scholarly journals The First Step of Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 Depends on an Alcohol Dehydrogenase-Type Enzyme

2021 ◽  
Vol 22 (4) ◽  
pp. 1552
Author(s):  
Arūnas Krikštaponis ◽  
Gintaras Urbelis ◽  
Rolandas Meškys
Keyword(s):  
Alcohol Dehydrogenase ◽  
Pyrone Ring ◽  
Qpcr Analysis ◽  
Pseudomonas Mandelii ◽  
Soil Isolate ◽  
Ring Transformations ◽  
Dna Fragment ◽  
Inducible Genes

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.

scholarly journals Engineering nucleosomes for generating diverse chromatin assemblies

2021 ◽  
Author(s):  
Zenita Adhireksan ◽  
Deepti Sharma ◽  
Phoi Leng Lee ◽  
Qiuye Bao ◽  
Sivaraman Padavattan ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Dna Nanostructures ◽  
Macromolecular Assemblies ◽  
Maximum Resolution ◽  
Different Types ◽  
Chromatin Structural ◽  
Dna Fragment

Abstract Structural characterization of chromatin is challenging due to conformational and compositional heterogeneity in vivo and dynamic properties that limit achievable resolution in vitro. Although the maximum resolution for solving structures of large macromolecular assemblies by electron microscopy has recently undergone profound increases, X-ray crystallographic approaches may still offer advantages for certain systems. One such system is compact chromatin, wherein the crystalline state recapitulates the crowded molecular environment within the nucleus. Here we show that nucleosomal constructs with cohesive-ended DNA can be designed that assemble into different types of circular configurations or continuous fibers extending throughout crystals. We demonstrate the utility of the method for characterizing nucleosome compaction and linker histone binding at near-atomic resolution but also advance its application for tackling further problems in chromatin structural biology and for generating novel types of DNA nanostructures. We provide a library of cohesive-ended DNA fragment expression constructs and a strategy for engineering DNA-based nanomaterials with a seemingly vast potential variety of architectures and histone chemistries.

New Inhibitors of Alcohol Dehydrogenase: Studies in Vivo and in Vitro in the Rat

1983 ◽  
Vol 7 (3) ◽  
pp. 264-270 ◽  
Author(s):  
Catherine Delmas ◽  
Georges de Saint Blanquat ◽  
Charles Freudenreich ◽  
Jean-FranCois Bielimann
Keyword(s):  
Alcohol Dehydrogenase

scholarly journals Dissection of Mechanisms of Realgar Intervening Telomere Protein POT1, TRF1, TRF2 Expression to Regulate Telomere Dynamics in THP-1 Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5896-5896
Author(s):  
Rui-Rong Xu ◽  
Nan-Xin Song ◽  
Xiao-Long Wu ◽  
Si-Yuan Cui ◽  
Zhen-Zhen Wang ◽  
...  
Keyword(s):  
Theoretical Basis ◽  
Cell Cycle Distribution ◽  
G1 Arrest ◽  
Mrna Levels ◽  
Protein Levels ◽  
Qpcr Analysis ◽  
Telomere Protein ◽  
Telomere Dynamics

Abstract Objective: To observe the mechanisms of realgar intervening telomere protein POT1, TRF1, TRF2 expression to regulate telomere dynamics in THP-1 cells, and elucidate the experimental and theoretical basis for realgar targeted threapy of AML. Methods: 1.In vitro: (1)Cultured human acute myeloid leukemia THP-1 cells. (2), The cells were incubated in absence or presence of increasing concentration of realgar for 24/48 h, from which determined the IC50. Cell viability was tested by CCK-8 assay. (3)Apoptotic rate and cell cycle distribution were tested by FCM. (4)Each group of POT1, TRF1, TRF2 protein levels were tested by western-blot analysis. (5)Each group of POT1,TRF1,TRF2 mRNA levels were tested by RT-qPCR analysis. 2.In vivo: (1)Established THP-1 model in NOD/SCID mice, and the mouse were treated by realgar. (2)All the mouse were killed by institutionally approved method after 3 weeks. The apoptotic rate and cell cycle distribution of mice spleen cells were examined by FCM. (3)The changes of POT1, TRF1, TRF2 protein levels in organic tissue were detected by IHC. Results: 1.In vitro: (1)CCK-8 assay showed that after treated by realgar, THP-1 cells growth were inhibited and the cell viability decreased in a dose- and time- dependent manner, IC50 was 0.023μg/mL. Therefore, we choose a medium concentration was 0.015μg/mL (lower than IC50) at 48h. (2)Our study demonstrated that after treatment for 48h, the apoptotic rate and the G1 arrest of these THP-1 cell increased, compared with control group. (3)As shown by western blot analysis, compared with controls, realgar group POT1, TRF1 protein levels significantly increaesed and TRF2 decreased(P<0.01). (4) As shown by by RT-qPCR analysis, POT1, TRF1, TRF2 mRNA levels were consistent with their protein levels. 2.In vivo: (1) The THP-1/NOD-SCID model was established, and survival curve showed that compared with controls, realgar group mouse had longer life span(P<0.01). (2) The results of FCM showed that after treatment of realgar, the rate of THP-1 cell apoptosis and G1 arrest significantly increased(P<0.01). (3) The results of IHC showed that compared with controls, POT1, TRF1 protein levels significantly increased and TRF2 decreased in realgar group mouse(P<0.01). Conclusion: In vitro and in vivo studies results indicated that realgar could significantly prolong the life span of the THP-1/NOD-SCID mice, inhibit proliferation and induce apoptosis of THP-1 cell, and regulate telomere dynamics through intervening THP-1 cell telomere protein POT1, TRF1, TRF2 expression. Our studies results provided experimental and theoretical basis for realgar targeted threapy of AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals The pathogenic biomarker alcohol dehydrogenase protein is involved in Bacillus cereus virulence and survival against host innate defence

