RESUMO
The UDP-glucose 4-epimerase (GALE) is a glycosyltransferase, which acts on protein and lipid glycosylation in normal and neoplastic cells. This study is aimed at investigating the differential tissue expression of GALE and its possible association with clinical-pathological parameters and the outcome of gastric adenocarcinoma patients. Seventy-one patients were evaluated in relation to GALE expression by immunohistochemistry. Our results showed that 48 (67.6%) patients were GALE positive and 23 (32.4%) negative. Positive staining was present on well-differentiated and moderate-differentiated histological grade of gastric adenocarcinomas (p < 0.0001). There was no significant association with outcome parameters (p > 0.05). Besides that, our results corroborated with the validation cohort analysis, where the expression of GALE mRNA was also associated with the histological grade (p < 0.001). These results suggest a possible use of this enzyme as a biomarker for well and moderately differentiated tumors.
Assuntos
Adenocarcinoma/secundário , Biomarcadores Tumorais/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias Gástricas/patologia , UDPglucose 4-Epimerase/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Terapia Combinada , Feminino , Seguimentos , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/terapia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/terapiaRESUMO
The aim of this work was to obtain insights about the factors that determine the lactose fermentative metabolism of Kluyveromyces marxianus UFV-3. K. marxianus UFV-3 and Kluyveromyces lactis JA6 were cultured in a minimal medium containing different lactose concentrations (ranging from 0.25 to 64 mmol l(-1)) under aerobic and hypoxic conditions to evaluate their growth kinetics, gene expression and enzymatic activity. The increase in lactose concentration and the decrease in oxygen level favoured ethanol yield for both yeasts but in K. marxianus UFV-3 the effect was more pronounced. Under hypoxic conditions, the activities of ß-galactosidase and pyruvate decarboxylase from K. marxianus UFV-3 were significantly higher than those in K. lactis JA6. The expression of the LAC4 (ß-galactosidase), RAG6 (pyruvate decarboxylase), GAL7 (galactose-1-phosphate uridylyltransferase) and GAL10 (epimerase) genes in K. marxianus UFV-3 was higher under hypoxic conditions than under aerobic conditions. The high expression of genes of the Leloir pathway, LAC4 and RAG6, associated with the high activity of ß-galactosidase and pyruvate decarboxylase contribute to the high fermentative flux in K. marxianus UFV-3. These data on the fermentative metabolism of K. marxianus UFV-3 will be useful for optimising the conversion of cheese whey lactose to ethanol.
Assuntos
Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Kluyveromyces/metabolismo , Lactose/metabolismo , Micologia/métodos , Aerobiose , Anaerobiose , Biomassa , Meios de Cultura , Laticínios , Indução Enzimática , Etanol/metabolismo , Fermentação , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Kluyveromyces/enzimologia , Kluyveromyces/genética , Kluyveromyces/crescimento & desenvolvimento , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , RNA Fúngico/genética , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da Espécie , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Thyroid nodules are a common clinical problem, and fine-needle aspiration biopsy (FNAB) is widely used for its evaluation. Only 5% are malignant, being papillary carcinoma (PC) the most frequent neoplasia. Approximately 20% are classified as indeterminate or suspicious for malignancy. Gene-expression pattern may be useful for diagnosing PC in difficult or ambiguous cases. In our prior study, we were able to apply RT-PCR method in a series of routinely performed FNAB of thyroid nodules using individual, residual samples. In this study, a total of 70 thyroid samples were evaluated for the expression of MPPED2, H/HBA2, MET, FN1, GALE, and QPCT genes, including 24 cases of frozen thyroid tissue, 12 nodular hyperplasia and 12 PC, and the 46 consecutive thyroid FNAB samples, previously analyzed (3 positive, 10 indeterminate and 32 negative for malignancy, and 1 insufficient). FN1, GALE, MET, and QPCT mRNA expression were significantly different in benign and malignant samples, with similar pattern of overexpression in aspirates compared to frozen tissue. H/HBA2 and MPPED2 expression varied. Histological correlation was possible in five indeterminate cases, revealing one PC and four benign lesions. In conclusion, FN1, GALE, MET, and QPCT were significantly overexpressed in thyroid PC. RT-PCR method could be applied to routine FNAB, showing a similar pattern of overexpression. Despite the small number of cases evaluated, our results suggest that molecular analysis may be of assistance in patients with indeterminate/suspicious cytology, adding elements for preoperative diagnosis and better management of these patients.
