RESUMO
All known field isolates of enterovirus 71 (EV71) can be divided into three distinct genogroups (A, B, C) and 10 subgenogroups (A, B1-5, C1-4) based on VP1 gene sequences. We examined VP1 gene sequences of 10, 12 and 11 EV71 strains isolated in peninsular Malaysia during the outbreaks of hand, foot and mouth disease in 1997, 2000 and 2005 respectively. Four EV71 strains isolated in the hand, foot and mouth disease outbreak of 2006 in Sarawak (Malaysian Borneo) were included to describe their genetic relationship. Four subgenogroups (C1, C2, B3 and B4) of EV71 co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two subgenogroups (C1 and B4) were noted to cause the outbreak in 2000. In the 2005 outbreak, besides EV71 strains of subgenogroup C1, EV71 strains belonged to subgenogroup B5 were isolated but formed a cluster which was distinct from EV71 strains of the subgenogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to subgenogroup B5. Phylogenetic analysis of the VP1 gene sequences showed that the four Sarawak EV71 isolates belonged to the same cluster as the EV71 strains that were isolated in peninsular Malaysia as early as May 2005. The data suggested that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.
Assuntos
Surtos de Doenças , Enterovirus Humano A/genética , Doença de Mão, Pé e Boca/genética , Doença de Mão, Pé e Boca/virologia , Sequência de Bases , Proteínas do Capsídeo/genética , Enterovirus Humano A/isolamento & purificação , Doença de Mão, Pé e Boca/epidemiologia , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15-30 micrograms/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 micrograms/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 micrograms/ml also inhibited phorbol myristate acetate-stimulated membrane potential change. In contrast, 30 micrograms/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 micrograms/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor-bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.
Assuntos
Actinas/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Fenobarbital/farmacologia , Explosão Respiratória/efeitos dos fármacos , Cálcio/metabolismo , Humanos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Polímeros/metabolismo , Receptores de Interleucina-2/análiseRESUMO
Six normal adult volunteers received 15 mg/kg of ethambutol (EMB) by mouth, once as an aqueous solution and again as the commercial tablet preparation. Each dose was separated by at least 7 days. Plasma and urine samples were collected at regular intervals for up to 24 and 72 hr, respectively. Peak plasma concentrations ranged from 3.25 to 5.62 mcg/ml, 2 to 4 hr after tablet dosage. Earlier peak times were found after administering the solution. For plasma concentrations up to 12 hr there was a distinct distribution phase followed by an apparent elimination phase with a mean half-life (t 1/2) (+/- SD) of 4.06 +/- 0.53 and 4.78 +/- 0.41 hr for the tablet and the solution, respectively. Excretion rate plots exhibited similar t 1/2 values for the apparent elimination phase. An even longer t 1/2 of approximately 10 hr was evident from 24-hr plasma samples and urinary excretion measurements up to 72 hr. Unchanged drug excreted in the urine averaged 61.1 +/- 3.8% of the dose for the tablet and 63.4 +/- 2.6% for the solution. Plasma protein binding for ethambutol determined by equilibrium dialysis and ultrafiltration was approximately 20% to 30%. The concentration ratio of ethambutol in erythrocytes to plasma ranged from 1.1 to 1.6.
Assuntos
Etambutol/metabolismo , Administração Oral , Adulto , Animais , Eritrócitos/metabolismo , Etambutol/administração & dosagem , Etambutol/sangue , Feminino , Meia-Vida , Haplorrinos , Humanos , Rim/metabolismo , Cinética , Macaca mulatta , Masculino , Ligação Proteica , Soluções , ComprimidosRESUMO
Platelet-derived growth factor (PDGF) is a potent stimulator of wound healing. PDGF gene therapy may promote greater periodontal regeneration than local protein application, due to sustained growth factor delivery to the target tissue. This investigation tested the ability of recombinant adenoviruses (rAds) encoding PDGF-A or PDGF-1308 (a PDGF-A dominant-negative mutant that disrupts endogenous PDGF bioactivity) to affect cells derived from the periodontium. Osteoblasts, periodontal ligament fibroblasts, and gingival fibroblasts were transduced with rAds, and gene expression, DNA synthesis, and cell proliferation were evaluated. The results revealed strong message for the PDGF-A gene for 7 days following gene delivery. Ad2/PDGF-A enhanced the mitogenic and proliferative response in all cell types, while Ad2/PDGF-1308 potently inhibited mitogenesis and proliferation. In conclusion, Ad2/PDGF can effectively transduce cells derived from the periodontium and promote biological activity equivalent to PDGF-AA. These studies support the potential use of gene therapy for sustained PDGF release in periodontal tissues.
