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1.
PLoS One ; 13(6): e0198207, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29856812

RESUMO

GALT is an important antigen of Actinobacillus pleuropneumoniae (APP), which was shown to provide partial protection against APP infection in a previous study in our lab. The main purpose of the present study is to investigate GALT induced cross-protection between different APP serotypes and elucidate key mechanisms of the immune response to GALT antigenic stimulation. Bioinformatic analysis demonstrated that galT is a highly conserved gene in APP, widely distributed across multiple pathogenic strains. Homologies between any two strains ranges from 78.9% to 100% regarding the galT locus. Indirect enzyme-linked immunosorbent assay (ELISA) confirmed that GALT specific antibodies could not be induced by inactivated APP L20 or MS71 whole cell bacterin preparations. A recombinant fusion GALT protein derived from APP L20, however has proven to be an effective cross-protective antigen against APP sevorar 1 MS71 (50%, 4/8) and APP sevorar 5b L20 (75%, 6/8). Histopathological examinations have confirmed that recombinant GALT vaccinated animals showed less severe pathological signs in lung tissues than negative controls after APP challenge. Immunohistochemical (IHC) analysis indicated that the infiltration of neutrophils in the negative group is significantly increased compared with that in the normal control (P<0.001) and that in surviving animals is decreased compared to the negative group. Anti-GALT antibodies were shown to mediate phagocytosis of neutrophils. After interaction with anti-GALT antibodies, survival rate of APP challenged vaccinated animals was significantly reduced (P<0.001). This study demonstrated that GALT is an effective cross-protective antigen, which could be used as a potential vaccine candidate against multiple APP serotypes.


Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/imunologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Pleuropneumonia/veterinária , Doenças dos Suínos/prevenção & controle , UDPglucose-Hexose-1-Fosfato Uridiltransferase/imunologia , Infecções por Actinobacillus/prevenção & controle , Actinobacillus pleuropneumoniae/classificação , Actinobacillus pleuropneumoniae/genética , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência Conservada , Avaliação Pré-Clínica de Medicamentos/veterinária , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização Secundária , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/imunologia , Fagocitose/imunologia , Pleuropneumonia/patologia , Pleuropneumonia/prevenção & controle , Distribuição Aleatória , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sorogrupo , Suínos , Doenças dos Suínos/imunologia , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Vacinação/veterinária
2.
Hum Mutat ; 39(1): 52-60, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28961353

RESUMO

Galactosemia Proteins Database 2.0 is a Web-accessible resource collecting information about the structural and functional effects of the known variations associated to the three different enzymes of the Leloir pathway encoded by the genes GALT, GALE, and GALK1 and involved in the different forms of the genetic disease globally called "galactosemia." It represents an evolution of two available online resources we previously developed, with new data deriving from new structures, new analysis tools, and new interfaces and filters in order to improve the quality and quantity of information available for different categories of users. We propose this new resource both as a landmark for the entire world community of galactosemia and as a model for the development of similar tools for other proteins object of variations and involved in human diseases.


Assuntos
Bases de Dados de Proteínas , Navegador , Galactosemias/genética , Galactosemias/metabolismo , Variação Genética , Humanos , Conformação Proteica , Relação Estrutura-Atividade , UDPglucose 4-Epimerase/química , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/química , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo
3.
J Inherit Metab Dis ; 40(3): 325-342, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28281081

RESUMO

Classic galactosemia is a rare inherited disorder of galactose metabolism caused by deficient activity of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme of the Leloir pathway. It presents in the newborn period as a life-threatening disease, whose clinical picture can be resolved by a galactose-restricted diet. The dietary treatment proves, however, insufficient in preventing severe long-term complications, such as cognitive, social and reproductive impairments. Classic galactosemia represents a heavy burden on patients' and their families' lives. After its first description in 1908 and despite intense research in the past century, the exact pathogenic mechanisms underlying galactosemia are still not fully understood. Recently, new important insights on molecular and cellular aspects of galactosemia have been gained, and should open new avenues for the development of novel therapeutic strategies. Moreover, an international galactosemia network has been established, which shall act as a platform for expertise and research in galactosemia. Herein are reviewed some of the latest developments in clinical practice and research findings on classic galactosemia, an enigmatic disorder with many unanswered questions warranting dedicated research.


