Production of Recombinant Human DNA Polymerase Delta in a Bombyx mori Bioreactor

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability

P50, the Small Subunit of DNA Polymerase Delta, Is Required for Mediation of the Interaction of Polymerase Delta Subassemblies with PCNA

Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits

Characterization of Biomechanical Properties of Mitral Valve Chordae Tendineae

Objective: Mitral regurgitation and prolapse are the two most common diseases of the mitral valve and result in the leakage of blood back into the left atrium during systole. Rupture of the chordae tendinae is the most common cause of mitral insufficiency. The goal of this study was to characterize the biomechanical properties of the mitral valve chordae tendineae of aged human and ovine hearts. Materials and Methods: A total of 115 chordae specimens from fresh ovine hearts (n=18, weight = 374.833 47.947g, age of 1-2 year old), and 152 from human hearts (n=14, weight = 516.538 125.718g, mean age of 76.29 10.35 years old) were subjected to uniaxial tensile tests using marker tracking technology. The elastic and failure properties of five types of chordae tendineae were characterized, namely the anterior strut, anterior marginal, anterior basal, posterior marginal, and posterior basal chordae. The elastic properties were fitted with the nonlinear hyperelastic Ogden material model. The microstructure of the chordae samples was assessed through histology. Results: Human anterior basal and strut chords were significantly larger than the corresponding ovine chords. The mechanical properties of human chords were all similar while there were variations in the mechanical properties among ovine chords of differing type. The human chords were significantly stiffer and less extensible, yet stronger, than the corresponding ovine chords. These findings can be explained by histology results: collagen fibers in the human chords were nearly straight which resulted in reduced extensibility, while the collagen fibers in the ovine chords were highly crimped which resulted in high extensibility. Conclusions: Aged human and ovine chordae tendinae have significantly different structure and material properties

doi: 10.1093/jpe/rtn005 Advanced Access published

available online at www.jpe.oxfordjournals.org GeoSVM: an efficient and effective tool to predict species ’ potential distribution

EFFECT OF NUMBER OF CROWNS ON THE CRUSH RESISTANCE IN OPEN-CELL STENT DESIGN

10.2140/jomms.2020.15.75JOURNAL OF MECHANICS OF MATERIALS AND STRUCTURES15175-8

Measurement of the Luminal Diameter of Peripheral Arterial Vasculature in YorkshirexLandrace Swine by Using Ultrasonography and Angiography

10.30802/AALAS-JAALAS-19-000153JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE594438-44