Assuntos
Expressão Gênica , Ligamento Periodontal/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Transfecção , Adenoviridae/genética , Análise de Variância , Animais , Northern Blotting , Divisão Celular/genética , DNA/biossíntese , DNA/genética , Fibroblastos/metabolismo , Citometria de Fluxo , Vetores Genéticos , Humanos , Imuno-Histoquímica , Camundongos , Mutação/genética , Osteoblastos/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/fisiologia , Plasmídeos , Regeneração , Estatística como Assunto , Transdução Genética , CicatrizaçãoRESUMO
BACKGROUND: A challenge in the reconstruction of periodontal structures is the targeted delivery of growth-promoting molecules to the tooth root surface. Polypeptide growth factors such as platelet-derived growth factor (PDGF) stimulate both cementogenesis and osteogenesis. Recent advances in gene therapy offer the advantage of delivering recombinant proteins to tissues for extended periods of time in vivo. METHODS: Recombinant adenoviral vectors encoding for the PDGF-A gene were constructed to allow delivery of PDGF transgenes to cells. The recombinant adenoviruses were assembled using the viral backbone of Ad2/CMV/EGFP and replacing GFP (reporter gene encoding green fluorescent protein driven by the cytomegalovirus promoter [CMV] within adenovirus type 2) with the PDGF-A gene. Root lining cells (cloned cementoblasts) were transduced with Ad2/PDGF-A and evaluated for gene expression, DNA synthesis, and cell proliferation. PDGF-inducible genes, c-myc and osteopontin, were also evaluated following gene delivery of Ad2/PDGF-A. RESULTS: The results revealed high level transduction of cementoblasts by gene transfer for 7 days as evidenced by flow cytometry and Northern blotting. Cementoblast DNA synthesis and subsequent proliferation were stimulated by Ad2/PDGF-A at levels equal to or greater than continuous rhPDGF-AA application. Strong message for the PDGF-A gene and protein as evidenced by Northern blotting and immunocytochemistry was noted. Furthermore, the potent induction of c-myc and osteopontin mRNA was found after PDGF gene delivery to cementoblasts. CONCLUSIONS: These findings demonstrate that gene delivery of platelet-derived growth factor stimulates cementoblast activity that is sustained above that of rhPDGF-AA application. The use of gene therapy as a mode of growth factor delivery offers a novel approach to periodontal tissue engineering.
Assuntos
Terapia Genética , Doenças Periodontais/terapia , Fator de Crescimento Derivado de Plaquetas/genética , Adenoviridae/genética , Fosfatase Alcalina/genética , Animais , Northern Blotting , Divisão Celular/genética , Células Cultivadas , Citomegalovirus/genética , DNA/biossíntese , Cemento Dentário/citologia , Cemento Dentário/fisiologia , Citometria de Fluxo , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter/genética , Genes myc/genética , Engenharia Genética , Vetores Genéticos , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Osteogênese/genética , Osteopontina , Regiões Promotoras Genéticas/genética , Sialoglicoproteínas/genética , Estatística como Assunto , Transdução Genética , Transgenes/genéticaRESUMO
This study was designed to determine the changes in the degree of conversion throughout composite resins of varying thicknesses after heat curing and to evaluate whether or not the thin wafer technique that was applied in this study was sensitive to changes in distance from the light source. A 5 mm-in-diameter hole was made in a 4 mm-thick Teflon plate, and composite resin was placed in the hole and light cured from the top for 60 seconds. Twenty samples were prepared; 10 of these were additionally heat cured in an inlay oven. After light curing or light and heat curing, the samples were sectioned into four parts and assigned to groups A, B, C, or D according to their distance from the light source. These sections were then thinned to 50-70 microns, and analyzed by use of a standard baseline technique with a Fourier Transform Infrared Spectrometer (FT-IR) to determine the degree of conversion. The degree of conversion diminished as the distance from the light source increased; however, once the samples were heat cured, significant increases in the degree of conversion were noted throughout the samples. In the heat-cured composites, the degree of conversion in the outer portion of the sample was higher than in the inner portion. The thin wafer technique with FT-IR is considered to be a reliable method for measuring the degree of conversion in a composite resin, because changes between groups were clearly noted.
