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1.
J Agric Food Chem ; 67(42): 11805-11814, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31566383

RESUMO

The impact of cross-breeding two low phytic acid (lpa) rice mutants on the content of phytic acid and the metabolite profile of the resulting double mutant was investigated. Progenies resulting from the cross of Os-lpa-XS110-1, a rice mutant carrying the myo-inositol kinase (OsMIK) mutated gene, and Os-lpa-XS110-2, with the multidrug resistance-associated protein ABC transporter gene 5 (OsMRP5) as the mutation target, were subjected to high-pressure ion chromatography. The reduction of the phytic acid content in the double mutant (-63%) was much more pronounced than in the single mutants (-26 and -47%). Gas chromatography-based metabolite profiling revealed a superimposition of the metabolite profiles inherited from the lpa progenitors in the double mutant progenies; the resulting metabolite signature was predominated by the OsMIK mutation effect. The study demonstrated that cross-breeding of two single lpa mutants can be employed to generate double lpa rice mutants showing both a significant reduction in the content of phytic acid and the imprinting of a specific mutation-induced metabolite signature.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Hibridização Genética , Oryza/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ácido Fítico/análise , Proteínas de Plantas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Mutação , Oryza/genética , Oryza/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácido Fítico/metabolismo , Proteínas de Plantas/metabolismo , Sementes/química , Sementes/genética , Sementes/metabolismo
2.
J Med Microbiol ; 68(10): 1534-1539, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368885

RESUMO

Introduction. Certain nontypeable Haemophilus influenzae cannot be assigned a sequence type (ST) by Multilocus Sequence Typing (MLST) due to the lack of the fucK gene, one of seven MLST loci in H. influenzae, which encodes a fucose-operon enzyme.Aims. To confirm whether the loss of fucK is also found in the encapsulated strains, we analysed clinical isolates of H. influenzae serotype e (Hie).Methodology. We conducted MLST, PFGE, and antimicrobial susceptibility tests of 45 Hie strains; the majority (n=43) were derived from respiratory samples of pediatric patients at Chiba Children's Hospital between 2000 and 2016. The two remaining strains were obtained from the blood of elderly patients with invasive H. influenzae diseases (IHiDs) between 2015 and 2016 at general hospitals. For the fucK-negative strains, PCR analysis for fucose operon was also performed.Results. Four STs (ST18, 122, 621 and 1758) were assigned to 13 strains, and remaining 32 (including one associated with IHiD) were fucK-negative, completely missing the fucose operon. The allelic profiles of six other loci were identical among 31 strains and to that of ST18, 122 and 621, and these strains were genetically closely related. Forty of 45 isolates were ampicillin-sensitive.Conclusions. The loss of fucK was frequently observed in clinical isolates of Hie from children. Moreover, fucK-negative Hie may be the cause of IHiD in adult patients. The majority of Hie, including fucK-negative strains, were shown to be clonally related and were ampicillin sensitive. This represents the first report examining fucK losses in encapsulated H. influenzae.


Assuntos
Proteínas de Bactérias/genética , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/isolamento & purificação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Adolescente , Ampicilina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Criança , Pré-Escolar , Feminino , Haemophilus influenzae/classificação , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Humanos , Japão , Masculino , Tipagem de Sequências Multilocus , Óperon , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Filogenia
3.
Microbiol Immunol ; 63(7): 285-288, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31166044

RESUMO

Phosphatidylinositol-4 kinase III ß (PI4KB) is a host factor that is required for enterovirus (EV) replication. In this study, the importance of host proteins that interact with PI4KB in EV replication was analyzed by trans complementation with PI4KB mutants in a PI4KB-knockout cell line. Ectopically expressed PI4KB mutants, which lack binding regions for ACBD3, RAB11, and 14-3-3 proteins, rescued replication of poliovirus and enterovirus 71. These findings suggest that interaction of PI4KB with these host proteins is not essential for EV replication once PI4KB has been expressed and that PI4KB is functionally independent from these host proteins regarding EV replication.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Enterovirus/metabolismo , Domínios e Motivos de Interação entre Proteínas , Replicação Viral/fisiologia , 1-Fosfatidilinositol 4-Quinase/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Linhagem Celular , Infecções por Enterovirus , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Poliovirus/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
4.
J Microbiol Biotechnol ; 29(6): 923-932, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154747

RESUMO

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce L-phenylalanine (LPhe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36- fold of the strain xllp1 (45.0 g/l). Moreover, the L-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/ g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.


Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Fenilalanina/biossíntese , Sistemas de Transporte de Aminoácidos Neutros/genética , Vias Biossintéticas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Glucose/metabolismo , Análise do Fluxo Metabólico , Mutação , Fenilalanina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regiões Promotoras Genéticas
5.
Appl Microbiol Biotechnol ; 103(13): 5259-5267, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31069485

RESUMO

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death among infectious diseases in the worldwide. Lack of more sensitive and effective diagnostic reagents has increased the awareness of rapid diagnosis for tuberculosis. In this study, T7 phage displayed genomic DNA library of M. tuberculosis was constructed to screen the antigens that specially bind with TB-positive serum from the whole genome of M. tuberculosis and to improve the sensitivity and specificity of tuberculosis serological diagnosis. After three rounds of biopanning, results of DNA sequencing and BLAST analysis showed that 19 positive phages displayed four different proteins and the occurrence frequency of the phage which displayed ribokinase was the highest. The results of indirect ELISA and dot immunoblotting indicated that representative phages could specifically bind to tuberculosis-positive serum. The prokaryotic expression vector containing the DNA sequence of ribokinase gene was then constructed and the recombinant protein was expressed and purified to evaluate the serodiagnosis value of ribokinase. The reactivity of the recombinant ribokinase with different clinical serum was detected and the sensitivities and specificities in tuberculosis serodiagnosis were 90% and 86%, respectively by screening serum from tuberculosis patients (n = 90) and uninfected individuals (n = 90) based on ELISA. Therefore, this study demonstrated that ribokinase had good potential for the serodiagnosis of tuberculosis.


Assuntos
Técnicas de Visualização da Superfície Celular , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriófago T7/genética , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Genoma Bacteriano , Biblioteca Genômica , Humanos , Immunoblotting , Lactente , Pessoa de Meia-Idade , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Testes Sorológicos , Tuberculose/sangue , Adulto Jovem
6.
J Clin Neurosci ; 66: 187-190, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088771

RESUMO

Pantothenate kinase-associated neurodegeneration (PKAN) is linked to brain iron accumulation caused by PANK2 gene mutation. Despite the importance of genetic testing to confirm PKAN and identify at risk parents, genetic screening is financially burdensome for developing countries like Thailand. Because genetic screeners are expensive and not reimbursed by the universal health care coverage system, they are not typically performed. To investigate clinical symptoms, radiological findings and mutation analysis for patients based in Thailand with unknown genetic status but suspected PKAN based on clinical symptoms. Genetic testing was performed for cases suspected for PKAN and their biological parents by direct genomic sequencing of PANK2 at Maharat Nakhon Ratchasima Hospital during 2017-2018. Clinical evaluation and documentation were performed by pediatric neurologists. Five children had classical onset form of PKAN. Most presented with gait dystonia. Three patients diagnosed after 4 years showed the eye-of-the-tiger sign in their brain MRI, whereas two younger patients revealed only isolated hyperintensity bilateral globus pallidus. However, PANK2 mutations were identified in all cases: the most common mutation was c.982-1G>C. This mutation was detected in four unrelated individuals but not reported in other studies. Genetic testing is recommended to confirm diagnoses in cases with supporting clinical features of PKAN with or without the classical 'eye-of-the-tiger-sign'. A novel PANK2 mutation (c.982-1G>C) was identified in South East Asian populations based in Thailand, suggesting that this genetic variant is a founder genotype in this population. Moreover, genetic diagnosis is helpful to provide appropriate genetic counseling to families.


