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Published: 9 March 2023
Last updated: 10 March 2023

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1

Li, Guo-qiang, Shan-shan Li, Ming-lu Zhang, Jun Wang, Lin Zhu, Feng-lai Liang, Ru-lin Liu, and Ting Ma. "Genetic Rearrangement Strategy for Optimizing the Dibenzothiophene Biodesulfurization Pathway in Rhodococcus erythropolis." Applied and Environmental Microbiology 74, no. 4 (December 28, 2007): 971–76. http://dx.doi.org/10.1128/aem.02319-07.

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ABSTRACT Dibenzothiophene (DBT) and its derivatives can be microbially desulfurized by enzymes DszC, DszA, and DszB, which are encoded by the operon dszABC and contribute to the conversion in tandem. We investigated the expression characteristics of the dsz operon. Our results revealed that the levels of transcription and translation of dszA, dszB, and dszC decreased according to the positions of the genes in the dsz operon. Furthermore, the translation of dszB was repressed by an overlapping structure in the dsz operon. In order to get better and steady expression of the Dsz enzymes and optimize the metabolic flux of DBT, we rearranged the dsz operon according to the catalytic capabilities of the Dsz enzymes and expressed the rearranged dsz operon, dszBCA, in Rhodococcus erythropolis. After rearrangement, the ratio of dszA, dszB, and dszC mRNAs in the cells was changed, from 11:3.3:1 to 1:16:5. Western blot analysis revealed that the levels of expression of dszB and dszC had been enhanced but that the expression of dszA had decreased. The desulfurization activity of resting cells prepared from R. erythropolis DRB, which carried the rearranged dsz operon, was about 12-fold higher than that of resting cells of R. erythropolis DRA, which carried the original operon in a similarly constructed vector.
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2

Sousa, João P. M., Pedro Ferreira, Rui P. P. Neves, Maria J. Ramos, and Pedro A. Fernandes. "The bacterial 4S pathway – an economical alternative for crude oil desulphurization that reduces CO2 emissions." Green Chemistry 22, no. 22 (2020): 7604–21. http://dx.doi.org/10.1039/d0gc02055a.

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We discuss structural and mechanistic aspects of the Dsz enzymes in the 4S pathway, with a focus on rational molecular strategies for enzyme engineering, aiming at enzyme catalytic rate and efficiency improvement to meet industrial demands.
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3

Pan, Jie, Fan Wu, Jia Wang, Linqing Yu, Naghmeh Hassanzadeh Khayyat, Benjamin C. Stark, and John J. Kilbane. "Enhancement of desulfurization activity by enzymes of the Rhodococcus dsz operon through coexpression of a high sulfur peptide and directed evolution." Fuel 112 (October 2013): 385–90. http://dx.doi.org/10.1016/j.fuel.2013.04.065.

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4

Tanaka, Yasuhiro, Osamu Yoshikawa, Kenji Maruhashi, and Ryuichiro Kurane. "The cbs mutant strain of Rhodococcus erythropolis KA2-5-1 expresses high levels of Dsz enzymes in the presence of sulfate." Archives of Microbiology 178, no. 5 (November 1, 2002): 351–57. http://dx.doi.org/10.1007/s00203-002-0466-7.

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5

Li, Lu, Lei Ye, Zhijie Guo, Wei Zhang, Xihao Liao, Ying Lin, and Shuli Liang. "A kinetic model to optimize and direct the dose ratio of Dsz enzymes in the 4S desulfurization pathway in vitro and in vivo." Biotechnology Letters 41, no. 11 (September 14, 2019): 1333–41. http://dx.doi.org/10.1007/s10529-019-02730-1.

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6

Duarte, Gabriela Frois, Alexandre Soares Rosado, Lucy Seldin, Welington de Araujo, and Jan Dirk van Elsas. "Analysis of Bacterial Community Structure in Sulfurous-Oil-Containing Soils and Detection of Species Carrying Dibenzothiophene Desulfurization (dsz) Genes." Applied and Environmental Microbiology 67, no. 3 (March 1, 2001): 1052–62. http://dx.doi.org/10.1128/aem.67.3.1052-1062.2001.

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ABSTRACT The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil. Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed. Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts. However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp.,Arthrobacter sp., and a bacterium of uncertain affiliation.dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment. PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonassp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures. Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively. Most of the strains carrying dszA or dszC were classified asR. erythropolis related, and all revealed the capacity to desulfurize DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8)dszA sequence and a large degree of internal conservation. The 37 sequences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil.
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7

Pan, Jie, Fan Wu, Jia Wang, Linqing Yu, Naghmeh Hassanzadeh Khayyat, Benjamin C. Stark, and John J. Kilbane II. "Corrigendum to “Enhancement of desulfurization activity by enzymes of the Rhodococcus dsz operon through coexpression of a high sulfur peptide and directed evolution” [Fuel 112 (2013) 385–390]." Fuel 113 (November 2013): 766. http://dx.doi.org/10.1016/j.fuel.2013.07.061.

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8

Elnashar, Magdy M. M., and Mohamed E. Hassan. "Novel Epoxy Activated Hydrogels for Solving Lactose Intolerance." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/817985.

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“Lactose intolerance” is a medical problem for almost 70% of the world population. Milk and dairy products contain 5–10% w/v lactose. Hydrolysis of lactose by immobilized lactase is an industrial solution. In this work, we succeeded to increase the lactase loading capacity to more than 3-fold to 36.3 U/g gel using epoxy activated hydrogels compared to 11 U/g gel using aldehyde activated carrageenan. The hydrogel’s mode of interaction was proven by FTIR, DSC, and TGA. The high activity of the epoxy group was regarded to its ability to attach to the enzyme’s –SH, –NH, and –OH groups, whereas the aldehyde group could only bind to the enzyme’s –NH2group. The optimum conditions for immobilization such as epoxy chain length and enzyme concentration have been studied. Furthermore, the optimum enzyme conditions were also deliberated and showed better stability for the immobilized enzyme and the Michaelis constants,KmandVmax, were doubled. Results revealed also that both free and immobilized enzymes reached their maximum rate of lactose conversion after 2 h, albeit, the aldehyde activated hydrogel could only reach 63% of the free enzyme. In brief, the epoxy activated hydrogels are more efficient in immobilizing more enzymes than the aldehyde activated hydrogel.
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9

Akhir, J., S. W. Budi, E. N. Herliyana, and Surono. "Lignocellulolytic enzyme potential of dark septate endophyte (dse) from Pinus merkusii roots in Dramaga Bogor Indonesia." IOP Conference Series: Earth and Environmental Science 959, no. 1 (January 1, 2022): 012031. http://dx.doi.org/10.1088/1755-1315/959/1/012031.

