The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol z) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol z alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol z. Pol z is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol z is substantially lower than that of homologous B family pols a, d and «. Pol z is particularly error prone for substitutions in specific sequence con-texts and generates multiple single base errors clustered in short patches at a rate that is unprece-dented in comparison with other polymerases. The unique error specificity of pol z in vitro is consistent with Pol z-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex muta-tions observed here, indicates that pol z contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them

Serum metabolomic analysis of the effect of exercise on nonalcoholic fatty liver disease

Objective: Exercise benefits people with nonalcoholic fatty liver disease (NAFLD). The aim of this study was to identify a panel of biomarkers and to provide the possible mechanism for the effect of exercise on NAFLD patients via an untargeted mass spectrometry-based serum metabolomics study. Methods: NAFLD patients were classified randomly into a control group (n = 74) and a 6-month vigorous exercise (n = 68) group. Differences in serum metabolic profiles were analyzed using untargeted ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technology. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to validate the differences between these two groups, and altered metabolites were obtained by ANOVA (fold change >2, P < 0.05) and identified with the online database Metlin and an in-house database. Results: Metabolic profiling and multiple statistical analyses of the serum samples indicated significant differences between the NAFLD patients in the control and the 6-month vigorous exercise groups. Finally, 36 metabolites were identified between the control vs exercise groups. These metabolites were mainly associated with glycerophospholipid- and sphingolipid-related pathways. Conclusion: Our study demonstrates that glycerophospholipid and sphingolip id alterations may contribute to the mechanism underlying the effect of exercise on NAFLD patients. A LC-MS-based metabolomics approach has a potential value for screening exercise-induced biomarkers

A study on the correlation between hemoglobin concentration and the storage quality of suspended red blood cells prepared from the whole blood of Tibetan male residents

BackgroundPrevious studies reported that the blood of Tibetans living at different altitudes may vary slightly. There is evidence that the harsh environmental conditions at high altitudes, such as low pressure and hypoxia, may affect the morphology and hemorheology of red blood cells (RBCs). Hypoxia would increase the levels of hemoglobin ([Hb]) and hematocrit (Hct), potentially increasing blood hyperviscosity and compromising blood collection and transfusions. Therefore, it is critical to investigate the in vitro storage quality of Tibetan RBCs.ObjectivesIn this study, the in vitro quality of suspended RBCs (SRBCs) prepared from whole blood (WB) of Tibetan residents with varying Hb concentrations ([Hb]) was measured during storage, and the relationship between the major factors in RBC storage and [Hb] was studied.Materials and methodsThe WB of Tibetan men was divided into three groups based on [Hb] levels (group A: 120 &lt; Hb ≤ 185 g/L; group B: 185 &lt; Hb ≤ 210 g/L; group C: Hb &gt; 210 g/L). The SRBCs prepared from WB were examined aseptically on days 1, 14, 21, and 35 after storage.Results[Hb] was not correlated with mean corpuscular volume (MCV), adenosine triphosphate (ATP), pH, P50, and hemolysis. There was a moderate or strong negative association between platelets (PLT) and [Hb] from days 1 to 35, and the PLT number of group C was lower than group A during storage. Group C had the highest change rates of electrolytes, glucose, and lactate, and there were moderate or strong positive correlations between lactate and [Hb] (r = 0.3772, p = 0.0045), glucose and [Hb] (r = 0.5845, p &lt; 0.0001), Na+ and [Hb] (r = 0.3966, p = 0.0027), and K+ and [Hb] (r = 0.4885, p = 0.0002). Group B had the highest change rates of 2,3-DPG on day 35, and there was a negative correlation between 2,3-DPG and [Hb] (r = −0.4933, p = 0.0001).ConclusionsThese new data on the [Hb] could have implications for researchers wishing to study the storage quality of Tibetan SRBCs, particularly in the context of erythrocyte metabolism, and we propose finding a new, suitable alternative solution for plateau SRBCs, particularly the blood with [Hb] greater than 185 g/L. Our results could have important implications for researchers wishing to study the potential framework of high-altitude-induced SRBC storage lesions

Structural insight into the substrate specificity of DNA polymerase μ.

DNA polymerase l (Pol l) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol l&apos;s unusual substrate specificity, we describe the 2.4 Å crystal structure of the polymerase domain of murine Pol l bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol l that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol l from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3¢ ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases. To accomplish the many DNA transactions involved in stably maintaining and replicating large genomes, mammals encode 16 DNA polymerases classified in families A, B, X and Y 1 . Clues to their functions include their substrate specificity, which can differ substantially, even for members of the same family. For example, two family X enzymes with intrinsic 5¢-deoxyribose 5¢-phosphate (dRP) lyase activities, DNA polymerase b (Pol b) and Pol l, are template dependent and efficiently fill short gaps, consistent with synthesis during short patch base excision repair 2,3 . Unlike Pol b, Pol l has a BRCT domain and probably participates in NHEJ of double-strand breaks (DSBs) in DNA 4-6 . Specifically, Pol l has a role in V(D)J recombination at immunoglobulin heavy-chain loci Pol m has an unusual primer-template specificity. Like Pol b and Pol l, Pol m can fill short gaps in a template-dependent manner 12 , yet it also shares with TdT the ability to catalyze template-independent synthesis 13 . Pol m has an unusually high capacity to extend misaligned primer termini 14 ; it can perform translesion synthesis (TLS) in vitro To test the relationship between substrate specificity and physiological function, one can compare structures of these polymerases bound to primer-templates. Structures already exist for Pol b 19-22 , Pol l 23-26 and TdT 27 , but not yet for Pol m. Here we fill this knowledge gap by describing a 2.4-Å crystal structure of a ternary complex of the polymerase domain of murine Pol m. This structure reveals substrate interactions unique to Pol m, compared with Pol b, Pol l and TdT. To test whether such differences are important for unusual substrate use by Pol m, we examined the properties of the wild-type enzyme and an H329A mutant that perturbs interactions with the DNA that are not found in Pol b and Pol l. A similar substitution was performed on the homologous histidine (H342A) in human TdT. These mutations were made to test whether the histidine is important for templateindependent synthesis and for template-dependent synthesis with substrates lacking a template nucleotide at the 3¢ primer terminus. RESULTS Overall structure of a ternary Pol l-DNA-ddTTP complex The polymerization domain (Pro132-Ala496) of murine Pol m was crystallized in a ternary complex with a gapped template-primer and a correctly paired nucleoside triphosphate bound in the nascent base pair-binding pocket. The DNA contained an 11-nucleotide (nt

An In Vitro Model of the Glomerular Capillary Wall Using Electrospun Collagen Nanofibres in a Bioartificial Composite Basement Membrane

The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease

siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells.</p> <p>Methods</p> <p>siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied <it>in vivo </it>by injection of the siRNA-transfected breast cancer cells into nude mice.</p> <p>The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined <it>in vitro </it>by MTT assay, FACS and SA-β-galactosidase staining.</p> <p>Results</p> <p>The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. <it>In vivo</it>, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 μM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 μM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments.</p> <p>Conclusion</p> <p>The study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer. When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.</p