Assuntos
Grupo com Ancestrais do Continente Asiático/etnologia , Grupo com Ancestrais do Continente Asiático/genética , Mutação/genética , Neurodegeneração Associada a Pantotenato-Quinase/etnologia , Neurodegeneração Associada a Pantotenato-Quinase/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Testes Genéticos/métodos , Globo Pálido/diagnóstico por imagem , Humanos , Imagem por Ressonância Magnética , Masculino , Neurodegeneração Associada a Pantotenato-Quinase/diagnóstico por imagem , Tailândia/etnologia
7.
BMC Vet Res ; 15(1): 155, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101115

RESUMO

BACKGROUND: Sphingosine kinase 1 (SPHK1) is an enzyme that converts pro-apoptotic ceramide and sphingosine into anti-apoptotic sphingosine-1-phosphate. There is growing evidence that SPHK1 activation promotes oncogenic transformation, tumor growth, chemotherapy resistance, and metastatic spread. High SPHK1 expression has been associated with a poor prognosis in several human cancers. RESULTS: In the present study, the expression level of SPHK1 was examined in feline mammary tumor (FMT) specimens, and the IHC expression level of SPHK1 was associated with the histological grade of FMTs. IHC analysis of 88 FMT cases revealed that the expression level of SPHK1 was upregulated in 53 tumor tissues (60.2%) compared to adjacent mammary tissues. SPHK1 expression in FMTs was significantly associated with histological grade, presence of lymphovascular invasion, and estrogen receptor negativity. Treatment of primary FMT cells with SPHK1 inhibitors reduced cell viability, indicating that SPHK1 acts to promote FMT cell survival. These results indicate that SPHK1 may play an important role in FMTs and may be a therapeutic target in cats with FMT. CONCLUSIONS: SPHK1 over-expression in breast cancer tissues is associated with a poor prognosis in humans. SPHK1 over-expression in more aggressive FMTs provides support for a potential role of SPHK1 inhibitors for the treatment of FMTs. Targeting SPHK1 has potent cytotoxic effects in primary FMT cells. These findings suggest that further examination of the role SPHK1 plays in FMTs will pave the way for the investigation of SPHK1 inhibitors in future clinical applications.


Assuntos
Doenças do Gato/patologia , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Vasos Sanguíneos/patologia , Doenças do Gato/enzimologia , Gatos , Feminino , Regulação Neoplásica da Expressão Gênica , Sistema Linfático/patologia , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Invasividade Neoplásica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo
8.
Mol Biotechnol ; 61(6): 432-441, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963480

RESUMO

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.


Assuntos
Trifosfato de Adenosina/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biotransformação , Carboidratos Epimerases/genética , Cátions Bivalentes , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/biossíntese , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Magnésio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polifosfatos/metabolismo , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismo
9.
Science ; 363(6431): 1088-1092, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30846598

RESUMO

Nicotinamide adenine dinucleotide phosphate (NADP+) is essential for producing NADPH, the primary cofactor for reductive metabolism. We find that growth factor signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces acute synthesis of NADP+ and NADPH. Akt phosphorylates NAD kinase (NADK), the sole cytosolic enzyme that catalyzes the synthesis of NADP+ from NAD+ (the oxidized form of NADH), on three serine residues (Ser44, Ser46, and Ser48) within an amino-terminal domain. This phosphorylation stimulates NADK activity both in cells and directly in vitro, thereby increasing NADP+ production. A rare isoform of NADK (isoform 3) lacking this regulatory region exhibits constitutively increased activity. These data indicate that Akt-mediated phosphorylation of NADK stimulates its activity to increase NADP+ production through relief of an autoinhibitory function inherent to its amino terminus.