Assuntos
Aminoaciltransferases/metabolismo , Carcinoma Papilar/metabolismo , Fibronectinas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , UDPglucose 4-Epimerase/metabolismo , Aminoaciltransferases/genética , Biópsia por Agulha Fina , Carcinoma , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/genética , Fibronectinas/genética , Expressão Gênica , Humanos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , UDPglucose 4-Epimerase/genética , alfa-Globinas/genética , alfa-Globinas/metabolismoRESUMO
A cluster of five genes, proposed to be involved in the formation of extracellular polysaccharide (EPS) precursors via the Leloir pathway, have been identified in the acidophilic autotroph Acidithiobacillus ferrooxidans. The order of the genes is luxA-galE-galK-pgm-galM, encoding a LuxA-like protein, UDP-glucose 4-epimerase, galactokinase, phosphoglucomutase, and galactose mutarotase, respectively. The gal cluster forms a single transcriptional unit and is therefore an operon. Two other putative genes of the Leloir pathway, galU, potentially encoding UDP-glucose pyrophosphorylase, and a gene designated galT-like, which may encode a galactose-1-phosphate uridylyltransferase-like activity, were found unlinked in the genome. Using semiquantitative reverse transcription-PCR, the genes of the gal operon were shown to be expressed more during growth in iron medium than in growth in sulfur medium. The functions of galE, pgm, galU, and the galT-like gene were validated by complementation of Escherichia coli mutants and by in vitro enzyme assays. The data suggest that A. ferrooxidans is capable of synthesizing the EPS precursors UDP-glucose and UDP-galactose. In addition, genes rfbA, -B, -C, and -D were identified in the genome of A. ferrooxidans, suggesting that it can also synthesize the EPS precursor dTDP-rhamnose. Since EPSs constitute the major bulk of biofilms, this study may provide an initial model for the metabolic pathways involved in biofilm formation in A. ferrooxidans and aid in understanding the role of biofilms in mineral leaching and the formation of acid mine drainage.
Assuntos
Acidithiobacillus/metabolismo , Proteínas de Bactérias/metabolismo , Família Multigênica , Polissacarídeos Bacterianos/biossíntese , Uridina Difosfato Galactose/biossíntese , Uridina Difosfato Glucose/biossíntese , Acidithiobacillus/enzimologia , Acidithiobacillus/genética , Acidithiobacillus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Dados de Sequência Molecular , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Análise de Sequência de DNA , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismoRESUMO
Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.
Assuntos
Arabinose/fisiologia , Azospirillum brasilense/crescimento & desenvolvimento , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/química , Arabinose/análise , Azospirillum/genética , Azospirillum brasilense/citologia , Azospirillum brasilense/metabolismo , Carboidratos/análise , Cinética , Microscopia Eletrônica de Varredura , Fosfotransferases (Fosfomutases)/genética , Polissacarídeos Bacterianos/ultraestrutura , Triticum/crescimento & desenvolvimento , UDPglucose 4-Epimerase/genéticaRESUMO
Guillain-Barre Syndrome (GBS) is a neuromuscular disorder and campylobacteriosis is known to trigger the onset of the disorder. A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 497-bp region of the UDP-galactose 4-epimerase (galE) gene sequence in campylobacters responsible for triggering the onset of GBS. The identity of the PCR product was confirmed by Hind III endonuclease restriction digestion, which produced the predicted 430 and 67-bp DNA fragments. The assay could detect the presence of the gene in Campylobacter suspensions containing as few as 5 cells ml(-1). The assay detected the presence of the gene in 17 of the 20 campylobacters isolated from chicken, 9 of the 13 campylobacters isolated from turkey and 7 of the 7 campylobacters isolated from human stools. All Campylobacter strains isolated from chicken, turkey and clinical samples were resistant to multiple antibiotics. The assay failed to detect the presence of the gene in five different microaerophilic strains of Helicobacter spp., E. coli and Salmonella spp. The entire diagnostic assay, including template preparation, amplification and electrophoresis, can be completed within 6 h.
