Reduction in hypoxia-reoxygenation-induced myocardial mitochondrial damage with exogenous methane

Albeit previous experiments suggest potential anti-inflammatory effect of exogenous methane (CH₄) in various organs, the mechanism of its bioactivity is not entirely understood. We aimed to investigate the potential mitochondrial effects and the underlying mechanisms of CH₄ in rat cardiomyocytes and mitochondria under simulated ischaemia/reperfusion (sI/R) conditions. Three-day-old cultured cardiomyocytes were treated with 2.2% CH₄-artificial air mixture during 2-hour-long reoxygenation following 4-hour-long anoxia (sI/R and sI/R + CH₄, n = 6-6), with normoxic groups serving as controls (SH and SH + CH₄; n = 6-6). Mitochondrial functions were investigated with high-resolution respirometry, and mitochondrial membrane injury was detected by cytochrome c release and apoptotic characteristics by using TUNEL staining. CH₄ admixture had no effect on complex II (CII)-linked respiration under normoxia but significantly decreased the complex I (CI)-linked oxygen consumption. Nevertheless, addition of CH₄ in the sI/R + CH4 group significantly reduced the respiratory activity of CII in contrast to CI and the CH₄ treatment diminished mitochondrial H₂O₂ production. Substrate-induced changes to membrane potential were partially preserved by CH₄, and additionally, cytochrome c release and apoptosis of cardiomyocytes were reduced in the CH₄-treated group. In conclusion, the addition of CH₄ decreases mitochondrial ROS generation via blockade of electron transport at CI and reduces anoxia-reoxygenation-induced mitochondrial dysfunction and cardiomyocyte injury in vitro.     

Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

The electrostatic potential in the secondary quinone (Q_B) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to Q_B. It is controlled by the ionization states of residues in a strongly interacting cluster around the Q_B site. Reduction of the Q_B induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the Q_B binding pocket. It binds to the Q_B site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the Q_B pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the Q_B site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the Q_B site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.     

Relationship between Fulton's condition factor and proximate body composition in three freshwater fish species

Morphometric‐based condition indices are widely used to assess proximate body composition and, collaterally, feeding and living conditions of fish. However, the exact relationship between condition indices and proximate body composition of fish and its relatedness to life history traits and seasonality has yet to be fully explored. Therefore, the aim of this study was to examine how the Fulton's condition factor (K‐factor) is related to the chemical composition (i.e. lipid, protein, water and carbon content, and molar carbon : nitrogen ratio), length and gonadal development of fish, and how these relationships are influenced by gender and seasonality in three freshwater fish species: Amur sleeper (Perccottus glenii), pumpkinseed (Lepomis gibbosus) and rudd (Scardinius erythrophthalmus). It was found that the strength and direction of association between the K‐factor and proximate body composition can vary markedly among fish species. The K‐factor correlated positively with gonadal development in pumpkinseed and Amur sleeper, while no such relationship existed in rudd. Condition factor can be a reliable measure of lipid content; however, the relationship was stronger in species with higher and more variable lipid contents. Moreover, a striking and consistently negative linkage was found between the K‐factor and water content of the fish body, which corresponds to the findings of several other studies. In turn, any relationship between the K‐factor and the protein content of fish was not detected. Gender seemed to exert a negligible effect on the relationship between the K‐factor and proximate body composition, while seasonal variance was obvious in most relationships.     

Scavenging behaviour and size-dependent carcass consumption of the black bullhead (Ameiurus melas)

This study examined the size-dependent scavenging behaviour of black bullheads Ameiurus melas under laboratory conditions, using common bleak Alburnus alburnus and pumpkinseed Lepomis gibbosus carcasses. Video camera observations showed that the activity of A. melas was higher at night, but substantial daytime activity was also recorded. Larger A. melas were more active than their smaller conspecifics, especially at night. All size classes exhibited a well-defined sequence of consuming different parts of the carcasses independent of size, but larger individuals tended to consume carcasses more efficiently. Carcasses of the softer-bodied A. alburnus were consumed more readily than those of the bonier L. gibbosus, independent of size. This scavenging behaviour of A. melas might facilitate the invasion success of the species.     

Oral phosphatidylcholine pretreatment alleviates the signs of experimental rheumatoid arthritis

Introduction  

Phosphatidylcholine and phosphatidylcholine-derived metabolites exhibit anti-inflammatory properties in various stress conditions. We hypothesized that dietary phosphatidylcholine may potentially function as an anti-inflammatory substance and may decrease inflammatory activation in a chronic murine model of rheumatoid arthritis (collagen-induced arthritis).     