Assuntos
Galactosemias/enzimologia , Galactosemias/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Animais , Galactose/metabolismo , Humanos
4.
J Inherit Metab Dis ; 40(1): 131-137, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27783170

RESUMO

Despite adequate dietary management, patients with classic galactosemia continue to have increased risks of cognitive deficits, speech dyspraxia, primary ovarian insufficiency, and abnormal motor development. A recent evaluation of a new galactose-1 phosphate uridylyltransferase (GALT)-deficient mouse model revealed reduced fertility and growth restriction. These phenotypes resemble those seen in human patients. In this study, we further assess the fidelity of this new mouse model by examining the animals for the manifestation of a common neurological sequela in human patients: cerebellar ataxia. The balance, grip strength, and motor coordination of GALT-deficient and wild-type mice were tested using a modified rotarod. The results were compared to composite phenotype scoring tests, typically used to evaluate neurological and motor impairment. The data demonstrated abnormalities with varying severity in the GALT-deficient mice. Mice of different ages were used to reveal the progressive nature of motor impairment. The varying severity and age-dependent impairments seen in the animal model agree with reports on human patients. Finally, measurements of the cerebellar granular and molecular layers suggested that mutant mice experience cerebellar hypoplasia, which could have resulted from the down-regulation of the PI3K/Akt signaling pathway.


Assuntos
Ataxia/genética , Galactosemias/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo/genética , Camundongos , Atividade Motora/genética , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/genética
5.
Carbohydr Res ; 413: 70-4, 2015 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-26101844

RESUMO

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme that catalyzes both acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthesis pathway. Our previous study demonstrated that the uridyltransferase domain of GlmU (tGlmU) exhibited a flexible substrate specificity, which could be further applied in unnatural sugar nucleotides preparation. However, the structural basis of tolerating variant substrates is still not clear. Herein, we further investigated the roles of several highly conserved amino acid residues involved in substrate binding and recognition by structure- and sequence-guided site-directed mutagenesis. Out of total 16 mutants designed, tGlmU Q76E mutant which had a novel catalytic activity to convert CTP and GlcNAc-1P into unnatural sugar nucleotide CDP-GlcNAc was identified. Furthermore, tGlmU Y103F and N169R mutants were also investigated to have enhanced uridyltransferase activities compared with wide-type tGlmU.


Assuntos
Domínio Catalítico , Sequência Conservada , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutagênese Sítio-Dirigida , UDPglucose-Hexose-1-Fosfato Uridiltransferase/química , Acetilglucosamina/metabolismo , Biocatálise , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complexos Multienzimáticos/genética , Mutação , Estrutura Secundária de Proteína
6.
Gene ; 559(2): 112-8, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25592817

RESUMO

Classical galactosemia is an autosomal recessive inborn error of metabolism due to mutations of the GALT gene leading to toxic accumulation of galactose and derived metabolites. With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. However, despite early diagnosis and treatment, the long term outcome for these patients is still unpredictable because they may go on to develop cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30years. No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications. A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of <1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype-phenotype correlation in classical galactosemia and underlines the importance of molecular diagnostic testing prior to making any treatment.


Assuntos
Galactosemias/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Galactosemias/diagnóstico , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Itália , Masculino , Mutação de Sentido Incorreto , Triagem Neonatal , Adulto Jovem
7.
Eur J Hum Genet ; 23(4): 500-6, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25052314

RESUMO

In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia.