Assuntos
NADP/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Animais , Cromatografia Líquida , Citosol/enzimologia , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Domínios Proteicos , Serina/genética , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem
10.
Viruses ; 11(3)2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30813555

RESUMO

African swine fever (ASF) is a hemorrhagic fever of wild and domestic pigs with a high rate of mortality. Originally endemic in Africa, this disease is currently disseminating in Europe and China, causing a large socioeconomic impact. ASF is caused by a DNA virus, African swine fever virus (ASFV). There is no vaccine available against ASFV, limiting the options for disease control. ASFV reorganizes intracellular membranes to generate viral factories (VFs) in order to amplify its genome. However, little is known about the process involved in the formation of these viral replication organelles. Membrane contact sites (MCSs) allow nonvesicular lipids and ion exchange between organelles. Lipid exchange to form VFs apparently requires a number of proteins at MCSs, such as the oxysterol-binding protein (OSBP), the acyl-coenzyme A binding domain containing 3 (ACBD3) and the phosphatidylinositol-phosphate-4-kinase III beta (PI4Kß). Itraconazole (ITZ) is an antifungal agent that targets sterol-transport molecules such as OSBP and OSBP-related protein 4 (ORP4). 25-Hydroxycholesterol (25-HC) inhibits lipid transport by high affinity binding OSBP. In this work, we analyzed the antiviral function of ITZ and 25-HC against ASFV in Vero cell cultures using the cell-adapted Ba71V isolate. ITZ and 25-HC decreased significantly ASFV replication. Our study revealed OSBP distribution in cytoplasmic membranes in uninfected Vero cells and to the periphery of VFs in infected cells. In addition, we showed that OSBP and OSBP-related proteins, PI4Kß and ACBD3 were recruited to VFs in the context ASFV infection.


Assuntos
Vírus da Febre Suína Africana/efeitos dos fármacos , Vírus da Febre Suína Africana/metabolismo , Interações entre Hospedeiro e Microrganismos , Metabolismo dos Lipídeos , Ligação Viral , Proteínas Adaptadoras de Transdução de Sinal/genética , Febre Suína Africana , Animais , Antivirais/farmacologia , Cercopithecus aethiops , Genoma Viral , Células HeLa , Humanos , Hidroxicolesteróis/farmacologia , Itraconazol/farmacologia , Proteínas de Membrana/genética , Antígenos de Histocompatibilidade Menor/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Esteroides/efeitos dos fármacos , Suínos , Células Vero
11.
Mech Dev ; 156: 20-31, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30904594

RESUMO

In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFß, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.


Assuntos
Desenvolvimento Embrionário/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Via de Sinalização Wnt/genética , Xenopus laevis/crescimento & desenvolvimento , Animais , Padronização Corporal/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Glicosilação , Humanos , Xenopus laevis/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
12.
Int J Mol Sci ; 20(5)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836629

RESUMO

Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.


Assuntos
Apoenzimas/genética , Simulação de Dinâmica Molecular , Fósforo-Oxigênio Liases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Apoenzimas/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Di-Hidroxiacetona/química , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Flavina-Adenina Dinucleotídeo/química , Gliceraldeído/química , Humanos , Fósforo-Oxigênio Liases/genética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Especificidade por Substrato
13.
BMB Rep ; 52(3): 220-225, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30885289

RESUMO

We have identified a mechanism to diminish the proliferative capacity of cells during cell expansion using human adiposederived stromal cells (hAD-SCs) as a model of replicative senescence. hAD-SCs of high-passage numbers exhibited a reduced proliferative capacity with accelerated cellular senescence. Levels of key bioactive sphingolipids were significantly increased in these senescent hAD-SCs. Notably, the transcription of sphingosine kinase 1 (SPHK1) was down-regulated in hAD-SCs at high-passage numbers. SPHK1 knockdown as well as inhibition of its enzymatic activity impeded the proliferation of hAD-SCs, with concomitant induction of cellular senescence and accumulation of sphingolipids, as seen in high-passage cells. SPHK1 knockdown-accelerated cellular senescence was attenuated by co-treatment with sphingosine-1-phosphate and an inhibitor of ceramide synthesis, fumonisin B1, but not by treatment with either one alone. Together, these results suggest that transcriptional down-regulation of SPHK1 is a critical inducer of altered sphingolipid profiles and enhances replicative senescence during multiple rounds of cell division. [BMB Reports 2019; 52(3): 220-225].


