RESUMO
BACKGROUND: γ-Aminobutyric acid-B receptor antibodies (GABA(B)R-ab) were recently described in 15 patients with limbic encephalitis (LE), associated with small-cell lung cancer (SCLC) or with concurrent glutamic acid decarboxylase (GAD) antibodies. We analyzed the frequency of GABA(B)R-ab in 147 patients with LE or neurologic syndromes associated with GAD-ab. METHODS: We examined the presence of GABA(B)R-ab in 70 patients with LE (33 paraneoplastic with onconeural antibodies, 18 paraneoplastic without onconeural antibodies [5 with Gad-ab], and 19 idiopathic with either GAD-ab [5 patients] or seronegative) and 77 patients with GAD-ab-associated neurologic syndromes other than LE (29 stiff-person syndrome, 28 cerebellar ataxia, 14 epilepsy, and 6 with diverse paraneoplastic neurologic syndromes). GABA(B)R-ab were analyzed in serum or CSF by indirect immunofluorescence on HEK293 cells transfected with GABA(B1) and GABA(B2) receptor subunits. RESULTS: GABA(B)R-ab were detected in 10 of the 70 patients with LE (14%). Eight had SCLC and 2 were idiopathic. One of the 8 patients with LE with SCLC had an additional onconeural antibody (Hu) and 2 GAD-ab. GABA(B)R-ab were identified in 7 (70%) of the 10 patients with LE and SCLC without onconeural antibodies. GABA(B)R-ab antibodies were not found in patients with GAD-ab and stiff-person syndrome, idiopathic cerebellar ataxia, or epilepsy. However, one patient with GAD-ab, paraneoplastic cerebellar ataxia, and anaplastic carcinoid of the thymus also presented GABA(B)R-ab. CONCLUSIONS: GABA(B)R-ab are the most common antibodies found in LE associated with SCLC previously considered "seronegative." In patients with GAD-ab, the frequency of GABA(B)R-ab is low and only observed in the context of cancer.
Assuntos
Anticorpos/sangue , Glutamato Descarboxilase/sangue , Encefalite Límbica/sangue , Receptores de GABA-B/sangue , Idoso , Anticorpos/imunologia , Feminino , Seguimentos , Glutamato Descarboxilase/imunologia , Células HEK293 , Humanos , Encefalite Límbica/imunologia , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/sangue , Doenças do Sistema Nervoso/imunologia , Receptores de GABA-B/imunologiaAssuntos
Anticorpos/sangue , Encéfalo/metabolismo , Encefalite/sangue , Encefalite/imunologia , Receptores de AMPA/imunologia , Idoso , Encéfalo/patologia , Encéfalo/fisiopatologia , Linhagem Celular Transformada , Encefalite/tratamento farmacológico , Encefalite/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Transfecção/métodosRESUMO
BACKGROUND: The sudden changes (increase in capillary permeability, edema formation, vasodilation and hypotension) observed in septic patients and the measurements taken in order to revert this situation make vancomycin concentrations difficult to interpret. Therapeutic drug monitoring (TDM) of vancomycin is routinely performed at steady state, target concentrations are peaks between 20 and 40 mg/l and troughs of 5-10 mg/l. Lately, continuous infusion of vancomycin (CIV) has been used as an alternative mode of administration mainly in critically ill patients with sepsis or septic shock. Despite this novel mode of administration, the need of drug monitoring in this population is under discussion. OBJECTIVE: The aim of our study was to test the usefulness of a multi-compartment model in order to understand the rapid changes that occur in critically ill patients. MATERIALS AND METHODS: A prospective, cohort study was carried out in the intensive care unit of the University Hospital. 25 intensive care unit adults patients with severe sepsis or septic shock receiving vancomycin in CIV modality for documented gram-positive infections were included in the study. Once the infusion was started, blood samples were drawn periodically and analyzed by fluorescence polarisation immunoassay (FPIA, TDx, Abbott Laboratories, Chicago, IL, USA). A multi-compartment model was used to predict vancomycin level evolution throughout the treatment of patients with sepsis. RESULTS: High doses of vancomycin were administered in order to rescue patients from septic shock. Plasma drug concentration dropped while clinical condition of patients worsened. Conversely, drug levels increased spontaneously once the infection was reverted. The theoretical model provided greater insight into pharmacokinetic features related with the use of vancomycin in septic patients. CONCLUSIONS: There was consistency between the model based prediction and the experimental data so dose adjustment was performed in order to reach target concentrations above 20 mg/l and an initial dose of 3 grams of vancomycin per day was recommended to reach these levels.