Assuntos
DNA Antissenso/farmacologia , Galactosemias/terapia , Processamento de RNA , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Animais , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Dicroísmo Circular , Fragmentação do DNA , Galactosemias/genética , Testes Genéticos , Variação Genética , Células HeLa , Humanos , Íntrons , Mutação , Oligonucleotídeos/farmacologia , Precursores de RNA/genética , Sítios de Splice de RNA , RNA Mensageiro/genética , Análise de Sequência de DNA
8.
Genetics ; 198(4): 1559-69, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25298520

RESUMO

Type III galactosemia is a metabolic disorder caused by reduced activity of UDP-galactose-4-epimerase, which participates in galactose metabolism and the generation of various UDP-sugar species. We characterized gale-1 in Caenorhabditis elegans and found that a complete loss-of-function mutation is lethal, as has been hypothesized for humans, whereas a nonlethal partial loss-of-function allele causes a variety of developmental abnormalities, likely resulting from the impairment of the glycosylation process. We also observed that gale-1 mutants are hypersensitive to galactose as well as to infections. Interestingly, we found interactions between gale-1 and the unfolded protein response.


Assuntos
Caenorhabditis elegans/genética , Galactosemias/genética , Galactosemias/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Desintegrinas/metabolismo , Hexosaminas/metabolismo , Redes e Vias Metabólicas , Metaloendopeptidases/metabolismo , Morfogênese/genética , Mutação , Fenótipo , Transporte Proteico , Transdução de Sinais , UDPglucose-Hexose-1-Fosfato Uridiltransferase/deficiência , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Resposta a Proteínas não Dobradas , Açúcares de Uridina Difosfato/metabolismo
9.
Clin Chim Acta ; 436: 298-302, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-24973740

RESUMO

BACKGROUND: The California newborn screening program uses newborns' dried blood spots (DBS) to screen for more than 45 genetic disorders. Deficiency of galactose-1-phosphate uridyl transferase (GALT) is one of the metabolic genetic disorders screened using newborn DBS. During follow-up tests, common mutations of the GALT gene have been identified using whole blood samples. To avoid the stress of drawing an additional blood sample from newborns who are identified as presumptive positive for galactosemia, we developed a method to test common mutations in the GALT gene using blood spots. METHODS: This method involves DNA extraction from DBS, followed by polymerase chain reaction (PCR), and single nucleotide extension (SNE). SNE products were detected by capillary electrophoresis. RESULTS: In a double-blind study, GALT gene common mutations/variants: IVS2-2A>G, p.S135L, p.T138M, p.Q188R, p.L195P, p.Y209C, p.L218L, p.K285N, and p.N314D were detected in seventy-three DBS which had previously been screened and confirmed as positive in the California Newborn Screening Program. Mutations found using blood spots gave 100% concordance with mutations from previously genotyped whole blood samples. CONCLUSIONS: This blood spot method decreases the genomic test turnaround time of GALT screened positive patients and potentially reduces emotional stress on families required to provide an additional blood draw.


Assuntos
Análise Mutacional de DNA/métodos , Teste em Amostras de Sangue Seco , Mutação , UDPglucose-Hexose-1-Fosfato Uridiltransferase/sangue , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Método Duplo-Cego , Técnicas de Genotipagem , Humanos , Recém-Nascido
10.
Appl Environ Microbiol ; 79(22): 7028-35, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24014529

RESUMO

The GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1, galT2, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.


Assuntos
Proteínas de Bactérias/metabolismo , Bifidobacterium/enzimologia , Oligossacarídeos/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , Bifidobacterium/crescimento & desenvolvimento , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosefosfatos/metabolismo , Deleção de Genes , Humanos , Leite Humano/química , Dados de Sequência Molecular , Família Multigênica , Plasmídeos/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Especificidade por Substrato
11.
Biochim Biophys Acta ; 1832(8): 1279-93, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23583749

RESUMO

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.