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0259386
Author(s):  
Devon W. Kavanaugh ◽  
Constance Porrini ◽  
Rozenn Dervyn ◽  
Nalini Ramarao
Keyword(s):  
Alcohol Dehydrogenase ◽  
Bacillus Cereus ◽  
Large Strain ◽  
Bacterial Species ◽  
Gene Encoding ◽  
Pathogenic Potential ◽  
Strain Collection ◽  
Opportunistic Human Pathogen

Bacillus cereus is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. B. cereus is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both in vitro and in vivo. Our results demonstrate that the adhB gene plays a significant role in resistance to nitric oxide and oxidative stress in vitro, as well as its pathogenic ability with regards to in vivo toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.

scholarly journals The monoclonal antibody Ca37, developed against Candida albicans alcohol dehydrogenase, inhibits the yeast in vitro and in vivo

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Aitziber Antoran ◽  
Leire Aparicio-Fernandez ◽  
Aize Pellon ◽  
Idoia Buldain ◽  
Leire Martin-Souto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Candida Albicans ◽  
Alcohol Dehydrogenase

Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase

2002 ◽  
Vol 363 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Tobias MODIG ◽  
Gunnar LIDÉN ◽  
Mohammad J. TAHERZADEH
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aldehyde Dehydrogenase ◽  
Pyruvate Dehydrogenase ◽  
Competitive Inhibition ◽  
Critical Role ◽  
Km Value ◽  
In Vivo Effects

The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated Km value of AlDH for furfural was found to be about 5μM, which was lower than that for acetaldehyde (10μM). For ADH, however, the estimated Km value for furfural (1.2mM) was higher than that for acetaldehyde (0.4mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition.

scholarly journals Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zheng Hu ◽  
Li Wang ◽  
Zhaoying Shi ◽  
Jing Jiang ◽  
Xiangning Li ◽  
...  
Keyword(s):  
Dna Assembly ◽  
Dna Templates ◽  
Core Fragment ◽  
Cas9 Nuclease ◽  
Overlap Extension ◽  
One Step ◽  
Dna Fragment ◽  
Polymerase Chain

Abstract Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

scholarly journals Defining the Regulatory Factors Required for Epidermal Gene Expression

2000 ◽  
Vol 20 (7) ◽  
pp. 2543-2555 ◽  
Author(s):  
Satrajit Sinha ◽  
Linda Degenstein ◽  
Cedith Copenhaver ◽  
Elaine Fuchs
Keyword(s):  
Transgenic Mice ◽  
Specific Activity ◽  
Regulatory Sequences ◽  
Mobility Shift ◽  
Heterologous Promoter ◽  
Hypersensitive Sites ◽  
Dna Fragment ◽  
High Level

ABSTRACT Keratins K5 and K14 are the hallmarks of mitotically active keratinocytes of stratified epithelia. They are transcribed at a high level and in a tissue-specific manner, enabling us to use the K14 gene to elucidate the regulatory mechanism underlying epidermis-specific transcription. We have identified four DNase I-hypersensitive sites (HSs) present in the 5′ regulatory sequences of the K14 gene under specific conditions where the gene is actively expressed. Two of these sites (HSsII and -III) are conserved in position and sequence within the human and mouse K14 genes. Using an in vivo transgenic approach and an in vitro keratinocyte culture approach, we have discovered that most of K14's transcriptional activity is restricted to a novel 700-bp regulatory domain encompassing these HSs. This enhancer is sufficient to confer epidermis-specific activity to a heterologous promoter in transfection assays in culture and in transgenic mice in vivo. A 125-bp DNA fragment encompassing HSsII harbors the majority of the transactivation activity in vitro, and electrophoretic mobility shift and mutational assays reveal a role for AP-1, ets, and AP-2 family members in orchestrating the keratinocyte-preferred expression of HSsII. The HSsII element also confers epidermal expressivity to a heterologous promoter in transgenic mice, although it is not sufficient on its own to fully restrict activity to keratinocytes. Within the HSsII element, the ets and AP-2 sites appear to be most critical in collaborating to regulate epidermal specificity in vivo.

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