Selection against genetic defects in conservation schemes while controlling inbreeding

We studied different genetic models and evaluation systems to select against a genetic disease with additive, recessive or polygenic inheritance in genetic conservation schemes. When using optimum contribution selection with a restriction on the rate of inbreeding (ΔF) to select against a disease allele, selection directly on DNA-genotypes is, as expected, the most efficient strategy. Selection for BLUP or segregation analysis breeding value estimates both need 1–2 generations more to halve the frequency of the disease allele, while these methods do not require knowledge of the disease mutation at the DNA level. BLUP and segregation analysis methods were equally efficient when selecting against a disease with single gene or complex polygene inheritance, i.e. knowledge about the mode of inheritance of the disease was not needed for efficient selection against the disease. Smaller schemes or schemes with a more stringent restriction on ΔF needed more generations to halve the frequency of the disease alleles or the fraction of diseased animals. Optimum contribution selection maintained ΔF at its predefined level, even when selection of females was at random. It is argued that in the investigated small conservation schemes with selection against a genetic defect, control of ΔF is very important.     

PNU-91325 increases fatty acid synthesis from glucose and mitochondrial long chain fatty acid degradation: a comparative tracer-based metabolomics study with rosiglitazone and pioglitazone in HepG2 cells

The mitochondrial membrane protein termed “mitoNEET,” is a putative secondary target for insulin-sensitizing thiazolidinedione (TZD) compounds but its role in regulating metabolic flux is not known. PNU-91325 is a thiazolidinedione derivative which exhibits high binding affinity to mitoNEET and lowers cholesterol, fatty acid and blood glucose levels in animal models. In this study we report the stable isotope-based dynamic metabolic profiles (SIDMAP) of rosiglitazone, pioglitazone and PNU-91325 in a dose-matching, dose-escalating study. One and 10 μM concentrations 1 and 10 μM drug concentrations were introduced into HepG2 cells in the presence of either [1,2−¹³C₂]-D-glucose or [U−¹³C₁₈]stearate, GC/MS used to determine positional tracer incorporation (mass isotopomer analysis) into multiple metabolites produced by the Krebs and pentose cycles, de novo fatty acid synthesis, long chain fatty acid oxidation, chain shortening and elongation. Rosiglitazone and pioglitazone (10 μM) increased pentose synthesis from [U−¹³C₁₈]stearate by 127% and 185%, respectively, while PNU-91325 rather increased glutamate synthesis in the Krebs cycle by 113% as compared to control vehicle treated cells. PNU-91325 also increased stearate chain shortening into palmitate by 59%. Glucose tracer-derived de novo palmitate and stearate synthesis were increased by 1 and 10 μM rosiglitazone by 41% and 83%, respectively, and by 63% and 75% by PNU-91325. Stearate uptake was also increased by 10 μM PNU-91325 by 15.8%. We conclude that the entry of acetyl Co-A derived from long-chain fatty acid β-oxidation into the mitochondria is facilitated by the mitoNEET ligand PNU-91325, which increases glucose-derived long chain fatty acid synthesis and breakdown via β-oxidation and anaplerosis in the mitochondria.     

Metabolic profile and quantification of deoxyribose synthesis pathways in HepG2 cells

Traditional tracer studies of cell proliferation fail to distinguish between label enrichment due to increased DNA repair versus DNA replication. We used the emerging stable (non-radiating) isotope-based dynamic metabolic profiling technique on HepG2 cells to determine synthesis pathways of nucleic acids from glucose and rates of proliferation using CG-MS assay of RNA and DNA enrichment. Comparing the isotopic enrichment curve in DNA with the theoretical curve based on cell growth, we observed that the measured tracer enrichment was significantly higher, indicating that surplus label was acquired during DNA repair. In particular, after the first duplication (3 days), 80.13% of the total enrichment observed corresponds to duplication and 19.87% corresponds to DNA repair as calculated from the [1, 2-¹³C₂]-glucose incorporation curve. Our data indicate contemporary measurements of cell proliferation rates relying on tracer incorporation may be overestimated. ¹³C label was distributed between m1 (m1/Σm = 80) and m2 (m2/Σm = 14) of deoxyribose, indicating that most of the glucose carbon was acquired via direct glucose oxidation in the pentose cycle. The stable isotope technique distinguishes rates of DNA synthesis and repair via the oxidative and non-oxidative pentose cycle, separately, in one test, without inhibition of either process. The contribution of DNA repair in malignant cells to isotope accumulation in deoxyribose remains to be investigated.