Assuntos
Infecções por Campylobacter/genética , Campylobacter/genética , Síndrome de Guillain-Barré/genética , Produtos Avícolas/microbiologia , UDPglucose 4-Epimerase/genética , Animais , Campylobacter/isolamento & purificação , Infecções por Campylobacter/complicações , Galinhas , Primers do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Fezes/microbiologia , Microbiologia de Alimentos , Síndrome de Guillain-Barré/complicações , Humanos , Reação em Cadeia da Polimerase , Mapeamento por Restrição , PerusRESUMO
AIMS: To evaluate the relationship between exopolysaccharide (EPS) production and the sugar nucleotide biosynthetic enzymes in Lactobacillus casei CRL 87 under optimum growth conditions for polymer formation: controlled pH on galactose or glucose. Studies with an EPS mutant were carried out to determine the key enzymes in EPS synthesis under the above culture conditions. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulphuric acid method, while the activities of the biosynthetic enzymes were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. An environmental pH of 5.0, using galactose as carbon source, markedly improved not only polymer production and yield but also, cell growth and lactic acid production. Analysis of the activities of the EPS precursor-forming enzymes revealed that polysaccharide synthesis was correlated with uridine-diphosphate (UDP)-glucose pyrophosphorylase and UDP-galactose 4-epimerase under these growth conditions. CONCLUSIONS: EPS synthesis by Lact. casei CRL 87 was considerably improved at a controlled pH of 5.0 with galactose as carbon source, and was correlated with the activity of UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase. The results obtained with the wild-type and EPS- strains suggest that UDP-galactose 4-epimerase plays an essential role in EPS formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Unravelling the key enzymes involved in EPS biosynthesis under optimum culture conditions for polymer production provides important information for the design of strategies, via genetic engineering, to enhance polysaccharide formation.
Assuntos
Lacticaseibacillus casei/enzimologia , Polissacarídeos Bacterianos/biossíntese , UDPglucose 4-Epimerase/metabolismo , Meios de Cultura , Fermentação , Galactose/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lacticaseibacillus casei/genética , Lacticaseibacillus casei/crescimento & desenvolvimento , Mutação/genética , Nucleotídeos/biossíntese , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus. These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production. The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain. The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors.
Assuntos
Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Polissacarídeos Bacterianos/biossíntese , Streptococcus/enzimologia , Streptococcus/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Mutação/fisiologia , Fenótipo , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/genética , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/genética , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismoRESUMO
Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.
Assuntos
Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Genoma Bacteriano , Proteínas Virais , Acetiltransferases/genética , Bactérias/efeitos dos fármacos , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , DNA Recombinante , Resistência a Múltiplos Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Deleção de Genes , Marcadores Genéticos , Vetores Genéticos , Gentamicinas/farmacologia , Integrases/genética , Canamicina/farmacologia , Canamicina Quinase/genética , Óperon Lac/genética , Mutagênese Insercional , Plasmídeos/genética , UDPglucose 4-Epimerase/genéticaRESUMO
Uridine 5'-diphosphate galacturonic acid (UDP-GalA) is a substrate for the galacturonosyltransferases that synthesize the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Pectin synthesis occurs in the Golgi and it is hypothesized that UDP-GalA is transported into the lumen of the Golgi by membrane-localized transporters. To study the transport and metabolism of UDP-GalA in the Golgi, UDP-GalA labeled in the uridine moiety is required. Here we present a high-yield method for the synthesis of [(3)H]UDP-GalA from [(3)H]UTP and Glc-1-P by sequential reactions catalyzed by UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and UDP-GlcA-4-epimerase and the separation of the reaction products over a Dionex CarboPac PA1 anion-exchange column using high-performance anion-exchange chromatography (HPAEC). Approximately half of the [(3)H]UTP was converted into [(3)H]UDP-GalA and the remaining 50% was recovered as [(3)H]UDP-GlcA. Both products were purified and the identity of the [(3)H]UDP-GalA was confirmed by its conversion into [(3)H]UDP-GlcA by UDP-GlcA-4-epimerase. The enzymatic synthesis of diverse nucleotide sugars radiolabeled in the nucleotide by the use of nucleotide-converting enzymes, combined with the high-resolution separation of the nucleotide sugars and their purification by HPAEC, can provide unique substrates required for the study of diverse nucleotide sugar transporters.