Problems utilizing an enzyme sensitive site assay for photorepair of exogenous DNA with cell-free extracts made from amphibian embryos

2000 ◽  
Vol 78 (10) ◽  
pp. 1869-1872 ◽  
Author(s):  
M Alex Smith
Keyword(s):  
Ultraviolet B ◽  
Signal Loss ◽  
Amphibian Species ◽  
Exogenous Dna ◽  
Free Protein ◽  
Amphibian Embryos ◽  
Damage Repair ◽  
Sensitive Site ◽  
Nuclease Digestion ◽  
Proper Estimation

Attempts were made to assay the ultraviolet-B (UVB) damage repair ability of seven Ontario amphibian species using an enzyme sensitive site restriction-enzyme (ESS) assay. Cell-free protein extracts of amphibian eggs caused the degradation of even high (9 mg/mL) exogenous DNA concentrations. This type of signal loss is characteristic of nuclease digestion. High endogenous concentrations of amphibian nucleases appear to preclude the use of plasmid DNA-ESS assays to determine the UVB damage repair abilities of amphibian eggs. Proper estimation of amphibian ultraviolet damage repair characteristics, using any assay, is reliant upon the generation of cell-free protein extracts created from amphibian embryos covered in protective jelly. The process of releasing the embryo from the glycoprotein-carbohydrate jelly ("dejellying") is achieved by shaking the jelly mass in a solution of 2% L-cysteine. Equivalent exposure to 2% L-cysteine results in a radically different end product with different amphibian taxa. These previously unreported phenomena have important implications for the production, standardization, and reporting of amphibian photorepair data.

scholarly journals hSSB2 (NABP1) is required for the recruitment of RPA during the cellular response to DNA UV damage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Didier Boucher ◽  
Ruvini Kariawasam ◽  
Joshua Burgess ◽  
Adrian Gimenez ◽  
Tristan E. Ocampo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Excision Repair ◽  
Protein A ◽  
Ultraviolet B ◽  
Repair System ◽  
Uvb Radiation ◽  
Exogenous Dna

AbstractMaintenance of genomic stability is critical to prevent diseases such as cancer. As such, eukaryotic cells have multiple pathways to efficiently detect, signal and repair DNA damage. One common form of exogenous DNA damage comes from ultraviolet B (UVB) radiation. UVB generates cyclobutane pyrimidine dimers (CPD) that must be rapidly detected and repaired to maintain the genetic code. The nucleotide excision repair (NER) pathway is the main repair system for this type of DNA damage. Here, we determined the role of the human Single-Stranded DNA Binding protein 2, hSSB2, in the response to UVB exposure. We demonstrate that hSSB2 levels increase in vitro and in vivo after UVB irradiation and that hSSB2 rapidly binds to chromatin. Depletion of hSSB2 results in significantly decreased Replication Protein A (RPA32) phosphorylation and impaired RPA32 localisation to the site of UV-induced DNA damage. Delayed recruitment of NER protein Xeroderma Pigmentosum group C (XPC) was also observed, leading to increased cellular sensitivity to UVB. Finally, hSSB2 was shown to have affinity for single-strand DNA containing a single CPD and for duplex DNA with a two-base mismatch mimicking a CPD moiety. Altogether our data demonstrate that hSSB2 is involved in the cellular response to UV exposure.

scholarly journals Nematode Parasites of Anurans from Three Cocoa Farms in Ondo State, Nigeria

2019 ◽  
pp. 1-11
Author(s):  
M. O. Oniya ◽  
A. L. Adeyekun
Keyword(s):  
Standard Procedure ◽  
Habitat Destruction ◽  
Species Level ◽  
Ultraviolet B ◽  
Pesticide Use ◽  
Amphibian Species ◽  
Ultraviolet B Radiation ◽  
Nematode Parasites ◽  
Ondo State ◽  
Cosmocerca Ornata