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Abstract The ability of Dark Septate Endophyte (DSE) derived from the roots of P. merkusii in decomposing organic matter is not much published. This study aims to determine the activity of lignolytic and cellulolytic enzymes, as well as the value of the cellulolytic index of Apls, isolates from P. merkusii roots taken from IPB Dramaga Bogor. Enzyme activity was tested by measuring the clear zone formed, while the cellulolytic index value was obtained by measuring the ratio of the clear zone formed around the colony. Data were analyzed descriptively. The results identified 10 isolates, 8 Apls isolates had potential enzyme activity that able to form clear zone, 5 isolates had lignolytic enzyme activity (Apls 1.4.1, Apls 2.3.1.1, Apls 3.4.2, Apls 2.1.1 and Apls 3.4 1.1) and 1 isolate had cellulolytic enzyme activity (Apls 3.1.3), and 2 isolates had lignocellulolytic enzyme activity (Apls 1.5.3 and Apls 3.4.3b), with the highest cellulolytic index value in Apls 1.5.3 isolates of 0,87 mm. 2 isolates had not lignocellulolytic enzymes activity (Apls 3.4.3 and Apls 3.1.4). The DSE roots of P. merkusii from IPB Dramaga Bogor had the potential for lignocellulolytic enzymes of 20 per cent of the total DSE found.
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10

Kloosterman, H., G. I. Hessels, J. W. Vrijbloed, G. J. Euverink, and L. Dijkhuizen. "(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica." Microbiology 149, no. 11 (November 1, 2003): 3321–30. http://dx.doi.org/10.1099/mic.0.26494-0.

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Prephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-d-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica. Deregulated, feedback-control-resistant mutants were isolated by incubation of A. methanolica on glucose mineral agar containing the toxic analogue p-fluoro-dl-phenylalanine (pFPhe). Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation. Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites. A. methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase). The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp. Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants. These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHP synthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are well integrated in A. methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.
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11

Bashirova, Anna, Subrata Pramanik, Pavel Volkov, Aleksandra Rozhkova, Vitaly Nemashkalov, Ivan Zorov, Alexander Gusakov, Arkady Sinitsyn, Ulrich Schwaneberg, and Mehdi Davari. "Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability." International Journal of Molecular Sciences 20, no. 7 (March 30, 2019): 1602. http://dx.doi.org/10.3390/ijms20071602.

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Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.
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12

Widiana, Dea Rizki, Sotharith Phon, Andriati Ningrum, and Lucia Dhiantika Witasari. "Purification and characterization of thermostable alpha‐amylase from Geobacillus sp. DS3 from Sikidang Crater, Central Java, Indonesia." Indonesian Journal of Biotechnology 27, no. 4 (December 30, 2022): 212. http://dx.doi.org/10.22146/ijbiotech.71643.

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Amylases are considered the most essential enzymes in biotechnology since they are widely utilized in the textile, food processing, and detergent industries. It is necessary to explore extracellular enzymatic activity in several microorganisms to discover a new potential application from amylases. In a previous study, thermophilic bacteria Geobacillus sp. DS3 isolated from Sikidang Crater, Dieng Plateau, Central Java, Indonesia showed amylase activity in starch medium at 70 °C. This study aimed to purify and characterize the thermostable alpha‐amylase from Geobacillus sp. DS3. The alpha‐amylase was produced and purified using ammonium sulfate and DEAE Sephadex A‐25 column. The enzyme activity was determined using the 3,5‐dinitrosalicylic acid (DNS) method. Geobacillus sp. DS3 optimally produced the alpha‐amylase at 60 °C for 15 h. The alpha‐amylase exhibited high enzymatic activity in 40–60% saturated ammonium sulfate extract. The molecular weight of the enzyme was estimated to be 58 kDa. The thermostable alpha‐amylase showed activity at the optimum temperature of 50 °C in 200 mM sodium phosphate buffer pH 7.0. The enzyme was inhibited by EDTA, PMSF, 2‐ME, and mostly by HgCl2. The Km and Vmax of the pure enzyme were 235.43 mM and 1428.57 U/mL, respectively. The result suggested that the purified thermostable alpha‐amylase from Geobacillus sp. DS3 offers potential application in areas of the food industry, such as the bakery industry.
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13

Kennamer, J. E., and A. M. Usmani. "DSC analysis of select diagnostic enzymes." Journal of Applied Polymer Science 42, no. 11 (June 5, 1991): 3073–74. http://dx.doi.org/10.1002/app.1991.070421128.

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14

Courilleau, Céline, Catherine Chailleux, Alain Jauneau, Fanny Grimal, Sébastien Briois, Elisa Boutet-Robinet, François Boudsocq, Didier Trouche, and Yvan Canitrot. "The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks." Journal of Cell Biology 199, no. 7 (December 24, 2012): 1067–81. http://dx.doi.org/10.1083/jcb.201205059.

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DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate–dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.
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15

Jiao, Yongqin, Patrik D'haeseleer, Brian D. Dill, Manesh Shah, Nathan C. VerBerkmoes, Robert L. Hettich, Jillian F. Banfield, and Michael P. Thelen. "Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community." Applied and Environmental Microbiology 77, no. 15 (June 17, 2011): 5230–37. http://dx.doi.org/10.1128/aem.03005-10.

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ABSTRACTIn microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ∼2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.
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16

Melati, I., G. Rahayu, Surono, H. Effendi, and C. Henny. "Decolourization of congo red synthetic dyes by dark septate endophytes." IOP Conference Series: Earth and Environmental Science 948, no. 1 (December 1, 2021): 012073. http://dx.doi.org/10.1088/1755-1315/948/1/012073.

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Abstract The use of fungi is known to be an eco-friendly and cost-competitive approach to degrade synthetic dyes such as Congo Red (CR) in industrial effluents. This research aimed to evaluate the potential of dark septate endophytes (DSE) fungi in decolourizing CR synthetic dyes. Two DSE strains, namely CPP and KSP, were studied to decolourize 50 mgL−1 CR based on the capability to produce the ligninolytic enzyme, dye decolourization efficiency, decolourization index, and fungal dry biomass weight after 7 and 14 days of incubation. CR decolourization was monitored spectrophotometry at 495 nm. The result indicated that CPP and KSP were successfully decolourized CR dye up to 97.00% and 85.00%, respectively, with decolourization index of 1.37 and 1.36 within 14 days. There is no significant difference in DSE growth with and without the addition of CR dye. In addition, these two DSE fungi (CPP and KSP) are able to produce ligninolytic enzymes. The results indicated that the DSE are potential to be used as decolourization agents for azo synthetic dyes. This is the first report on the ability of DSE to decolourize azo synthetic dyes.
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17

Walden, Patricia M., Andrew E. Whitten, Lakshmanane Premkumar, Maria A. Halili, Begoña Heras, Gordon J. King, and Jennifer L. Martin. "The atypical thiol–disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase." Acta Crystallographica Section D Structural Biology 75, no. 3 (February 26, 2019): 283–95. http://dx.doi.org/10.1107/s2059798318018442.

