Entry of [(1,2-13C2)acetyl]-L-carnitine in liver tricarboxylic acid cycle and lipogenesis: a study by 13C NMR spectroscopy in conscious, freely moving rats.

The biochemical pathways involved in acetyl-L-carnitine utilization were investigated in conscious, freely moving rats by 13C NMR spectroscopy. Following 4-h [(1,2-13C2)acetyl]-L-carnitine infusion in fasted animals, the free carnitine levels in serum were increased, and an efflux of unlabelled acetyl-L-carnitine from tissues was observed. [(1,2-13C2)Acetyl]-L-carnitine was found to enter biosynthetic pathways in liver, and the acetyl moiety was incorporated into both cholesterol and 3-hydroxybutyrate carbon skeleton. In accord with the entry of [(1,2-13C2)acetyl]-L-carnitine in the mitochondrial acetylCoA pool associated with tricarboxylic acid cycle, the 13C label was also found in liver glutamate, glutamine, and glutathione. The analysis of the 13C-labelling pattern in 3-hydroxybutyrate and cholesterol carbon skeleton provided evidence that the acetyl-L-carnitine-derived acetylCoA pool used for ketone bodies synthesis in mitochondria was homogeneous, whereas cholesterol was synthesized from two different acetylCoA pools located in the extra- and intramitochondrial compartment, respectively. Furthermore, cholesterol molecules were shown to be preferentially synthesized by the metabolic route involving the direct channelling of CoA-activated mitochondria-derived ketone bodies into 3-hydroxy-3-methylglutarylCoA pathway, prior to equilibration of their acyl groups with extramitochondrial acetylCoA pool via acetoacetylCoA thiolase.     

Tryptophan environments in glutathione transferase of human placenta from temperature-dependent phosphorescence studies

An investigation of the tryptophan emission properties of glutathione transferase from human placenta was conducted in order to characterize the environments of the two aromatic residues. The low-temperature phosphorescence spectra and temperature dependence of the phosphorescence quantum yield of the tryptophan residues revealed a difference in the chemical nature and dynamical structure of the surrounding protein matrix. Thus, one tryptophan residue seems to be deeply embedded within the polypeptide in a rigid weakly polar environment, characteristic of a beta-type secondary structure. The other is located in a more polar site, probably near the surface, in a rather flexible region of the macromolecule. At high temperature, the heterogeneity in the triplet lifetime of the internal residue attests to the presence of multiple conformers which are not in rapid equilibrium in the phosphorescence time scale. The anisotropy of the phosphorescence emission of glutathione transferase indicates that no energy transfer occurs between the two residues, and measurement of the rotational correlation time yields an hydrodynamic volume which is in good agreement with the molecular weight reported in the literature for the dimer.     

Glutathione redox cycle enzyme activities in erythrocytes of multiple sclerosis patients

The erythrocytes of multiple sclerosis patients with elevated superoxide dismutase levels were tested for the activities of glutathione redox cycle enzymes. No differences were observed between multiple sclerosis and normal control erythrocytes when the activities were referred to either hemoglobin concentration or lactate dehydrogenase content. Our results indicate that no adaptative changes occur in the activities of glutathione redox cycle enzymes in erythrocytes of multiple sclerosis subjects as a consequence of an elevated superoxide dismutase level.     

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl- choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μ M 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μ M TDGA CPT activity dropped more rapidly than between 10 and 40 μ M. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl- dl - aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

Le ionophore A23187

The acrosomic status of spermatozoa prepared for IVF has been evaluated by means of immunofluorescence test from Fenichel and Hsi using calcium A 23187 ionophore as inductor of acrosome reaction (AR). The spontaneous AR remains slight, even after 6 hour-incubation in Menezo B2 (6,8+2,7%). The response to ionophore, moderate before (11,2+9%), frankly increases after a 6h-capacitation (28,9+8,3%) in a group of 25 IVF couples (tubal indication, normal semen, positive fertilization). Nevertheless, it remains slight or null in 4 cases of unexplained repeated failure of fertilization. The response to ionophore A 23187 allows to explore the kinetics of capacitation of spermatozoa and their ability to perform AR. Its significance in terms of fecondance remains to be precised.     

