scholarly journals Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

2005 ◽  
Vol 71 (10) ◽  
pp. 5920-5928 ◽  
Author(s):  
Corinna Stansen ◽  
Davin Uy ◽  
Stephane Delaunay ◽  
Lothar Eggeling ◽  
Jean-Louis Goergen ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Specific Activity ◽  
Temperature Shift ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Reverse Transcription Pcr ◽  
Glutamate Production ◽  
Lactate Utilization

ABSTRACT Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

Ontogeny of the expression and regulation of interleukin-6 (IL-6) and IL-1 mRNAs by human trophoblast cells during differentiation in vitro

1995 ◽  
Vol 147 (3) ◽  
pp. 487-496 ◽  
Author(s):  
A Stephanou ◽  
L Myatt ◽  
A L W Eis ◽  
N Sarlis ◽  
H Jikihara ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Internal Control ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Cytokine Mrna ◽  
Human Trophoblast ◽  
Reverse Transcription Pcr ◽  
Cytotrophoblast Cells ◽  
Almost All

Abstract During human placental differentiation, mononuclear cytotrophoblast cells fuse and differentiate into syncytiotrophoblast cells. Although syncytiotrophoblast cells have been shown to express interleukin-1α (IL-1α), IL-1β and IL-6, the pattern of expression of these cytokines during placental differentiation is unknown. We have examined the expression of IL-1α, IL-1β and IL-6 mRNA during differentiation of cytotrophoblast cells in culture. IL-1α, IL-1β and IL-6 mRNA levels were determined by semiquantitative reverse transcription-PCR analysis using glyceraldehyde phosphate dehydrogenase as an internal control. All three cytokine mRNA levels decreased markedly during trophoblast differentiation. After 6 days in culture, when almost all the cytotrophoblast cells had fused and differentiated into syncytiotrophoblast cells, the amounts of IL-1α, IL-1β and IL-6 mRNA were decreased by 87·1, 72·1 and 60·9% respectively. Exogenous IL-6 had differential effects on cytokine mRNA expression. When added to placental cultures during the first 6 days of culture, IL-6 markedly inhibited IL-6, IL-1α and IL-1β mRNA expression. However, when added to the cells during days 6–9 of culture, when most of the cells were syncytiotrophoblast cells, IL-6 stimulated IL-lα and IL-1β mRNA expression. The results of these studies indicate that IL-1α, IL-1β and IL-6 mRNA expression decreases markedly during cytotrophoblast differentiation in vitro and that the regulation of trophoblast cytokine mRNA levels changes during differentiation. Journal of Endocrinology (1995) 147, 487–496

scholarly journals The Global Repressor SugR Controls Expression of Genes of Glycolysis and of the l-Lactate Dehydrogenase LdhA in Corynebacterium glutamicum

2008 ◽  
Vol 190 (24) ◽  
pp. 8033-8044 ◽  
Author(s):  
Verena Engels ◽  
Steffen N. Lindner ◽  
Volker F. Wendisch
Keyword(s):  
Lactate Dehydrogenase ◽  
Corynebacterium Glutamicum ◽  
Target Genes ◽  
Lactate Production ◽  
Oxygen Deprivation ◽  
Mrna Levels ◽  
Phosphotransferase System ◽  
Gel Retardation ◽  
Activity Measurements

ABSTRACT The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), and NAD-dependent l-lactate dehydrogenase (ldhA). The deletion of sugR resulted in increased mRNA levels of eno, pyk, and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, l-lactate production by C. glutamicum was determined. The overexpression of sugR reduced l-lactate production by about 25%, and sugR deletion increased l-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative l-lactate dehydrogenase in C. glutamicum.

scholarly journals Desulfovibrio gigas Flavodiiron Protein Affords Protection against Nitrosative Stress In Vivo

2006 ◽  
Vol 188 (8) ◽  
pp. 2745-2751 ◽  
Author(s):  
Rute Rodrigues ◽  
João B. Vicente ◽  
Rute Félix ◽  
Solange Oliveira ◽  
Miguel Teixeira ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Nitrosative Stress ◽  
Reductase Activity ◽  
Anaerobic Growth ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Desulfovibrio Gigas ◽  
Reverse Transcription Pcr

ABSTRACT Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O2. Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas Δroo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O2 reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.

scholarly journals The DeoR-Type Regulator SugR Represses Expression of ptsG in Corynebacterium glutamicum

2007 ◽  
Vol 189 (8) ◽  
pp. 2955-2966 ◽  
Author(s):  
Verena Engels ◽  
Volker F. Wendisch
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Dna Microarray ◽  
Sole Carbon Source ◽  
Glucose Utilization ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Phosphotransferase System ◽  
High Fructose ◽  
Promoter Dna