Assuntos
Galactosemias/enzimologia , Deficiências na Proteostase/enzimologia , UDPglucose-Hexose-1-Fosfato Uridiltransferase/química , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Galactosemias/etiologia , Galactosemias/genética , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Desnaturação Proteica , Deficiências na Proteostase/etiologia , Deficiências na Proteostase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética
12.
Dis Model Mech ; 6(1): 84-94, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22773758

RESUMO

Classic galactosemia is a genetic disorder that results from profound loss of galactose-1P-uridylyltransferase (GALT). Affected infants experience a rapid escalation of potentially lethal acute symptoms following exposure to milk. Dietary restriction of galactose prevents or resolves the acute sequelae; however, many patients experience profound long-term complications. Despite decades of research, the mechanisms that underlie pathophysiology in classic galactosemia remain unclear. Recently, we developed a Drosophila melanogaster model of classic galactosemia and demonstrated that, like patients, GALT-null Drosophila succumb in development if exposed to galactose but live if maintained on a galactose-restricted diet. Prior models of experimental galactosemia have implicated a possible association between galactose exposure and oxidative stress. Here we describe application of our fly genetic model of galactosemia to the question of whether oxidative stress contributes to the acute galactose sensitivity of GALT-null animals. Our first approach tested the impact of pro- and antioxidant food supplements on the survival of GALT-null and control larvae. We observed a clear pattern: the oxidants paraquat and DMSO each had a negative impact on the survival of mutant but not control animals exposed to galactose, and the antioxidants vitamin C and α-mangostin each had the opposite effect. Biochemical markers also confirmed that galactose and paraquat synergistically increased oxidative stress on all cohorts tested but, interestingly, the mutant animals showed a decreased response relative to controls. Finally, we tested the expression levels of two transcripts responsive to oxidative stress, GSTD6 and GSTE7, in mutant and control larvae exposed to galactose and found that both genes were induced, one by more than 40-fold. Combined, these results implicate oxidative stress and response as contributing factors in the acute galactose sensitivity of GALT-null Drosophila and, by extension, suggest that reactive oxygen species might also contribute to the acute pathophysiology in classic galactosemia.


Assuntos
Galactosemias/genética , Galactosemias/metabolismo , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Cisteína/metabolismo , Dimetil Sulfóxido/toxicidade , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Galactose/metabolismo , Galactose/toxicidade , Galactosemias/tratamento farmacológico , Galactosemias/etiologia , Galactosefosfatos/metabolismo , Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Genes de Insetos , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Mutação , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Espécies Reativas de Oxigênio/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/deficiência , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Xantonas/farmacologia
13.
Adv Nutr ; 3(3): 422S-9S, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22585921

RESUMO

Intestinal colonization of bifidobacteria is important for the health of infants. Human milk oligosaccharides (HMO) have been identified as growth factors for bifidobacteria. Recently, a bifidobacterial enzymatic system to metabolize HMO was identified. 1,3-ß-Galactosyl-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which catalyzes the reversible phosphorolysis of galacto-N-biose (GNB) (Galß1→3GalNAc)] and lacto-N-biose I (LNB) (Galß1→3GlcNAc), is a key enzyme to explain the metabolism of HMO. Infant-type bifidobacteria possess the intracellular pathway to specifically metabolize GNB and LNB (GNB/LNB pathway). Bifidobacterium bifidum possesses extracellular enzymes to liberate LNB from HMO. However, Bifidobacterium longum subsp. infantis imports intact HMO to be hydrolyzed by intracellular enzymes. Bifidobacterial enzymes related to the metabolism of HMO are useful tools for preparing compounds related to HMO. For instance, LNB and GNB were produced from sucrose and GlcNAc/GalNAc in 1 pot using 4 bifidobacterial enzymes, including GLNBP. LNB is expected to be a selective bifidus factor for infant-type strains.