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Disulfide-bond-forming (DSB) oxidative folding enzymes are master regulators of virulence that are localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K-12 consists of a dithiol-oxidizing redox-relay pair (DsbA/B), a disulfide-isomerizing redox-relay pair (DsbC/D) and the specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast, the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these, α-DsbA1 and α-DsbB, form a redox-relay pair analogous to DsbA/B from E. coli. The third enzyme, α-DsbA2, incorporates a DsbA-like sequence but does not interact with α-DsbB. In comparison to other DsbA enzymes, α-DsbA2 has ∼50 extra N-terminal residues (excluding the signal peptide). The crystal structure of α-DsbA2ΔN, an N-terminally truncated form in which these ∼50 residues are removed, confirms the DsbA-like nature of this domain. However, α-DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. Firstly, α-DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase: i.e. its role is to shuffle rather than to introduce disulfide bonds. Moreover, small-angle X-ray scattering (SAXS) of α-DsbA2 reveals a homotrimeric arrangement that differs from those of the other characterized bacterial disulfide isomerases DsbC from Escherichia coli (homodimeric) and ScsC from Proteus mirabilis (PmScsC; homotrimeric with a shape-shifter peptide). α-DsbA2 lacks the shape-shifter motif and SAXS data suggest that it is less flexible than PmScsC. These results allow conclusions to be drawn about the factors that are required for functionally equivalent disulfide isomerase enzymatic activity across structurally diverse protein architectures.
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18

Lawrence, Ben M., Liza O’Donnell, Lee B. Smith, and Diane Rebourcet. "New Insights into Testosterone Biosynthesis: Novel Observations from HSD17B3 Deficient Mice." International Journal of Molecular Sciences 23, no. 24 (December 8, 2022): 15555. http://dx.doi.org/10.3390/ijms232415555.

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Androgens such as testosterone and dihydrotestosterone (DHT) are essential for male sexual development, masculinisation, and fertility. Testosterone is produced via the canonical androgen production pathway and is essential for normal masculinisation and testis function. Disruption to androgen production can result in disorders of sexual development (DSD). In the canonical pathway, 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) is viewed as a critical enzyme in the production of testosterone, performing the final conversion required. HSD17B3 deficiency in humans is associated with DSD due to low testosterone concentration during development. Individuals with HSD17B3 mutations have poorly masculinised external genitalia that can appear as ambiguous or female, whilst having internal Wolffian structures and testes. Recent studies in mice deficient in HSD17B3 have made the surprising finding that testosterone production is maintained, male mice are masculinised and remain fertile, suggesting differences between mice and human testosterone production exist. We discuss the phenotypic differences observed and the possible other pathways and enzymes that could be contributing to testosterone production and male development. The identification of alternative testosterone synthesising enzymes could inform the development of novel therapies to endogenously regulate testosterone production in individuals with testosterone deficiency.
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19

Rosato, Antonella, Angela Romano, Grazia Totaro, Annamaria Celli, Fabio Fava, Giulio Zanaroli, and Laura Sisti. "Enzymatic Degradation of the Most Common Aliphatic Bio-Polyesters and Evaluation of the Mechanisms Involved: An Extended Study." Polymers 14, no. 9 (April 30, 2022): 1850. http://dx.doi.org/10.3390/polym14091850.

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Commercial hydrolytic enzymes belonging to different subclasses (several lipases, proteinase k, cutinase) were investigated for their ability to degrade different aliphatic polyesters, i.e., poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBSA), two poly(caprolactone), having two different molecular weights, poly(lactic acid) (PLA) and poly(propylene carbonate) (PPC). The enzyme screening was first carried out by investigating the capacity of fully degrading the target polymers in 24 h, then weight loss measurements of selected polyesters and target enzymes were performed. Solid residues after enzyme degradation were characterized by proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and thermogravimetry (TGA). Liquid fractions were studied via GPC, 1H NMR and high-performance liquid chromatography (HPLC). PCL and PBSA were found to be the most biodegradable polyesters, under the conditions used in this study. PBS was fully degraded only by cutinase, whereas none of the tested enzymes were able to completely degrade PLA and PPC, in the conditions assessed here. Cutinase exhibited the highest hydrolytic activity on PBSA, while lipase from Candida sp. (CALB) on low molecular weight PCL. Chemical analyses on residual solids showed that the enzymatic degradation occurred homogeneously from the surface through an erosion mechanism and did not significantly affect the macromolecular structure and thermal stability. Cleaving action mode for each enzyme (endo- and/or exo-type) on the different polyesters were also proposed based on the evaluation of the degradation products in the liquid fraction.
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20

Cheng, Shi, Zitao Guo, Chaojuan Liang, Yi Shi, Peng Geng, Yu Xin, Zhenghua Gu, and Liang Zhang. "Immobilization of Phospholipase A1 Using a Protein-Inorganic Hybrid System." Polymers 13, no. 17 (August 26, 2021): 2865. http://dx.doi.org/10.3390/polym13172865.

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In this study, four kinds of phospholipase A1-metal (Al/Co/Cu/Mn) hybrid nanostructures were prepared for enhancing the stability of the free PLA1. The formed hybrid complexes were characterized by scanning electron microscope (SEM), Fourier infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The stability and substrate specificity of immobilized enzymes were subsequently determined. After immobilization, the temperature tolerance of PLA1–metal hybrid nanostructures was enhanced. The relative activity of PLA1–Al/Co/Cu hybrid nanostructures remained above 60% at 50 °C, while that of free enzyme was below 5%. The thermal transition temperature measured by differential scanning calorimetry (DSC) was found to increase from 65.59 °C (free enzyme) to 173.14 °C, 123.67 °C, 96.31 °C, and 114.79 °C, referring to PLA1–Cu/Co/Al/Mn hybrid nanostructures, respectively. Additionally, after a storage for fourteen days at 4 °C, the immobilized enzymes could exhibit approximately 60% of the initial activity, while the free PLA1 was inactivated after four days of storage. In brief, using Co2+, Cu2+, Al3+, and Mn2+ as the hybridization materials for immobilization could improve the catalytic properties and stability of the free PLA1, suggesting a promising method for a wider application of PLA1 in many fields such as food, cosmetics, and the pharmaceutical industry.
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21

DE GASPAR, IGNACIO, MARIA JOSE BLANQUEZ, BENITO FRAILE, RICARDO PANIAGUA, and MARIA ISABEL ARENAS. "The hatching gland cells of trout embryos: characterisation of N- and O-linked oligosaccharides." Journal of Anatomy 194, no. 1 (January 1999): 109–18. http://dx.doi.org/10.1017/s0021878298004488.