Assuntos
Células-Tronco Mesenquimais/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Apoptose/fisiologia , Proliferação de Células/genética , Senescência Celular/genética , Regulação para Baixo , Humanos , Lisofosfolipídeos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingolipídeos/genética , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
14.
Can J Ophthalmol ; 54(1): 51-59, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30851774

RESUMO

OBJECTIVE: To identify the disease-causing variants in 2 families with autosomal recessive inherited retinal dystrophies (IRDs) and to characterize phenotypic variability across the affected family members. DESIGN: Exome sequencing and ophthalmic clinical examination study. PARTICIPANTS: Six members from 2 consanguineous Jordanian families with IRD. METHODS: Ophthalmic examinations and whole-exome sequencing (WES) were performed to identify IRD-causing variants in affected individuals from each family, followed by segregation analysis of candidate variants in affected and unaffected family members by Sanger sequencing. RESULTS: We identified 2 different homozygous deletion variants in CERKL in each family: a novel pathogenic variant, c.450_451delAT, and a known variant, c.1187_1188delTG. Both variants co-segregated with the disease in all affected family members. The resulting phenotypes further supported that CERKL is associated with cone-rod dystrophy (CRD) rather than retinitis pigmentosa (RP), as originally established. CONCLUSION: Our study expands the genotypic spectra of CERKL variants, providing insights into the relevant pathogenesis of RP/CRD. We also confirm that the WES approach is a valuable tool for the molecular diagnosis of retinopathies.


Assuntos
Consanguinidade , DNA/genética , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Distrofias Retinianas/genética , Adolescente , Adulto , Análise Mutacional de DNA , Exoma , Feminino , Genótipo , Humanos , Jordânia , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Distrofias Retinianas/congênito , Distrofias Retinianas/metabolismo , Adulto Jovem
15.
J Med Food ; 22(4): 325-336, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30864855

RESUMO

Nonalcoholic fatty liver disease is a progressive disease involving the accumulation of lipid droplets in the liver. In this study, we investigated the anti-hepatosteatosis effects of fermented Cordyceps militaris extract (CME) in AML-12 hepatocytes. Although the levels of adenosine and cordycepin were reduced in the extracts of CM grown on germinated soybean (GSCE) and fermented CM grown on germinated soybean (GSC) by Pediococcus pentosaceus ON188 (ON188E), the expression of fatty acid oxidation (FAO) genes were upregulated only by GSC-ON188E treatment in a dose-dependent manner. In contrast, a lipogenic gene, stearoyl Coenzyme A desaturase 1, was downregulated by ON188E. Formation of intracellular lipid droplets by the addition of oleic acid was reduced by ON188E to levels observed in WY14643-treated cells. When cells were treated with ON188E, sphingosine kinase 2 mainly responsible for hepatic sphingosine 1-phosphate (S1P) synthesis was upregulated and S1P was elevated. Collectively, the fermented GSC extract activates FAO through elevation of S1P synthesis and has potential as a therapeutic for hepatosteatosis.


Assuntos
Cordyceps/química , Ácidos Graxos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Extratos Vegetais/farmacologia , Animais , Linhagem Celular , Cordyceps/metabolismo , Fermentação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Oxirredução/efeitos dos fármacos , Pediococcus pentosaceus/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo
16.
J Basic Microbiol ; 59(5): 542-551, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30747439