Assuntos
Bifidobacterium/enzimologia , Bifidobacterium/crescimento & desenvolvimento , Leite Humano/química , Oligossacarídeos/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Galactosiltransferases/metabolismo , Humanos , Lactente , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Fosforilases/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo
14.
Eur J Med Chem ; 53: 150-8, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22521370

RESUMO

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the formation of UDP-GlcNAc, a fundamental precursor in cell wall biosynthesis. GlmU represents an attractive target for new antibacterial agents. In this study, a theoretical three-dimensional (3D) structure of GlmU from Xanthomonas oryzae pv. oryzae (Xo-GlmU) was generated, and the ligand-receptor interaction was investigated by molecular docking. Then a structure-based virtual screening was performed, three hit compounds were identified as specific inhibitors of the uridyltransferase activity of Xo-GlmU, with IC(50) values in the 0.81-23.21 µM range. Subsequently, the mode-of-inhibition and K(i) values of the three inhibitors were confirmed. The minimum inhibitory concentrations (MICs) of the candidate compounds for X. oryzae pv. oryzae (Xoo) were also determined. The research provided novel chemical scaffolds for antimicrobial drug discovery.


Assuntos
Inibidores Enzimáticos/farmacologia , UDPglucose-Hexose-1-Fosfato Uridiltransferase/antagonistas & inibidores , UDPglucose-Hexose-1-Fosfato Uridiltransferase/química , Interface Usuário-Computador , Xanthomonas/enzimologia , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Proteica , Homologia de Sequência de Aminoácidos , Xanthomonas/efeitos dos fármacos
15.
Antonie Van Leeuwenhoek ; 101(3): 541-50, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22068918

RESUMO

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/metabolismo
16.
Carbohydr Res ; 346(15): 2432-6, 2011 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-21955790

RESUMO

2-Acetamido-2-deoxy-D-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-D-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.


Assuntos
Acetilgalactosamina/síntese química , Acetilglucosamina/química , Biocatálise , Acetilgalactosamina/isolamento & purificação , Acetilglucosamina/isolamento & purificação , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Bifidobacterium/enzimologia , Cromatografia por Troca Iônica , Ensaios Enzimáticos , Cinética , Fosfotransferases (Aceptor do Grupo Álcool)/química , UDPglucose 4-Epimerase/química , UDPglucose-Hexose-1-Fosfato Uridiltransferase/química , Uridina Difosfato Glucose/química , Uridina Difosfato N-Acetilglicosamina/química
17.
Acta Ophthalmol ; 89(5): 489-94, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20222886

RESUMO

PURPOSE: Observations of multiple ocular malformations together with heterozygosity for galactosaemia in siblings and homozygosity in one child are highly unusual. In these case histories, a series of investigations in one family are reported. METHODS: Members of a family of two brothers and one sister and their children were pre- and post-surgically examined over several years. Blood examination was carried out in a laboratory specializing in investigation into genetic diseases (Dr Podskarbi, Munich). RESULTS: Two brothers and one sister suffered from cataract-induced visual deterioration at 38, 34 and 35 years of age, respectively. All three siblings reported having had bilateral poor vision since early childhood. The three siblings' parents had no congenital ocular malformations, nor was there any parental consanguinity. One child, the 10-year-old son of the 35-year-old sister, exhibited classic galactosaemia and normal ocular findings. This sister's other child was healthy. All three siblings presented congenital lens luxation, axial myopia, cataract and iridodonesis. In addition, the 34-year-old brother showed unilateral right corectopia and left coloboma adjacent to the optic disc. The 38-year-old brother revealed myopic fundus changes, but no coloboma. The three siblings experienced a distinct increase in visual acuity after cataract surgery. Both eyes of the patients were partially or distinctly amblyopic, respectively. We assume an autosomal-recessive transmission. Molecular genetic examination of the 10-year-old child with classic galactosaemia showed homozygosity for the mutation Q188R with a complete galactose-1-phosphate-uridyltransferase (GALT) deficiency. Because of his galactose-free diet, the child showed normal values for galactose-1-phosphate. The 35-year-old mother showed compound heterozygosity for Q188R and G1391A (D2/G). The 10-year-old boy's father also revealed heterozygosity for galactosaemia caused by GALT deficiency. The two children of the 38-year-old brother were heterozygous for G1391A. They did not show any clinical abnormality. None of the family members had clinical signs of Marfan's syndrome or homocysteinuria. The three siblings' parents were not consanguineous. CONCLUSIONS: Patients with worsening cataracts occurring at a pre-senile age should be examined for galactosaemia. We describe for the first time the molecular genetic findings in congenital ectopia lentis et pupillae. Early treatment in conjunction with a galactose-free diet is mandatory in patients with galactosaemia. Members of a family with heterozygosity for galactosaemia should be advised to attend a human genetic consultation.