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A histochemical, light and electron microscopy study of the hatching gland cells (HGCs) in incubated 50-d-old trout embryos is reported. The distribution of carbohydrate residues in the glycoconjugates of these cells was studied by means of a battery of 13 different lectins conjugated with horseradish peroxidase (PNA, ConA, LCA, WGA, SBA, UEA-I, HPA, DBA) or digoxigenin (DSA, MAA, AAA, SNA, GNA). Identification of N- and O-linked oligosaccharides in HGCs was performed by application of both chemical and enzymatic treatments. Present results suggest that HGCs are seromucous cells which store both high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), and that their cytoplasmic granules, endoplasmic reticulum and Golgi complex contain additional sialic acid-rich glycoproteins. The negative charge of these glycoproteins might be responsible for the rapid expansion of mucin to form a highly hydrated gel, which would facilite the action of these enzymes in programmed cell death and might play a major role during the morphogenic events.
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Álvarez, J., Carolina Herrero Filgueira, Alexandre González, Cristóbal Colón Mejeras, Andrés Beiras Iglesias, Shunji Tomatsu, José Blanco Méndez, Asteria Luzardo Álvarez, María Couce, and Francisco Otero Espinar. "Enzyme-Loaded Gel Core Nanostructured Lipid Carriers to Improve Treatment of Lysosomal Storage Diseases: Formulation and In Vitro Cellular Studies of Elosulfase Alfa-Loaded Systems." Pharmaceutics 11, no. 10 (October 11, 2019): 522. http://dx.doi.org/10.3390/pharmaceutics11100522.

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Mucopolysaccharidosis IVA (Morquio A) is a rare inherited metabolic disease caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS). Until now, treatments employed included hematopoietic stem cell transplantation and enzyme replacement therapy (ERT); the latter being the most commonly used to treat mucopolysaccharidoses, but with serious disadvantages due to rapid degradation and clearance. The purpose of this study was to develop and evaluate the potential of nanostructured lipid carriers (NLCs) by encapsulating elosulfase alfa and preserving its enzyme activity, leading to enhancement of its biological effect in chondrocyte cells. A pegylated elosulfase alfa-loaded NLC was characterized in terms of size, ζ potential, structural lipid composition (DSC and XRD), morphology (TEM microscopy), and stability in human plasma. The final formulation was freeze-dried by selecting the appropriate cryoprotective agent. Viability assays confirmed that NLCs were non-cytotoxic to human fibroblasts. Imaging techniques (confocal and TEM) were used to assess the cellular uptake of NLCs loaded with elosulfase alfa. This study provides evidence that the encapsulated drug exhibits enzyme activity inside the cells. Overall, this study provides a new approach regarding NLCs as a promising delivery system for the encapsulation of elosulfase alfa or other enzymes and the preservation of its activity and stability to be used in enzymatic replacement therapy (ERT).
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Chailleux, Catherine, Sandrine Tyteca, Christophe Papin, François Boudsocq, Nadine Puget, Céline Courilleau, Mikhaïl Grigoriev, Yvan Canitrot, and Didier Trouche. "Physical interaction between the histone acetyl transferase Tip60 and the DNA double-strand breaks sensor MRN complex." Biochemical Journal 426, no. 3 (February 24, 2010): 365–71. http://dx.doi.org/10.1042/bj20091329.

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Chromatin modifications and chromatin-modifying enzymes are believed to play a major role in the process of DNA repair. The histone acetyl transferase Tip60 is physically recruited to DNA DSBs (double-strand breaks) where it mediates histone acetylation. In the present study, we show, using a reporter system in mammalian cells, that Tip60 expression is required for homology-driven repair, strongly suggesting that Tip60 participates in DNA DSB repair through homologous recombination. Moreover, Tip60 depletion inhibits the formation of Rad50 foci following ionizing radiation, indicating that Tip60 expression is necessary for the recruitment of the DNA damage sensor MRN (Mre11–Rad50–Nbs1) complex to DNA DSBs. Moreover, we found that endogenous Tip60 physically interacts with endogenous MRN proteins in a complex which is distinct from the classical Tip60 complex. Taken together, our results describe a physical link between a DNA damage sensor and a histone-modifying enzyme, and provide important new insights into the role and mechanism of action of Tip60 in the process of DNA DSB repair.
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Liu, Yangjin, Fan Jiang, Chunwei Du, Mengqing Li, Zhifu Leng, Xiuzhu Yu, and Shuang-Kui Du. "Optimization of Corn Resistant Starch Preparation by Dual Enzymatic Modification Using Response Surface Methodology and Its Physicochemical Characterization." Foods 11, no. 15 (July 26, 2022): 2223. http://dx.doi.org/10.3390/foods11152223.

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Corn starch was dually modified using thermostable α-amylase and pullulanase to prepare resistant starch (RS). The concentration of starch liquid, the amount of added thermostable α-amylase, the duration of enzymatic hydrolysis and the amount of added pullulanase were optimized using RSM to increase RS content of the treated sample. The optimum pretreatment conditions were 15% starch liquid, 3 U/g thermostable α-amylase, 35 min of enzymatic hydrolysis and 8 U/g pullulanase. The maximum RS content of 10.75% was obtained, and this value was significantly higher than that of native corn starch. The degree of polymerization (DP) of the enzyme-modified starch decreased compared with that of native starch. The scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were performed to assess structural changes in native and pretreated starch. The effect of dual enzyme pretreatment on the structure and properties of corn starch was significant. Unlike the untreated one, the pretreated corn starch showed clear pores and cracks. Significant differences in RS contents and structural characterization between starch pretreated and untreated with dual enzymes demonstrated that the dual enzyme modification of corn was effective in enhancing RS contents.
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Gorton, R. L., P. L. White, E. Bagkeris, D. Cotterall, R. Desai, T. McHugh, and C. C. Kibbler. "Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System." Journal of Clinical Microbiology 53, no. 7 (April 15, 2015): 2072–78. http://dx.doi.org/10.1128/jcm.00157-15.

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The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, −0.02; limits of agreement [LOA], −0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, −0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01;P= 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%;P< 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples.
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Mizumoto, Shuji, and Shuhei Yamada. "Histories of Dermatan Sulfate Epimerase and Dermatan 4-O-Sulfotransferase from Discovery of Their Enzymes and Genes to Musculocontractural Ehlers-Danlos Syndrome." Genes 14, no. 2 (February 16, 2023): 509. http://dx.doi.org/10.3390/genes14020509.

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Dermatan sulfate (DS) and its proteoglycans are essential for the assembly of the extracellular matrix and cell signaling. Various transporters and biosynthetic enzymes for nucleotide sugars, glycosyltransferases, epimerase, and sulfotransferases, are involved in the biosynthesis of DS. Among these enzymes, dermatan sulfate epimerase (DSE) and dermatan 4-O-sulfotranserase (D4ST) are rate-limiting factors of DS biosynthesis. Pathogenic variants in human genes encoding DSE and D4ST cause the musculocontractural type of Ehlers-Danlos syndrome, characterized by tissue fragility, joint hypermobility, and skin hyperextensibility. DS-deficient mice exhibit perinatal lethality, myopathy-related phenotypes, thoracic kyphosis, vascular abnormalities, and skin fragility. These findings indicate that DS is essential for tissue development as well as homeostasis. This review focuses on the histories of DSE as well as D4ST, and their knockout mice as well as human congenital disorders.
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Sathe, Manisha, Shruti Srivastava, Sumit Agrawal, and Ramrao Ghorpade. "Effect of Spacer and the Enzyme-Linked Immunosorbent Assay." Defence Science Journal 66, no. 5 (September 30, 2016): 471. http://dx.doi.org/10.14429/dsj.66.10700.