Assuntos
Resinas Compostas/química , Análise de Variância , Temperatura Alta , Luz , Teste de Materiais/métodos , Polímeros/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Tecnologia Odontológica/métodosRESUMO
The prevalence of HFMD as well as the causative agents was unknown in peninsular Malaysia prior to May 1997. From May 1997 to June 2001, 585 patients suspected to have enterovirus infections, with 467 patients clinically diagnosed as having HFMD, were investigated in the diagnostic virology unit of the University Malaya Medical Centre. Data from this study showed that HFMD is endemic in Malaysia with the occurrence of two outbreaks during the study period. In each outbreak, a number of viruses were isolated but enterovirus 71 was the main virus isolated in both outbreaks. Echovirus 7 (Eo7) was isolated from 5 patients with HFMD in the second outbreak, a clinical entity that has not been attributed to it previously. Children aged 4 years and below, particularly those between 1 and 2 years of age, were in the main group of patients affected by the illness. HFMD by itself and without neurological involvement was relatively benign and self-limiting. There was no significant difference in the virus isolation rate with respect to gender and ethnic groups. Virus isolation was attempted in a total of 764 clinical specimens consisting of 342 stool specimens, 285 oral secretions specimens and 137 vesicular fluid specimens. Oral specimens gave the highest virus isolation rate (33.3%) followed by vesicular specimens (27.0%). Stool specimens only yielded an isolation rate of 14.0%.
Assuntos
Doença de Mão, Pé e Boca/epidemiologia , Centros Médicos Acadêmicos/estatística & dados numéricos , Criança , Pré-Escolar , Feminino , Doença de Mão, Pé e Boca/virologia , Humanos , Lactente , Malásia/epidemiologia , Masculino , Estudos RetrospectivosRESUMO
Various modifications of alginate-poly-L-lysine microcapsules were made, such as the inclusion of polyethylenimine (PEI) or carboxyl methyl cellulose (CMC) in the core and the coating of bis(polyoxyethylene bis[amine]) (PEGA) onto the microcapsule membrane surface. A characterization of the modified microcapsules in terms of mechanical and mass transfer properties as well as their chemical composition was performed. The PEI treatment greatly enhanced the mechanical stability of the microcapsules, and this treatment did not affect the coating process of poly-L-lysine or PEGA. PEGA was found to be able to coat the microcapsules while the mass transfer property was similar to the poly-L-lysine coated ones.
Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Cápsulas , Polilisina/química , Carboximetilcelulose Sódica/química , Portadores de Fármacos/química , Composição de Medicamentos , Géis , Ácido Glucurônico , Ácidos Hexurônicos , Polietilenoglicóis/química , Polietilenoimina/química , Estresse Mecânico , Propriedades de Superfície , Resistência à TraçãoRESUMO
Cells in the secretion of involuting and non-lactating mammary glands of the ewe were studied. Light microscopy studies revealed that the majority of the cells were mononucleated, but it was impossible to distinguish macrophages from sloughed epithelial cells. Electron microscopy studies showed that the majority of the cells were macrophages and that they phagocytosed polystyrene latex spheres. Very few epithelial cells were found and they were distinguished from macrophages by numerous microfilaments and blunt microvilli. Epithelial cells could be further distinguished from macrophages by the presence of tight junctions. Tissue cultures of cells in secretion of mammary glands revealed the predominant cell type to be the macrophage. After 3 days in culture these macrophages formed multinucleated giant cells which contained acid-phosphatase granules. Fluorescein-labelled antiserum prepared against the cell cultures, stained cells in the lumens of alveoli and ducts, but not epithelial cells, in frozen sections of mammary tissue. The study confirms previous findings that the large majority of cells from mammary glands in late involution are macrophages. It also points to the usefulness of non-lactating mammary glands as a source of sheep and cattle macrophages.
