RESUMEN

The non-conventional yeast Komagataella phaffii, formerly Pichia pastoris, is a popular host for recombinant protein production. The relatively lower gene targeting efficiency observed in this species occurs due to high levels of non-homologous recombination activity. In the current study, we explored the function of the K. phaffii homolog of DNA ligase IV (Dnl4p) by creating a DNL4-disrupted strain. To assess the roles of non-homologous end joining (NHEJ)-related proteins in this species, strains deleted for either or both genes encoding Dnl4p or the telomeric Ku complex subunit (Ku70p) were generated. These deletions were constructed by either of two distinct marker-recycling methods (yielding either a seamless gene deletion or a Cre-loxP-mediated gene deletion). The resulting dnl4- and/or ku70-deleted K. phaffii strains were used to evaluate gene targeting efficiency in gene knock-out and gene knock-in experiments. The Dnl4p-defective strain showed improved gene targeting efficiency for homologous recombination compared to the wild-type and Ku70p-deffective strains. The dnl4 ku70 double knock-out strain exhibited a further improvement in gene targeting efficiency. Thus, the K. phaffii dnl4 and dnl4 ku70 deletion strains are expected to serve as useful platforms for functional analysis and strain development in this species.

Asunto(s)

ADN Ligasa (ATP)/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Marcación de Gen/métodos , Saccharomycetales/genética , ADN Ligasa (ATP)/metabolismo , Proteínas Fúngicas/metabolismo , Recombinación Homóloga , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo

RESUMEN

The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

Asunto(s)

Centrómero/genética , Vectores Genéticos/genética , Pichia/genética , Plásmidos/genética , Secuencia de Bases , Cromosomas Fúngicos/genética , Replicación del ADN , ADN de Hongos/genética , Secuencias Invertidas Repetidas

RESUMEN

Clove (Syzygium aromaticum flower buds) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-A(y) mice. In-vitro evaluation showed the extract had human peroxisome proliferator-activated receptor (PPAR)-γ ligand-binding activity in a GAL4-PPAR-γ chimera assay. Bioassay-guided fractionation of the EtOH extract resulted in the isolation of eight compounds, of which dehydrodieugenol (2) and dehydrodieugenol B (3) had potent PPAR-γ ligand-binding activities, whereas oleanolic acid (4), a major constituent in the EtOH extract, had moderate activity. Furthermore, 2 and 3 were shown to stimulate 3T3-L1 preadipocyte differentiation through PPAR-γ activation. These results indicate that clove has potential as a functional food ingredient for the prevention of type 2 diabetes and that 2-4 mainly contribute to its hypoglycemic effects via PPAR-γ activation.

Asunto(s)

Diabetes Mellitus/tratamiento farmacológico , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Syzygium/química , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Femenino , Hipoglucemiantes/farmacología , Lignanos/química , Ratones , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Extractos Vegetales/farmacocinética , Triterpenos/química

RESUMEN

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. Turmeric oleoresin, an extract of turmeric, is often used for flavoring and coloring. Curcuminoids and turmeric essential oil are both contained in turmeric oleoresin, and both of these fractions have hypoglycemic effects. In the present study, we comprehensively assessed the effect of turmeric oleoresin on hepatic gene expression in obese diabetic KK-Ay mice using DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR). Female KK-Ay mice aged 6 weeks (n = 6/group) were fed a high-fat diet containing turmeric oleoresin, curcuminoids, and essential oil for 5 weeks. The same diet without any of these fractions was used as a control diet. Ingestion of turmeric oleoresin and essential oil inhibited the development of increased blood glucose and abdominal fat mass, while curcuminoids only inhibited the increase in blood glucose. DNA microarray analysis indicated that turmeric oleoresin ingestion up-regulated the expression of genes related to glycolysis, beta-oxidation, and cholesterol metabolism in the liver of KK-Ay mice, while expression of gluconeogenesis-related genes was down-regulated. Real-time PCR analysis was conducted to assess the contribution of the curcuminoids and essential oil in turmeric oleoresin to the changes in expression of representative genes selected by DNA microarray analysis. This analysis suggested that curcuminoids regulated turmeric oleoresin ingestion-induced expression of glycolysis-related genes and also that curcuminoids and turmeric essential oil acted synergistically to regulate the peroxisomal beta-oxidation-related gene expression induced by turmeric oleoresin ingestion. These changes in gene expression were considered to be the mechanism by which the turmeric oleoresin affected the control of both blood glucose levels and abdominal adipose tissue masses. All of these results suggest that the use of whole turmeric oleoresin is more effective than the use of either curcuminoids or the essential oil alone.