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The effect of spacers and the enzyme-linked immunosorbent assay (ELISA) formats on the functional parameters of assays such as lower detection limit, inhibitory concentration at 50 per cent (IC50), and specificity were studied. Enzyme conjugates having hydrophobic and hydrophilic spacers were prepared using O-isopropyl methylphosphonic acid (IMPA) and horseradish peroxidase (HRP) as an enzyme label. Comparison was made with reference to enzyme conjugate without any spacer. The present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate significantly improves the sensitivity of assays. An enhanced IC50 value achieved was 0.01 μg mL−1 for free antigen detection by direct immunoassay using hydrophilic spacers and precoating of ELISA plates by secondary antibody. The use of a hydrophilic spacer might have helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay.
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Arbizu, Shirley, Susanne U. Mertens-Talcott, Stephen Talcott, and Giuliana D. Noratto. "Dark Sweet Cherry (Prunus avium) Supplementation Reduced Blood Pressure and Pro-Inflammatory Interferon Gamma (IFNγ) in Obese Adults without Affecting Lipid Profile, Glucose Levels and Liver Enzymes." Nutrients 15, no. 3 (January 29, 2023): 681. http://dx.doi.org/10.3390/nu15030681.

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Dark sweet cherries (DSC) are rich in fiber and polyphenols that decrease risk factors associated with obesity. This single-blind randomized placebo-controlled study investigated DSC effects on inflammation, cardiometabolic, and liver health biomarkers in obese adults. Participants (>18 years, body mass index (BMI) = 30–40 kg/m2) consumed 200 mL of DSC drink (juice supplemented with DSC powder) (n = 19) or a placebo drink (n = 21) twice/day for 30 days. Anthropometric and physiological biomarkers were monitored at baseline (D1), mid-point (D15), and endpoint (D30) visits. Blood inflammatory biomarkers were assessed at D1, D15, and D30, and blood lipids, glucose, and liver enzymes at D1 and D30. DSC consumption lowered systolic blood pressure (SBP) (p = 0.05) and decreased diastolic blood pressure (DBP) compared to placebo (p = 0.04). Stratification of participants by BMI revealed a greater (p = 0.008) SBP reduction in BMI > 35 participants. DSC lowered pro-inflammatory interferon-gamma (IFNγ) (p = 0.001), which correlated with SBP changes. The interleukin (IL)-1RA and SBP changes were correlated in the placebo group, as well as triglycerides (TG) with DBP. The increased IL-10 levels in the placebo group suggested a compensatory mechanism to counteract elevated IFNγ levels. No significant between-group differences were detected for blood lipids, glucose, and liver enzymes. In conclusion, DSC helped to decrease blood pressure levels and inflammation in obese adults.
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Shakoor, Hira, Fatima Abdelfattah, Khaula Albadi, Mentalla Adib, Jaleel Kizhakkayil, and Carine Platat. "Inhibition of Digestive Enzyme and Stimulation of Human Liver Cells (HepG2) Glucose Uptake by Date Seeds Extract." Evidence-Based Complementary and Alternative Medicine 2020 (July 29, 2020): 1–10. http://dx.doi.org/10.1155/2020/4290702.

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Type 2 diabetes mellitus is increasing worldwide, and the United Arab Emirates is presenting one of the world’s highest prevalence rates. Dietary polyphenols exert an antidiabetic effect by modulating carbohydrates digestion and cellular glucose uptake. Due to their particularly high content in polyphenols, date seeds represent a potential antidiabetic agent. This study aims to determine if date seed polyphenols inhibit the activity of the enzymes (α-amylase and α-glucosidase), responsible for the digestion of carbohydrates and modulating the glucose uptake by human liver cells. In vitro activity of the intestinal α-glucosidase, pancreatic α-amylase, the glucose uptake by HepG2 cells, and the expression of GLUT4 and AMPK analyzed by western blotting (with and without date seeds extract). Our result showed that the maximum enzymes inhibition was obtained with 400 μg/mL and 900 μg/mL DSE for α-amylase and α-glucosidase, respectively. The HepG2 cell viability significantly decreased up to 80% at 4000 μg/mL DSE. The expression of GLUT4 was higher at 100 μg/mL DSE (with insulin and without insulin). However, the expressions of P-AMPK and AMPK were increased by DSE, mainly in a non-insulin-dependent manner. Therefore, DSE, by inhibiting carbohydrate digestion and stimulating glucose uptake by HepG2, can potentially demonstrate the therapeutic potential for diabetes management.
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Rai, G. P., and K. S. Venkateswaran. "Limitations and Practical Problems in Enzyme Linked Immunosorbent Assays ." Defence Science Journal 42, no. 2 (January 1, 1992): 71–84. http://dx.doi.org/10.14429/dsj.42.4353.

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Radha, Remya, and Sathyanarayana N. Gummadi. "pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase." Protein & Peptide Letters 26, no. 10 (September 30, 2019): 743–50. http://dx.doi.org/10.2174/0929866526666190617092944.

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Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.
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Qu, Jia, Wenyi Sun, Jie Zhong, Hao Lv, Mingrui Zhu, Jun Xu, Nan Jin, et al. "Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells." Journal of Cell Biology 216, no. 2 (January 25, 2017): 409–24. http://dx.doi.org/10.1083/jcb.201607008.

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Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet their molecular mechanisms remain poorly understood. Phosphoglycerate mutase 1 (PGAM1) is an important glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway, and serine biosynthesis in cancer cells. Herein, we report that PGAM1 is required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs) caused by DNA-damaging agents. Mechanistically, PGAM1 facilitates DSB end resection by regulating the stability of CTBP-interacting protein (CtIP). Knockdown of PGAM1 in cancer cells accelerates CtIP degradation through deprivation of the intracellular deoxyribonucleotide triphosphate pool and associated activation of the p53/p73 pathway. Enzymatic inhibition of PGAM1 decreases CtIP protein levels, impairs HR repair, and hence sensitizes BRCA1/2-proficient breast cancer to poly(ADP-ribose) polymerase (PARP) inhibitors. Together, this study identifies a metabolically dependent function of PGAM1 in promoting HR repair and reveals a potential therapeutic opportunity for PGAM1 inhibitors in combination with PARP inhibitors.
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Weterings, Eric, and David J. Chen. "DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?" Journal of Cell Biology 179, no. 2 (October 15, 2007): 183–86. http://dx.doi.org/10.1083/jcb.200705106.