Assuntos
Anormalidades do Olho/genética , Galactosemias/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Adulto , Catarata/complicações , Catarata/genética , Anormalidades do Olho/complicações , Saúde da Família , Feminino , Galactosemias/complicações , Humanos , Subluxação do Cristalino/complicações , Subluxação do Cristalino/genética , Masculino , Miopia/complicações , Miopia/genética , Linhagem , Mutação Puntual , Distúrbios Pupilares/complicações , Distúrbios Pupilares/genética , Irmãos
18.
Heredity (Edinb) ; 104(2): 148-54, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19639008

RESUMO

Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.


Assuntos
Galactosemias/enzimologia , Frequência do Gene , Mutação de Sentido Incorreto , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Europa (Continente) , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Galactosemias/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , UDPglucose-Hexose-1-Fosfato Uridiltransferase/deficiência
19.
Nat Cell Biol ; 11(9): 1157-63, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19701194

RESUMO

Mounting an effective host immune response without incurring inflammatory injury requires the precise regulation of cytokine expression. To achieve this, cytokine mRNAs are post-transcriptionally regulated by diverse RNA-binding proteins and microRNAs (miRNAs) targeting their 3' untranslated regions (UTRs). Zcchc11 (zinc-finger, CCHC domain-containing protein 11) contains RNA-interacting motifs, and has been implicated in signalling pathways involved in cytokine expression. The nature of the Zcchc11 protein and how it influences cytokine expression are unknown. Here we show that Zcchc11 directs cytokine expression by uridylating cytokine-targeting miRNAs. Zcchc11 is a ribonucleotidyltransferase with a preference for uridine and is essential for maintaining the poly(A) tail length and stability of transcripts for interleukin-6 (IL-6) and other specific cytokines. The miR-26 family of miRNAs targets IL-6, and the addition of terminal uridines to the miR-26 3' end abrogates IL-6 repression. Whereas 78% of miR-26a sequences in control cells contained 1-3 uridines on their 3' ends, less than 0.1% did so in Zcchc11-knockdown cells. Thus, Zcchc11 fine tunes IL-6 production by uridylating miR-26a, which we propose is an enzymatic modification of the terminal nucleotide sequence of mature miRNA as a means to regulate gene expression.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-6/genética , MicroRNAs/metabolismo , Uridina Monofosfato/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação a DNA/química , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Interleucina-6/metabolismo , Camundongos , Dados de Sequência Molecular , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo
20.
Carbohydr Res ; 344(12): 1449-52, 2009 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-19560126

RESUMO

All monodeoxygenated galactoses were treated with galactokinase, and for the 2-, 3-, and 4-deoxy compounds, transformation into the corresponding galactopyranosyl phosphates could be observed. In case of the 2-deoxy derivative, further reaction via UDP-2-deoxy-D-lyxo-hexose (UDP-2-deoxygalactose), which was also obtained chemically, the multiple enzymatic system could be employed to prepare 2'-deoxy-N-acetyllactosamine.


Assuntos
Galactosiltransferases/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Uridina Difosfato Galactose/análogos & derivados , Amino Açúcares/química , Amino Açúcares/metabolismo , Galactoquinase/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Especificidade por Substrato , Uridina Difosfato Galactose/química , Uridina Difosfato Galactose/metabolismo
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