Assuntos
Antibacterianos/farmacocinética , Monitoramento de Medicamentos/métodos , Unidades de Terapia Intensiva , Sepse/sangue , Vancomicina/farmacocinética , Antibacterianos/uso terapêutico , Imunoensaio de Fluorescência por Polarização , Humanos , Infusões Intravenosas , Modelos Biológicos , Estudos Prospectivos , Sepse/tratamento farmacológico , Índice de Gravidade de Doença , Choque Séptico/sangue , Choque Séptico/tratamento farmacológico , Fatores de Tempo , Vancomicina/uso terapêuticoRESUMO
Atrophy of the upper jaw poses problems for fixed rehabilitation of implants. Of the different management possibilities that have been developed, transzygomatic implant placement is a good option and affords a high success rate. These implants are positioned in zygomatic bone by using 2 possible techniques. We present a 48-year-old woman with severe posterior maxillary atrophy in which the canine and frontozygomatic prominence were used to place 6 anterior and 2 transzygomatic implants by using the sinus slot technique for fixed bridge rehabilitation. The advantages of the sinus slot technique over the sinus window technique are discussed.
Assuntos
Implantes Dentários , Prótese Dentária Fixada por Implante , Maxila/cirurgia , Seio Maxilar/cirurgia , Ligas Metalo-Cerâmicas , Aumento do Rebordo Alveolar , Atrofia , Transplante Ósseo , Implantação Dentária Endóssea/instrumentação , Implantação Dentária Endóssea/métodos , Planejamento de Dentadura , Prótese Total Superior , Revestimento de Dentadura , Feminino , Humanos , Maxila/patologia , Pessoa de Meia-Idade , Zigoma/cirurgiaRESUMO
El objetivo de este estudio fue establecer pautas para el monitoreo de Vancomicina cuando la misma es administrada intraventricularmente en lactantes. En este estudio se incluyeron once pacientes con derivaciones ventrículo-peritoneal. Todos los pacientes recibieron una dosis de Vancomicina intraventricular (IVT). Las muestras de líquido cefalorraquídeo (LCR) fueron analizadas y se calcularon los parámetros farmacocinéticos: constante de velocidad de eliminación (ke) y volumen de distribución (Vd) para poder llevar a cabo un ajuste posológico. Las medias referidas al Vd y a la semivida de eliminación para estos pacientes fueron de 244(±162) mL y 37.1(±23.3) horas respectivamente. En algunos pacientes se observó una gran variabilidad en el Vd. Este cambio en Vd se correlacionó con problemas en el tamaño ventricular o con ventrículos septados. En todos los casos, se propuso una nueva dosis de acuerdo a los parámetros calculados. Debido a variaciones en el sistema a lo largo de la terapia, se propuso un protocolo para la recolección de muestras de LCR de forma tal de individualizar la dosis de Vancomicina con los parámetros farmacocinéticos obtenidos
The objective of this study was to establish guidelines for the monitoring of intraventricularly administered Vancomycin in infants. Eleven patients with ventriculo-peritoneal shunts who developed ventriculitis were included in the study. All patients were given an intraventricular (IVT) dose of Vancomycin. Cerebrospinal fluid (CSF) samples were analysed and pharmacokinetic parameters: elimination rate constant (ke) and distribution volume (Vd) were calculated in order to adjust the dose. Mean values of Vd and elimination half-life for these patients were 244 (± 162) mL and 37.1 (±23.3) hours respectively. A great variability in the Vd was observed in some patients. This change in Vd correlates with problems in the ventricle size or with septated ventricles. In all cases a new dose was suggested according to the calculated parameters. Due to variations in the system throughout therapy, a protocol for CSF samples collection was proposed in order to individualise Vancomycin dosage according to pharmacokinetic parameters
Assuntos
Masculino , Feminino , Lactente , Humanos , Vancomicina/administração & dosagem , Vancomicina/antagonistas & inibidores , Líquido Cefalorraquidiano , Ventrículo de Músculo Esquelético/patologia , Ventrículos do Coração , Ventrículos do Coração/patologia , Monitoramento de Medicamentos/métodos , Infecções/tratamento farmacológico , Staphylococcus epidermidis , Vancomicina/metabolismo , Vancomicina/farmacologia , Vancomicina/farmacocinética , Staphylococcus epidermidis/isolamento & purificação , Técnica Indireta de Fluorescência para Anticorpo/métodosRESUMO
Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.