A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate.

Mutants of sperm whale myoglobin were constructed at position 29 (B10 in helix notation) to examine the effects of distal pocket size on the rates of ligand binding and autooxidation. Leu29 was replaced with Ala, Val, and Phe using the synthetic gene and Escherichia coli expression system of Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8961-8965). Structures of the ferric forms of Val29 and Phe29, and the oxy form of Phe29 myoglobin were determined to 1.7 A by x-ray crystallography. The ferric mutant proteins are remarkably isomorphous with the wild type protein except in the immediate vicinity of residue 29. Thus, the protein structure in the distal pocket of myoglobin can accommodate either a large "hole" (i.e. Ala or Val) or a large side chain (i.e. Phe) at position 29 without perturbation of tertiary structure. Phe29 oxymyoglobin is also identical to the native oxy protein in terms of overall structure and interactions between the bound O2 and His64, Val68, Phe43, and Ile107. The distance between the nearest side chain atom of residue 29 and the second atom of the bound oxygen molecule is 3.2 A in the Phe29 protein and 4.9 A in native myoglobin. The equilibrium constants for O2 binding to Ala29, Val29, and Leu29 (native) myoglobin are the same, approximately 1.0 x 10(6) M-1 at 20 degrees C, whereas that for the Phe29 protein is markedly greater, 15 x 10(6) M-1. This increase in affinity is due primarily to a 10-fold decrease in the O2 dissociation rate constant for the Phe29 mutant and appears to be the result of stabilizing interactions between the negative portion of the bound O2 dipole and the partially positive edge of the phenyl ring. Increasing the size of residue 29 causes large decreases in the rate of autooxidation of myoglobin: k(ox) = 0.24, 0.23, 0.055, and 0.005 h-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, in air at 37 degrees C. Thus, the Leu29----Phe mutation produces a reduced protein that is remarkably stable and is expressed in E. coli as 100% MbO2. The selective pressure to conserve Leu29 at the B10 position probably represents a compromise between reducing the rate of autooxidation and maintaining a large enough O2 dissociation rate constant to allow rapid oxygen release during respiration.     

Behavioural and chemical mechanisms behind a Mediterranean ant–ant association

1. Interspecific competition among ants is common, and so is competitive exclusion among dominant ant species. In contrast, specific associations between non‐parasitic ant species are rare, especially in the temperate zones. As an exception, the subordinate ant Camponotus lateralis frequently co‐occurs with the dominant Crematogaster scutellaris but rarely with other dominant ants. 2. This association is one of various associations between Camponotus and Crematogaster species across the world. However, the mechanisms behind these co‐occurences are largely unknown. 3. In the present study, we therefore investigated the association of Ca. lateralis and Cr. scutellaris. We studied the spatial association of the nests, interspecific aggression, both species' cuticular hydrocarbon profiles, and their propensity to follow the other species' pheromone trails. 4. Crematogaster scutellaris usually attacked and displaced the generally submissive Ca. lateralis, but was significantly less aggressive at jointly used trails. Camponotus nests were always in close proximity to Crematogaster nests. 5. The cuticular hydrocarbons of both species consisted of alkanes with chain lengths between C21 and C35. The two species had 25 hydrocarbons in common, including mono‐, di‐, and tetramethyl alkanes. Despite this qualitative similarity, however, the quantitative hydrocarbon composition differed between the two species. 6. Camponotus lateralis followed artificial trails containing trail pheromones of Cr. scutellaris, but the latter did not follow Ca. lateralis trail pheromones. Interspecific trail‐following by Camponotus, but not vice versa, has been observed in another Camponotus–Crematogaster association and may be a more general mechanism that facilitates associations between the two ant genera.     

Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age

Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n = 40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.