Asunto(s)

Curcuma , Hipoglucemiantes/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Fitoterapia , Extractos Vegetales/farmacología , Animales , Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Ratones , Ratones Obesos , Análisis de Secuencia por Matrices de Oligonucleótidos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Aceites de Plantas/administración & dosificación , Aceites de Plantas/química , Aceites de Plantas/farmacología , Aceites de Plantas/uso terapéutico , Rizoma

RESUMEN

The turmeric (Curcuma longa L. rhizomes) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-A(y) mice. In an in vitro evaluation, the extract stimulated human adipocyte differentiation in a dose-dependent manner and showed human peroxisome proliferator-activated receptor (PPAR)-gamma ligand-binding activity in a GAL4-PPAR-gamma chimera assay. The main constituents of the extract were identified as curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone, which had also PPAR-gamma ligand-binding activity. These results indicate that turmeric is a promising ingredient of functional food for the prevention and/or amelioration of type 2 diabetes and that curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone mainly contribute to the effects via PPAR-gamma activation.

Asunto(s)

Curcuma , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/genética , Hipoglucemiantes/uso terapéutico , Rizoma , Animales , Relación Dosis-Respuesta a Droga , Femenino , Hipoglucemiantes/química , Hipoglucemiantes/aislamiento & purificación , Ratones

RESUMEN

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. The chemistry includes curcuminoids and sesquiterpenoids as components, which are known to have antioxidative, anticarcinogenic, and antiinflammatory activities. In this study, we investigated the effects of three turmeric extracts on blood glucose levels in type 2 diabetic KK-A(y) mice (6 weeks old, n = 5/group). These turmeric extracts were obtained by ethanol extraction (E-ext) to yield both curcuminoids and sesquiterpenoids, hexane extraction (H-ext) to yield sesquiterpenoids, and ethanol extraction from hexane-extraction residue (HE-ext) to yield curcuminoids. The control group was fed a basal diet, while the other groups were fed a diet containing 0.1 or 0.5 g of H-ext or HE-ext/100 g of diet or 0.2 or 1.0 g of E-ext/100 g of diet for 4 weeks. Although blood glucose levels in the control group significantly increased (P < 0.01) after 4 weeks, feeding of 0.2 or 1.0 g of E-ext, 0.5 g of H-ext, and 0.5 g of HE-ext/100 g of diet suppressed the significant increase in blood glucose levels. Furthermore, E-ext stimulated human adipocyte differentiation, and these turmeric extracts had human peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand-binding activity in a GAL4-PPAR-gamma chimera assay. Also, curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone had PPAR-gamma ligand-binding activity. These results indicate that both curcuminoids and sesquiterpenoids in turmeric exhibit hypoglycemic effects via PPAR-gamma activation as one of the mechanisms, and suggest that E-ext including curcuminoids and sesquiterpenoids has the additive or synergistic effects of both components.

Asunto(s)

Glucemia/análisis , Curcuma/química , Curcumina/análisis , Diabetes Mellitus Tipo 2/sangre , Hipoglucemiantes/análisis , Sesquiterpenos/análisis , Adipocitos/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Curcumina/administración & dosificación , Diabetes Mellitus Tipo 2/terapia , Etanol , Humanos , Hipoglucemiantes/administración & dosificación , Ratones , PPAR gamma/metabolismo , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Sesquiterpenos/administración & dosificación

RESUMEN

Licorice, the root of the Glycyrrhiza species, is one of the most frequently employed botanicals in traditional medicines. In this study, we investigated the effects of hydrophobic flavonoids from Glycyrrhiza glabra LINNE on abdominal fat accumulation and blood glucose level in obese diabetic KK-A(y) mice. In order to enrich a fraction of hydrophobic flavonoids, licorice flavonoid oil (LFO) was prepared by further extracting licorice ethanolic extract with medium-chain triglycerides (MCT), and adjusting the concentration of glabridin, the major flavonoid of licorice, to 1.2% in oil. KK-A(y) mice aged 6 weeks were assigned to 5 groups (n=6 each), and fed a high-fat diet containing 0 (control), 0.5%, 1%, or 2% LFO, or 0.5% conjugated linoleic acid (CLA) for 4 weeks. Compared with the control, body weight gain and weights of abdominal adipose tissues were suppressed (p<0.05) by feeding the diet containing 2% LFO, and blood glucose levels after 2 and 4 weeks were suppressed by all of the diets containing LFO. Although CLA feeding suppressed (p<0.05) body weight gain, it increased (p<0.05) blood glucose level after 2 weeks compared with the control level. Furthermore, LFO and licorice ethanolic extract stimulated human adipocyte differentiation in vitro. These results indicate that licorice hydrophobic flavonoids have abdominal fat-lowering and hypoglycemic effects, possibly mediated via activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma).