Assuntos
Macrófagos/ultraestrutura , Glândulas Mamárias Animais/ultraestrutura , Ovinos/anatomia & histologia , Fosfatase Ácida/análise , Animais , Células Epiteliais , Epitélio/ultraestrutura , Feminino , Imunofluorescência , Lactação , Látex , Linfócitos/ultraestrutura , Macrófagos/enzimologia , Glândulas Mamárias Animais/citologia , Microesferas , Fagocitose , GravidezRESUMO
The purpose of this study was to evaluate apical extrusion of root canal content using ultrasonic and hand instrumentation. Forty-nine tooth models were fabricated with clear resin. Each model contained a canal in the center. Each tooth model was mounted in a plastic cube (1 x 1 x 2 cm) with white dental plaster so that the coronal 2-3 mm of the model was exposed for instrumentation. Methylene blue dye with glycerin was used as a marker for root canal content. The study consisted of three groups. In group I, Enac ultrasonic instrumentation was used 1 mm from the apex; in group II, Enac ultrasonic instrumentation was used 3 mm from the apex; in group III, K files were used with a push-pull instrumentation technique, 1 mm from the apex. After instrumentation the resin models were extracted and the plaster blocks were sectioned through the long axis of the models. Photographs were made of the area of apical leakage and the amount of dye penetration was measured using a planimeter. There were no differences between hand instrumentation and both ultrasonic groups. At p less than 0.05, ultrasonic instrumentation 3 mm from the apex leaked significantly more than the ultrasonic instrumentation 1 mm from the apex.
Assuntos
Preparo da Cavidade Dentária/instrumentação , Infiltração Dentária , Tratamento do Canal Radicular/instrumentação , Terapia por Ultrassom , Irrigantes do Canal Radicular , Tratamento do Canal Radicular/efeitos adversos , Raiz Dentária/anatomia & histologiaRESUMO
The concept of microfluidics has significantly influenced the design and the implementation of modern bioanalytical systems due to the fact that these miniaturized devices can handle and manipulate samples in a much more efficient way than conventional instruments. In an analogy to the development of microelectronics, increasingly sophisticated devices with greater functionalities have become one of the major goals being pursued in the area of micrototal analysis systems. The incorporation of polymeric membranes into microfluidic networks has therefore been employed in an effort to enhance the functionalities of these microfabricated devices. These commercially available membranes are porous, flexible, mechanically robust and compatible with plastic microfluidic networks. The large surface area-to-volume ratio of porous membrane media is particularly important for achieving rapid buffer exchange during microdialysis and obtaining ultrahigh concentration of adsorbed enzymes for various biochemical reactions. Furthermore, the membrane pore diameter in the sub-microm range eliminates the constraints of diffusional mass-transfer resistance for performing chiral separation using adsorbed protein as the chiral stationary phase. A review on the recent advancement in the integration of polymeric membranes with microfluidic networks is presented for their widespread applications in bioanalytical chemistry.
Assuntos
Bioquímica/instrumentação , Técnicas de Química Analítica/instrumentação , Membranas Artificiais , Polímeros/química , Adsorção , Aflatoxinas/análise , Aflatoxinas/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Cromatografia/instrumentação , Cromatografia/métodos , Difusão , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Contaminação de Alimentos , Microdiálise/instrumentação , Microdiálise/métodos , Miniaturização , Porosidade , Proteínas/isolamento & purificação , Reologia , Estereoisomerismo , Ultrafiltração/instrumentação , Ultrafiltração/métodosRESUMO
For this work, two different plastic microfluidic devices are designed and fabricated for applications in high-throughput residue analysis of food contaminants and drug screening of small-molecule libraries. Microfluidic networks on copolyester and poly(dimethylsiloxane) substrates are fabricated by silicon template imprinting and capillary molding techniques. The first device is developed to perform affinity capture, concentration, and direct identification of targeted compounds using electrospray ionization mass spectrometry. Poly(vinylidene fluoride) membranes sandwiched between the imprinted copolyester microchannels in an integrated platform provide continuous affinity dialysis and concentration of a reaction mixture containing aflatoxin B1 antibody and aflatoxins. The second microfluidic device is composed of microchannels on the poly(dimethylsiloxane) substrates. The device is designed to perform miniaturized ultrafiltration of affinity complexes of phenobarbital antibody and barbiturates, including the sequential loading, washing, and dissociation steps. These microfabricated devices not only significantly reduce dead volume and sample consumption but also increase the detection sensitivity by at least 1-2 orders of magnitude over those reported previously. Improvements in detection sensitivity are attributed to analyte preconcentration during the affinity purification step, limited analyte dilution in the microdialysis junction, minimal sample loss, and the amenability of ESI-MS to nanoscale sample flow rates.
Assuntos
Aflatoxinas/análise , Anticorpos Monoclonais/análise , Barbitúricos/análise , Avaliação Pré-Clínica de Medicamentos , Resíduos de Drogas/análise , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Aflatoxina B1/análise , Filtração/instrumentação , Filtração/métodos , Contaminação de Alimentos/análise , Membranas Artificiais , Fenobarbital/análise , Poliésteres , Siloxanas , Espectrometria de Massas por Ionização por Electrospray/métodos , Compostos de VinilaRESUMO
The application of the field-effect for direct control of electroosmosis in a polydimethylsiloxane (PDMS)-based microfluidic system, constructed on a silicon wafer with a 2.0 microm electrically insulating layer of silicon dioxide, is demonstrated. This microfluidic system consists of a 2.0 cm open microchannel fabricated on a PDMS slab, which can reversibly adhere to the silicon wafer to form a hybrid microfluidic device. Aside from mechanically serving as a robust bottom substrate to seal the channel and support the microfluidic system, the silicon wafer is exploited to achieve field-effect flow control by grounding the semiconductive silicon medium. When an electric field is applied through the channel, a radial electric potential gradient is created across the silicon dioxide layer that allows for direct control of the zeta potential and the resulting electroosmotic flow (EOF). By configuring this microfluidic system with two power supplies at both ends of the microchannel, the applied electric potentials can be varied for manipulating the polarity and the magnitude of the radial electric potential gradient across the silicon dioxide layer. At the same time, the longitudinal potential gradient through the microchannel, which is used to induce EOF, is held constant. The results of EOF control in this hybrid microfluidic system are presented for phosphate buffer at pH 3 and pH 5. It is also demonstrated that EOF control can be performed at higher solution pH of 6 and 7.4 by modifying the silicon wafer surface with cetyltrimethylammonium bromide (CTAB) prior to assembly of the hybrid microfluidic system. Results of EOF control from this study are compared with those reported in the literature involving the use of other microfluidic devices under comparable solution conditions.
Assuntos
Dimetilpolisiloxanos/química , Eletroforese Capilar/instrumentação , Microquímica/instrumentação , Silicones/química , Cetrimônio , Compostos de Cetrimônio/farmacologia , Eletroforese Capilar/métodos , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Microquímica/métodos , Reologia , Silício , Tensoativos/farmacologia , Moldes GenéticosRESUMO
Polyvinylchloride (PVC) resins are widely used in industry. Asthma due to the thermal degradation products of PVC are well documented. In this first case of occupational asthma due to unheated PVC resin dust the patient was exposed to PVC resin dust during the mixing of chemicals used for making plastic seals for bottle caps.
Assuntos
Asma/induzido quimicamente , Poeira/efeitos adversos , Doenças Profissionais/induzido quimicamente , Cloreto de Polivinila/efeitos adversos , Polivinil/efeitos adversos , Resinas Vegetais/efeitos adversos , Adulto , Humanos , MasculinoRESUMO
An in vitro fluid dynamic study was performed to compare the hemodynamic characteristics of a rigid and a flexible total artificial heart. The artificial ventricles were incorporated into a mock circulatory system, and pressure signals within the ventricular chamber, proximal to the inflow valve and distal to the outflow valve, were obtained. The instantaneous flow rate through the inflow and outflow valves was measured with electromagnetic flow probes. Flow visualization studies performed on the flexible ventricle suggested a vortical motion within the chamber with a smooth washout of fluid in the next pumping phase, but flow disturbances were observed near the wall of the ventricle as well as near the outflow valve. The rate of pressure increase (dP/dt) was smaller in the flexible ventricle as compared with the rigid ventricle for comparable flows and heart rates. The results of the present study indicated that the flexible ventricle with polyurethane valves, having the advantage of ease of implantation and cost savings, can be a viable alternative as a bridge to transplant.
Assuntos
Coração Artificial , Hemodinâmica/fisiologia , Humanos , Técnicas In Vitro , Modelos Cardiovasculares , Modelos Estruturais , Poliuretanos , Desenho de PróteseRESUMO
Human enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae) has been responsible for sporadic cases and outbreaks of hand-foot-and-mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like disease in Europe, the U.S.A., Australia and Asia. Recently, there has been an increase in EV71 activity in the Asia-Pacific region, with many outbreaks of HFMD associated with brainstem encephalitis manifesting as neurogenic pulmonary oedema with a high case fatality rate. In 1997, and again in 2000, EV71 outbreaks occurred in peninsular Malaysia. Variations in VP1 gene sequences have been shown to divide all known EV71 field isolates into three distinct genogroups (A, B and C). Consequently we examined the VP1 gene sequences of 43 EV71 strains isolated in peninsular Malaysia between 1997 and 2000 in order to determine the genogroup prevalence over the period. In this study we show that four subgenogroups (B3, B4, C1 and C2) of EV71 circulated in peninsular Malaysia between 1997 and 2000. Subgenogroups B3, B4 and C1 have been identified as the primary cause of the outbreaks of EV71 in peninsular Malaysia. Subgenogroup C1 also displayed endemic circulation from 1997 to 2000 and subgenogroup C2 was present at a low level during the 1997 outbreak.
Assuntos
Infecções por Enterovirus/epidemiologia , Enterovirus/genética , Doença de Mão, Pé e Boca/epidemiologia , Sequência de Aminoácidos , Sequência de Bases , Sequência Consenso , Primers do DNA , Surtos de Doenças , Enterovirus/classificação , Enterovirus/patogenicidade , Genótipo , Geografia , Doença de Mão, Pé e Boca/mortalidade , Humanos , Incidência , Malásia/epidemiologia , Epidemiologia Molecular/métodos , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
An unusual lon mutation (called lonR9) is dominant over the wild-type gene, which encodes the ATP-dependent protease La (Lon) in Escherichia coli, when present in multicopy plasmids. Here, we cloned and sequenced lonR9, and showed that the mutant gene carries a single point mutation in its open reading frame, which leads to replacement of Glu614 by Lys. The LonR9 protein and its poly-His-tagged form were purified to apparent homogeneity. Both of the purified proteins were capable of inhibiting the ATP-dependent proteolysis and the protein-activated ATP hydrolysis by protease La. Furthermore, the His-tagged LonR9 protein was found to form mixed oligomeric complexes with protease La, upon analysis by chromatography on a metal-chelating column. These results suggest that the phenotypic dominance of the lonR9 mutant is due to the formation of mixed oligomeric complexes between LonR9 and protease La, in which the defective components prevent the function of the wild-type subunits.