativadores/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Parede Celular/microbiologia , DNA Helicases/química , Dickeya chrysanthemi/enzimologia , Dickeya chrysanthemi/patogenicidade , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Plantas/microbiologia , Polissacarídeo-Liases/biossíntese , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/química , Virulência/genética
20.
Mol Microbiol ; 29(6): 1391-405, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9781877

RESUMO

The plant pathogen Erwinia chrysanthemi produces three acyl-homoserine lactones (acyl-HSLs). One has been identified as N-(3-oxohexanoyl)-homoserine lactone (OHHL), and the two others were supposed to be N (hexanoyl)-homoserine lactone (HHL) and N-(decanoyl)-homoserine lactone (DHL). The genes for a quorum-sensing signal generator (expI) and a response regulator (expR) were cloned. These genes are convergently transcribed and display high similarity to the expI-expR genes of Erwinia carotovora. ExpI is responsible for both OHHL and HHL production. Inactivation of expl had little effect on pectinase synthesis in E. chrysanthemi, as expression of only two of the pectate lyase genes, pelA and pelB, was decreased. E. chrysanthemi expR mutants still produced acyl-HSL and pectinases. However, gel shift and DNAse I footprinting experiments showed that the purified E. chrysanthemi ExpR protein binds specifically to the promoter regions of the five major pel genes. Addition of OHHL modified the ExpR-DNA bandshift profiles, indicating that ExpR interacts with OHHL and binds to DNA in different ways, depending on the OHHL concentration. Localization of the ExpR binding sites just upstream of promoter regions suggests that ExpR functions as an activator of pel expression in the presence of OHHL. The absence of a phenotype in expR mutants strongly suggests that at least an additional interchangeable ExpR homologue exists in E. chrysanthemi. Finally, transcription of expI::uidA and expR::uidA fusions is dependent on the population density, suggesting the existence of a quorum-sensing hierarchy in E. chrysanthemi. These results suggest that the expI-expR locus is part of a complex autoregulatory system that controls quorum sensing in E. chrysanthemi.


Assuntos
4-Butirolactona/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Genes Bacterianos , Homosserina/análogos & derivados , Transativadores , 4-Butirolactona/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Dickeya chrysanthemi/patogenicidade , Homosserina/biossíntese , Dados de Sequência Molecular , Mutação , Pectobacterium carotovorum/genética , Feromônios/biossíntese , Plantas/microbiologia , Poligalacturonase/biossíntese , Polissacarídeo-Liases/genética , Transdução de Sinais , Especificidade da Espécie
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