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The DNA-dependent protein kinase (DNA-PK) is one of the central enzymes involved in DNA double-strand break (DSB) repair. It facilitates proper alignment of the two ends of the broken DNA molecule and coordinates access of other factors to the repair complex. We discuss the latest findings on DNA-PK phosphorylation and offer a working model for the regulation of DNA-PK during DSB repair.
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Chen, Pin, Xiaoqian Chen, Wei Yu, Bo Zhou, Lihua Liu, Yuzhuo Yang, Peng Du, Libo Liu, and Chun Li. "Ciprofloxacin stress changes key enzymes and intracellular metabolites of Lactobacillus plantarum DNZ-4." Food Science and Human Wellness 11, no. 2 (March 2022): 332–40. http://dx.doi.org/10.1016/j.fshw.2021.11.007.

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Davis, Anthony J., Benjamin P. C. Chen, and David J. Chen. "DNA-PK: A dynamic enzyme in a versatile DSB repair pathway." DNA Repair 17 (May 2014): 21–29. http://dx.doi.org/10.1016/j.dnarep.2014.02.020.

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Peek, James, and Dinesh Christendat. "Structural studies on dehydroshikimate dehydratase." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C475. http://dx.doi.org/10.1107/s2053273314095242.

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The soil bacterium, Pseudomonas putida, is capable of using the alicyclic compound quinate as a sole carbon source. During this process, quinate is converted to 3-dehydroshikimate, which subsequently undergoes a dehydration to form protocatechuate. The latter transformation is performed by the enzyme dehydroshikimate dehydratase (DSD). We have recombinantly produced DSD from P. putida and are currently performing x-ray crystallographic studies on the enzyme to gain structural insight into its catalytic mechanism and mode of substrate recognition. Initial crystals of DSD diffracted to 2.7 Ä resolution, but exhibited strong twinning. A redesigned construct has recently yielded crystals that diffract to similar resolution, but with a significantly reduced tendency toward twinning. Interestingly, sequence analysis of P. putida DSD reveals that the protein is in fact a fusion of two distinct domains: an N-terminal sugar phosphate isomerase-like domain associated with DSD activity, and a C-terminal hydroxyphenylpyruvate dioxygenase (HPPD)-like domain with unknown functional significance. Structural characterization of the protein may provide novel insight into the functional relevance of the unusual HPPD-like domain.
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Stenson, Trevor H., and Alison A. Weiss. "DsbA and DsbC Are Required for Secretion of Pertussis Toxin by Bordetella pertussis." Infection and Immunity 70, no. 5 (May 2002): 2297–303. http://dx.doi.org/10.1128/iai.70.5.2297-2303.2002.

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ABSTRACT The Dsb family of enzymes catalyzes disulfide bond formation in the gram-negative periplasm, which is required for folding and assembly of many secreted proteins. Pertussis toxin is arguably the most complex toxin known: it is assembled from six subunits encoded by five genes (for subunits S1 to S5), with 11 intramolecular disulfide bonds. To examine the role of the Dsb enzymes in assembly and secretion of pertussis toxin, we identified and mutated the Bordetella pertussis dsbA, dsbB, and dsbC homologues. Mutations in dsbA or dsbB resulted in decreased levels of S1 (the A subunit) and S2 (a B-subunit protein), demonstrating that DsbA and DsbB are required for toxin assembly. Mutations in dsbC did not impair assembly of periplasmic toxin but resulted in decreased toxin secretion, suggesting a defect in the formation of the Ptl secretion complex.
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Evdokimova, Viktoria, Manoj Gandhi, Jayanagendra Rayapureddi, James R. Stringer, and Yuri E. Nikiforov. "Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases." Endocrine-Related Cancer 19, no. 3 (February 9, 2012): 271–81. http://dx.doi.org/10.1530/erc-11-0314.

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Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such asRET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiationin vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads toRET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducingRET/PTCby electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved inRET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100 U) or StuI (100 and 250 U). The predominant rearrangement type wasRET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that producedRET/PTChad restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that producedRET/PTChad restriction sites present in clusters, which was not the case for RE that failed to induceRET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantlyRET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility thatRET/PTCrearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.
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Gnügge, Robert, and Lorraine S. Symington. "Efficient DNA double-strand break formation at single or multiple defined sites in the Saccharomyces cerevisiae genome." Nucleic Acids Research 48, no. 20 (October 14, 2020): e115-e115. http://dx.doi.org/10.1093/nar/gkaa833.

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Abstract DNA double-strand breaks (DSBs) are common genome lesions that threaten genome stability and cell survival. Cells use sophisticated repair machineries to detect and heal DSBs. To study DSB repair pathways and associated factors, inducible site-specific endonucleases have proven to be fundamental tools. In Saccharomyces cerevisiae, galactose-inducible rare-cutting endonucleases are commonly used to create a single DSB at a unique cleavage site. Galactose induction requires cell cultivation in suboptimal growth media, which is tedious especially when working with slow growing DSB repair mutants. Moreover, endonucleases that simultaneously create DSBs in multiple defined and unique loci of the yeast genome are not available, hindering studies of DSB repair in different genomic regions and chromatin contexts. Here, we present new tools to overcome these limitations. We employ a heterologous media-independent induction system to express the yeast HO endonuclease or bacterial restriction enzymes for single or multiple DSB formation, respectively. The systems facilitate tightly controlled and efficient DSB formation at defined genomic sites and will be valuable tools to study DSB repair at a local and genome-wide scale.
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Bedinskaya, V. V., L. A. Stepanenko, E. V. Simonova, A. G. Atlas, E. B. Rakova, and V. I. Zlobin. "Characterization of CRISPR/CAS System in Pseudomonas aeruginosa DSM 50071 Based on Bioinformatic Analysis of its Structures." Bulletin of Irkutsk State University. Series Biology. Ecology 40 (2022): 3–14. http://dx.doi.org/10.26516/2073-3372.2022.40.3.