RESUMO

Xylulose kinase is an important enzyme involved in xylose metabolism, which is considered as essential biocatalyst for sustainable lignocellulosic-derived pentose utilization. Bacillus coagulans IPE22 is an ideal bacterium for refinery due to its strong ability to ferment xylose at high temperature. However, the B. coagulans xylose utilization mechanism remains unclear and the related promising enzymes need to be developed. In the present study, the gene coding for xylulose kinase from B. coagulans IPE22 (Bc-XK) was expressed in Escherichia coli BL21 (DE3). Bc-XK has a 1536 bp open reading frame, encoding a protein of 511 amino acids (56.15 kDa). Multiple sequence alignments were performed and a phylogenetic tree was built to evaluate differences among Bc-XK and other bacteria homologs. Bc-XK showed a broad adaptability to high temperature and the enzyme displayed its best performance at pH 8.0 and 60 °C. Bc-XK was activated by Mg2+ , Mn2+ , and Co2+ . Meanwhile, the enzyme could keep activity at 60 °C for at least 180 min. KM values of Bc-XK for xylulose and ATP were 1.29 mM and 0.76 mM, respectively. The high temperature stability of Bc-XK implied that it was an attractive candidate for industrial application.


Assuntos
Bacillus coagulans/enzimologia , Proteínas de Bactérias/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Xilose/metabolismo , Sequência de Aminoácidos , Bacillus coagulans/classificação , Bacillus coagulans/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Filogenia , Alinhamento de Sequência , Temperatura Ambiente
17.
Genetics ; 211(4): 1283-1295, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30782598

RESUMO

The transcription factor Nrf2 plays a critical role in the organism-wide regulation of the antioxidant stress response. The Nrf2 homolog SKN-1 functions in the intestinal cells nonautonomously to negatively regulate neuromuscular junction (NMJ) function in Caenorhabditis elegans To identify additional molecules that mediate SKN-1 signaling to the NMJ, we performed a candidate screen for suppressors of aldicarb resistance caused by acute treatment with the SKN-1 activator arsenite. We identified two receptor tyrosine kinases, EGL-15 (fibroblast growth factor receptor, FGFR) and DAF-2 (insulin-like peptide receptor), that are required for NMJ regulation in response to stress. Through double-mutant analysis, we found that EGL-15 functions downstream of, or parallel to, SKN-1 and SPHK-1 (sphingosine kinase), and that the EGL-15 ligand EGL-17 FGF and canonical EGL-15 effectors are required for oxidative stress-mediated regulation of NMJ function. DAF-2 also functions downstream of or parallel to SKN-1 to regulate NMJ function. Through tissue-specific rescue experiments, we found that FGFR signaling functions primarily in the hypodermis, whereas insulin-like peptide receptor signaling is required in multiple tissues. Our results support the idea that the regulation of NMJ function by SKN-1 occurs via a complex organism-wide signaling network involving receptor tyrosine kinase signaling in multiple tissues.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Junção Neuromuscular/metabolismo , Estresse Oxidativo , Receptor de Insulina/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Junção Neuromuscular/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptor de Insulina/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Mol Biotechnol ; 61(4): 274-285, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30747382

RESUMO

One of the most widespread pathogens worldwide is methicillin-resistant Staphylococcus aureus, a bacterium that provokes severe life-threatening illnesses both in hospitals and in the community. The principal challenge lies in the resistance of MRSA to current treatments, which encourages the study of different molecular targets that could be used to develop new drugs against this infectious agent. With this goal, a detailed characterization of shikimate kinase from this microorganism (SaSK) is described. The results showed that SaSK has a Km of 0.153 and 224 µM for shikimate and ATP, respectively, and a global reaction rate of 13.4 µmol/min/mg; it is suggested that SaSK utilizes the Bi-Bi Ping Pong reaction mechanism. Furthermore, the physicochemical data indicated that SaSK is an unstable, hydrophilic, and acidic protein. Finally, structural information showed that SaSK presented folding that is typical of its homologous counterparts and contains the typical domains of this family of proteins. Amino acids that have been shown to be important for SaSK protein function are conserved. Therefore, this study provides fundamental information that may aid in the design of inhibitors that could be used to develop new antibacterial agents.


