Effects of diet enriched with restructured meats,containing Himanthalia elongata, on hypercholesterolaemic induction,CYP7A1 expression and antioxidant enzyme activity and expression in growing rats

Meat and pork consumptions are very high in Spain. Seaweeds are rich in fibre, minerals, and bioactive substances. Due to the growing demand for healthier meats, this work studied the effect of diets containing restructured pork (RP) enriched with Himanthalia elongata (Sea Spaghetti) on: (1) cholesterolaemia; (2) liver cytochrome P450 7A1 (CYP7A1) expression; (3) liver antioxidant enzyme activities and gene expression; (4) the liver antioxidant substrate concentrations. Four groups of 10 Wistar rats each were fed a mix of 85% AIN-93 M rodent diet and 15% freeze-dried RM for 35 days. The control group (C) consumed control RP; the Sea Spaghetti (SS) group, RP with 5% Sea Spaghetti. Animals on added cholesterol diets (CholC and CholSS) consumed their basal C and SS diets enriched with cholesterol and cholic acid as hypercholesterolaemic agent. Food intake was significantly affected by the alga × cholesterol interaction and by dietary cholesterol (both p < 0.001). Plasma cholesterol was significantly affected by the cholesterol × alga interaction (p < 0.05). CholC rats showed significantly higher plasma cholesterol (p < 0.001) than did their C counterparts, whilst serum cholesterol of CholSS was significantly lower (p < 0.05) than that of CholC. The glutathione peroxide (GSSG) concentrations and all mRNA expressions were significantly affected by the cholesterol × alga interaction (at least p < 0.05). SS vs C group showed significant (at least p < 0.05) increases in superoxide dismutase (Mn-SOD and Cu,Zn-SOD), glutathione peroxidase (GPx) and decrease of glutathione reductase (GR) expressions, and increased GR activity, GSSG and the redox index. CholSS vs CholC showed significant (at least p < 0.05) increases of CYP7A1, GR and Cu,Zn-SOD expression but decreases in catalase, Mn-SOD and GPx expression, and increase of GR activity. In conclusion, Sea Spaghetti could be widely used in RP design. Its addition to non-cholesterol enriched RP diet reduced oxidation mechanisms. SS-RP partially blocked the effect of the hypercholesterolaemic agent, giving rise to a new balance of the antioxidant enzyme expression.     

A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.     

Kinetics of the DNA polymerase pyrococcus kodakaraensis

The polymerase chain reaction is one of the most important reactions in molecular biology. Single stranded DNA is copied in a complex series of steps, at the core of which lies the action of the DNA polymerase. At each nucleotide along the template, the polymerase screens the dNTP pool until it finds the complementary dNTP. The insertion of each dNMP is a balance between high fidelity and rapid elongation. In this study the kinetics of the β type polymerase pyrococcus kodakaraensis (KOD) is analyzed. The kinetics is influenced by reaction conditions such as the dNTP pool composition and temperature. In a previous study by Viljoen et al. [2005, A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection. Computational Biology and Chemistry 29, 101-110], a macroscopic kinetics expression of the polymerase chain reaction has been derived. The model contains four parameters that are intrinsic to a specific polymerase. The experiments to measure the temperature-dependence of the parameters for KOD DNA polymerase are reported. The results indicate that the optimal temperature for an equimolar dNTP pool is and the optimum temperature shifts to lower temperatures when the dNTP pool composition is biased.     

Diallyl sulfides: Selective inhibitors of family X DNA polymerases from garlic (Allium sativum L.)

Diallyl sulfides, organosulfur compounds isolated from garlic (Allium sativum L.), selectively inhibit the activities of mammalian family X DNA polymerases (pols), such as pol β, pol λ and terminal deoxynucleotidyl transferase (TdT), in vitro. The purified fraction (i.e., Sample-A) consisted of diallyl trisulfide, diallyl tetrasulfide and diallyl pentasulfide (molecular ratio: 5.3:3:1). Commercially purchased diallyl sulfides also inhibited the activities of family X pols, and the order of their effect was as follows: Sample-A > diallyl trisulfide > diallyl disulfide > diallyl monosulfide, suggesting that the number of sulfur atoms in the compounds might play an important structural role in enzyme inhibition. The suppression of human cancer cell (promyelocytic leukaemia cell line, HL-60) growth had the same tendency as the inhibition of pol X family among the compounds. Diallyl sulfides were suggested to bind to the pol β-like region of family X pols.     

Varied active-site constraints in the klenow fragment of E. coli DNA polymerase I and the lesion-bypass Dbh DNA polymerase

We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.