Significant impact of divalent metal ions on the fidelity,sugar selectivity,and drug incorporation efficiency of human PrimPol

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979 nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4 × 10⁻² to 3.8 × 10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57–1800 with Mn²⁺ and 150–4500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).     

Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

Contrasting patterns of density‐dependent selection at different life stages can create more than one fast–slow axis of life‐history variation

There has been much recent research interest in the existence of a major axis of life‐history variation along a fast–slow continuum within almost all major taxonomic groups. Eco‐evolutionary models of density‐dependent selection provide a general explanation for such observations of interspecific variation in the "pace of life." One issue, however, is that some large‐bodied long‐lived “slow” species (e.g., trees and large fish) often show an explosive “fast” type of reproduction with many small offspring, and species with “fast” adult life stages can have comparatively “slow” offspring life stages (e.g., mayflies). We attempt to explain such life‐history evolution using the same eco‐evolutionary modeling approach but with two life stages, separating adult reproductive strategies from offspring survival strategies. When the population dynamics in the two life stages are closely linked and affect each other, density‐dependent selection occurs in parallel on both reproduction and survival, producing the usual one‐dimensional fast–slow continuum (e.g., houseflies to blue whales). However, strong density dependence at either the adult reproduction or offspring survival life stage creates quasi‐independent population dynamics, allowing fast‐type reproduction alongside slow‐type survival (e.g., trees and large fish), or the perhaps rarer slow‐type reproduction alongside fast‐type survival (e.g., mayflies—short‐lived adults producing few long‐lived offspring). Therefore, most types of species life histories in nature can potentially be explained via the eco‐evolutionary consequences of density‐dependent selection given the possible separation of demographic effects at different life stages.     

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques

Summary Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO₂ and HCO ₃ ^– -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO₂ and HCO ₃ ^– -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.     

Hepatic DNAJB9 Drives Anabolic Biasing to Reduce Steatosis and Obesity

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