Assuntos
Staphylococcus aureus Resistente à Meticilina/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trifosfato de Adenosina/metabolismo , Desenho de Drogas , Estabilidade Enzimática , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Ácido Chiquímico/metabolismo , Homologia Estrutural de Proteína
19.
Metab Brain Dis ; 34(2): 557-563, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30637540

RESUMO

D-glycerate 2 kinase (DGK) is an enzyme that mediates the conversion of D-glycerate, an intermediate metabolite of serine and fructose metabolism, to 2-phosphoglycerate. Deficiency of DGK leads to accumulation of D-glycerate in various tissues and its massive excretion in urine. D-glyceric aciduria (DGA) is an autosomal recessive metabolic disorder caused by mutations in the GLYCTK gene. The clinical spectrum of DGA is highly variable, ranging from severe progressive infantile encephalopathy to a practically asymptomatic condition. We describe a male patient from a consanguineous Arab family with infantile onset of DGA, characterized by profound psychomotor retardation, progressive microcephaly, intractable seizures, cortical blindness and deafness. Consecutive brain MR imaging showed an evolving brain atrophy, thinning of the corpus callosum and diffuse abnormal white matter signals. Whole exome sequencing identified the homozygous missense variant in the GLYCTK gene [c.455 T > C, NM_145262.3], which affected a highly conserved leucine residue located at a domain of yet unknown function of the enzyme [p.Leu152Pro, NP_660305]. In silico analysis of the variant supported its pathogenicity. A review of the 15 previously reported patients, together with the current one, confirms a clear association between DGA and severe neurological impairment. Yet, future studies of additional patients with DGA are required to better understand the clinical phenotype and pathogenesis.


Assuntos
Encefalopatias/metabolismo , Epilepsia/metabolismo , Hiperoxalúria Primária/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Encefalopatias/genética , Criança , Epilepsia/diagnóstico , Epilepsia/genética , Ácidos Glicéricos/metabolismo , Humanos , Hiperoxalúria Primária/genética , Lactente , Masculino , Mutação/genética , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo
20.
Microb Cell Fact ; 18(1): 4, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626394

RESUMO

BACKGROUND: As an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum. As an alternative, bio-production of isoprene has attracted much attention in recent years. Previous researches were mostly focused on key enzymes to improve isoprene production. In this research, besides screening of key enzymes, we also paid attention to expression intensity of non-key enzymes. RESULTS: Firstly, screening of key enzymes, IDI, MK and IspS, from other organisms and then RBS optimization of the key enzymes were carried out. The strain utilized IDIsa was firstly detected to produce more isoprene than other IDIs. IDIsa expression was improved after RBS modification, leading to 1610-fold increase of isoprene production. Secondly, RBS sequence optimization was performed to reduce translation initiation rate value of non-key enzymes, ERG19 and MvaE. Decreased ERG19 and MvaE expression and increased isoprene production were detected. The final strain showed 2.6-fold increase in isoprene production relative to the original strain. Furthermore, for the first time, increased key enzyme expression and decreased non-key enzyme expression after RBS sequence optimization were obviously detected through SDS-PAGE analysis. CONCLUSIONS: This study prove that desired enzyme expression and increased isoprene production were obtained after RBS sequence optimization. RBS optimization of genes could be a powerful strategy for metabolic engineering of strain. Moreover, to increase the production of engineered strain, attention should not only be focused on the key enzymes, but also on the non-key enzymes.


Assuntos
Enzimas/metabolismo , Escherichia coli/metabolismo , Hemiterpenos/biossíntese , Engenharia Metabólica , Ribossomos/metabolismo , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Técnicas de Cultura Celular por Lotes , Sítios de Ligação , Butadienos/análise , Carboxiliases/genética , Carboxiliases/metabolismo , Cromatografia Gasosa , Enzimas/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hemiterpenos/análise , Isomerases/genética , Isomerases/metabolismo , Redes e Vias Metabólicas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ribossomos/química , Ribossomos/genética
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