Asunto(s)

Tejido Adiposo/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Glucemia/efectos de los fármacos , Flavonoides/farmacología , Glycyrrhiza , Hipoglucemiantes/farmacología , Abdomen , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Femenino , Glycyrrhiza/química , Ratones , Ratones Obesos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/química

RESUMEN

The EtOAc extract of licorice (Glycyrrhiza uralensis roots) exhibited considerable PPAR-gamma ligand-binding activity. Bioassay-guided fractionation of the extract using a GAL-4-PPAR-gamma chimera assay method resulted in the isolation of two isoflavenes, one of which is a new compound named dehydroglyasperin D, an isoflavan, two 3-arylcoumarins, and an isoflavanone as the PPAR-gamma ligand-binding active ingredients of licorice. The isoprenyl group at C-6 and the C-2' hydroxyl group in the aromatic ring-C part in the isoflavan, isoflavene, or arylcoumarin skeleton were found to be the structural requirements for PPAR-gamma ligand-binding activity. Glycyrin, one of the main PPAR-gamma ligands of licorice, significantly decreased the blood glucose levels of genetically diabetic KK-A(y) mice.

Asunto(s)

Cumarinas/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/genética , Glycyrrhiza , Hipoglucemiantes/uso terapéutico , Fenoles/farmacología , Fitoterapia , Extractos Vegetales/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Línea Celular , Chlorocebus aethiops , Ligandos , Ratones , Ratones Mutantes , Fenoles/farmacocinética , Fenoles/uso terapéutico , Pioglitazona , Extractos Vegetales/farmacocinética , Extractos Vegetales/uso terapéutico , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Tiazolidinedionas/uso terapéutico , Factores de Transcripción/genética

RESUMEN

The metabolic syndrome, including type 2 diabetes, insulin resistance, obesity/abdominal obesity, hypertension and dyslipidemia, is a major public health problem. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands such as thiazolidinediones are effective against this syndrome. In this study, we showed that nonaqueous fractions of licorice (Glycyrrhiza uralensis Fisher) extracted with ethanol, ethyl acetate and acetone, but not an aqueous extract, had PPAR-gamma ligand-binding activity with a GAL4-PPAR-gamma chimera assay. Some prenylflavonoids including glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D, a newly found compound, were identified as active compounds with PPAR-gamma ligand-binding activity in the nonaqueous fraction of licorice. A licorice ethanolic extract contained these four active compounds at a total concentration of 16.7 g/100 g extract. Feeding the licorice ethanolic extract at 0.1-0.3 g/100 g diet [approximately 100 to 300 mg/(kg body x d)] for 4 wk decreased (P < 0.05) blood glucose level in younger (6 wk old) and older (13 wk old) diabetic KK-Ay mice and reduced (P < 0.05) weights of intra-abdominal adipose tissues in high fat diet-induced obese C57BL mice. An increase in blood pressure in spontaneously hypertensive rats was suppressed (P < 0.01) by 3 wk of oral administration of the licorice ethanolic extract at 300 mg/(kg body x d). These findings indicate that licorice ethanolic extract is effective in preventing and ameliorating diabetes, ameliorating abdominal obesity and preventing hypertension, and suggest that licorice ethanolic extract would be effective in preventing and/or ameliorating the metabolic syndrome.

Asunto(s)

Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glycyrrhiza , Extractos Vegetales/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Tiazolidinedionas/farmacología , Factores de Transcripción/fisiología , Acetatos , Acetona , Animales , Glucemia/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/prevención & control , Grasas de la Dieta , Etanol , Femenino , Flavonoides/farmacología , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/prevención & control , Raíces de Plantas , Plásmidos , Ratas , Ratas Endogámicas SHR , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Proteínas Recombinantes de Fusión/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Transfección

RESUMEN

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes alpha,beta-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5'-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the alpha,beta-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D-and L-beta-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.

Asunto(s)

Amoníaco-Liasas/aislamiento & purificación , Amoníaco-Liasas/metabolismo , Escherichia coli/enzimología , beta-Alanina/análogos & derivados , beta-Alanina/metabolismo , Secuencia de Aminoácidos , Amoníaco-Liasas/química , Amoníaco-Liasas/genética , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido