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1.
J Microbiol Biotechnol ; 34(6): 1299-1306, 2024 Jun 28.
Article in English | MEDLINE | ID: mdl-38755001

ABSTRACT

Antibiotics are used to control infectious diseases. However, adverse effects of antibiotics, such as devastation of the gut microbiota and enhancement of the inflammatory response, have been reported. Health benefits of fermented milk are established and can be enhanced by the addition of probiotic strains. In this study, we evaluated effects of fermented milk containing Lacticaseibacillus rhamnosus (L. rhamnosus) SNUG50430 in a mouse model with antibiotic treatment. Fermented milk containing 2 × 105 colony-forming units of L. rhamnosus SNUG50430 was administered to six week-old female BALB/c mice for 1 week. Interleukin (IL)-10 levels in colon samples were significantly increased (P < 0.05) compared to water-treated mice, whereas interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) were decreased, of mice treated with fermented milk containing L. rhamnosus SNUG50430-antibiotics-treated (FM+LR+Abx-treated) mice. Phylum Firmicutes composition in the gut was restored and the relative abundances of several bacteria, including the genera Coprococcus and Lactobacillus, were increased in FM+LR+Abx-treated mice compared to PBS+Abx-treated mice. Interestingly, abundances of genus Coprococcus and Lactobacillus were positively correlated with IL-5 and IL-10 levels (P < 0.05) in colon samples and negative correlated with IFN-γ and TNF-α levels in serum samples (P < 0.001). Acetate and butyrate were increased in mice with fermented milk and fecal microbiota of FM+LR+Abx-treated mice were highly enriched with butyrate metabolism pathway compared to water-treated mice (P < 0.05). Thus, fermented milk containing L. rhamnosus SNUG50430 was shown to ameliorate adverse health effects caused by antibiotics through modulating immune responses and the gut microbiota.


Subject(s)
Anti-Bacterial Agents , Cultured Milk Products , Gastrointestinal Microbiome , Interleukin-10 , Lacticaseibacillus rhamnosus , Mice, Inbred BALB C , Probiotics , Animals , Gastrointestinal Microbiome/drug effects , Female , Mice , Probiotics/administration & dosage , Anti-Bacterial Agents/pharmacology , Interleukin-10/metabolism , Cultured Milk Products/microbiology , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/blood , Interferon-gamma/metabolism , Colon/microbiology , Fermentation , Cytokines/metabolism , Cytokines/blood , Feces/microbiology
2.
Biomolecules ; 10(5)2020 05 06.
Article in English | MEDLINE | ID: mdl-32384794

ABSTRACT

Fermented vegetable juices have gained attention due to their various beneficial effects on human health. In this study, we employed gas chromatography-mass spectrometry, direct infusion-mass spectrometry, and liquid chromatography-mass spectrometry to identify useful metabolites, lipids, and carotenoids in vegetable juice (VJ) fermented with Lactobacillus plantarum HY7712, Lactobacillus plantarum HY7715, Lactobacillus helveticus HY7801, and Bifidobacterium animalis ssp. lactis HY8002. A total of 41 metabolites, 24 lipids, and 4 carotenoids were detected in the fermented and non-fermented VJ (control). The lycopene, α-carotene, and ß-carotene levels were higher in VJ fermented with L. plantarum strains (HY7712 and HY7715) than in the control. Proline content was also elevated in VJ fermented with HY7715. Uracil, succinic acid, and α-carotene concentration was increased in VJ fermented with HY7801, while glycine and lycopene levels were raised in VJ fermented with HY8002. This study confirmed that each probiotic strain has distinctive characteristics and produces unique changes to metabolic profiles of VJ during fermentation. Our results suggest that probiotic-fermented VJ is a promising functional beverage that contains more beneficial metabolites and carotenoids than commercial non-fermented VJ.


Subject(s)
Fermentation , Fruit and Vegetable Juices/microbiology , Probiotics/metabolism , Carotenoids/metabolism , Lactobacillus/metabolism , Lipidomics , Metabolome
3.
Biotechnol Lett ; 42(4): 519-528, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31970557

ABSTRACT

OBJECTIVES: To investigate the preventive effect of Lactobacillus casei HY2782 on toxicity induced by particulate matter (PM, inhalable particles less than 10 µm in diameter) in human intestinal CCD-18Co cells and a model animal Caenorhabditis elegans. RESULTS: L. casei HY2782 treatment prevented PM-induced intestinal cell death via cellular reactive oxygen species production and membrane disruption attenuation. PM significantly decreased the total number of eggs laid and the body bending activity of C. elegans, demonstrating PM toxicity. L. casei HY2782 treatment restored the reproductive toxicity and decline in locomotion activity induced by PM in C. elegans. Overall, L. casei HY2782 attenuated PM toxicity in vitro in cultured intestinal cells and in vivo in the model nematode. CONCLUSION: Our study provides a potential clue for developing L. casei HY2782 probiotics that attenuate PM-induced cellular and physiological toxicity; however, further in-depth preclinical trials using mammalian animal models and clinical trials are required.


Subject(s)
Caenorhabditis elegans/physiology , Intestines/cytology , Lacticaseibacillus casei/physiology , Particulate Matter/toxicity , Animals , Caenorhabditis elegans/drug effects , Cell Line , Cell Proliferation , Cell Survival , Humans , Intestines/drug effects , Reactive Oxygen Species/metabolism
4.
Article in English | MEDLINE | ID: mdl-27516803

ABSTRACT

To evaluate the pharmacokinetics of compound K after oral administration of HYFRG and RG in humans, an open-label, randomized, single-dose, fasting, and one-period pharmacokinetic study was conducted. After oral administration of a single 3 g dose of HYFRG and RG to 24 healthy Korean males, the mean (±SD) of AUC0-t and C max of compound K from HYFRG were 1466.83 ± 295.89 ng·h/mL and 254.45 ± 51.20 ng/mL, being 115.2- and 80-fold higher than those for RG (12.73 ± 7.83 ng·h/mL and 3.18 ± 1.70 ng/mL), respectively; in case of Sprague Dawley rats the mean (±SD) of AUC0-t and C max of compound K from HYFRG was 58.03 ± 32.53 ng·h/mL and 15.19 ± 10.69 ng/mL, being 6.3- and 6.0-fold higher than those from RG (9.21 ± 7.52 ng·h/mL and 2.55 ± 0.99 ng/mL), respectively. T max of compound K in humans and rats was 2.54 ± 0.92 and 3.33 ± 0.50 h for HYFRG and 9.11 ± 1.45 and 6.75 ± 3.97 hours for RG, respectively. In conclusion, the administration of HYFRG resulted in a higher and faster absorption of compound K in both humans and rats compared to RG.

5.
J Microbiol Biotechnol ; 26(6): 1035-45, 2016 Jun 28.
Article in English | MEDLINE | ID: mdl-26975767

ABSTRACT

To evaluate the effects of lactic acid bacteria (LAB) on Peyer's patch cells, mice were treated with a high dose of kanamycin to disturb the gut microbial environment. The overarching goal was to explore the potential of LAB for use as a dietary probiotic that buffers the negative consequences of antibiotic treatment. In vitro, LAB stimulated the production of immunoglobulin A (IgA) from isolated Peyer's patch cells. Inflammation-related genes (TNF-α, IL-1ß, and IL-8) were up-regulated in Caco-2 cells stimulated with lipopolysaccharide (LPS), while tight-junction-related genes (ZO-1 and occludin) were down-regulated; the effects of LPS on inflammatory gene and tight-junction gene expression were reversed by treatment with LAB. Mice treated with a high dose of kanamycin showed increased serum IgE levels and decreases in serum IgA and fecal IgA levels; the number of Peyer's patch cells decreased with kanamycin treatment. However, subsequent LAB treatment was effective in reducing the serum IgE level and recovering the serum IgA and fecal IgA levels, as well as the number of Peyer's patch cells. In addition, ZO-1 and occludin mRNA levels were up-regulated in the ileum tissues of mice receiving LAB treatment. Lactic acid bacteria can enhance the intestinal immune system by improving the integrity of the intestinal barrier and increasing the production of IgA in Peyer's patches. Lactic acid bacteria should be considered a potential probiotic candidate for improving intestinal immunity, particularly in mitigating the negative consequences of antibiotic use.


Subject(s)
Gastrointestinal Microbiome , Lactobacillus/physiology , Peyer's Patches/immunology , Peyer's Patches/physiology , Probiotics/therapeutic use , Tight Junctions/genetics , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/adverse effects , Caco-2 Cells , Humans , Immunoglobulin A/biosynthesis , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin E/blood , Interleukin-1beta/genetics , Interleukin-8/genetics , Kanamycin/administration & dosage , Kanamycin/adverse effects , Lipopolysaccharides/pharmacology , Mice , Occludin/genetics , Peyer's Patches/cytology , Peyer's Patches/microbiology , Tumor Necrosis Factor-alpha/genetics , Zonula Occludens-1 Protein/genetics
6.
J Microbiol Biotechnol ; 26(3): 483-7, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26699746

ABSTRACT

The triglyceride-lowering effect of probiotics Lactobacillus plantarum KY1032 and Lactobacillus curvatus HY7601 were investigated. Male SD Wistar rats were randomly divided into three groups and fed high-fat diet (HFD), HFD and probiotics (5 X 10(9) CFU/day of L. plantarum KY1032 and 5 X 10(9) CFU/day of L. curvatus HY7601), or normal diet for 6 weeks. Probiotic treatment significantly lowered the elevated plasma triglyceride and increased plasma free fatty acid, glycerol, and plasma apolipoprotein A-V (ApoA-V) levels. The probiotic-treated group showed elevated hepatic mRNA expression of PPARα, bile acid receptor (FXR), and ApoA-V. These results demonstrate that L. plantarum KY1032 and L. curvatus HY7601 lower triglycerides in hypertriglyceridemic rats by upregulating ApoA-V, PPARα, and FXR.


Subject(s)
Hypertriglyceridemia/drug therapy , Lactobacillus plantarum/physiology , Lactobacillus/physiology , Probiotics/administration & dosage , Triglycerides/metabolism , Animals , Diet, High-Fat/adverse effects , Dietary Supplements , Disease Models, Animal , Humans , Hypertriglyceridemia/metabolism , Liver/metabolism , Male , Rats , Rats, Wistar
7.
J Microbiol Biotechnol ; 25(12): 2160-8, 2015 Dec 28.
Article in English | MEDLINE | ID: mdl-26428734

ABSTRACT

The beneficial effects of probiotics are now widely reported, although there are only a few studies on their anti-aging effects. We have found that Lactobacillus plantarum HY7714 (HY7714) improves skin hydration and has anti-photoaging effects, and in the present study, we have further evaluated the anti-aging effect of HY7714 via a randomized, double blind, placebo-controlled clinical trial. The trial included 110 volunteers aged 41 and 59 years who have dry skin and wrinkles. Participants took 1 × 10(10) CFU/day of HY7714 (probiotic group) or a placebo (placebo group) for 12 weeks. Skin hydration, wrinkles, skin gloss, and skin elasticity were measured every 4 weeks during the study period. There were significant increases in the skin water content in the face (p < 0.01) and hands (p < 0.05) at week 12 in the probiotic group. Transepidermal water loss decreased significantly in both groups at weeks 4, 8, and 12 (p < 0.001 compared with baseline), and was suppressed to a greater extent in the face and forearm in the probiotic group at week 12. Volunteers in the probiotic group had a significant reduction in wrinkle depth at week 12, and skin gloss was also significantly improved by week 12. Finally, skin elasticity in the probiotic group improved by 13.17% (p < 0.05 vs. controls) after 4 weeks and by 21.73% (p < 0.01 vs. controls) after 12 weeks. These findings are preliminary confirmation of the anti-aging benefit to the skin of L. plantarum HY7714 as a nutricosmetic agent.


Subject(s)
Lactobacillus plantarum/physiology , Probiotics/administration & dosage , Skin Aging , Double-Blind Method , Female , Humans , Middle Aged , Placebos/administration & dosage , Skin Physiological Phenomena , Treatment Outcome
8.
J Med Food ; 18(12): 1380-6, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26331671

ABSTRACT

Ursolic acid is a lipophilic pentacyclic triterpenoid found in many fruits and herbs and is used in several herbal folk medicines for diabetes. In this study, we evaluated the effects of apple pomace extract (APE; ursolic acid content, 183 mg/g) on skeletal muscle atrophy. To examine APE therapeutic potential in muscle atrophy, we investigated APE effects on the expression of biomarkers associated with muscle atrophy and hypertrophy. We found that APE inhibited atrophy, while inducing hypertrophy in C2C12 myotubes by decreasing the expression of atrophy-related genes and increasing the expression of hypertrophy-associated genes. The in vivo experiments using mice fed a diet with or without APE showed that APE intake increased skeletal muscle mass, as well as grip strength and exercise capacity. In addition, APE significantly improved endurance in the mice, as evidenced by increased exhaustive running time and muscle weight, and reduced the expression of the genes involved in the development of muscle atrophy. APE also decreased the concentration of serum lactate and lactate dehydrogenase, inorganic phosphate, and creatinine, the indicators of accumulated fatigue and exercise-induced stress. These results suggest that APE may be useful as an ergogenic functional food or dietary supplement.


Subject(s)
Malus/chemistry , Muscle Strength/drug effects , Muscle, Skeletal/drug effects , Muscular Atrophy/prevention & control , Physical Endurance/drug effects , Running/physiology , Triterpenes/pharmacology , Animals , Biomarkers/blood , Cell Line , Dietary Supplements , Exercise Tolerance , Fatigue/blood , Fatigue/prevention & control , Fruit/chemistry , Gene Expression/drug effects , Hypertrophy , Male , Mice, Inbred C57BL , Muscle Fibers, Skeletal/drug effects , Muscle Strength/genetics , Muscle, Skeletal/cytology , Muscle, Skeletal/physiology , Muscular Atrophy/blood , Muscular Atrophy/genetics , Physical Endurance/physiology , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Triterpenes/therapeutic use , Ursolic Acid
9.
Atherosclerosis ; 241(2): 649-56, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26117402

ABSTRACT

OBJECTIVE: Previous studies have indicated that supplementation with probiotics might improve lipid metabolism. The objective of the study was to evaluate the effect of supplementation with probiotic strains Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032 on triglyceride (TG) and apolipoprotein A-V (apo A-V) levels. METHODS: A randomized, double-blinded, placebo-controlled study was conducted with 128 non-diabetic subjects with hypertriglyceridemia. Over a 12-week test period, the probiotic group consumed 2 g/day of a powdered supplement containing L. curvatus HY7601 and L. plantarum KY1032, whereas the placebo group consumed a powder lacking probiotics. RESULTS: After the treatment, the probiotic group showed an 18.3% (P < 0.001) reduction in TGs and increases of 21.1% (P = 0.001) and 15.6% (P < 0.001) in the apo A-V and LDL particle size, respectively. The probiotic group had a significant reduction in TGs (P = 0.040) and increases in the plasma apo A-V (P = 0.003) and LDL particle size (P < 0.001) compared with the placebo group. In the probiotic group, the reduction in the TG levels was negatively correlated with changes in the apo A-V and baseline TGs, regardless of the APOA5 -1131T > C genotype. CONCLUSION: The consumption of two probiotic strains for 12 weeks reduced TGs and increased the apo A-V and LDL particle size in hypertriglyceridemic subjects. This effect was more pronounced in subjects with higher levels of fasting TGs regardless of their APOA5 -1131T > C genotype.


Subject(s)
Apolipoproteins A/chemistry , Hypertriglyceridemia/therapy , Lactobacillus plantarum , Lactobacillus , Probiotics/therapeutic use , Anthropometry , Apolipoprotein A-V , Apolipoproteins A/genetics , Blood Glucose/analysis , Blood Pressure , C-Reactive Protein/chemistry , Cholesterol, LDL/blood , Cohort Studies , Dietary Supplements , Double-Blind Method , Fasting , Fatty Acids, Nonesterified/blood , Female , Genotype , Humans , Hypertriglyceridemia/microbiology , Lipoproteins, LDL/chemistry , Male , Middle Aged , Particle Size , Triglycerides/blood
10.
J Microbiol Biotechnol ; 24(12): 1736-43, 2014 Dec 28.
Article in English | MEDLINE | ID: mdl-25179898

ABSTRACT

In this study, we evaluated the effect of Lactobacillus plantarum HY7714 on skin hydration in human dermal fibroblasts and in hairless mice. In Hs68 cells, L. plantarum HY7714 not only increased the serine palmitoyltransferase (SPT) mRNA level, but also decreased the ceramidase mRNA level. In order to confirm the hydrating effects of L. plantarum HY7714 in vivo, we orally administered vehicle or L. plantarum HY7714 at a dose of 1 × 10(9) CFU/day to hairless mice for 8 weeks. In hairless mice, L. plantarum HY7714 decreased UVB-induced epidermal thickness. In addition, we found that L. plantarum HY7714 administration suppressed the increase in transepidermal water loss and decrease in skin hydration, which reflects barrier function fluctuations following UV irradiation. In particular, L. plantarum HY7714 administration increased the ceramide level compared with that in the UVB group. In the experiment on SPT and ceramidase mRNA expressions, L. plantarum HY7714 administration improved the reduction in SPT mRNA levels and suppressed the increase in ceramidase mRNA levels caused by UVB in the hairless mice skins. Collectively, these results suggest that L. plantarum HY7714 can be a potential candidate for preserving skin hydration levels against UV irradiation.


Subject(s)
Lactobacillus plantarum/growth & development , Probiotics/administration & dosage , Skin Physiological Phenomena/radiation effects , Skin/radiation effects , Ultraviolet Rays , Administration, Oral , Animals , Cell Line , Ceramidases/biosynthesis , Fibroblasts/physiology , Gene Expression Profiling , Humans , Mice, Hairless , RNA, Messenger/analysis , RNA, Messenger/genetics , Serine C-Palmitoyltransferase/biosynthesis , Skin/enzymology
11.
Biochem Biophys Res Commun ; 424(3): 385-90, 2012 Aug 03.
Article in English | MEDLINE | ID: mdl-22771327

ABSTRACT

Cadmium is a toxic metal, and the mechanism of cadmium toxicity in living organisms has been well studied. Here, we used Saccharomyces cerevisiae as a model system to examine the detailed molecular mechanism of cell growth defects caused by cadmium. Using a plate assay of a yeast deletion mutant collection, we found that deletion of SML1, which encodes an inhibitor of Rnr1, resulted in cadmium resistance. Sml1 protein levels increased when cells were treated with cadmium, even though the mRNA levels of SML1 remained unchanged. Using northern and western blot analyses, we found that cadmium inhibited Sml1 degradation by inhibiting Sml1 phosphorylation. Sml1 protein levels increased when cells were treated with cadmium due to disruption of the dependent protein degradation pathway. Furthermore, cadmium promoted cell cycle progression into the G2 phase. The same result was obtained using cells in which SML1 was overexpressed. Deletion of SML1 delayed cell cycle progression. These results are consistent with Sml1 accumulation and with growth defects caused by cadmium stress. Interestingly, although cadmium treatment led to increase Sml1 levels, intracellular dNTP levels also increased because of Rnr3 upregulation due to cadmium stress. Taken together, these results suggest that cadmium specifically affects the phosphorylation of Sml1 and that Sml1 accumulates in cells.


Subject(s)
Cadmium/toxicity , Proteolysis/drug effects , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Cell Cycle/drug effects , DNA Damage , Phosphorylation/drug effects , Ribonucleotide Reductases/antagonists & inhibitors , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics , Sequence Deletion , Up-Regulation
12.
Biochem J ; 431(2): 257-65, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20670216

ABSTRACT

Cadmium is a toxic metal and the mechanism of its toxicity has been studied in various model systems from bacteria to mammals. We employed Saccharomyces cerevisiae as a model system to study cadmium toxicity at the molecular level because it has been used to identify the molecular mechanisms of toxicity found in higher organisms. cDNA microarray and Northern blot analyses revealed that cadmium salts inhibited the expression of genes related to copper metabolism. Western blotting, Northern blotting and chromatin immunoprecipitation experiments indicated that CTR1 expression was inhibited at the transcriptional level through direct inhibition of the Mac1 transcriptional activator. The decreased expression of CTR1 results in cellular copper deficiency and inhibition of Fet3 activity, which eventually impairs iron uptake. In this way, cadmium exhibits a negative effect on both iron and copper homoeostasis.


Subject(s)
Cadmium/toxicity , Copper/metabolism , Homeostasis/drug effects , Nuclear Proteins/antagonists & inhibitors , Regulon/genetics , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae/genetics , Trans-Activators/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Copper/pharmacology , Down-Regulation/drug effects , Down-Regulation/genetics , Gene Expression Regulation, Fungal/drug effects , Genes, Fungal/genetics , Iron/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Transport/drug effects , Regulon/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Up-Regulation/drug effects , Up-Regulation/genetics
13.
Biochem J ; 422(1): 181-91, 2009 Jul 29.
Article in English | MEDLINE | ID: mdl-19469713

ABSTRACT

Aft1 is a transcriptional activator in Saccharomyces cerevisiae that responds to iron availability and regulates the expression of genes in the iron regulon, such as FET3, FTR1 and the ARN family. Using a two-hybrid screen, we found that Aft1 physically interacts with the FOB (ferrioxamine B) transporter Arn3. This interaction modulates the ability of Arn3 to take up FOB. The interaction between Arn3 and Aft1 was confirmed by beta-galactosidase, co-immunoprecipitation and SPR (surface plasmon resonance) assays. Truncated Aft1 had a stronger interaction with Arn3 and caused a higher FOB-uptake activity than full-length Aft1. Interestingly, only full-length Aft1 induced the correct localization of Arn3 in response to FOB. Furthermore, we found Aft1 affected Arn3 ubiquitination. These results suggest that Aft1 interacts with Arn3 and may regulate the ubiquitination of Arn3 in the cytosolic compartment.


Subject(s)
Deferoxamine/metabolism , Ferric Compounds/metabolism , Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Ubiquitination , Cell Membrane/metabolism , Ceruloplasmin/metabolism , Endoplasmic Reticulum/metabolism , Lysine/metabolism , Mutation/genetics , Protein Binding , Protein Processing, Post-Translational , Protein Transport , Reproducibility of Results , Surface Plasmon Resonance , Two-Hybrid System Techniques
14.
Biochem Biophys Res Commun ; 377(2): 395-399, 2008 Dec 12.
Article in English | MEDLINE | ID: mdl-18851946

ABSTRACT

Using cDNA microarray analysis, we found that the mRNA of YJL217W and several other genes related to cell wall organization and biogenesis were up-regulated by galactose in Saccharomyces cerevisiae early during the induction process. YJL217W is also known as REE1 (Regulation of Enolase I). Both the Gal4 regulatory region and the Mac1 binding domain were found on the upstream region of REE1, and the expression of REE1 was up-regulated by galactose but not by glucose. The up-regulation of REE1 by galactose was not observed in the Deltagal4 strain. From the two-hybrid analysis, we found that Ree1 physically interacted with Gal83. Furthermore, from 2-D gel electrophoresis we found that the deletion of REE1 resulted in the up-regulation of Eno1. From Western blotting, we learned that the expression of Eno1 in the Deltaree1 strain was different from that in wild-type strains and that Eno1 expression was not changed by glucose stimulation. Taken together, these results suggest that Ree1p functions in the galactose metabolic pathway via the Gal83 protein and that it may control the level of Eno1p, which is also affected by the Snf1 complex, in S. cerevisiae.


Subject(s)
Galactose/metabolism , Phosphopyruvate Hydratase/biosynthesis , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Base Sequence , Galactose/pharmacology , Gene Deletion , Gene Expression Regulation, Fungal , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Protein Biosynthesis , Repressor Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Two-Hybrid System Techniques
15.
Biosci Biotechnol Biochem ; 70(5): 1089-101, 2006 May.
Article in English | MEDLINE | ID: mdl-16717408

ABSTRACT

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.


Subject(s)
Bacterial Proteins/chemistry , Geobacillus stearothermophilus/enzymology , Protein Serine-Threonine Kinases/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Base Sequence , Cations, Divalent/chemistry , Cloning, Molecular , Fructosediphosphates/chemistry , Geobacillus stearothermophilus/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Phosphates/chemistry , Phosphorylases/chemistry , Phosphorylases/genetics , Phosphorylases/isolation & purification , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/isolation & purification , Sequence Analysis, Protein
16.
Fungal Genet Biol ; 43(4): 273-82, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16464625

ABSTRACT

The ScFTR1 gene encodes an iron permease in Saccharomyces cerevisiae. Its homologues, FgFtr1 and FgFtr2, were identified from filamentous pathogenic plant fungus, Fusarium graminearum. Homologies between the deduced amino acid sequences of ScFtr1p and FgFtr1 and FgFtr2 were 56 and 54%, respectively, and both had REXXE sequences, which form the conserved amino acid sequence of ScFtr1p. FgFtr1 expression increased under iron depletion, and although FgFtr2 mRNA was not detected in the wild-type strain, it was detected in the deltafgftr1 strain in the iron-depleted condition. When the FgFtr1 and FgFtr2 were deleted, the amount of growth was found not to be different from the wild-type in iron-depleted media. However, the mRNA of FgSid, a homologue of the SIDA of Aspergillus fumigatus, was dramatically increased in the deltafgftr1/deltafgftr2 strain and in an iron-depleted condition. FgFtr1 and FgFtr2 genes act as functional complements when they are introduced into the S. cerevisiae deltaScftr1 strain. The deltaScftr1 strain, which contains either the FgFtr1 or FgFtr2, grew well in iron-depleted media. Moreover, specific alteration of the REXXE consensus sequence of FgFtr1 and FgFtr2 did not allow for sustained growth of the deltaScftr1 strain on iron-depleted medium. The iron uptake activity was recovered when FgFtr1 and FgFtr2 genes were introduced into the deltaScftr1 strain. Though the Fet3p in S. cerevisiae was found on the intracellular vesicle in the deltaScftr1 strain, Fet3p was found on the plasma membrane when FgFtr1 or FgFtr2 was introduced into the deltaftr1 strain. An infection test was carried out with deletion strains; however, no change in the ability of these strains to cause disease was observed. These results suggest that FgFtr1 and FgFtr2 may function as iron permeases in the reductive iron uptake pathway and that they do not play major roles in the pathogenicity of F. graminearum.


Subject(s)
Fungal Proteins/physiology , Fusarium/enzymology , Iron/metabolism , Membrane Transport Proteins/physiology , Amino Acid Motifs/genetics , Amino Acid Sequence , Blotting, Southern , Conserved Sequence , DNA, Fungal/genetics , Fungal Proteins/genetics , Fusarium/genetics , Fusarium/metabolism , Fusarium/pathogenicity , Gene Deletion , Gene Expression Regulation, Fungal , Genetic Complementation Test , Hordeum/microbiology , Membrane Transport Proteins/genetics , Molecular Sequence Data , Plant Diseases/microbiology , RNA, Fungal/analysis , RNA, Messenger/analysis , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic , Virulence
17.
Biosci Biotechnol Biochem ; 68(7): 1414-23, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15277745

ABSTRACT

The gene encoding the catabolite control protein A (CcpA) of Bacillus stearothermophilus No. 236, a strong xylanolytic bacterium, was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the ccpA gene corresponded to an open reading frame of 1,005 bp that encodes a polypeptide of 334 amino acid residues with a calculated molecular mass of 36,902 kDa. The CcpA protein belonging to the LacI/GalR family of transcriptional regulators was produced by a recombinant E. coli strain expressing the B. stearothermophilus No. 236 ccpA gene and purified to apparent homogeneity. The transcription start site was mapped at a position 63 nucleotides upstream of the translation initiation codon, and a presumed promoter sequence was also identified. The deduced amino acid sequence of the ccpA gene product contained the helix-turn-helix motif found in many DNA-binding proteins, and showed the highest identity (62%) with CcpA from B. subtilis. The B. stearothermophilus No. 236 ccpA gene was demonstrated to be able to complement a B. subtilis ccpA mutant that exhibited two distinct mutant phenotypes: a growth defect and a release of carbon catabolite repression (CCR). These results indicate that the ccpA gene product of B. stearothermophilus No. 236 is functionally active also in B. subtilis. Electrophoretic mobility shift assay with the purified CcpA revealed that the CcpA of B. stearothermophilus No. 236 bound specifically to the xynA creB (catabolite responsive element B) sequence. Taken together, these results strongly suggest that the CcpA protein participates in CCR of B. stearothermophilus No. 236 xynA gene.


Subject(s)
Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Geobacillus stearothermophilus/genetics , Repressor Proteins/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Blotting, Northern , Chromatography, Agarose , Cloning, Molecular , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Genetic Complementation Test , Geobacillus stearothermophilus/metabolism , Molecular Sequence Data , Molecular Weight , Open Reading Frames , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Transcription, Genetic/physiology
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