The research was designed to investigate the parasitic fauna of Anurans from cocoa farms in Ondo state. Amphibians are one of the most threatened groups of vertebrates. Many reasons are attributed to the decline of amphibian species such as global warming, habitat destruction and modification, others include: exploitation, pesticide use, introduced species, ultraviolet-B radiation (UV-B), pollution, parasites and diseases. A total of 31 frogs from 4 genera, Hemisus, Ptychadena, Rana and Xenopus and 7 toads from 1 genus, Sclerophrys were examined. 9 frogs were collected from Oluwateru farm at Iwoye Village; 7 frogs were collected from Folorunso farm at Ako-Igbatoro and 15 frogs from Obodulu farm in Idanre. 5 toads were collected from Oluwateru farm at Iwoye Village and 2 toads from Obodulu farm in Idanre. In all cases collection was done between 20:00 hrs and 05:00 hrs. Collected specimens were transported in sealed but ventilated containers to the laboratory where identification was done to species level. The frogs were anaesthetized until death in absolute chloroform soaked in cotton wool placed inside kill-jar for 3 minutes in the laboratory.  The gastrointestinal tracts were cut open and the contents of the various sections were put into separate Petri dishes containing normal saline.  The skin and the bladder were observed directly under a dissecting microscope for the presence of cysts and monogeneans. The parasites were fixed and preserved in 70% alcohol following standard procedure. Parasites recovered from the gastrointestinal tracts of the anurans include Cosmocerca ornata, Deising, 1861, Cosmocerca cummutata, Diesing, 1851 Paracosmocerca mucronata, Kung and Wu, 1945, Ampliceacum africanum, Taylor, 1924, Gendria liberrei Bain and Philipon, 1969 and Chenospirura asturi Hsu, 1957 Others were Procamallus brevis Kung, 1948 and Camallanus dimitrovi   Durette- Desset and Batcharov, 1974. Some of the parasites are zoonotic while a few others are established parasites of African fishes and water Birds raising probable public health concerns from the findings. Further works aimed at unravelling the biodiversity of hosts and parasites in the lush ecosystem of Ondo state, as well as identification of organisms involved in the life cycle are noted.

Isoflavones in aglycone solution enhance ultraviolet B-induced DNA damage repair efficiency

2014 ◽  
Vol 39 (3) ◽  
pp. 391-394 ◽  
Author(s):  
B. Iovine ◽  
M. Garofalo ◽  
M. Orefice ◽  
V. Giannini ◽  
F. Gasparri ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Ultraviolet B ◽  
Damage Repair ◽  
Repair Efficiency

Correlated trait response: comparing amphibian defense strategies across a stress gradient

2009 ◽  
Vol 87 (1) ◽  
pp. 41-49 ◽  
Author(s):  
T. S. Garcia ◽  
D. J. Paoletti ◽  
A. R. Blaustein
Keyword(s):  
Multiple Stressors ◽  
Species Difference ◽  
Elevation Gradient ◽  
Stress Gradient ◽  
Ultraviolet B ◽  
Correlated Response ◽  
Body Color ◽  
Multiple Traits ◽  
Amphibian Species ◽  
Rana Aurora

Animals inhabiting complex environments often contend with multiple stressors that can select for conflicting responses. Individuals can mediate these conflicts by utilizing correlated responses across multiple traits. In aquatic habitats, larval amphibians often face conflicting, simultaneous pressures, such as ultraviolet-B (UV-B) radiation and predators. UV-B radiation and predation risk influence behavior and body color in many amphibian species, altering activity rates, refuge use, and coloration. When both UV-B and predators are present, individuals can avoid conflicts by coupling behavior with body color to form a correlated response. UV-B exposure rates vary along an elevation gradient, thus trait combinations may also vary. We quantified changes in activity rates and body color in two anuran species, the red-legged frog ( Rana aurora Baird and Girard, 1852) (low elevations) and the cascades frog ( Rana cascadae Slater, 1939) (high elevations), during exposure to predator chemical cues (rough-skinned newt, Taricha granulosa (Skilton, 1849)) and UV-B radiation. Rana aurora decreased activity in response to UV-B and became more cryptic over time, while R. cascadae coupled decreased activity rates in response to predators with dark body coloration to screen out UV-B. Both species responded with a correlated trait response, yet employed opposite strategies. This observed species difference may be reflective of differences in stress across habitats and availability of alternative defenses.

scholarly journals Exogenous damage causes cell DNA damage through mediated reactive oxygen levels

2019 ◽  
Vol 131 ◽  
pp. 01018
Author(s):  
Peiyan Guo ◽  
Ning Ma ◽  
Jingbo Shan ◽  
Techang Chen ◽  
Yujie Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Recognition Mechanism ◽  
Exogenous Dna ◽  
Damage Recognition ◽  
Damage Repair ◽  
Intracellular Ros ◽  
Accumulation Of Damage ◽  
Tumor Drugs

Many anti-tumor drugs can induce tumor apoptosis by increasing intracellular ROS. In the present study, we build a model which did not directly cause DNA damage, but simulated damage products. The model of this injury was transferred into the cell so that the cell’s damage recognition mechanism mistakenly recognized that its own DNA was damaged, which in turn induced a response. Based on this model, the damaged plasmids (exogenous DNA damage) were transferred into the cells and the amount of reactive oxygen in the cells was improved, and DNA damage of the cells was increased. Therefore, exogenous DNA damage can affect the accumulation of damage in cells by affecting the level of reactive oxygen species, which provides a reference for DNA damage repair research.

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