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An algorithm for bioinformatic search and analysis of the structures of CRISPR/Cas systems of bacteria and screening of phages and plasmids through spacer sequences of CRISPR cassettes in the genome of the Pseudomonas aeruginosa strain DSM 50071 is presented.Using several search algorithms in the CRISPR/Cas system of the studied strain, the presence of three CRISPR loci and a group of Cas genes characteristic of Type-I Subtype-I-F was determined.Analysis of the spacer composition of CRISPR cassettes showed the presence of 31 to 43 spacers and a universal consensus repeat in all cassettes.Screening of the spacer sequences of the CRISPR cassettes of the studied strain showed their correspondence to the protospacers of phages and plasmids of bacteria of the families Pseudomonadaceae and Enterobacteriaceae. A complete characterization of bacteriophages to which this strain is resistant is given with their accession number in NCBI. A complete identification of spacers to protospacers of phages specific for bacteria of the Pseudomonadaceae family, most often isolated from the lungs of patients with bronchiectasis, pneumonia, as well as from hospitals and reservoirs, has been established.Full correspondence between spacers and protospacers of bacterial plasmids with pan-resistance and causing the development of respiratory failure and pneumonia was revealed. Correspondence of a segment of one spacer with protospacers of several bacterial phages of the same family was noted. This may indicate that the bacterium “expediently” acquires new spacers from DNA regions that are conserved for phages of bacteria of the same family.Genes that have phage protospacers in their structure have been identified.It has been established that these genes are responsible for the synthesis of enzymes that regulate the processes of virus reproduction.Therefore, activation of the CRISPR/Cas system in the genome of this strain will allow the restriction endonuclease to introduce breaks into unmethylated DNA, which will lead to disruption of the synthesis of this enzyme, and, consequently, disruption of bacteriophage replication.Correspondences of spacer sequences with protospacers of plasmids included in the structure of genes responsible for the synthesis of conjugative transfer enzymes were revealed.These results suggested that activation of the CRISPR/Cas system of this strain would disrupt the processes replication of bacteriophage and conjugation.The proposed algorithm made it possible to obtain information about the structure of the CRISPR/Cas system of the P. aeruginosa DSM 50071 strain, about its resistance to certain phages and plasmids. In the future, this will serve as the basis for creating approaches for targeted therapy of infectious diseases.
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Baronio, Ortolano, Menabò, Cassio, Baldazzi, Di Natale, Tonti, Vestrucci, and Balsamo. "46,XX DSD due to Androgen Excess in Monogenic Disorders of Steroidogenesis: Genetic, Biochemical, and Clinical Features." International Journal of Molecular Sciences 20, no. 18 (September 17, 2019): 4605. http://dx.doi.org/10.3390/ijms20184605.

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The term ‘differences of sex development’ (DSD) refers to a group of congenital conditions that are associated with atypical development of chromosomal, gonadal, or anatomical sex. Disorders of steroidogenesis comprise autosomal recessive conditions that affect adrenal and gonadal enzymes and are responsible for some conditions of 46,XX DSD where hyperandrogenism interferes with chromosomal and gonadal sex development. Congenital adrenal hyperplasias (CAHs) are disorders of steroidogenesis that mainly involve the adrenals (21-hydroxylase and 11-hydroxylase deficiencies) and sometimes the gonads (3-beta-hydroxysteroidodehydrogenase and P450-oxidoreductase); in contrast, aromatase deficiency mainly involves the steroidogenetic activity of the gonads. This review describes the main genetic, biochemical, and clinical features that apply to the abovementioned conditions. The activities of the steroidogenetic enzymes are modulated by post-translational modifications and cofactors, particularly electron-donating redox partners. The incidences of the rare forms of CAH vary with ethnicity and geography. The elucidation of the precise roles of these enzymes and cofactors has been significantly facilitated by the identification of the genetic bases of rare disorders of steroidogenesis. Understanding steroidogenesis is important to our comprehension of differences in sexual development and other processes that are related to human reproduction and fertility, particularly those that involve androgen excess as consequence of their impairment.
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Shui-Zhong, Luo, Wu Xiang-Zhi, Xu Pei-Lin, Pan Li-Hua, Zheng Zhi, Cao Li-Li, Zhao Yan-Yan, and Jiang Shao-Tong. "Enzyme-Resistant Dextrin from Chinese Yam Starch for Potential Application in Beverage Industry: Preparation, Physicochemical Properties and In Vitro Digestion." Current Topics in Nutraceutical Research 17, no. 2 (June 1, 2018): 140–47. http://dx.doi.org/10.37290/ctnr2641-452x.17:140-147.

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Preparation conditions, physicochemical properties and in vitro digestibility of enzyme-resistant dextrin from Chinese yam starches, which are native, easily accessible and cheap raw materials in China, were investigated in the current study. The results showed that the enzyme-resistant fraction content in enzyme-resistant dextrin increased and the whiteness of the enzyme-resistant dextrin decreased with the increasing concentrations of hydrochloric and citric acids and the rising heating temperatures, and the prolonged heating times. Considering the manufacturing cost and the beneficial usage, the enzyme-resistant dextrin produced by heating of Chinese yam starch in the presence of hydrochloric (0.11% dsb) and citric (0.2% dsb) acids at 150°C for 60min was selected. Enzyme-resistant dextrin had a low molecular weight of about 6800 Da and rich “non-digestible” α-1,2 glycosidic bond and was well-soluble in water, stable at pH 3.0–7.0 or after heating at 85°C for 0.5 h showed, high thermal stability and high resistant to the artificial gastric juice and small intestinal fluids. These characteristics make the selected enzyme-resistant dextrin suitable for use in the soft drink industry as the soluble dietary fibres and prebiotics in the beverages. This would provide an excellent opportunity to reduce the caloric value of the beverages and exert a beneficial effect on the intestinal microflora of people consuming beverages enriched with enzyme-resistant dextrins.
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43

Tuizer, Sapir, Manoj Pun, Iris Yedidia, and Zohar Kerem. "Disalicylic Acid Provides Effective Control of Pectobacterium brasiliense." Microorganisms 10, no. 12 (December 19, 2022): 2516. http://dx.doi.org/10.3390/microorganisms10122516.

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Bis(2-carboxyphenyl) succinate (disalicylic acid; DSA) is composed of two salicylic acids connected by a succinyl linker. Here, we propose its use as a new, synthetic plant-protection agent. DSA was shown to control Pectobacterium brasiliense, an emerging soft-rot pathogen of potato and ornamental crops, at minimal inhibitory concentrations (MIC) lower than those of salicylic acid. Our computational-docking analysis predicted that DSA would inhibit the quorum-sensing (QS) synthase of P. brasiliense ExpI more strongly than SA would. In fact, applying DSA to P. brasiliense inhibited its biofilm formation, secretion of plant cell wall-degrading enzymes, motility and production of acyl–homoserine lactones (AHL) and, subsequently, impaired its virulence. DSA also inhibited the production of AHL by a QS-negative Escherichia coli strain (DH5α) that had been transformed with P. brasiliense AHL synthase, as demonstrated by the biosensors Chromobacterium violaceaum CV026 and E. coli pSB401. Inhibition of the QS machinery appears to be one of the mechanisms by which DSA inhibits specific virulence determinants. A new route is proposed for the synthesis of DSA, which holds greater potential for use as an anti-virulence agent than its precursor SA. Based on these findings, DSA is an excellent candidate for repurposing for new applications.
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44

Ng, Philip, and Mark D. Baker. "Mechanisms of Double-Strand-Break Repair During Gene Targeting in Mammalian Cells." Genetics 151, no. 3 (March 1, 1999): 1127–41. http://dx.doi.org/10.1093/genetics/151.3.1127.

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AbstractIn the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin μ-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal μ-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal μ-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process: The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an “end” bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence.Formation of hDNA was frequently associated with gene targeting and, in most cases, began ∼645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.
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45

Inglingstad, Ragnhild Aabøe, Tove G. Devold, Ellen K. Eriksen, Halvor Holm, Morten Jacobsen, Kristian H. Liland, Elling O. Rukke, and Gerd E. Vegarud. "Comparison of the digestion of caseins and whey proteins in equine, bovine, caprine and human milks by human gastrointestinal enzymes." Dairy Science & Technology 90, no. 5 (April 7, 2010): 549–63. http://dx.doi.org/10.1051/dst/2010018.

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46

Habib, Hosam M., Esmail M. El-Fakharany, Usama D. Souka, Fatma M. Elsebaee, Mohamed G. El-Ziney, and Wissam H. Ibrahim. "Polyphenol-Rich Date Palm Fruit Seed (Phoenix Dactylifera L.) Extract Inhibits Labile Iron, Enzyme, and Cancer Cell Activities, and DNA and Protein Damage." Nutrients 14, no. 17 (August 27, 2022): 3536. http://dx.doi.org/10.3390/nu14173536.

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Date palm fruit seed (Phoenix dactylifera L.) extract (DSE), an under-utilized resource, is a rich source of polyphenols with high potency for disease prevention and antioxidative activities. For the first time, the present study demonstrated that DSE inhibits labile iron activity and DNA and BSA damage and inhibits acetylcholinesterase and tyrosinase activities. Moreover, DSE reduces the proliferation of hepatic, colorectal, and breast cancer cells dose-dependently through apoptotic mechanisms. Furthermore, DSE significantly suppressed the expression of both BCl-2 and P21 genes and increased the P53 expression level when compared with the untreated cells and the 5-FU treated cells. These findings suggest a strong potential for DSE in protecting against the iron-catalyzed ferroptosis that results in programmed cell death. The results also confirm the efficacy of DSE against cancer cells. Therefore, DSE constitutes a valuable candidate for developing functional foods and for natural compound-based chemotherapy for the pharmaceutical and nutraceutical industries.
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47

Krishnam Raju, Kovoru, Beeravelli Sudhakar, and Kolapalli Venkata Ramana Murthy. "Factorial Design Studies and Biopharmaceutical Evaluation of Simvastatin Loaded Solid Lipid Nanoparticles for Improving the Oral Bioavailability." ISRN Nanotechnology 2014 (February 13, 2014): 1–8. http://dx.doi.org/10.1155/2014/951016.

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Statins are HMG-CoA reductase inhibitors, which lower the cholesterol level through reversible and competitive inhibition; they are involved in the biosynthesis of cholesterol and other sterols. Simvastatin exhibits poor oral bioavailability (<5%) and undergoes extensive microsomal metabolism by CYP enzymes. CYP3A4 is the major metabolizing enzyme that metabolizes lactone form of simvastatin and significantly lowers intestinal uptake. The hydrophobic properties of simvastatin prevent complete dissolution of the drug in the intestinal fluid which also contributes to its lower bioavailability. SLNs are alternative carrier system to polymeric nanoparticles. SLNs are in submicron size range (1–1000 nm). To overcome the hepatic first pass metabolism and to enhance the bioavailability, intestinal lymphatic transport of drugs can be exploited. In the present study, attempt has been made to prepare solid lipid nanoparticles of simvastatin to improve the bioavailability. SLNs of simvastatin were prepared with Trimyristin by hot homogenization followed by ultrasonication method. The SLNs were characterized for various physicochemical properties and analytical techniques like PXRD, DSC to study thermal nature and morphology of formulation and excipients. Promising results of the study indicated the applicability of simvastatin solid lipid nanoparticles as potential tools for improvement of bioavailability of poorly soluble drugs.
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48

Deng, Xun, Xiaoshuang Song, Saiyaremu Halifu, Wenjing Yu, and Ruiqing Song. "Effects of Dark Septate Endophytes Strain A024 on Damping-off Biocontrol, Plant Growth and the Rhizosphere Soil Enviroment of Pinus sylvestris var. mongolica Annual Seedlings." Plants 9, no. 7 (July 20, 2020): 913. http://dx.doi.org/10.3390/plants9070913.

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Dark septate endophytes (DSEs) exert a vital role in promoting plant growth, improving mineral absorption, biological disease control, and enhancing plant stress resistance. The effects of dark septate endophyte strain, Phialocephala bamuru A024 on damping-off biocontrol, plant development, nutrients within the rhizosphere soil, as well as bacterial communities in the annual seedlings of P. sylvestris var. Mongolica were studied. According to our findings, following P. bamuru A024 inoculation, the damping-off disease morbidity decreased significantly compared with control, some physiological indices such as β-1,3-glucanase, chitinase enzyme activity as well as a soluble protein and proline content in P. sylvestris var. mongolica were elevated under R. solani stress. After inoculation with P. bamuru A024, the biomass in seedlings, nutrients in soil, root structure index, together with activities of soil enzymes were remarkably up-regulated relative to control (p < 0.05). As suggested by the results of high-throughput sequencing, the microbial structure in the rhizosphere soil of the P. sylvestris var. mongolica showed significant differences (p < 0.05) after P. bamuru A024 inoculation compared to control treatment and the rhizosphere soil bacterial community structure after DSE A024 inoculation was positively correlated to the main soil nutrition indices.
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Kelly, Vincent P., Takafumi Suzuki, Osamu Nakajima, Tsuyoshi Arai, Yoshitaka Tamai, Satoru Takahashi, Susumu Nishimura, and Masayuki Yamamoto. "The Distal Sequence Element of the Selenocysteine tRNA Gene Is a Tissue-Dependent Enhancer Essential for Mouse Embryogenesis." Molecular and Cellular Biology 25, no. 9 (May 1, 2005): 3658–69. http://dx.doi.org/10.1128/mcb.25.9.3658-3669.2005.

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ABSTRACT Appropriate expression of the selenocysteine tRNA (tRNASec) gene is necessary for the production of an entire family of selenoprotein enzymes. This study investigates the consequence of disrupting an upstream enhancer region of the mouse tRNASec gene (Trsp) known as the distal sequence element (DSE) by use of a conditional repair gene targeting strategy, in which a 3.2-kb insertion was introduced into the promoter of the gene. In the absence of DSE activity, homozygous mice failed to develop in utero beyond embryonic day 7.5 and had severely decreased levels of selenoprotein transcript. Cre-mediated removal of the selection cassette recovered DSE regulation of Trsp, restoring wild-type levels of tRNASec expression and allowing the generation of viable rescued mice. Further analysis of targeted heterozygous adult mice revealed that the enhancer activity of the DSE is tissue dependent since, in contrast to liver, heart does not require the DSE for normal expression of Trsp. Similarly, in mouse cell lines we showed that the DSE functions as a cell-line-specific inducible element of tRNASec. Together, our data demonstrate that the DSE is a tissue-dependent regulatory element of tRNASec expression and that its activity is vital for sufficient tRNASec production during mouse embryogenesis.
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50

Frigerio, Chiara, Elena Di Nisio, Michela Galli, Chiara Vittoria Colombo, Rodolfo Negri, and Michela Clerici. "The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice." International Journal of Molecular Sciences 24, no. 4 (February 7, 2023): 3248. http://dx.doi.org/10.3390/ijms24043248.

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DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.
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