ABSTRACT Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids. Uptake of the preferred carbon source glucose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is reduced during coutilization of glucose with acetate, sucrose, or fructose compared to growth on glucose as the sole carbon source. Here we show that the DeoR-type regulator SugR (NCgl1856) represses expression of ptsG, which encodes the glucose-specific PTS enzyme II. Overexpression of sugR resulted in reduced ptsG mRNA levels, decreased glucose utilization, and perturbed growth on media containing glucose. In mutants lacking sugR, expression of the ptsG′-′cat fusion was increased two- to sevenfold during growth on gluconeogenic carbon sources but remained similar during growth on glucose or other sugars. As shown by DNA microarray analysis, SugR also regulates expression of other genes, including ptsS and the putative NCgl1859-fruK-ptsF operon. Purified SugR bound to DNA regions upstream of ptsG, ptsS, and NCgl1859, and a 75-bp ptsG promoter fragment was sufficient for SugR binding. Fructose-6-phosphate interfered with binding of SugR to the ptsG promoter DNA. Thus, while during growth on gluconeogenic carbon sources SugR represses ptsG, ptsG expression is derepressed during growth on glucose or under other conditions characterized by high fructose-6-phosphate concentrations, representing one mechanism which allows C. glutamicum to adapt glucose uptake to carbon source availability.

scholarly journals Regulation of l-Lactate Utilization by the FadR-Type Regulator LldR of Corynebacterium glutamicum

2007 ◽  
Vol 190 (3) ◽  
pp. 963-971 ◽  
Author(s):  
Tobias Georgi ◽  
Verena Engels ◽  
Volker F. Wendisch
Keyword(s):  
Energy Source ◽  
Corynebacterium Glutamicum ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Carbon Sources ◽  
Upstream Region ◽  
Growth Defect ◽  
Mrna Levels ◽  
Lactate Utilization ◽  
In Cells

ABSTRACT Corynebacterium glutamicum can grow on l-lactate as a sole carbon and energy source. The NCgl2816-lldD operon encoding a putative transporter (NCgl2816) and a quinone-dependent l-lactate dehydrogenase (LldD) is required for l-lactate utilization. DNA affinity chromatography revealed that the FadR-type regulator LldR (encoded by NCgl2814) binds to the upstream region of NCgl2816-lldD. Overexpression of lldR resulted in strongly reduced NCgl2816-lldD mRNA levels and strongly reduced LldD activity, and as a consequence, a severe growth defect was observed in cells grown on l-lactate as the sole carbon and energy source, but not in cells grown on glucose, ribose, or acetate. Deletion of lldR had no effect on growth on these carbon sources but resulted in high NCgl2816-lldD mRNA levels and high LldD activity in the presence and absence of l-lactate. Purified His-tagged LldR bound to a 54-bp fragment of the NCgl2816-lldD promoter, which overlaps with the transcriptional start site determined by random amplification of cDNA ends-PCR and contains a putative operator motif typical of FadR-type regulators, which is −1TNGTNNNACNA10. Mutational analysis revealed that this motif with hyphenated dyad symmetry is essential for binding of LldD to the NCgl2816-lldD promoter. l-Lactate, but not d-lactate, interfered with binding of LldRHis to the NCgl2816-lldD promoter. Thus, during growth on media lacking l-lactate, LldR represses expression of NCgl2816-lldD. In the presence of l-lactate in the growth medium or under conditions leading to intracellular l-lactate accumulation, the l-lactate utilization operon is induced.

scholarly journals Analysis of Genes Involved in Arsenic Resistance in Corynebacterium glutamicum ATCC 13032

2005 ◽  
Vol 71 (10) ◽  
pp. 6206-6215 ◽  
Author(s):  
Efrén Ordóñez ◽  
Michal Letek ◽  
Noelia Valbuena ◽  
José A. Gil ◽  
Luis M. Mateos
Keyword(s):  
Corynebacterium Glutamicum ◽  
Bacterial Resistance ◽  
Regulatory Protein ◽  
Arsenate Reductase ◽  
Pcr Analysis ◽  
Arsenic Resistance ◽  
Recombinant Strains ◽  
Reverse Transcription Pcr ◽  
Insertional Mutant ◽  
Genes Encoding

ABSTRACT Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1′) located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1′, arsR2, arsB2, and arsC2 being inducible by arsenite.

scholarly journals The ldhA Gene, Encoding Fermentative l-Lactate Dehydrogenase of Corynebacterium glutamicum, Is under the Control of Positive Feedback Regulation Mediated by LldR

2009 ◽  
Vol 191 (13) ◽  
pp. 4251-4258 ◽  
Author(s):  
Koichi Toyoda ◽  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Lactate Dehydrogenase ◽  
Corynebacterium Glutamicum ◽  
Promoter Region ◽  
Lactate Production ◽  
Feedback Regulation ◽  
Gene Encoding ◽  
Key Enzyme ◽  
In The Wild ◽  
Lactate Utilization ◽  
Ldha Gene

ABSTRACT Corynebacterium glutamicum ldhA encodes l-lactate dehydrogenase, a key enzyme that couples l-lactate production to reoxidation of NADH formed during glycolysis. We previously showed that in the absence of sugar, SugR binds to the ldhA promoter region, thereby repressing ldhA expression. In this study we show that LldR is another protein that binds to the ldhA promoter region, thus regulating ldhA expression. LldR has hitherto been characterized as an l-lactate-responsive transcriptional repressor of l-lactate utilization genes. Transposon mutagenesis of a reporter strain carrying a chromosomal ldhA promoter-lacZ fusion (PldhA-lacZ) revealed that ldhA disruption drastically decreased expression of PldhA-lacZ. PldhA-lacZ expression in the ldhA mutant was restored by deletion of lldR, suggesting that LldR acts as a repressor of ldhA in the absence of l-lactate and the LldR-mediated repression is not relieved in the ldhA mutant due to its inability to produce l-lactate. lldR deletion did not affect PldhA-lacZ expression in the wild-type background during growth on either glucose, acetate, or l-lactate. However, it upregulated PldhA-lacZ expression in the sugR mutant background during growth on acetate. The binding sites of LldR and SugR are located around the −35 and −10 regions of the ldhA promoter, respectively. C. glutamicum ldhA expression is therefore primarily repressed by SugR in the absence of sugar. In the presence of sugar, SugR-mediated repression of ldhA is alleviated, and ldhA expression is additionally enhanced by LldR inactivation in response to l-lactate produced by LdhA.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals Activation of Apoptosis in a βB1-CTGF Transgenic Mouse Model

2021 ◽  
Vol 22 (4) ◽  
pp. 1997
Author(s):  
Maximilian Weiss ◽  
Sabrina Reinehr ◽  
Ana M. Mueller-Buehl ◽  
Johanna D. Doerner ◽  
Rudolf Fuchshofer ◽  
...  
Keyword(s):  
Open Angle Glaucoma ◽  
Bipolar Cells ◽  
Nerve Degeneration ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Pcr Analysis ◽  
Progression Rate ◽  
Suitable Model ◽  
Mrna Levels ◽  
Gabaergic Synapse ◽  
Slow Progression

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in βB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5–9/group) and quantitative real-time PCR analysis (n = 5–7/group) in 5- and 10-week-old βB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in βB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the βB1-CTGF mice a suitable model to study primary open-angle glaucoma.

scholarly journals MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 1202-1212
Author(s):  
Aichun Zhang ◽  
Yangzi Jin
Keyword(s):  
Allergic Rhinitis ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Specific Ige ◽  
Inflammatory Factors ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Pathway Activation ◽  
Nasal Lavage Fluid

AbstractAllergic rhinitis (AR) is one of the most common chronic diseases. This study examined whether microRNA (miR)-182-5p plays a role in AR by regulating toll-like receptor 4 (TLR4). First, data demonstrated that TLR4 was a target of miR-182-5p. Subsequently, AR mouse model was established to explore the role of miR-182-5p and TLR4 in AR in vivo. Initially, quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that miR-182-5p was downregulated, while TLR4 expression was upregulated in AR mice. Then we found that miR-182-5p mimic reduced the frequency of sneezing and nose rubbing of the AR mice. In addition, miR-182-5p mimic significantly increased ovalbumin (OVA)-specific IgE and leukotriene C4 expression levels in nasal lavage fluid (NLF) and serum of AR mice. miR-182-5p mimic decreased the number of inflammatory cells in NLF of AR mice. It also reduced the levels of inflammatory factors in the serum of AR mice, such as interleukin (IL)-4, IL-5, IL-13, IL-17 and tumor necrosis factor (TNF)-α, while increasing the release of IFN-γ and IL-2. Finally, miR-182-5p mimic inhibited NF-κB signaling pathway activation in AR mice. However, all effects of miR-182-5p mimic on AR mice were reversed by TLR4-plasmid. In conclusion, miR-182-5p/TLR4 axis may represent a novel therapeutic target for AR.

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