Assuntos
Citosol/enzimologia , Malato Desidrogenase/química , Catálise , Guanidina/química , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , NAD/química , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Proteínas Recombinantes/química , Sais , Temperatura , Termodinâmica , Thermus/enzimologiaRESUMO
The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described transketolase-like enzyme. To identify key residues for DXS activity, we compared the amino acid sequence of Escherichia coli DXS with that of E. coli and yeast transketolase (TK). Alignment showed a previously undetected conserved region containing an invariant histidine residue that has been described to participate in proton transfer during TK catalysis. The possible role of the conserved residue in E. coli DXS (H49) was examined by site-directed mutagenesis. Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity. These findings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity.
Assuntos
Eritritol/análogos & derivados , Eritritol/metabolismo , Escherichia coli/enzimologia , Açúcares de Poli-Isoprenil Fosfato/biossíntese , Fosfatos Açúcares/metabolismo , Transferases/química , Transferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico/genética , Sequência Conservada , Primers do DNA/genética , Escherichia coli/genética , Histidina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transferases/genética , Transcetolase/genéticaRESUMO
The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Fosfomicina/análogos & derivados , Complexos Multienzimáticos/biossíntese , Oxirredutases/biossíntese , Plastídeos/metabolismo , Solanum lycopersicum/fisiologia , Terpenos/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , DNA Complementar , Fosfomicina/farmacologia , Hibridização In Situ , Solanum lycopersicum/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Oxirredutases/química , Oxirredutases/genética , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
We report a novel fluorometric end-point assay for the determination of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity based on the reaction of 1-deoxy-D-xylulose 5-phosphate (DX5P) with 3,5-diaminobenzoic acid in an acidic medium to form a highly fluorescent quinaldine derivative. The assay was validated in three ways: (a) for a fixed amount of DXS in the reaction mixture the emitted fluorescence increased linearly with the reaction time, (b) for a fixed reaction time fluorescence intensity increased with the concentration of DXS in the reaction mixture, and (c) the increase in fluorescence intensity correlated (r = 0.99; P < 0.002) with the amount of DX5P formed in the reaction mixture determined radiometrically. The sensitivity of the fluorometric assay is similar to that of the previously described radiometric methods. This assay can be useful for the functional characterization of DXS as well as for the screening of DXS inhibitors with potential antibiotic, herbicidal, or antimalarial action.
Assuntos
Fluorometria/métodos , Frutose/análogos & derivados , Quinaldinas/análise , Transferases/isolamento & purificação , Aminobenzoatos/análise , Aminobenzoatos/química , Escherichia coli/enzimologia , Frutose/análise , Frutose/química , Quinaldinas/química , Proteínas Recombinantes/análise , Sensibilidade e Especificidade , Transferases/metabolismoRESUMO
Activation of V gamma 9/V delta 2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites. We suggested earlier that compounds synthesized by the 2-C:-methyl-D-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the V gamma 9/V delta 2 T cell reactivity of many pathogens. Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E. coli extracts to stimulate gamma delta T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome.
Assuntos
Enzimas , Eritritol/metabolismo , Hemiterpenos , Ativação Linfocitária , Compostos Organofosforados/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Fosfatos Açúcares/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Aldose-Cetose Isomerases/genética , Proteínas de Bactérias/genética , Fracionamento Celular , Eritritol/análogos & derivados , Eritritol/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Deleção de Genes , Humanos , Peso Molecular , Complexos Multienzimáticos/genética , Oxirredutases/genética , Engenharia de Proteínas , Transdução de Sinais/imunologia , Fosfatos Açúcares/fisiologia , Subpopulações de Linfócitos T/microbiologiaRESUMO
The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most eubacteria and plants and has remarkable biotechnological interest. However, only the first steps of this pathway have been determined. Using bioinformatic and genetic approaches, we have identified gcpE as a novel gene of the MEP pathway. The distribution of this gene in bacteria and plants strictly parallels that of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, which catalyses the first committed step of the MEP pathway. Our data demonstrate that the gcpE gene is essential for the MEP pathway in Escherichia coli and indicate that this gene is required for the trunk line of the isoprenoid biosynthetic route.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enzimas , Eritritol/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos/genética , Fosfatos Açúcares/metabolismo , Proteínas de Bactérias/química , Clonagem Molecular , Eritritol/análogos & derivados , Eritritol/química , Escherichia coli/química , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Fases de Leitura Aberta/genética , Homologia de Sequência , Fosfatos Açúcares/químicaRESUMO
Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.
Assuntos
Eritritol/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Hemiterpenos , Ácido Mevalônico/metabolismo , Compostos Organofosforados/metabolismo , Fósforo-Oxigênio Liases , Fosfatos Açúcares/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Carboxiliases/genética , Cromossomos Bacterianos/genética , DNA Recombinante/genética , Eritritol/análogos & derivados , Escherichia coli/enzimologia , Deleção de Genes , Genes Bacterianos/genética , Genes Essenciais/genética , Genes Fúngicos/genética , Genes Letais/genética , Humanos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Óperon/genética , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfatos de Poli-Isoprenil/metabolismo , Recombinação Genética/genéticaRESUMO
Plant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-specific isoform of phytoene synthase) in wild-type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.
Assuntos
Carotenoides/biossíntese , Solanum lycopersicum/enzimologia , Transferases/genética , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Northern Blotting , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Geranil-Geranildifosfato Geranil-Geraniltransferase , Hibridização In Situ , Isoenzimas/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Dados de Sequência Molecular , RNA de Plantas/análise , Transferases/metabolismoRESUMO
An alternative mevalonate-independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet. In this work, E. coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Escherichia coli/metabolismo , Hemiterpenos , Compostos Organofosforados/metabolismo , Arabinose/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Escherichia coli/genética , Ácido Mevalônico/metabolismo , Mutagênese Insercional , Reação em Cadeia da PolimeraseRESUMO
Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5' ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and influorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5' region of the MVK gene to the beta-glucuronidase (GUS) reporter gene, indicated that the MVK 5'-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions -295 and -194 of the MVK 5'-flanking region are crucial for high-level MVK gene expression.
Assuntos
Arabidopsis/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA de Plantas/química , DNA de Plantas/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucuronidase/genética , Glucuronidase/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Distribuição Tecidual , Transcrição GênicaRESUMO
Escherichia coli synthesizes its isoprenoids via the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MC4100dxs::CAT strain, defective in deoxyxylulose-5-phosphate synthase, which is the first enzyme in this metabolic route, exclusively synthesizes its isoprenoids from exogenous 2-C-methyl-D-erythritol (ME) added to the culture medium. The fate of the hydrogen atoms in the MEP pathway was followed by the incorporation of [1,1-(2)H(2)]ME and [3,5,5,5-(2)H(4)]ME. The two C-1 hydrogen atoms of ME were found without any loss in the prenyl chain of menaquinone and/or ubiquinone on the carbon atoms derived from C-4 of isopentenyl diphosphate (IPP) and on the E-methyl group of dimethylallyl diphosphate (DMAPP), the C-5 hydrogen atoms on the methyl groups derived from IPP C-5 methyl group and the Z-methyl group of DMAPP. This showed that no changes in the oxidation state of these carbon atoms occurred in the reaction sequence between MEP and IPP. Furthermore, no deuterium scrambling was observed between the carbon atoms derived from C-4 and C-5 of IPP or DMAPP, suggesting a completely stereoselective IPP isomerase or no significant activity of this enzyme. The C-3 deuterium atom of [3,5,5,5-(2)H(4)]ME was preserved only in the DMAPP starter unit and was completely missing from all those derived from IPP. This finding, aided by the non-essential role of the IPP isomerase gene, suggests the presence in E. coli of two different routes towards IPP and DMAPP, starting from a common intermediate derived from MEP.
Assuntos
Eritritol/análogos & derivados , Fosfatos Açúcares/metabolismo , Terpenos/metabolismo , Sequência de Bases , Primers do DNA , Deutério , Eritritol/química , Eritritol/metabolismo , Escherichia coli/genética , Genes Bacterianos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Fosfatos Açúcares/químicaRESUMO
Farnesyl diphosphate synthase (FPS), the enzyme that catalyses the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), is considered a regulatory enzyme of plant isoprenoid biosynthesis. The promoter regions of the FPS1 and FPS2 genes controlling the expression of isoforms FPS1S and FPS2, respectively, were fused to the beta-glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana plants. The FPS1S:GUS gene is widely expressed in all plant tissues throughout development, thus supporting a role for FPS1S in the synthesis of isoprenoids serving basic plant cell functions. In contrast, the FPS2:GUS gene shows a pattern of expression restricted to specific organs at particular stages of development. The highest levels of GUS activity are detected in flowers, especially in pollen grains, from the early stages of flower development. After pollination, much lower levels of GUS activity are detected in the rest of floral organs, with the exception of the ovary valves, which remain unstained throughout flower development. GUS activity is also detected in developing and mature seeds. In roots, GUS expression is primarily detected at sites of lateral root initiation and in junctions between primary and secondary roots. No GUS activity is detected in root apical meristems. GUS expression is also observed in junctions between primary and secondary stems. Overall, the pattern of expression of FPS2:GUS suggests a role for FPS2 in the synthesis of particular isoprenoids with specialized functions. Functional FPS2 gene promoter deletion analysis in transfected protoplasts and transgenic A. thaliana plants indicate that all the cis-acting elements required to establish the full pattern of expression of the FPS2 gene are contained in a short region extending from positions -111 to +65. The potential regulatory role of specific sequences within this region is discussed.
Assuntos
Alquil e Aril Transferases/genética , Arabidopsis/genética , Glucuronidase/genética , Regiões Promotoras Genéticas/genética , Arabidopsis/crescimento & desenvolvimento , Sequência de Bases , DNA de Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Geraniltranstransferase , Glucuronidase/metabolismo , Histocitoquímica , Isoenzimas/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Protoplastos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , TransfecçãoRESUMO
OBJECTIVE: To compare the diagnostic prevalence of type-2 Diabetes Mellitus (DM2) according to the criteria of the American Diabetes Association (ADA-97) and the WHO. DESIGN: Crossover descriptive study. SETTING: Urban Health Centre, with a total of 34,234 clinical histories (CH). PATIENTS: 782 patients, selected through simple random sampling from medical records, aged 45 or over, with a minimum of 5 attendances recorded in their CH and at least 3 attendances in the previous 2 years, were studied. MEASUREMENTS AND MAIN RESULTS: Through review of the CH, the variables age, sex and presence of obesity (BMI3 30) were collected. DM2 patients were classified according to WHO criteria, in the three ADA-97 categories: DM2, basal glucaemia altered (BGA) and normal glucaemia (NG) and other situations with altered glucaemia. Mean age was 62.6 (SD 10.8), with 56.4% women. 95 patients (12.1%) had no glucaemia recorded. 15.6% of the patients with WHO criteria (10.7% through oral overload) were diagnosed with DM2, proportion that went up to 18.3% (CI 95% of difference: -1 to 6.4%) on application of the ADA-97 criteria. 12.8% had BGA, 54.1% NG and 2.7% other situations. Additional DM2 cases diagnosed with ADA-97 criteria had a greater proportion of obese people (p = 0.03), with no differences for age and sex found. CONCLUSIONS: We found a 2.7% increase in the prevalence of DM2 diagnosis in the 45 or over population on applying the ADA-97 criteria. These criteria simplify the diagnosis and will enable an appreciable number of patients not diagnosed under WHO criteria (since oral overload is little used) to be diagnosed. This will foment early action to retard micro and macrovascular complications caused by the disease.